Evolution and Diversity of the Ras Superfamily of Small GTPases in Prokaryotes

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Wuichet, Kristin; Søgaard-Andersen, Lotte

2015-01-01

The Ras superfamily of small GTPases are single domain nucleotide-dependent molecular switches that act as highly tuned regulators of complex signal transduction pathways. Originally identified in eukaryotes for their roles in fundamental cellular processes including proliferation, motility, polarity, nuclear transport, and vesicle transport, recent studies have revealed that single domain GTPases also control complex functions such as cell polarity, motility, predation, development and antibiotic resistance in bacteria. Here, we used a computational genomics approach to understand the abundance, diversity, and evolution of small GTPases in prokaryotes. We collected 520 small GTPase sequences present in 17% of 1,611 prokaryotic genomes analyzed that cover diverse lineages. We identified two discrete families of small GTPases in prokaryotes that show evidence of three distinct catalytic mechanisms. The MglA family includes MglA homologs, which are typically associated with the MglB GTPase activating protein, whereas members of the Rup (Ras superfamily GTPase of unknown function in prokaryotes) family are not predicted to interact with MglB homologs. System classification and genome context analyses support the involvement of small GTPases in diverse prokaryotic signal transduction pathways including two component systems, laying the foundation for future experimental characterization of these proteins. Phylogenetic analysis of prokaryotic and eukaryotic GTPases supports that the last universal common ancestor contained ancestral MglA and Rup family members. We propose that the MglA family was lost from the ancestral eukaryote and that the Ras superfamily members in extant eukaryotes are the result of vertical and horizontal gene transfer events of ancestral Rup GTPases. PMID:25480683

The non-receptor tyrosine kinase Lyn controls neutrophil adhesion by recruiting the CrkL–C3G complex and activating Rap1 at the leading edge

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He, Yuan; Kapoor, Ashish; Cook, Sara; Liu, Shubai; Xiang, Yang; Rao, Christopher V.; Kenis, Paul J. A.; Wang, Fei

2011-01-01

Establishing new adhesions at the extended leading edges of motile cells is essential for stable polarity and persistent motility. Despite recent identification of signaling pathways that mediate polarity and chemotaxis in neutrophils, little is known about molecular mechanisms governing cell–extracellular-matrix (ECM) adhesion in these highly polarized and rapidly migrating cells. Here, we describe a signaling pathway in neutrophils that is essential for localized integrin activation, leading edge attachment and persistent migration during chemotaxis. This pathway depends upon Gi-protein-mediated activation and leading edge recruitment of Lyn, a non-receptor tyrosine kinase belonging to the Src kinase family. We identified the small GTPase Rap1 as a major downstream effector of Lyn to regulate neutrophil adhesion during chemotaxis. Depletion of Lyn in neutrophil-like HL-60 cells prevented chemoattractant-induced Rap1 activation at the leading edge of the cell, whereas ectopic expression of Rap1 largely rescued the defects induced by Lyn depletion. Furthermore, Lyn controls spatial activation of Rap1 by recruiting the CrkL–C3G protein complex to the leading edge. Together, these results provide novel mechanistic insights into the poorly understood signaling network that controls leading edge adhesion during chemotaxis of neutrophils, and possibly other amoeboid cells. PMID:21628423

Topological and functional properties of the small GTPases protein interaction network.

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Anna Delprato

Full Text Available Small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran regulate key cellular processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. A great deal of experimental evidence supports the existence of signaling cascades and feedback loops within and among the small GTPase subfamilies suggesting that these proteins function in a coordinated and cooperative manner. The interplay occurs largely through association with bi-partite regulatory and effector proteins but can also occur through the active form of the small GTPases themselves. In order to understand the connectivity of the small GTPases signaling routes, a systems-level approach that analyzes data describing direct and indirect interactions was used to construct the small GTPases protein interaction network. The data were curated from the Search Tool for the Retrieval of Interacting Genes (STRING database and include only experimentally validated interactions. The network method enables the conceptualization of the overall structure as well as the underlying organization of the protein-protein interactions. The interaction network described here is comprised of 778 nodes and 1943 edges and has a scale-free topology. Rac1, Cdc42, RhoA, and HRas are identified as the hubs. Ten sub-network motifs are also identified in this study with themes in apoptosis, cell growth/proliferation, vesicle traffic, cell adhesion/junction dynamics, the nicotinamide adenine dinucleotide phosphate (NADPH oxidase response, transcription regulation, receptor-mediated endocytosis, gene silencing, and growth factor signaling. Bottleneck proteins that bridge signaling paths and proteins that overlap in multiple small GTPase networks are described along with the functional annotation of all proteins in the network.

Stage-specific functions of the small Rho GTPases Cdc42 and Rac1 for adult hippocampal neurogenesis

DEFF Research Database (Denmark)

Vadodaria, Krishna C; Brakebusch, Cord; Suter, Ueli

2013-01-01

The molecular mechanisms underlying the generation, maturation, and integration of new granule cells generated throughout life in the mammalian hippocampus remain poorly understood. Small Rho GTPases, such as Cdc42 and Rac1, have been implicated previously in neural stem/progenitor cell (NSPC......) proliferation and neuronal maturation during embryonic development. Here we used conditional genetic deletion and virus-based loss-of-function approaches to identify temporally distinct functions for Cdc42 and Rac1 in adult hippocampal neurogenesis. We found that Cdc42 is involved in mouse NSPC proliferation......, initial dendritic development, and dendritic spine maturation. In contrast, Rac1 is dispensable for early steps of neuronal development but is important for late steps of dendritic growth and spine maturation. These results establish cell-autonomous and stage-specific functions for the small Rho GTPases...

The Ins and Outs of Small GTPase Rac1 in the Vasculature

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Marinković, Goran; Heemskerk, Niels; van Buul, Jaap D.; de Waard, Vivian

2015-01-01

The Rho family of small GTPases forms a 20-member family within the Ras superfamily of GTP-dependent enzymes that are activated by a variety of extracellular signals. The most well known Rho family members are RhoA (Ras homolog gene family, member A), Cdc42 (cell division control protein 42), and

Development and application of a quantitative multiplexed small GTPase activity assay using targeted proteomics.

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Zhang, Cheng-Cheng; Li, Ru; Jiang, Honghui; Lin, Shujun; Rogalski, Jason C; Liu, Kate; Kast, Juergen

2015-02-06

Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.

Nucleophosmin1 is a negative regulator of the small GTPase Rac1

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Zoughlami, Younes; van Stalborgh, Anne M.; van Hennik, Paula B.; Hordijk, Peter L.

2013-01-01

The Rac1 GTPase is a critical regulator of cytoskeletal dynamics and controls many biological processes, such as cell migration, cell-cell contacts, cellular growth and cell division. These complex processes are controlled by Rac1 signaling through effector proteins. We have previously identified

Targeting and localized signalling by small GTPases

NARCIS (Netherlands)

ten Klooster, Jean Paul; Hordijk, Peter L.

2007-01-01

Polarized cellular responses, for example, cell migration, require the co-ordinated assembly of signalling complexes at a particular subcellular location, such as the leading edge of cells. Small GTPases of the Ras superfamily play central roles in many (polarized) responses to growth factors,

RAP-1a is the main rhoptry-associated-protein-1 (RAP-1) recognized during infection with Babesia sp. BQ1 (Lintan) (B. motasi-like phylogenetic group), a pathogen of sheep in China.

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Niu, Qingli; Bonsergent, Claire; Rogniaux, Hélène; Guan, Guiquan; Malandrin, Laurence; Moreau, Emmanuelle

2016-12-15

Babesia sp. BQ1 (Lintan) is one of the parasites isolated from infected sheep in China that belongs to the B. motasi-like phylogenetic group. The rhoptry-associated-protein 1 (rap-1) locus in this group consists of a complex organization of 12 genes of three main types: 6 rap-1a variants intercalated with 5 identical copies of rap-1b and a single 3' ending rap-1c gene. In the present study, transcription analysis performed by standard RT-PCR demonstrated that the three different rap-1 gene types and the four rap-1a variants were transcribed by the parasite cultivated in vitro. Peptides, specific for each rap-1 type gene, were selected in putative linear B-epitopes and used to raise polyclonal rabbit antisera. Using these sera, the same expression pattern of RAP-1 proteins was found in parasites cultivated in vitro or collected from acute infection whereas only RAP-1a67 was detectable in merozoite extracts. However, ELISA performed with recombinant RAP-1a67, RAP-1b or RAP-1c and sera from infected sheep demonstrated that RAP-1a67 is the main RAP-1 recognized during infection, even if some infected sheep also recognized RAP-1b and/or RAP-1c. Copyright © 2016 Elsevier B.V. All rights reserved.

Leucine-rich repeat kinase-1 regulates osteoclast function by modulating RAC1/Cdc42 Small GTPase phosphorylation and activation.

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Zeng, Canjun; Goodluck, Helen; Qin, Xuezhong; Liu, Bo; Mohan, Subburaman; Xing, Weirong

2016-10-01

Leucine-rich repeat kinase-1 (Lrrk1) consists of ankyrin repeats (ANK), leucine-rich repeats (LRR), a GTPase-like domain of Roc (ROC), a COR domain, a serine/threonine kinase domain (KD), and WD40 repeats (WD40). Previous studies have revealed that knockout (KO) of Lrrk1 in mice causes severe osteopetrosis, and a human mutation of Lrrk1 leads to osteosclerotic metaphysial dysplasia. The molecular mechanism by which Lrrk1 regulates osteoclast function is unknown. In this study, we generated a series of Lrrk1 mutants and evaluated their ability to rescue defective bone resorption in Lrrk1-deficient osteoclasts by use of pit formation assays. Overexpression of Lrrk1 or LRR-truncated Lrrk1, but not ANK-truncated Lrrk1, WD40-truncated Lrrk1, Lrrk1-KD, or K651A mutant Lrrk1, rescued bone resorption function of Lrrk1 KO osteoclasts. We next examined whether RAC1/Cdc42 small GTPases are direct substrates of Lrrk1 in osteoclasts. Western blot and pull-down assays revealed that Lrrk1 deficiency in osteoclasts resulted in reduced phosphorylation and activation of RAC1/Cdc42. In vitro kinase assays confirmed that recombinant Lrrk1 phosphorylated RAC1-GST protein, and immunoprecipitation showed that the interaction of Lrrk1 with RAC1 occurred within 10 min after RANKL treatment. Overexpression of constitutively active Q61L RAC1 partially rescued the resorptive function of Lrrk1-deficient osteoclasts. Furthermore, lack of Lrrk1 in osteoclasts led to reduced autophosphorylation of p21 protein-activated kinase-1 at Ser 144 , catalyzed by RAC1/Cdc42 binding and activation. Our data indicate that Lrrk1 regulates osteoclast function by directly modulating phosphorylation and activation of small GTPase RAC1/Cdc42 and that its function depends on ANK, ROC, WD40, and kinase domains. Copyright © 2016 the American Physiological Society.

Different roles of the small GTPases Rac1, Cdc42, and RhoG in CALEB/NGC-induced dendritic tree complexity.

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Schulz, Jana; Franke, Kristin; Frick, Manfred; Schumacher, Stefan

2016-10-01

Rho GTPases play prominent roles in the regulation of cytoskeletal reorganization. Many aspects have been elaborated concerning the individual functions of Rho GTPases in distinct signaling pathways leading to cytoskeletal rearrangements. However, major questions have yet to be answered regarding the integration and the signaling hierarchy of different Rho GTPases in regulating the cytoskeleton in fundamental physiological events like neuronal process differentiation. Here, we investigate the roles of the small GTPases Rac1, Cdc42, and RhoG in defining dendritic tree complexity stimulated by the transmembrane epidermal growth factor family member CALEB/NGC. Combining gain-of-function and loss-of-function analysis in primary hippocampal neurons, we find that Rac1 is essential for CALEB/NGC-mediated dendritic branching. Cdc42 reduces the complexity of dendritic trees. Interestingly, we identify the palmitoylated isoform of Cdc42 to adversely affect dendritic outgrowth and dendritic branching, whereas the prenylated Cdc42 isoform does not. In contrast to Rac1, CALEB/NGC and Cdc42 are not directly interconnected in regulating dendritic tree complexity. Unlike Rac1, the Rac1-related GTPase RhoG reduces the complexity of dendritic trees by acting upstream of CALEB/NGC. Mechanistically, CALEB/NGC activates Rac1, and RhoG reduces the amount of CALEB/NGC that is located at the right site for Rac1 activation at the cell membrane. Thus, Rac1, Cdc42, and RhoG perform very specific and non-redundant functions at different levels of hierarchy in regulating dendritic tree complexity induced by CALEB/NGC. Rho GTPases play a prominent role in dendritic branching. CALEB/NGC is a transmembrane member of the epidermal growth factor (EGF) family that mediates dendritic branching, dependent on Rac1. CALEB/NGC stimulates Rac1 activity. RhoG inhibits CALEB/NGC-mediated dendritic branching by decreasing the amount of CALEB/NGC at the plasma membrane. Palmitoylated, but not prenylated form

Comparative phylogenetic and expression analysis of small GTPases families in legume and non-legume plants.

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Flores, Ana Claudia; Via, Virginia Dalla; Savy, Virginia; Villagra, Ulises Mancini; Zanetti, María Eugenia; Blanco, Flavio

2018-02-01

Small monomeric GTPases act as molecular switches in several processes that involve polar cell growth, participating mainly in vesicle trafficking and cytoskeleton rearrangements. This gene superfamily has largely expanded in plants through evolution as compared with other Kingdoms, leading to the suggestion that members of each subfamily might have acquired new functions associated to plant-specific processes. Legume plants engage in a nitrogen-fixing symbiotic interaction with rhizobia in a process that involves polar growth processes associated with the infection throughout the root hair. To get insight into the evolution of small GTPases associated with this process, we use a comparative genomic approach to establish differences in the Ras GTPase superfamily between legume and non-legume plants. Phylogenetic analyses did not show clear differences in the organization of the different subfamilies of small GTPases between plants that engage or not in nodule symbiosis. Protein alignments revealed a strong conservation at the sequence level of small GTPases previously linked to nodulation by functional genetics. Interestingly, one Rab and three Rop proteins showed conserved amino acid substitutions in legumes, but these changes do not alter the predicted conformational structure of these proteins. Although the steady-state levels of most small GTPases do not change in response to rhizobia, we identified a subset of Rab, Rop and Arf genes whose transcript levels are modulated during the symbiotic interaction, including their spatial distribution along the indeterminate nodule. This study provides a comprehensive study of the small GTPase superfamily in several plant species. The genetic program associated to root nodule symbiosis includes small GTPases to fulfill specific functions during infection and formation of the symbiosomes. These GTPases seems to have been recruited from members that were already present in common ancestors with plants as distant as monocots

Epidermal activation of the small GTPase Rac1 in psoriasis pathogenesis.

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Winge, Mårten C G; Marinkovich, M Peter

2017-01-05

The small GTPase Ras-related C3 botulinum toxin substrate 1 (RAC1) plays a central role in skin homeostasis, including barrier function, wound healing and inflammatory responses. Psoriasis is a common skin disease characterized by deregulation of these functions, and affected skin exhibit keratinocyte hyperproliferation, inflammation and immune cell infiltration. Although psoriasis is often triggered by environmental stimulus, there is a strong genetic association with genes expressed in both immune cells and keratinocytes, of which several are linked to Rac1 signaling. Rac1 is highly active in human psoriatic lesional skin and keratinocytes, and keratinocyte-specific overexpression of an activated mutant of Rac1, Rac1 V12 , in a transgenic mouse model closely mimics the presentation of human psoriasis. Both Rac1 activation in keratinocytes and immune derived stimulus are required to drive psoriasiform signaling in transgenic mouse and human xenograft models of psoriasis. Therefore, understanding how increased Rac1 activation in psoriatic epidermis is regulated is central to understanding how the abnormal crosstalk between keratinocytes and immune cells is maintained.

Inhibition of endothelial cell proliferation by targeting Rac1 GTPase with small interference RNA in tumor cells

International Nuclear Information System (INIS)

Xue Yan; Bi Feng; Zhang Xueyong; Pan Yanglin; Liu Na; Zheng Yi; Fan Daiming

2004-01-01

Hypoxia-induced angiogenesis plays an important role in the malignancy of solid tumors. A number of recent studies including our own have suggested that Rho family small GTPases are involved in this process, and Rac1, a prominent member of the Rho family, may be critical in regulating hypoxia-induced gene activation of several angiogenesis factors and tumor suppressors. To further define Rac1 function in angiogenesis and to explore novel approaches to modulate angiogenesis, we employed the small interference RNA technique to knock down gene expression of Rac1 in gastric cancer cell line AGS that expresses a high level of Rac1. Both the mRNA and protein levels of Rac1 in the AGS cells were decreased dramatically after transfection with a Rac1-specific siRNA vector. When the conditioned medium derived from the Rac1 downregulated AGS cells was applied to the human endothelial cells, it could significantly inhibit the cell proliferation. Further study proved that, VEGF and HIF-1α, two angiogenesis promoting factors, were found to be downregulated whereas p53 and VHL, which are tumor suppressors and angiogenesis inhibitors, were upregulated in the Rac1 siRNA transfected cells. Our results suggest that Rac1 may be involved in angiogenesis by controlling the expression of angiogenesis-related factors and provide a possible strategy for the treatment of tumor angiogenesis by targeting the Rac1 GTPase

Maturation and integration of adult born hippocampal neurons: signal convergence onto small Rho GTPases

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Krishna eVadodaria

2013-08-01

Full Text Available Adult neurogenesis, restricted to specific regions in the mammalian brain, represents one of the most interesting forms of plasticity in the mature nervous system. Adult-born hippocampal neurons play important roles in certain forms of learning and memory, and altered hippocampal neurogenesis has been associated with a number of neuropsychiatric diseases such as major depression and epilepsy. Newborn neurons go through distinct developmental steps from a dividing neurogenic precursor to a synaptically integrated mature neuron. Previous studies have uncovered several molecular signaling pathways involved in distinct steps of this maturational process. In this context, the small Rho GTPases, Cdc42, Rac1 and RhoA have recently been shown to regulate the morphological and synaptic maturation of adult-born dentate granule cells in vivo. Distinct upstream regulators, including several growth factors that modulate maturation and integration of newborn neurons have been shown to also recruit the small Rho GTPases. Here we review recent findings and highlight the possibility that small Rho GTPases may act as central assimilators, downstream of critical input onto adult-born hippocampal neurons contributing to their maturation and integration into the existing dentate gyrus circuitry.

Nucleotide sequence of a human cDNA encoding a ras-related protein (rap1B)

Energy Technology Data Exchange (ETDEWEB)

Pizon, V; Lerosey, I; Chardin, P; Tavitian, A [INSERM, Paris (France)

1988-08-11

The authors have previously characterized two human ras-related genes rap1 and rap2. Using the rap1 clone as probe they isolated and sequenced a new rap cDNA encoding the 184aa rap1B protein. The rap1B protein is 95% identical to rap1 and shares several properties with the ras protein suggesting that it could bind GTP/GDP and have a membrane location. As for rap1, the structural characteristics of rap1B suggest that the rap and ras proteins might interact on the same effector.

Rho GTPases and cancer

DEFF Research Database (Denmark)

Li, Hui; Peyrollier, Karine; Kilic, Gülcan

2014-01-01

Rho GTPases are a family of small GTPases, which play an important role in the regulation of the actin cytoskeleton. Not surprisingly, Rho GTPases are crucial for cell migration and therefore highly important for cancer cell invasion and the formation of metastases. In addition, Rho GTPases...... are involved in growth and survival of tumor cells, in the interaction of tumor cells with their environment, and they are vital for the cancer supporting functions of the tumor stroma. Recent research has significantly improved our understanding of the regulation of Rho GTPase activity, the specificity of Rho...

Small GTPases are involved in sprout formation in human granulosa lutein cells.

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Franz, Maximilian B; Daube, Stefanie; Keck, Christoph; Sator, Michael; Pietrowski, Detlef

2013-04-01

The corpus luteum (CL), develops from the ruptured follicle after gonadotropin stimulation. Based on intracellular reorganization of the cytoskeleton an human chorionic gonadotropin (hCG) dependent sprouting and migration of luteinizing granulosa cells (LGCs) and endothelial cells is observed. Rho-GTPases are shown to be key regulators of cytoskeletal restructuring. In the present study we analyzed the role of Rho-GTPases in the sprouting activity of LGCs. We used the Rho-GTPase-inhibitors Toxin A and -B and the Cdc42-activator Bradykinin in a LGC-spheroid sprouting assay to determine the effect of these modulators in LGCs. Toxin A and Toxin B reduces sprout formation in LGC spheroids. However, the reduction is less than in hCG treated cells. The usage of Bradykinin demonstrates both, a reduction of sprouts in untreated spheroids and an increase of sprouting in previous hCG treated spheroids. The presented results let us suggest that small Rho-GTPases may regulate the sprouting activity of LGCs after stimulation by hCG and that this mechanism may play a role in CL formation.

Effect of Rap1 binding on DNA distortion and potassium permanganate hypersensitivity.

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Le Bihan, Yann-Vaï; Matot, Béatrice; Pietrement, Olivier; Giraud-Panis, Marie-Josèphe; Gasparini, Sylvaine; Le Cam, Eric; Gilson, Eric; Sclavi, Bianca; Miron, Simona; Le Du, Marie-Hélène

2013-03-01

Repressor activator protein 1 (Rap1) is an essential factor involved in transcription and telomere stability in the budding yeast Saccharomyces cerevisiae. Its interaction with DNA causes hypersensitivity to potassium permanganate, suggesting local DNA melting and/or distortion. In this study, various Rap1-DNA crystal forms were obtained using specifically designed crystal screens. Analysis of the DNA conformation showed that its distortion was not sufficient to explain the permanganate reactivity. However, anomalous data collected at the Mn edge using a Rap1-DNA crystal soaked in potassium permanganate solution indicated that the DNA conformation in the crystal was compatible with interaction with permanganate ions. Sequence-conservation analysis revealed that double-Myb-containing Rap1 proteins all carry a fully conserved Arg580 at a position that may favour interaction with permanganate ions, although it is not involved in the hypersensitive cytosine distortion. Permanganate reactivity assays with wild-type Rap1 and the Rap1[R580A] mutant demonstrated that Arg580 is essential for hypersensitivity. AFM experiments showed that wild-type Rap1 and the Rap1[R580A] mutant interact with DNA over 16 successive binding sites, leading to local DNA stiffening but not to accumulation of the observed local distortion. Therefore, Rap1 may cause permanganate hypersensitivity of DNA by forming a pocket between the reactive cytosine and Arg580, driving the permanganate ion towards the C5-C6 bond of the cytosine.

miR-518b Enhances Human Trophoblast Cell Proliferation Through Targeting Rap1b and Activating Ras-MAPK Signal

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Ming Liu

2018-03-01

Full Text Available Preeclampsia is a pregnancy-specific complication defined as newly onset gestational hypertension and proteinuria. Deficiency in placental development is considered as the predominant cause of preeclampsia. Our previous study found that the expression of miR-518b increased significantly in the preeclamptic placentas, indicating the potential participation of this small RNA in the occurrence of preeclampsia. In this study, data analysis using multiple databases predicted Rap1b as a candidate target of miR-518b. An evident decrease in Rap1b expression was observed in preeclamptic placentas when compared with the control placentas, which was negatively correlated with the level of miR-518b. Based on the data of in situ hybridization and immunohistochemistry showing that Rap1b exhibited similar localization with miR-518b in villous cytotrophoblast cells and column trophoblasts, we further explored their function in regulating trophoblast cell proliferation. In HTR8/SVneo cells, exogenous transfection of miR-518b reduced the expression of Rap1b, and dual-luciferase reporter assay validated Rap1b as the direct target of miR-518b. The small RNA could increase the BrdU incorporation and the ratio of cells at S phase, and enhance the phosphorylation of Raf-1 and ERK1/2. Such growth-promoting effect could be efficiently reversed by Rap1b overexpression. The data indicate that miR-518b can promote trophoblast cell proliferation via Rap1b–Ras–MAPK pathway, and the aberrant upregulation of miR-518b in preeclamptic placenta may contribute to the excessive trophoblast proliferation. The study provides new evidence to further understand the etiology of preeclampsia.

Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication.

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Greicy H Goto

2015-08-01

Full Text Available Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.

Sequence and organization of the rhoptry-associated-protein-1 (rap-1) locus for the sheep hemoprotozoan Babesia sp. BQ1 Lintan (B. motasi phylogenetic group).

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Niu, Qingli; Bonsergent, Claire; Guan, Guiquan; Yin, Hong; Malandrin, Laurence

2013-11-15

Babesiosis is a frequent infection of animals worldwide by tick borne pathogen Babesia, and several species are responsible for ovine babesiosis. Recently, several Babesia motasi-like isolates were described in sheep in China. In this study, we sequenced the multigenic rap-1 gene locus of one of these isolates, Babesia sp. BQ1 Lintan. The RAP-1 proteins are involved in the process of red blood cells invasion and thus represent a potential target for vaccine development. A complex composition and organization of the rap-1 locus was discovered with: (1) the presence of 3 different types of rap-1 sequences (rap-1a, rap-1b and rap-1c); (2) the presence of multiple copies of rap-1a and rap-1b; (3) polymorphism among the rap-1a copies, with two classes (named rap-1a61 and rap-1a67) having a similarity of 95.7%, each class represented by two close variants; (4) polymorphism between rap-1a61-1 and rap-1a61-2 limited to three nucleotide positions; (5) a difference of eight nucleotides between rap-1a67-1 and rap-1a67-2 from position 1270 to the putative stop site of rap-1a67-1 which might produce two putative proteins of slightly different sizes; (6) the ratio of rap-1a copies corresponding to one rap-1a67, one rap-1a61-1 and one rap-1a61-2; (7) the presence of three different intergenic regions separating rap-1a, rap-1b and rap-1c; (8) interspacing of the rap-1a copies with rap-1b copies; and (9) the terminal position of rap-1c in the locus. A 31kb locus composed of 6 rap-1a sequences interspaced with 5 rap-1b sequences and with a terminal rap-1c copy was hypothesized. A strikingly similar sequence composition (rap-1a, rap-1b and rap-1c), as well as strong gene identities and similar locus organization with B. bigemina were found and highlight the conservation of synteny at this locus in this phylogenetic clade. Copyright © 2013 Elsevier B.V. All rights reserved.

Spatio-temporal manipulation of small GTPase activity at subcellular level and on timescale of seconds in living cells.

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DeRose, Robert; Pohlmeyer, Christopher; Umeda, Nobuhiro; Ueno, Tasuku; Nagano, Tetsuo; Kuo, Scot; Inoue, Takanari

2012-03-09

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events(1, 2). Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time(3). In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac(4), Cdc42(4), RhoA(4) and Ras(5), in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells(6). Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level(6).

Recycling domains in plant cell morphogenesis: small GTPase effectors, plasma membrane signalling and the exocyst.

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Zárský, Viktor; Potocký, Martin

2010-04-01

The Rho/Rop small GTPase regulatory module is central for initiating exocytotically ACDs (active cortical domains) in plant cell cortex, and a growing array of Rop regulators and effectors are being discovered in plants. Structural membrane phospholipids are important constituents of cells as well as signals, and phospholipid-modifying enzymes are well known effectors of small GTPases. We have shown that PLDs (phospholipases D) and their product, PA (phosphatidic acid), belong to the regulators of the secretory pathway in plants. We have also shown that specific NOXs (NADPH oxidases) producing ROS (reactive oxygen species) are involved in cell growth as exemplified by pollen tubes and root hairs. Most plant cells exhibit several distinct plasma membrane domains (ACDs), established and maintained by endocytosis/exocytosis-driven membrane protein recycling. We proposed recently the concept of a 'recycling domain' (RD), uniting the ACD and the connected endosomal recycling compartment (endosome), as a dynamic spatiotemporal entity. We have described a putative GTPase-effector complex exocyst involved in exocytic vesicle tethering in plants. Owing to the multiplicity of its Exo70 subunits, this complex, along with many RabA GTPases (putative recycling endosome organizers), may belong to core regulators of RD organization in plants.

Requirement of the Caenorhabditis elegans RapGEF pxf-1 and rap-1 for epithelial integrity

Czech Academy of Sciences Publication Activity Database

Pellis-van Berkel, W.; Verheijen, M. H. G.; Cuppen, E.; Asahina, Masako; de Rooij, J.; Jansen, G.; Plasterk, R. H. A.; Bos, J. L.; Zwartkruis, F. J. T.

2005-01-01

Roč. 16, č. 1 (2005), s. 106-116 ISSN 1059-1524 R&D Projects: GA AV ČR KJB5022303 Institutional research plan: CEZ:AV0Z60220518 Keywords : Rap signaling pathway * epidermis * Caenorhabditis elegans Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.520, year: 2005

Ageing management studies of RAPS-1

International Nuclear Information System (INIS)

Bohra, A.K.; Jain, L.K.; Joshi, K.M.

2006-01-01

Unit-l of Rajasthan Atomic Power Station (RAPS-1) is the first nuclear power plant of India with pressurized heavy water reactor. The construction of Unit-l of Rajasthan Atomic Power Station (RAPS-1) was started in the year 1966 in collaboration with Canada. The Unit-1 achieved first criticality on August 1972 and was first synchronized to Grid on November 1972. During initial operation of the Unit, several problems were faced in its various systems and these were addressed by incorporating various engineering changes and procedures. In this unit various major innovative repairs were done like end shield leak repair, OPRD leak repair. Considering the operation of various systems of Unit-1, since year 1971 it was imperative to study ageing degradation mechanisms and mitigating measures were to be taken. Although the ageing management is a continuous process the opportunity of Unit-1 shutdown for upgradations from 30-04-2002 to 08-02-2004 was utilized for inspection and assessment of health of various SSC, which otherwise could not have been done with unit in operational state. This paper contains the following in detail. (1) Ageing management programme, its objectives and scope (2) Methodology of ageing management studies - Replacement and upgradation -Additional inspection programme based on ageing management review - Statistical analysis of ageing degradation occurrence - Estimation of residual life span of cables and relays (3) Criteria for selection of components for ageing management programme (4) Findings of ageing management studies-case studies. The ageing study done for RAPS-1 indicated that appropriate ageing monitoring methods and procedures exist in the station for taking timely mitigating measures. The technological obsoleteness has been overcome by installing new components of latest technology. On overall assessment, the Unit-1 was considered fit for further service. (author)

Ablation of p120-Catenin Altering the Activity of Small GTPase in Human Lung Cancer Cells

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Nan LIU

2009-05-01

Full Text Available Background and objective p120-catenin (p120ctn, a member of the Armadillo gene family, has emerged as an important modulator of small GTPase activities. Therefore, it plays novel roles in tumor malignant phenotype, such as invasion and metastasis, whose mechanism are not well clarified yet. The aim of this study is to explore the roles of p120ctn on the regulation of small GTP family members in lung cancer and the effects to lung cancer invasions andmetastasis. Methods After p120ctn was knocked down by siRNA, in vivo and in vitro analysis was applied to investigate the role and possible mechanism of p120ctn in lung cancer, such as Western Blot, pull-down analysis, and nude mice models. Results p120ctn depletion inactivated RhoA, with the the activity of Cdc42 and Rac1 increased, the invasiveness of lung cancer cells was promoted both in vitro and in vivo . Conclusion p120ctn gene knockdown enhances the metastasis of lung cancer cells, probably by altering expression of small GTPase, such as inactivation of RhoA and activation of Cdc42/Rac1.

Identification of the functional domains of the telomere protein Rap1 in Schizosaccharomyces pombe.

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Ikumi Fujita

Full Text Available The telomere at the end of a linear chromosome plays crucial roles in genome stability. In the fission yeast Schizosaccharomyces pombe, the Rap1 protein, one of the central players at the telomeres, associates with multiple proteins to regulate various telomere functions, such as the maintenance of telomere DNA length, telomere end protection, maintenance of telomere heterochromatin, and telomere clustering in meiosis. The molecular bases of the interactions between Rap1 and its partners, however, remain largely unknown. Here, we describe the identification of the interaction domains of Rap1 with its partners. The Bqt1/Bqt2 complex, which is required for normal meiotic progression, Poz1, which is required for telomere length control, and Taz1, which is required for the recruitment of Rap1 to telomeres, bind to distinct domains in the C-terminal half of Rap1. Intriguingly, analyses of a series of deletion mutants for rap1(+ have revealed that the long N-terminal region (1-456 a.a. [amino acids] of Rap1 (full length: 693 a.a. is not required for telomere DNA length control, telomere end protection, and telomere gene silencing, whereas the C-terminal region (457-693 a.a. containing Poz1- and Taz1-binding domains plays important roles in those functions. Furthermore, the Bqt1/Bqt2- and Taz1-binding domains are essential for normal spore formation after meiosis. Our results suggest that the C-terminal half of Rap1 is critical for the primary telomere functions, whereas the N-terminal region containing the BRCT (BRCA1 C-terminus and Myb domains, which are evolutionally conserved among the Rap1 family proteins, does not play a major role at the telomeres.

Stage-specific control of neural crest stem cell proliferation by the small rho GTPases Cdc42 and Rac1

DEFF Research Database (Denmark)

Fuchs, Sebastian; Herzog, Dominik; Sumara, Grzegorz

2009-01-01

-renewal and proliferation of later stage, but not early migratory NCSCs. This stage-specific requirement for small Rho GTPases is due to changes in NCSCs that, during development, acquire responsiveness to mitogenic EGF acting upstream of both Cdc42 and Rac1. Thus, our data reveal distinct mechanisms for growth control......The neural crest (NC) generates a variety of neural and non-neural tissues during vertebrate development. Both migratory NC cells and their target structures contain cells with stem cell features. Here we show that these populations of neural crest-derived stem cells (NCSCs) are differentially...

Rap phosphatase of virulence plasmid pXO1 inhibits Bacillus anthracis sporulation.

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Bongiorni, Cristina; Stoessel, Ricarda; Shoemaker, Dorinda; Perego, Marta

2006-01-01

This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis.

Rap Phosphatase of Virulence Plasmid pXO1 Inhibits Bacillus anthracis Sporulation†

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Bongiorni, Cristina; Stoessel, Ricarda; Shoemaker, Dorinda; Perego, Marta

2006-01-01

This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis. PMID:16385039

GTPases and the origin of the ribosome

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Smith Temple F

2010-05-01

Full Text Available Abstract Background This paper is an attempt to trace the evolution of the ribosome through the evolution of the universal P-loop GTPases that are involved with the ribosome in translation and with the attachment of the ribosome to the membrane. The GTPases involved in translation in Bacteria/Archaea are the elongation factors EFTu/EF1, the initiation factors IF2/aeIF5b + aeIF2, and the elongation factors EFG/EF2. All of these GTPases also contain the OB fold also found in the non GTPase IF1 involved in initiation. The GTPase involved in the signal recognition particle in most Bacteria and Archaea is SRP54. Results 1 The Elongation Factors of the Archaea based on structural considerations of the domains have the following evolutionary path: EF1→ aeIF2 → EF2. The evolution of the aeIF5b was a later event; 2 the Elongation Factors of the Bacteria based on the topological considerations of the GTPase domain have a similar evolutionary path: EFTu→ IF→2→EFG. These evolutionary sequences reflect the evolution of the LSU followed by the SSU to form the ribosome; 3 the OB-fold IF1 is a mimic of an ancient tRNA minihelix. Conclusion The evolution of translational GTPases of both the Archaea and Bacteria point to the evolution of the ribosome. The elongation factors, EFTu/EF1, began as a Ras-like GTPase bringing the activated minihelix tRNA to the Large Subunit Unit. The initiation factors and elongation factor would then have evolved from the EFTu/EF1 as the small subunit was added to the evolving ribosome. The SRP has an SRP54 GTPase and a specific RNA fold in its RNA component similar to the PTC. We consider the SRP to be a remnant of an ancient form of an LSU bound to a membrane. Reviewers This article was reviewed by George Fox, Leonid Mirny and Chris Sander.

The small GTPase, Rap1, mediates CD31-induced integrin adhesion

NARCIS (Netherlands)

Reedquist, K. A.; Ross, E.; Koop, E. A.; Wolthuis, R. M.; Zwartkruis, F. J.; van Kooyk, Y.; Salmon, M.; Buckley, C. D.; Bos, J. L.

2000-01-01

Integrin-mediated leukocyte adhesion is a critical aspect of leukocyte function that is tightly regulated by diverse stimuli, including chemokines, antigen receptors, and adhesion receptors. How cellular signals from CD31 and other adhesion amplifiers are integrated with those from classical

A photocleavable rapamycin conjugate for spatiotemporal control of small GTPase activity.

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Umeda, Nobuhiro; Ueno, Tasuku; Pohlmeyer, Christopher; Nagano, Tetsuo; Inoue, Takanari

2011-01-12

We developed a novel method to spatiotemporally control the activity of signaling molecules. A newly synthesized photocaged rapamycin derivative induced rapid dimerization of FKBP (FK-506 binding protein) and FRB (FKBP-rapamycin binding protein) upon UV irradiation. With this system and the spatially confined UV irradiation, we achieved subcellularly localized activation of Rac, a member of small GTPases. Our technique offers a powerful approach to studies of dynamic intracellular signaling events.

Essential role of the small GTPase Ran in postnatal pancreatic islet development.

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Fang Xia

Full Text Available The small GTPase Ran orchestrates pleiotropic cellular responses of nucleo-cytoplasmic shuttling, mitosis and subcellular trafficking, but whether deregulation of these pathways contributes to disease pathogenesis has remained elusive. Here, we generated transgenic mice expressing wild type (WT Ran, loss-of-function Ran T24N mutant or constitutively active Ran G19V mutant in pancreatic islet β cells under the control of the rat insulin promoter. Embryonic pancreas and islet development, including emergence of insulin(+ β cells, was indistinguishable in control or transgenic mice. However, by one month after birth, transgenic mice expressing any of the three Ran variants exhibited overt diabetes, with hyperglycemia, reduced insulin production, and nearly complete loss of islet number and islet mass, in vivo. Deregulated Ran signaling in transgenic mice, adenoviral over-expression of WT or mutant Ran in isolated islets, or short hairpin RNA (shRNA silencing of endogenous Ran in model insulinoma INS-1 cells, all resulted in decreased expression of the pancreatic and duodenal homeobox transcription factor, PDX-1, and reduced β cell proliferation, in vivo. These data demonstrate that a finely-tuned balance of Ran GTPase signaling is essential for postnatal pancreatic islet development and glucose homeostasis, in vivo.

The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication.

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Emilie Fugier

2009-06-01

Full Text Available The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER-derived replicative organelle named the "Brucella-containing vacuole" (BCV. Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.

Small GTPases and formins in mammalian oocyte maturation: cytoskeletal organizers.

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Kwon, Sojung; Lim, Hyunjung J

2011-03-01

The maturation process of mammalian oocytes accompanies an extensive rearrangement of the cytoskeleton and associated proteins. As this process requires a delicate interplay between the cytoskeleton and its regulators, it is often targeted by various external and internal adversaries that affect the congression and/or segregation of chromosomes. Asymmetric cell division in oocytes also requires specific regulators of the cytoskeleton, including formin-2 and small GTPases. Recent literature providing clues regarding how actin filaments and microtubules interact during spindle migration in mouse oocytes are highlighted in this review.

The small Rho GTPase Rac1 controls normal human dermal fibroblasts proliferation with phosphorylation of the oncoprotein c-myc

International Nuclear Information System (INIS)

Nikolova, Ekaterina; Mitev, Vanio; Zhelev, Nikolai; Deroanne, Christophe F.; Poumay, Yves

2007-01-01

Proliferation of dermal fibroblasts is crucial for the maintenance of skin. The small Rho GTPase, Rac1, has been identified as a key transducer of proliferative signals in various cell types, but in normal human dermal fibroblasts its significance to cell growth control has not been studied. In this study, we applied the method of RNA interference to suppress endogenous Rac1 expression and examined the consequences on human skin fibroblasts. Rac1 knock-down resulted in inhibition of DNA synthesis. This effect was not mediated by inhibition of the central transducer of proliferative stimuli, ERK1/2 or by activation of the pro-apoptotic p38. Rather, as a consequence of the suppressed Rac1 expression we observed a significant decrease in phosphorylation of c-myc, revealing for the first time that in human fibroblasts Rac1 exerts control on proliferation through c-myc phosphorylation. Thus Rac1 activates proliferation of normal fibroblasts through stimulation of c-myc phosphorylation without affecting ERK1/2 activity

A Pan-GTPase Inhibitor as a Molecular Probe.

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Lin Hong

Full Text Available Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.

Remote Area Power Supply (RAPS) load and resource profiles.

Energy Technology Data Exchange (ETDEWEB)

Giles, Lauren (Energetics, Inc., Washington, DC); Skolnik, Edward G. (Energetics, Inc., Washington, DC); Marchionini, Brian (Energetics, Inc., Washington, DC); Fall, Ndeye K. (Energetics, Inc., Washington, DC)

2007-07-01

In 1997, an international team interested in the development of Remote Area Power Supply (RAPS) systems for rural electrification projects around the world was organized by the International Lead Zinc Research Organization (ILZRO) with the support of Sandia National Laboratories (SNL). The team focused on defining load and resource profiles for RAPS systems. They identified single family homes, small communities, and villages as candidates for RAPS applications, and defined several different size/power requirements for each. Based on renewable energy and resource data, the team devised a ''strawman'' series of load profiles. A RAPS system typically consists of a renewable and/or conventional generator, power conversion equipment, and a battery. The purpose of this report is to present data and information on insolation levels and load requirements for ''typical'' homes, small communities, and larger villages around the world in order to facilitate the development of robust design practices for RAPS systems, and especially for the storage battery component. These systems could have significant impact on areas of the world that would otherwise not be served by conventional electrical grids.

Catalysis of GTP Hydrolysis by Small GTPases at Atomic Detail by Integration of X-ray Crystallography, Experimental, and Theoretical IR Spectroscopy*

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Rudack, Till; Jenrich, Sarah; Brucker, Sven; Vetter, Ingrid R.; Gerwert, Klaus; Kötting, Carsten

2015-01-01

Small GTPases regulate key processes in cells. Malfunction of their GTPase reaction by mutations is involved in severe diseases. Here, we compare the GTPase reaction of the slower hydrolyzing GTPase Ran with Ras. By combination of time-resolved FTIR difference spectroscopy and QM/MM simulations we elucidate that the Mg2+ coordination by the phosphate groups, which varies largely among the x-ray structures, is the same for Ran and Ras. A new x-ray structure of a Ran·RanBD1 complex with improved resolution confirmed this finding and revealed a general problem with the refinement of Mg2+ in GTPases. The Mg2+ coordination is not responsible for the much slower GTPase reaction of Ran. Instead, the location of the Tyr-39 side chain of Ran between the γ-phosphate and Gln-69 prevents the optimal positioning of the attacking water molecule by the Gln-69 relative to the γ-phosphate. This is confirmed in the RanY39A·RanBD1 crystal structure. The QM/MM simulations provide IR spectra of the catalytic center, which agree very nicely with the experimental ones. The combination of both methods can correlate spectra with structure at atomic detail. For example the FTIR difference spectra of RasA18T and RanT25A mutants show that spectral differences are mainly due to the hydrogen bond of Thr-25 to the α-phosphate in Ran. By integration of x-ray structure analysis, experimental, and theoretical IR spectroscopy the catalytic center of the x-ray structural models are further refined to sub-Å resolution, allowing an improved understanding of catalysis. PMID:26272610

Strong conservation of rhoptry-associated-protein-1 (RAP-1) locus organization and sequence among Babesia isolates infecting sheep from China (Babesia motasi-like phylogenetic group).

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Niu, Qingli; Valentin, Charlotte; Bonsergent, Claire; Malandrin, Laurence

2014-12-01

Rhoptry-associated-protein 1 (RAP-1) is considered as a potential vaccine candidate due to its involvement in red blood cell invasion by parasites in the genus Babesia. We examined its value as a vaccine candidate by studying RAP-1 conservation in isolates of Babesia sp. BQ1 Ningxian, Babesia sp. Tianzhu and Babesia sp. Hebei, responsible for ovine babesiosis in different regions of China. The rap-1 locus in these isolates has very similar features to those described for Babesia sp. BQ1 Lintan, another Chinese isolate also in the B. motasi-like phylogenetic group, namely the presence of three types of rap-1 genes (rap-1a, rap-1b and rap-1c), multiple conserved rap-1b copies (5) interspaced with more or less variable rap-1a copies (6), and the 3' localization of one rap-1c. The isolates Babesia sp. Tianzhu, Babesia sp. BQ1 Lintan and Ningxian were almost identical (average nucleotide identity of 99.9%) over a putative locus of about 31 Kb, including the intergenic regions. Babesia sp. Hebei showed a similar locus organization but differed in the rap-1 locus sequence, for each gene and intergenic region, with an average nucleotide identity of 78%. Our results are in agreement with 18S rDNA phylogenetic studies performed on these isolates. However, in extremely closely related isolates the rap-1 locus seems more conserved (99.9%) than the 18S rDNA (98.7%), whereas in still closely related isolates the identities are much lower (78%) compared with the 18S rDNA (97.7%). The particularities of the rap-1 locus in terms of evolution, phylogeny, diagnosis and vaccine development are discussed. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Regulation of ER-Golgi Transport Dynamics by GTPases in Budding Yeast

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Yasuyuki Suda

2018-01-01

Full Text Available A large number of proteins are synthesized de novo in the endoplasmic reticulum (ER. They are transported through the Golgi apparatus and then delivered to their proper destinations. The ER and the Golgi play a central role in protein processing and sorting and show dynamic features in their forms. Ras super family small GTPases mediate the protein transport through and between these organelles. The ER-localized GTPase, Sar1, facilitates the formation of COPII transport carriers at the ER exit sites (ERES on the ER for the transport of cargo proteins from the ER to the Golgi. The Golgi-localized GTPase, Arf1, controls intra-Golgi, and Golgi-to-ER transport of cargo proteins by the formation of COPI carriers. Rab GTPases localized at the Golgi, which are responsible for fusion of membranes, are thought to establish the identities of compartments. Recent evidence suggests that these small GTPases regulate not only discrete sites for generation/fusion of transport carriers, but also membrane dynamics of the organelles where they locate to ensure the integrity of transport. Here we summarize the current understandings about the membrane traffic between these organelles and highlight the cutting-edge advances from super-resolution live imaging of budding yeast, Saccharomyces cerevisiae.

Genetic diversity and natural selection in the rhoptry-associated protein 1 (RAP-1) of recent Plasmodium knowlesi clinical isolates from Malaysia.

Science.gov (United States)

Rawa, Mira Syahfriena Amir; Fong, Mun-Yik; Lau, Yee-Ling

2016-02-05

The Plasmodium rhoptry-associated protein 1 (RAP-1) plays a role in the formation of the parasitophorous vacuole following the parasite's invasion of red blood cells. Although there is some evidence that the protein is recognized by the host's immune system, study of Plasmodium falciparum RAP-1 (PfRAP-1) suggests that it is not under immune pressure. A previous study on five old (1953-1962) P. knowlesi strains suggested that RAP-1 has limited genetic polymorphism and might be under negative selection. In the present study, 30 recent P. knowlesi isolates were studied to obtain a better insight into the polymorphism and natural selection of PkRAP-1. Blood samples from 30 knowlesi malaria patients were used. These samples were collected between 2010 and 2014. The PkRAP-1 gene, which contains two exons, was amplified by PCR, cloned into Escherichia coli and sequenced. Genetic diversity and phylogenetic analyses were performed using MEGA6 and DnaSP ver. 5.10.00 programs. Thirty PkRAP-1 sequences were obtained. The nucleotide diversity (π) of exons 1, 2 and the total coding region (0.00915, 0.01353 and 0.01298, respectively) were higher than those of the old strains. Further analysis revealed a lower rate of non-synonymous (dN) than synonymous (dS) mutations, suggesting negative (purifying) selection of PkRAP-1. Tajima's D test and Fu and Li's D test values were not significant. At the amino acid level, 22 haplotypes were established with haplotype H7 having the highest frequency (7/34, 20.5 %). In the phylogenetic analysis, two distinct haplotype groups were observed. The first group contained the majority of the haplotypes, whereas the second had fewer haplotypes. The present study found higher genetic polymorphism in the PkRAP-1 gene than the polymorphism level reported in a previous study. This observation may stem from the difference in sample size between the present (n = 30) and the previous (n = 5) study. Synonymous and non-synonymous mutation analysis indicated

Gain-of-function mutant p53 activates small GTPase Rac1 through SUMOylation to promote tumor progression.

Science.gov (United States)

Yue, Xuetian; Zhang, Cen; Zhao, Yuhan; Liu, Juan; Lin, Alan W; Tan, Victor M; Drake, Justin M; Liu, Lianxin; Boateng, Michael N; Li, Jun; Feng, Zhaohui; Hu, Wenwei

2017-08-15

Tumor suppressor p53 is frequently mutated in human cancer. Mutant p53 often promotes tumor progression through gain-of-function (GOF) mechanisms. However, the mechanisms underlying mutant p53 GOF are not well understood. In this study, we found that mutant p53 activates small GTPase Rac1 as a critical mechanism for mutant p53 GOF to promote tumor progression. Mechanistically, mutant p53 interacts with Rac1 and inhibits its interaction with SUMO-specific protease 1 (SENP1), which in turn inhibits SENP1-mediated de-SUMOylation of Rac1 to activate Rac1. Targeting Rac1 signaling by RNAi, expression of the dominant-negative Rac1 (Rac1 DN), or the specific Rac1 inhibitor NSC23766 greatly inhibits mutant p53 GOF in promoting tumor growth and metastasis. Furthermore, mutant p53 expression is associated with enhanced Rac1 activity in clinical tumor samples. These results uncover a new mechanism for Rac1 activation in tumors and, most importantly, reveal that activation of Rac1 is an unidentified and critical mechanism for mutant p53 GOF in tumorigenesis, which could be targeted for therapy in tumors containing mutant p53. © 2017 Yue et al.; Published by Cold Spring Harbor Laboratory Press.

Catalysis of GTP hydrolysis by small GTPases at atomic detail by integration of X-ray crystallography, experimental, and theoretical IR spectroscopy.

Science.gov (United States)

Rudack, Till; Jenrich, Sarah; Brucker, Sven; Vetter, Ingrid R; Gerwert, Klaus; Kötting, Carsten

2015-10-02

Small GTPases regulate key processes in cells. Malfunction of their GTPase reaction by mutations is involved in severe diseases. Here, we compare the GTPase reaction of the slower hydrolyzing GTPase Ran with Ras. By combination of time-resolved FTIR difference spectroscopy and QM/MM simulations we elucidate that the Mg(2+) coordination by the phosphate groups, which varies largely among the x-ray structures, is the same for Ran and Ras. A new x-ray structure of a Ran·RanBD1 complex with improved resolution confirmed this finding and revealed a general problem with the refinement of Mg(2+) in GTPases. The Mg(2+) coordination is not responsible for the much slower GTPase reaction of Ran. Instead, the location of the Tyr-39 side chain of Ran between the γ-phosphate and Gln-69 prevents the optimal positioning of the attacking water molecule by the Gln-69 relative to the γ-phosphate. This is confirmed in the RanY39A·RanBD1 crystal structure. The QM/MM simulations provide IR spectra of the catalytic center, which agree very nicely with the experimental ones. The combination of both methods can correlate spectra with structure at atomic detail. For example the FTIR difference spectra of RasA18T and RanT25A mutants show that spectral differences are mainly due to the hydrogen bond of Thr-25 to the α-phosphate in Ran. By integration of x-ray structure analysis, experimental, and theoretical IR spectroscopy the catalytic center of the x-ray structural models are further refined to sub-Å resolution, allowing an improved understanding of catalysis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Guanine nucleotide exchange factor αPIX leads to activation of the Rac 1 GTPase/glycogen phosphorylase pathway in interleukin (IL)-2-stimulated T cells

DEFF Research Database (Denmark)

Llavero, Francisco; Urzelai, Bakarne; Osinalde, Nerea

2015-01-01

Recently, we have reported that the active form of Rac 1 GTPase binds to the glycogen phosphorylase muscle isoform (PYGM) and modulates its enzymatic activity leading to T cell proliferation. In the lymphoid system, Rac 1 and in general other small GTPases of the Rho family participate...... in the signaling cascades that are activated after engagement of the T cell antigen receptor. However, little is known about the IL-2-dependent Rac 1 activator molecules. For the first time, a signaling pathway leading to the activation of Rac 1/PYGM in response to IL-2-stimulated T cell proliferation is described....... More specifically, αPIX, a known guanine nucleotide exchange factor for the small GTPases of the Rho family, preferentially Rac 1, mediates PYGM activation in Kit 225 T cells stimulated with IL-2. Using directed mutagenesis, phosphorylation of αPIX Rho-GEF serines 225 and 488 is required for activation...

In situ detection of the activation of Rac1 and RalA small GTPases in mouse adipocytes by immunofluorescent microscopy following in vivo and ex vivo insulin stimulation.

Science.gov (United States)

Takenaka, Nobuyuki; Nihata, Yuma; Ueda, Sho; Satoh, Takaya

2017-11-01

Rac1 has been implicated in insulin-dependent glucose uptake by mechanisms involving plasma membrane translocation of the glucose transporter GLUT4 in skeletal muscle. Although the uptake of glucose is also stimulated by insulin in adipose tissue, the role for Rac1 in adipocyte insulin signaling remains controversial. As a step to reveal the role for Rac1 in adipocytes, we aimed to establish immunofluorescent microscopy to detect the intracellular distribution of activated Rac1. The epitope-tagged Rac1-binding domain of a Rac1-specific target was utilized as a probe that specifically recognizes the activated form of Rac1. Rac1 activation in response to ex vivo and in vivo insulin stimulations in primary adipocyte culture and mouse white adipose tissue, respectively, was successfully observed by immunofluorescent microscopy. These Rac1 activations were mediated by phosphoinositide 3-kinase. Another small GTPase RalA has also been implicated in insulin-stimulated glucose uptake in skeletal muscle and adipose tissue. Similarly to Rac1, immunofluorescent microscopy using an activated RalA-specific polypeptide probe allowed us to detect intracellular distribution of insulin-activated RalA in adipocytes. These novel approaches to visualize the activation status of small GTPases in adipocytes will largely contribute to the understanding of signal transduction mechanisms particularly for insulin action. Copyright © 2017 Elsevier Inc. All rights reserved.

A role for Sar1 and ARF1 GTPases during Golgi biogenesis in the protozoan parasite Trypanosoma brucei

Science.gov (United States)

Yavuz, Sevil; Warren, Graham

2017-01-01

A single Golgi stack is duplicated and partitioned into two daughter cells during the cell cycle of the protozoan parasite Trypanosoma brucei. The source of components required to generate the new Golgi and the mechanism by which it forms are poorly understood. Using photoactivatable GFP, we show that the existing Golgi supplies components directly to the newly forming Golgi in both intact and semipermeabilized cells. The movement of a putative glycosyltransferase, GntB, requires the Sar1 and ARF1 GTPases in intact cells. In addition, we show that transfer of GntB from the existing Golgi to the new Golgi can be recapitulated in semipermeabilized cells and is sensitive to the GTP analogue GTPγS. We suggest that the existing Golgi is a key source of components required to form the new Golgi and that this process is regulated by small GTPases. PMID:28495798

Rac and Rho GTPases in cancer cell motility control

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Parri Matteo

2010-09-01

Full Text Available Abstract Rho GTPases represent a family of small GTP-binding proteins involved in cell cytoskeleton organization, migration, transcription, and proliferation. A common theme of these processes is a dynamic reorganization of actin cytoskeleton which has now emerged as a major switch control mainly carried out by Rho and Rac GTPase subfamilies, playing an acknowledged role in adaptation of cell motility to the microenvironment. Cells exhibit three distinct modes of migration when invading the 3 D environment. Collective motility leads to movement of cohorts of cells which maintain the adherens junctions and move by photolytic degradation of matrix barriers. Single cell mesenchymal-type movement is characterized by an elongated cellular shape and again requires extracellular proteolysis and integrin engagement. In addition it depends on Rac1-mediated cell polarization and lamellipodia formation. Conversely, in amoeboid movement cells have a rounded morphology, the movement is independent from proteases but requires high Rho GTPase to drive elevated levels of actomyosin contractility. These two modes of cell movement are interconvertible and several moving cells, including tumor cells, show an high degree of plasticity in motility styles shifting ad hoc between mesenchymal or amoeboid movements. This review will focus on the role of Rac and Rho small GTPases in cell motility and in the complex relationship driving the reciprocal control between Rac and Rho granting for the opportunistic motile behaviour of aggressive cancer cells. In addition we analyse the role of these GTPases in cancer progression and metastatic dissemination.

The Rho-family GTPase Rac1 regulates integrin localization in Drosophila immunosurveillance cells.

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Miguel J Xavier

Full Text Available BACKGROUND: When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila β-integrin Myospheroid (Mys is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of α and β subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and we have previously shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. We wanted to test the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response. RESULTS: In the current study we demonstrate that Rac1 is required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results we conclude that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization. SIGNIFICANCE: We show for the first time that the small GTPase Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila we quite often raise larvae at 29°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes.

The small GTPase RhoH is an atypical regulator of haematopoietic cells

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Kubatzky Katharina F

2008-09-01

Full Text Available Abstract Rho GTPases are a distinct subfamily of the superfamily of Ras GTPases. The best-characterised members are RhoA, Rac and Cdc42 that regulate many diverse actions such as actin cytoskeleton reorganisation, adhesion, motility as well as cell proliferation, differentiation and gene transcription. Among the 20 members of that family, only Rac2 and RhoH show an expression restricted to the haematopoietic lineage. RhoH was first discovered in 1995 as a fusion transcript with the transcriptional repressor LAZ3/BCL6. It was therefore initially named translation three four (TTF but later on renamed RhoH due to its close relationship to the Ras/Rho family of GTPases. Since then, RhoH has been implicated in human cancer as the gene is subject to somatic hypermutation and by the detection of RHOH as a translocation partner for LAZ3/BCL6 or other genes in human lymphomas. Underexpression of RhoH is found in hairy cell leukaemia and acute myeloid leukaemia. Some of the amino acids that are crucial for GTPase activity are mutated in RhoH so that the protein is a GTPase-deficient, so-called atypical Rho GTPase. Therefore other mechanisms of regulating RhoH activity have been described. These include regulation at the mRNA level and tyrosine phosphorylation of the protein's unique ITAM-like motif. The C-terminal CaaX box of RhoH is mainly a target for farnesyl-transferase but can also be modified by geranylgeranyl-transferase. Isoprenylation of RhoH and changes in subcellular localisation may be an additional factor to fine-tune signalling. Little is currently known about its signalling, regulation or interaction partners. Recent studies have shown that RhoH negatively influences the proliferation and homing of murine haematopoietic progenitor cells, presumably by acting as an antagonist for Rac1. In leukocytes, RhoH is needed to keep the cells in a resting, non-adhesive state, but the exact mechanism has yet to be elucidated. RhoH has also been

PI3-kinase γ promotes Rap1a-mediated activation of myeloid cell integrin α4β1, leading to tumor inflammation and growth.

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Michael C Schmid

Full Text Available Tumor inflammation, the recruitment of myeloid lineage cells into the tumor microenvironment, promotes angiogenesis, immunosuppression and metastasis. CD11b+Gr1lo monocytic lineage cells and CD11b+Gr1hi granulocytic lineage cells are recruited from the circulation by tumor-derived chemoattractants, which stimulate PI3-kinase γ (PI3Kγ-mediated integrin α4 activation and extravasation. We show here that PI3Kγ activates PLCγ, leading to RasGrp/CalDAG-GEF-I&II mediated, Rap1a-dependent activation of integrin α4β1, extravasation of monocytes and granulocytes, and inflammation-associated tumor progression. Genetic depletion of PLCγ, CalDAG-GEFI or II, Rap1a, or the Rap1 effector RIAM was sufficient to prevent integrin α4 activation by chemoattractants or activated PI3Kγ (p110γCAAX, while activated Rap (RapV12 promoted constitutive integrin activation and cell adhesion that could only be blocked by inhibition of RIAM or integrin α4β1. Similar to blockade of PI3Kγ or integrin α4β1, blockade of Rap1a suppressed both the recruitment of monocytes and granulocytes to tumors and tumor progression. These results demonstrate critical roles for a PI3Kγ-Rap1a-dependent pathway in integrin activation during tumor inflammation and suggest novel avenues for cancer therapy.

Molecular characterization of the Babesia caballi rap-1 gene and epidemiological survey in horses in Israel.

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Rapoport, Adi; Aharonson-Raz, Karin; Berlin, Dalia; Tal, Saar; Gottlieb, Yuval; Klement, Eyal; Steinman, Amir

2014-04-01

Equine piroplasmosis imposes great concerns for the equine industry regarding international horse movement, and therefore requires reliable diagnostic tools. Recent studies from South Africa and Jordan, including a preliminary study in Israel, reported extremely low seroprevalence to Babesia caballi (B. caballi) (0-1%) using the acceptable rhoptry-associated protein-1 (RAP-1) cELISA. In accordance with the study from South Africa demonstrating a significant heterogeneity in the rap-1 gene sequence of South African B. caballi isolates, the objectives of this study were to phylogenetically characterize the rap-1 gene of the Israeli isolates and determine the prevalence of B. caballi in horses in Israel. Out of 273 horses tested using the RAP-1 cELISA, only one was sero-positive, while 9.3% were positive on PCR performed on the rap-1 gene. Phylogenetic analysis of the rap-1 gene grouped the Israeli isolates in a cluster together with the South African strains (99% nt identity), but in a separate cluster from the American/Caribbean strains (81-82% nt identity). These findings support the existence of heterogeneity in the RAP-1 amino-acid sequences of the Israeli and South African isolates as compared to that used in the cELISA commercial kit and raise doubts as to the ability of this assay to serve as a sole regulatory test for international horse movement. Risk factor analysis found management and age to significantly associate with prevalence of B. caballi, as higher prevalence was noted in horses held out on pasture and a negative association was recorded with age. In addition, B. caballi was not detected in horses in the steppe-arid and extreme-arid climatic regions as compared to the wetter regions. Findings of this study emphasize the need to combine several detection methods to ameliorate the control and spread of the disease. Copyright © 2014 Elsevier B.V. All rights reserved.

Rap Poetry and Postmodernism

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Kirill Molokov

2017-10-01

Full Text Available This article observes several most significant rap albums of this decade within postmodern literature. Today rap culture ceased to be a sort of “outsider” in academic opinion, because of its influences on the culture and art innovations. We study albums as literary objects according to literary aesthetic theories and principles, display the main postmodern features they have, and analyze the role of rap poetry within postmodernism in general. The results suggest that rap poetry is postmodern not only musically, but also lyrically, as an object of literature. The rap music embodies all the postmodern traits and synthesizes them within the syntheses of music and literature and high art and pop culture.

Cobalt production in RAPS-1

International Nuclear Information System (INIS)

Krishnan, P.D.; Purandare, H.D.

1978-01-01

At present in RAPS-1 radioisotope Co 60 is produced by irradiating Co 59 in the adjusters which perform the function of regulation of reactivity, power and xenon override. But the manrem expenditure of the crew handling the charge and discharge of the adjusters is going to be prohibitively high. It is therefore proposed to irradiate Co 59 in the fuel channel positions. The physics optimisation study for such irradiation is presented. The burnup penalty and loss of power are estimated to produce the required quantity of Co 60 after optimising the number of cobalt pencils in a bundle and the positions of the cobalt producing channels in the reactor core. (author)

Two Components of Aversive Memory in Drosophila, Anesthesia-Sensitive and Anesthesia-Resistant Memory, Require Distinct Domains Within the Rgk1 Small GTPase.

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Murakami, Satoshi; Minami-Ohtsubo, Maki; Nakato, Ryuichiro; Shirahige, Katsuhiko; Tabata, Tetsuya

2017-05-31

Multiple components have been identified that exhibit different stabilities for aversive olfactory memory in Drosophila These components have been defined by behavioral and genetic studies and genes specifically required for a specific component have also been identified. Intermediate-term memory generated after single cycle conditioning is divided into anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM), with the latter being more stable. We determined that the ASM and ARM pathways converged on the Rgk1 small GTPase and that the N-terminal domain-deleted Rgk1 was sufficient for ASM formation, whereas the full-length form was required for ARM formation. Rgk1 is specifically accumulated at the synaptic site of the Kenyon cells (KCs), the intrinsic neurons of the mushroom bodies, which play a pivotal role in olfactory memory formation. A higher than normal Rgk1 level enhanced memory retention, which is consistent with the result that Rgk1 suppressed Rac-dependent memory decay; these findings suggest that rgk1 bolsters ASM via the suppression of forgetting. We propose that Rgk1 plays a pivotal role in the regulation of memory stabilization by serving as a molecular node that resides at KC synapses, where the ASM and ARM pathway may interact. SIGNIFICANCE STATEMENT Memory consists of multiple components. Drosophila olfactory memory serves as a fundamental model with which to investigate the mechanisms that underlie memory formation and has provided genetic and molecular means to identify the components of memory, namely short-term, intermediate-term, and long-term memory, depending on how long the memory lasts. Intermediate memory is further divided into anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM), with the latter being more stable. We have identified a small GTPase in Drosophila , Rgk1, which plays a pivotal role in the regulation of olfactory memory stability. Rgk1 is required for both ASM and ARM. Moreover, N

The transcription factor Rap1p is required for tolerance to cell-wall perturbing agents and for cell-wall maintenance in Saccharomyces cerevisiae.

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Azad, Gajendra Kumar; Singh, Vikash; Baranwal, Shivani; Thakare, Mayur Jankiram; Tomar, Raghuvir S

2015-01-02

Yeast repressor activator protein (Rap1p) is involved in genomic stability and transcriptional regulation. We explored the function of Rap1p in yeast physiology using Rap1p truncation mutants. Our results revealed that the N-terminal truncation of Rap1p (Rap1ΔN) leads to hypersensitivity towards elevated temperature and cell-wall perturbing agents. Cell wall analysis showed an increase in the chitin and glucan content in Rap1ΔN cells as compared with wild type cells. Accordingly, mutant cells had a twofold thicker cell wall, as observed by electron microscopy. Furthermore, Rap1ΔN cells had increased levels of phosphorylated Slt2p, a MAP kinase of the cell wall integrity pathway. Mutant cells also had elevated levels of cell wall integrity response transcripts. Taken together, our findings suggest a connection between Rap1p and cell wall homeostasis. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Role of Rab family GTPases and their effectors in melanosomal logistics.

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Ohbayashi, Norihiko; Fukuda, Mitsunori

2012-04-01

Rab GTPases constitute a family of small GTPases that regulate a variety of membrane trafficking events in all eukaryotic cells by recruiting their specific effector molecules. Recent accumulating evidence indicates that members of the mammalian Rab small GTPase family are involved in certain physiological and pathological processes. In particular, functional impairments of specific Rab proteins, e.g. Rab38 and Rab27A, their regulators or their effectors cause pigmentation disorders in humans and coat colour variations in mice because such impairments cause defects in melanosomal logistics, i.e. defects in melanosome biogenesis and transport. Genetic and biochemical analyses of the gene products responsible for mammalian pigmentation disorders in the past decade have revealed that Rab-mediated endosomal transport systems and melanosome transport systems play crucial roles in the efficient darkening of mammalian hair and skin. In this article, we review current knowledge regarding melanosomal logistics, with particular focus on the roles of Rab small GTPases and their effectors.

Glutaminase 2 is a novel negative regulator of small GTPase Rac1 and mediates p53 function in suppressing metastasis

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Zhang, Cen; Liu, Juan; Zhao, Yuhan; Yue, Xuetian; Zhu, Yu; Wang, Xiaolong; Wu, Hao; Blanco, Felix; Li, Shaohua; Bhanot, Gyan; Haffty, Bruce G; Hu, Wenwei; Feng, Zhaohui

2016-01-01

Glutaminase (GLS) isoenzymes GLS1 and GLS2 are key enzymes for glutamine metabolism. Interestingly, GLS1 and GLS2 display contrasting functions in tumorigenesis with elusive mechanism; GLS1 promotes tumorigenesis, whereas GLS2 exhibits a tumor-suppressive function. In this study, we found that GLS2 but not GLS1 binds to small GTPase Rac1 and inhibits its interaction with Rac1 activators guanine-nucleotide exchange factors, which in turn inhibits Rac1 to suppress cancer metastasis. This function of GLS2 is independent of GLS2 glutaminase activity. Furthermore, decreased GLS2 expression is associated with enhanced metastasis in human cancer. As a p53 target, GLS2 mediates p53’s function in metastasis suppression through inhibiting Rac1. In summary, our results reveal that GLS2 is a novel negative regulator of Rac1, and uncover a novel function and mechanism whereby GLS2 suppresses metastasis. Our results also elucidate a novel mechanism that contributes to the contrasting functions of GLS1 and GLS2 in tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.10727.001 PMID:26751560

WAVE regulatory complex activation by cooperating GTPases Arf and Rac1

DEFF Research Database (Denmark)

Koronakis, Vassilis; Hume, Peter J; Humphreys, Daniel

2011-01-01

The WAVE regulatory complex (WRC) is a critical element in the control of actin polymerization at the eukaryotic cell membrane, but how WRC is activated remains uncertain. While Rho GTPase Rac1 can bind and activate WRC in vitro, this interaction is of low affinity, suggesting other factors may...... be important. By reconstituting WAVE-dependent actin assembly on membrane-coated beads in mammalian cell extracts, we found that Rac1 was not sufficient to engender bead motility, and we uncovered a key requirement for Arf GTPases. In vitro, Rac1 and Arf1 were individually able to bind weakly to recombinant...... be central components in WAVE signalling, acting directly, alongside Rac1....

Influence of slope and gradation on rip rap stability and degradation mechanisms

International Nuclear Information System (INIS)

Lefebvre, G.; Rohan, K.; Belfahdel, M. B.

1997-01-01

A major investigation was undertaken at the La Grande hydroelectric complex with some 220 dikes and dams to study rip rap stability and repair. Degradation mechanisms were also studied under laboratory conditions to verify the main field study conclusions and to test different repair techniques. The result of both laboratory and field observation was that rip rap gradation has only marginal effect on slope stability and degradation mechanisms. On the other hand, the inclusion of even a small fraction of fine blocks (as little as 10 per cent) into the rip rap was shown to be very detrimental to the stability of steep rip rap but only marginally effective on flat slopes. 15 refs., 8 figs

[Cloning of VH and VL Gene of Human anti-IL1RAP McAb and Construction of Recombinant Chimeric Receptor].

Science.gov (United States)

Yin, Ling-Ling; Ruan, Su-Hong; Tian, Yu; Zhao, Kai; Xu, Kai Lin

2015-10-01

To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs). The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells. The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired. Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.

GCR1, a transcriptional activator in Saccharomyces cerevisiae, complexes with RAP1 and can function without its DNA binding domain.

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Tornow, J; Zeng, X; Gao, W; Santangelo, G M

1993-01-01

In Saccharomyces cerevisiae, efficient expression of glycolytic and translational component genes requires two DNA binding proteins, RAP1 (which binds to UASRPG) and GCR1 (which binds to the CT box). We generated deletions in GCR1 to test the validity of several different models for GCR1 function. We report here that the C-terminal half of GCR1, which includes the domain required for DNA binding to the CT box in vitro, can be removed without affecting GCR1-dependent transcription of either the glycolytic gene ADH1 or the translational component genes TEF1 and TEF2. We have also identified an activation domain within a segment of the GCR1 protein (the N-terminal third) that is essential for in vivo function. RAP1 and GCR1 can be co-immunoprecipitated from whole cell extracts, suggesting that they form a complex in vivo. The data are most consistent with a model in which GCR1 is attracted to DNA through contact with RAP1. Images PMID:8508768

Microgravity simulation activates Cdc42 via Rap1GDS1 to promote vascular branch morphogenesis during vasculogenesis

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Shouli Wang

2017-12-01

Full Text Available Gravity plays an important role in normal tissue maintenance. The ability of stem cells to repair tissue loss in space through regeneration and differentiation remains largely unknown. To investigate the impact of microgravity on blood vessel formation from pluripotent stem cells, we employed the embryoid body (EB model for vasculogenesis and simulated microgravity by clinorotation. We first differentiated mouse embryonic stem cells into cystic EBs containing two germ layers and then analyzed vessel formation under clinorotation. We observed that endothelial cell differentiation was slightly reduced under clinorotation, whereas vascular branch morphogenesis was markedly enhanced. EB-derived endothelial cells migrated faster, displayed multiple cellular processes, and had higher Cdc42 and Rac1 activity when subjected to clinorotation. Genetic analysis and rescue experiments demonstrated that Cdc42 but not Rac1 is required for microgravity-induced vascular branch morphogenesis. Furthermore, affinity pull-down assay and mass spectrometry identified Rap1GDS1 to be a Cdc42 guanine nucleotide exchange factor, which was upregulated by clinorotation. shRNA-mediated knockdown of Rap1GDS1 selectively suppressed Cdc42 activation and inhibited both baseline and microgravity-induced vasculogenesis. This was rescued by ectopic expression of constitutively active Cdc42. Taken together, these results support the notion that simulated microgravity activates Cdc42 via Rap1GDS1 to promote vascular branch morphogenesis.

Novel Activities of Select NSAID R-Enantiomers against Rac1 and Cdc42 GTPases.

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Tudor I Oprea

Full Text Available Rho family GTPases (including Rac, Rho and Cdc42 collectively control cell proliferation, adhesion and migration and are of interest as functional therapeutic targets in numerous epithelial cancers. Based on high throughput screening of the Prestwick Chemical Library® and cheminformatics we identified the R-enantiomers of two approved drugs (naproxen and ketorolac as inhibitors of Rac1 and Cdc42. The corresponding S-enantiomers are considered the active component in racemic drug formulations, acting as non-steroidal anti-inflammatory drugs (NSAIDs with selective activity against cyclooxygenases. Here, we show that the S-enantiomers of naproxen and ketorolac are inactive against the GTPases. Additionally, more than twenty other NSAIDs lacked inhibitory action against the GTPases, establishing the selectivity of the two identified NSAIDs. R-naproxen was first identified as a lead compound and tested in parallel with its S-enantiomer and the non-chiral 6-methoxy-naphthalene acetic acid (active metabolite of nabumetone, another NSAID as a structural series. Cheminformatics-based substructure analyses-using the rotationally constrained carboxylate in R-naproxen-led to identification of racemic [R/S] ketorolac as a suitable FDA-approved candidate. Cell based measurement of GTPase activity (in animal and human cell lines demonstrated that the R-enantiomers specifically inhibit epidermal growth factor stimulated Rac1 and Cdc42 activation. The GTPase inhibitory effects of the R-enantiomers in cells largely mimic those of established Rac1 (NSC23766 and Cdc42 (CID2950007/ML141 specific inhibitors. Docking predicts that rotational constraints position the carboxylate moieties of the R-enantiomers to preferentially coordinate the magnesium ion, thereby destabilizing nucleotide binding to Rac1 and Cdc42. The S-enantiomers can be docked but are less favorably positioned in proximity to the magnesium. R-naproxen and R-ketorolac have potential for rapid

IQ-domain GTPase-activating protein 1 promotes the malignant phenotype of invasive ductal breast carcinoma via canonical Wnt pathway.

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Zhao, Huan-Yu; Han, Yang; Wang, Jian; Yang, Lian-He; Zheng, Xiao-Ying; Du, Jiang; Wu, Guang-Ping; Wang, En-Hua

2017-06-01

IQ-domain GTPase-activating protein 1 is a scaffolding protein with multidomain which plays a role in modulating dishevelled (Dvl) nuclear translocation in canonical Wnt pathway. However, the biological function and mechanism of IQ-domain GTPase-activating protein 1 in invasive ductal carcinoma (IDC) remain unknown. In this study, we found that IQ-domain GTPase-activating protein 1 expression was elevated in invasive ductal carcinoma, which was positively correlated with tumor grade, lymphatic metastasis, and poor prognosis. Coexpression of IQ-domain GTPase-activating protein 1 and Dvl in the nucleus and cytoplasm of invasive ductal carcinoma was significantly correlated but not in the membrane. Postoperative survival in the patients with their coexpression in the nucleus and cytoplasm was obviously lower than that without coexpression. The positive expression rates of c-myc and cyclin D1 were significantly higher in the patients with nuclear coexpression of Dvl and IQ-domain GTPase-activating protein 1 than that with cytoplasmic coexpression, correlating with poor prognosis. IQ-domain GTPase-activating protein 1 significantly enhanced cell proliferation and invasion in invasive ductal carcinoma cell lines by interacting with Dvl in cytoplasm to promote Dvl nuclear translocation so as to upregulate the expression of c-myc and cyclin D1. Collectively, our data suggest that IQ-domain GTPase-activating protein 1 may promote the malignant phenotype of invasive ductal carcinoma via canonical Wnt signaling, and it could be used as a potential prognostic biomarker for breast cancer patients.

Rho GTPases in ameloblast differentiation

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Keishi Otsu

2016-05-01

Full Text Available During tooth development, ameloblasts differentiate from inner enamel epithelial cells to enamel-forming cells by modulating the signal pathways mediating epithelial–mesenchymal interaction and a cell-autonomous gene network. The differentiation process of epithelial cells is characterized by marked changes in their morphology and polarity, accompanied by dynamic cytoskeletal reorganization and changes in cell–cell and cell–matrix adhesion over time. Functional ameloblasts are tall, columnar, polarized cells that synthesize and secrete enamel-specific proteins. After deposition of the full thickness of enamel matrix, ameloblasts become smaller and regulate enamel maturation. Recent significant advances in the fields of molecular biology and genetics have improved our understanding of the regulatory mechanism of the ameloblast cell life cycle, mediated by the Rho family of small GTPases. They act as intracellular molecular switch that transduce signals from extracellular stimuli to the actin cytoskeleton and the nucleus. In our review, we summarize studies that provide current evidence for Rho GTPases and their involvement in ameloblast differentiation. In addition to the Rho GTPases themselves, their downstream effectors and upstream regulators have also been implicated in ameloblast differentiation.

Merkel Cell Polyomavirus Small T Antigen Drives Cell Motility via Rho-GTPase-Induced Filopodium Formation.

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Stakaitytė, Gabrielė; Nwogu, Nnenna; Dobson, Samuel J; Knight, Laura M; Wasson, Christopher W; Salguero, Francisco J; Blackbourn, David J; Blair, G Eric; Mankouri, Jamel; Macdonald, Andrew; Whitehouse, Adrian

2018-01-15

Cell motility and migration is a complex, multistep, and multicomponent process intrinsic to progression and metastasis. Motility is dependent on the activities of integrin receptors and Rho family GTPases, resulting in the remodeling of the actin cytoskeleton and formation of various motile actin-based protrusions. Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high likelihood of recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is associated with the majority of MCC cases, and MCPyV-induced tumorigenesis largely depends on the expression of the small tumor antigen (ST). Since the discovery of MCPyV, a number of mechanisms have been suggested to account for replication and tumorigenesis, but to date, little is known about potential links between MCPyV T antigen expression and the metastatic nature of MCC. Previously, we described the action of MCPyV ST on the microtubule network and how it impacts cell motility and migration. Here, we demonstrate that MCPyV ST affects the actin cytoskeleton to promote the formation of filopodia through a mechanism involving the catalytic subunit of protein phosphatase 4 (PP4C). We also show that MCPyV ST-induced cell motility is dependent upon the activities of the Rho family GTPases Cdc42 and RhoA. In addition, our results indicate that the MCPyV ST-PP4C interaction results in the dephosphorylation of β 1 integrin, likely driving the cell motility pathway. These findings describe a novel mechanism by which a tumor virus induces cell motility, which may ultimately lead to cancer metastasis, and provides opportunities and strategies for targeted interventions for disseminated MCC. IMPORTANCE Merkel cell polyomavirus (MCPyV) is the most recently discovered human tumor virus. It causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer. However, the molecular mechanisms implicating MCPyV-encoded proteins in cancer development are yet to be fully elucidated. This study builds

"Everybody Gotta Have a Dream": Rap-centered Aspirations among Young Black Males Involved in Rap Music Production - A Qualitative Study.

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Foster, B Brian

Youth express diverse desires for their educational and occupational futures. Sometimes these aspirations are directed towards somewhat unconventional careers such as rapping and other types of involvement in rap music production. Although many studies have examined traditional educational and occupational aspirations, less is known about the factors that give rise to rap-centered aspirations and how individuals pursue them, particularly as they transition to early adulthood. Drawing on 54 semi- and unstructured interviews with 29 black young men involved in rap music production, I find that rap-centered aspirations are shaped by a range of factors, most notably feedback regarding one's rap skills, access to recording and production equipment, and the financial means to maintain involvement in rap music production while also ensuring personal and family economic stability. The young men in the study attached different meanings to their aspirations and sometimes recast their motivations for participating in rap music production in response to various social and economic factors.

Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein

International Nuclear Information System (INIS)

Klinglmayr, Eva; Wenger, Julia; Mayr, Sandra; Bossy-Wetzel, Ella; Puehringer, Sandra

2012-01-01

The crystallization and initial diffraction analysis of human Drp1 GTPase-GED fusion protein are reported. The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 Å resolution. The hexagonal-shaped crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis

Role of the Small GTPase Rho3 in Golgi/Endosome trafficking through functional interaction with adaptin in Fission Yeast.

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Ayako Kita

Full Text Available BACKGROUND: We had previously identified the mutant allele of apm1(+ that encodes a homolog of the mammalian µ1A subunit of the clathrin-associated adaptor protein-1 (AP-1 complex, and we demonstrated the role of Apm1 in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we isolated rho3(+, which encodes a Rho-family small GTPase, an important regulator of exocystosis, as a multicopy-suppressor of the temperature-sensitive growth of the apm1-1 mutant cells. Overexpression of Rho3 suppressed the Cl(- sensitivity and immunosuppressant sensitivity of the apm1-1 mutant cells. Overexpression of Rho3 also suppressed the fragmentation of vacuoles, and the accumulation of v-SNARE Syb1 in Golgi/endosomes and partially suppressed the defective secretion associated with apm1-deletion cells. Notably, electron microscopic observation of the rho3-deletion cells revealed the accumulation of abnormal Golgi-like structures, vacuole fragmentation, and accumulation of secretory vesicles; these phenotypes were very similar to those of the apm1-deletion cells. Furthermore, the rho3-deletion cells and apm1-deletion cells showed very similar phenotypic characteristics, including the sensitivity to the immunosuppressant FK506, the cell wall-damaging agent micafungin, Cl(-, and valproic acid. Green fluorescent protein (GFP-Rho3 was localized at Golgi/endosomes as well as the plasma membrane and division site. Finally, Rho3 was shown to form a complex with Apm1 as well as with other subunits of the clathrin-associated AP-1 complex in a GTP- and effector domain-dependent manner. CONCLUSIONS/SIGNIFICANCE: Taken together, our findings reveal a novel role of Rho3 in the regulation of Golgi/endosome trafficking and suggest that clathrin-associated adaptor protein-1 and Rho3 co-ordinate in intracellular transport in fission yeast. To the best of our knowledge, this study provides the first evidence

Rhoptry-associated protein (rap-1) genes in the sheep pathogen Babesia sp. Xinjiang: Multiple transcribed copies differing by 3' end repeated sequences.

Science.gov (United States)

Niu, Qingli; Marchand, Jordan; Yang, Congshan; Bonsergent, Claire; Guan, Guiquan; Yin, Hong; Malandrin, Laurence

2015-07-30

Sheep babesiosis occurs mainly in tropical and subtropical areas. The sheep parasite Babesia sp. Xinjiang is widespread in China, and our goal is to characterize rap-1 (rhoptry-associated protein 1) gene diversity and expression as a first step of a long term goal aiming at developing a recombinant subunit vaccine. Seven different rap-1a genes were amplified in Babesia sp. Xinjiang, using degenerate primers designed from conserved motifs. Rap-1b and rap-1c gene types could not be identified. In all seven rap-1a genes, the 5' regions exhibited identical sequences over 936 nt, and the 3' regions differed at 28 positions over 147 nt, defining two types of genes designated α and β. The remaining 3' part varied from 72 to 360 nt in length, depending on the gene. This region consists of a succession of two to ten 36 nt repeats, which explains the size differences. Even if the nucleotide sequences varied, 6 repeats encoded the same stretch of amino acids. Transcription of at least four α and two β genes was demonstrated by standard RT-PCR. Copyright © 2015 Elsevier B.V. All rights reserved.

Opposing effects of actin signaling and LFA-1 on establishing the affinity threshold for inducing effector T-cell responses in mice

Czech Academy of Sciences Publication Activity Database

Palmer, E.; Drobek, Aleš; Štěpánek, Ondřej

2016-01-01

Roč. 46, č. 8 (2016), s. 1887-1901 ISSN 0014-2980 R&D Projects: GA ČR GJ16-09208Y Institutional support: RVO:68378050 Keywords : Actin cytoskeleton * Antigen affinity treshold * LFA-1 * Rap1 * Rho-family GTPases * T-cell receptor signaling Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.227, year: 2016

The small GTPase Rac1 is required for smooth muscle contraction

DEFF Research Database (Denmark)

Rahman, Awahan; Davis, Benjamin; Lövdahl, Cecilia

2014-01-01

The role of the small GTP-binding protein Rac1 in smooth muscle contraction was examined using small molecule inhibitors (EHT1864, NSC23766) and a novel smooth muscle-specific, conditional, Rac1 knockout mouse strain. EHT1864, which affects nucleotide binding and inhibits Rac1 activity, concentra...

Mutations in the small GTPase gene RAB39B are responsible for X-linked mental retardation associated with autism, epilepsy, and macrocephaly.

Science.gov (United States)

Giannandrea, Maila; Bianchi, Veronica; Mignogna, Maria Lidia; Sirri, Alessandra; Carrabino, Salvatore; D'Elia, Errico; Vecellio, Matteo; Russo, Silvia; Cogliati, Francesca; Larizza, Lidia; Ropers, Hans-Hilger; Tzschach, Andreas; Kalscheuer, Vera; Oehl-Jaschkowitz, Barbara; Skinner, Cindy; Schwartz, Charles E; Gecz, Jozef; Van Esch, Hilde; Raynaud, Martine; Chelly, Jamel; de Brouwer, Arjan P M; Toniolo, Daniela; D'Adamo, Patrizia

2010-02-12

Human Mental Retardation (MR) is a common and highly heterogeneous pediatric disorder affecting around 3% of the general population; at least 215 X-linked MR (XLMR) conditions have been described, and mutations have been identified in 83 different genes, encoding proteins with a variety of function, such as chromatin remodeling, synaptic function, and intracellular trafficking. The small GTPases of the RAB family, which play an essential role in intracellular vesicular trafficking, have been shown to be involved in MR. We report here the identification of mutations in the small GTPase RAB39B gene in two male patients. One mutation in family X (D-23) introduced a stop codon seven amino acids after the start codon (c.21C > A; p.Y7X). A second mutation, in the MRX72 family, altered the 5' splice site (c.215+1G > A) and normal splicing. Neither instance produced a protein. Mutations segregate with the disease in the families, and in some family members intellectual disabilities were associated with autism spectrum disorder, epileptic seizures, and macrocephaly. We show that RAB39B, a novel RAB GTPase of unknown function, is a neuronal-specific protein that is localized to the Golgi compartment. Its downregulation leads to an alteration in the number and morphology of neurite growth cones and a significant reduction in presynaptic buttons, suggesting that RAB39B is required for synapse formation and maintenance. Our results demonstrate developmental and functional neuronal alteration as a consequence of downregulation of RAB39B and emphasize the critical role of vesicular trafficking in the development of neurons and human intellectual abilities. Copyright (c) 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Species-Specific Expression of Full-Length and Alternatively Spliced Variant Forms of CDK5RAP2.

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John S Y Park

Full Text Available CDK5RAP2 is one of the primary microcephaly genes that are associated with reduced brain size and mental retardation. We have previously shown that human CDK5RAP2 exists as a full-length form (hCDK5RAP2 or an alternatively spliced variant form (hCDK5RAP2-V1 that is lacking exon 32. The equivalent of hCDK5RAP2-V1 has been reported in rat and mouse but the presence of full-length equivalent hCDK5RAP2 in rat and mouse has not been examined. Here, we demonstrate that rat expresses both a full length and an alternatively spliced variant form of CDK5RAP2 that are equivalent to our previously reported hCDK5RAP2 and hCDK5RAP2-V1, repectively. However, mouse expresses only one form of CDK5RAP2 that is equivalent to the human and rat alternatively spliced variant forms. Knowledge of this expression of different forms of CDK5RAP2 in human, rat and mouse is essential in selecting the appropriate model for studies of CDK5RAP2 and primary microcephaly but our findings further indicate the evolutionary divergence of mouse from the human and rat species.

MiR-203 involves in neuropathic pain development and represses Rap1a expression in nerve growth factor differentiated neuronal PC12 cells.

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Li, Haixia; Huang, Yuguang; Ma, Chao; Yu, Xuerong; Zhang, Zhiyong; Shen, Le

2015-01-01

Although microRNAs (miRNAs) have been shown to play a role in numerous biological processes, their function in neuropathic pain is not clear. The rat bilateral sciatic nerve chronic constriction injury (bCCI) is an established model of neuropathic pain, so we examined miRNA expression and function in the spinal dorsal horn in bCCI rats. Microarray and real-time polymerase chain reaction were used to examine the expression of miRNA in nerve system of bCCI rats, and the targets of miRNA were predicted by bioinformatic approaches. The function of specific miRNA was estimated through the methods of gene engineering. This study revealed substantially (∼10-fold) decreased miR-203 expression in the spinal dorsal horns but not the dorsal root ganglions, hippocampus, or anterior cingulate cortexes of bCCI rats. Rap1a protein expression was upregulated in bCCI rat spinal dorsal horns. We further verified that miR-203 directly targeted the 3'-untranslated region of the rap1a gene, thereby decreasing rap1a protein expression in neuron-like cells. Rap1a has diverse neuronal functions and their perturbation is responsible for several mental disorders. For example, Rap1a/MEK/ERK is involved in peripheral sensitization. These data suggest a potential role for miR-203 in regulating neuropathic pain development, and Rap1a is a validated target gene in vitro. Results from our study and others indicate the possibility that Rap1a may be involved in pain. We hope that these results can provide support for future research into miR-203 in gene therapy for neuropathic pain.

Mutations in CDK5RAP2 cause Seckel syndrome.

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Yigit, Gökhan; Brown, Karen E; Kayserili, Hülya; Pohl, Esther; Caliebe, Almuth; Zahnleiter, Diana; Rosser, Elisabeth; Bögershausen, Nina; Uyguner, Zehra Oya; Altunoglu, Umut; Nürnberg, Gudrun; Nürnberg, Peter; Rauch, Anita; Li, Yun; Thiel, Christian Thomas; Wollnik, Bernd

2015-09-01

Seckel syndrome is a heterogeneous, autosomal recessive disorder marked by prenatal proportionate short stature, severe microcephaly, intellectual disability, and characteristic facial features. Here, we describe the novel homozygous splice-site mutations c.383+1G>C and c.4005-9A>G in CDK5RAP2 in two consanguineous families with Seckel syndrome. CDK5RAP2 (CEP215) encodes a centrosomal protein which is known to be essential for centrosomal cohesion and proper spindle formation and has been shown to be causally involved in autosomal recessive primary microcephaly. We establish CDK5RAP2 as a disease-causing gene for Seckel syndrome and show that loss of functional CDK5RAP2 leads to severe defects in mitosis and spindle organization, resulting in cells with abnormal nuclei and centrosomal pattern, which underlines the important role of centrosomal and mitotic proteins in the pathogenesis of the disease. Additionally, we present an intriguing case of possible digenic inheritance in Seckel syndrome: A severely affected child of nonconsanguineous German parents was found to carry heterozygous mutations in CDK5RAP2 and CEP152. This finding points toward a potential additive genetic effect of mutations in CDK5RAP2 and CEP152.

Mutations in CDK5RAP2 cause Seckel syndrome

Science.gov (United States)

Yigit, Gökhan; Brown, Karen E; Kayserili, Hülya; Pohl, Esther; Caliebe, Almuth; Zahnleiter, Diana; Rosser, Elisabeth; Bögershausen, Nina; Uyguner, Zehra Oya; Altunoglu, Umut; Nürnberg, Gudrun; Nürnberg, Peter; Rauch, Anita; Li, Yun; Thiel, Christian Thomas; Wollnik, Bernd

2015-01-01

Seckel syndrome is a heterogeneous, autosomal recessive disorder marked by prenatal proportionate short stature, severe microcephaly, intellectual disability, and characteristic facial features. Here, we describe the novel homozygous splice-site mutations c.383+1G>C and c.4005-9A>G in CDK5RAP2 in two consanguineous families with Seckel syndrome. CDK5RAP2 (CEP215) encodes a centrosomal protein which is known to be essential for centrosomal cohesion and proper spindle formation and has been shown to be causally involved in autosomal recessive primary microcephaly. We establish CDK5RAP2 as a disease-causing gene for Seckel syndrome and show that loss of functional CDK5RAP2 leads to severe defects in mitosis and spindle organization, resulting in cells with abnormal nuclei and centrosomal pattern, which underlines the important role of centrosomal and mitotic proteins in the pathogenesis of the disease. Additionally, we present an intriguing case of possible digenic inheritance in Seckel syndrome: A severely affected child of nonconsanguineous German parents was found to carry heterozygous mutations in CDK5RAP2 and CEP152. This finding points toward a potential additive genetic effect of mutations in CDK5RAP2 and CEP152. PMID:26436113

Rho GTPase function in tumorigenesis

DEFF Research Database (Denmark)

Karlsson, R; Pedersen, Esben Ditlev Kølle; Wang, Zhipeng

2009-01-01

, for that reason, Rho GTPases, their regulators, and their effectors have been suggested to control tumor formation and progression in humans. However, while the tumor-relevant functions of Rho GTPases are very well documented in vitro, we are only now beginning to assess their contribution to cancer in human...... patients and in animal models. This review will give a very brief overview of Rho GTPase function in general and then focus on in vivo evidence for a role of Rho GTPases in malignant tumors, both in human patients and in genetically modified mice....

Insight into temperature dependence of GTPase activity in human guanylate binding protein-1.

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Anjana Rani

Full Text Available Interferon-γ induced human guanylate binding protein-1(hGBP1 belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried out at 15°C. The hydrolysis reaction in hGBP1 is believed to involve with a number of catalytic steps. To investigate the effect of temperature in the product formation and on the different catalytic complexes of hGBP1, we carried out temperature dependent GTPase assays, mutational analysis, chemical and thermal denaturation studies. The Arrhenius plot for both GDP and GMP interestingly showed nonlinear behaviour, suggesting that the product formation from the GTP-bound enzyme complex is associated with at least more than one step. The negative activation energy for GDP formation and GTPase assay with external GDP together indicate that GDP formation occurs through the reversible dissociation of GDP-bound enzyme dimer to monomer, which further reversibly dissociates to give the product. Denaturation studies of different catalytic complexes show that unlike other complexes the free energy of GDP-bound hGBP1 decreases significantly at lower temperature. GDP formation is found to be dependent on the free energy of the GDP-bound enzyme complex. The decrease in the free energy of this complex at low temperature compared to at high is the reason for higher GDP formation at low temperature. Thermal denaturation studies also suggest that the difference in the free energy of the GTP-bound enzyme dimer compared to its monomer plays a crucial role in the product formation; higher stability favours GMP but lower favours GDP. Thus, this study provides the first thermodynamic insight into the effect of temperature in the product formation of hGBP1.

Effects of using nursing home residents to serve as group activity leaders: lessons learned from the RAP project.

Science.gov (United States)

Skrajner, Michael J; Haberman, Jessica L; Camp, Cameron J; Tusick, Melanie; Frentiu, Cristina; Gorzelle, Gregg

2014-03-01

Previous research has demonstrated that persons with early to moderate stage dementia are capable of leading small group activities for persons with more advanced dementia. In this study, we built upon this previous work by training residents in long-term care facilities to fill the role of group activity leaders using a Resident-Assisted Programming (RAP) training regimen. There were two stages to the program. In the first stage, RAP training was provided by researchers. In the second stage, RAP training was provided to residents by activities staff members of long-term care facilities who had been trained by researchers. We examine the effects of RAP implemented by researchers and by activities staff member on long-term care resident with dementia who took part in these RAP activities. We also examined effects produced by two types of small group activities: two Montessori-based activities and an activity which focuses on persons with more advanced dementia, based on the work of Jitka Zgola. Results demonstrate that levels of positive engagement seen in players during RAP (resident-led activities) were typically higher than those observed during standard activities programming led by site staff. In general, Montessori-Based Dementia Programming® produced more constructive engagement than Zgola-based programming (ZBP), though ZBP did increase a positive form of engagement involving observing activities with interest. In addition, RAP implemented by activities staff members produced effects that were, on the whole, similar to those produced when RAP was implemented by researchers. Implications of these findings for providing meaningful social roles for persons with dementia residing in long-term care, and suggestions for further research in this area, are discussed.

Structural Basis of Rap Phosphatase Inhibition by Phr Peptides

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Gallego del Sol, Francisca; Marina, Alberto

2013-01-01

Two-component systems, composed of a sensor histidine kinase and an effector response regulator (RR), are the main signal transduction devices in bacteria. In Bacillus, the Rap protein family modulates complex signaling processes mediated by two-component systems, such as competence, sporulation, or biofilm formation, by inhibiting the RR components involved in these pathways. Despite the high degree of sequence homology, Rap proteins exert their activity by two completely different mechanisms of action: inducing RR dephosphorylation or blocking RR binding to its target promoter. However the regulatory mechanism involving Rap proteins is even more complex since Rap activity is antagonized by specific signaling peptides (Phr) through a mechanism that remains unknown at the molecular level. Using X-ray analyses, we determined the structure of RapF, the anti-activator of competence RR ComA, alone and in complex with its regulatory peptide PhrF. The structural and functional data presented herein reveal that peptide PhrF blocks the RapF-ComA interaction through an allosteric mechanism. PhrF accommodates in the C-terminal tetratricopeptide repeat domain of RapF by inducing its constriction, a conformational change propagated by a pronounced rotation to the N-terminal ComA-binding domain. This movement partially disrupts the ComA binding site by triggering the ComA disassociation, whose interaction with RapF is also sterically impaired in the PhrF-induced conformation of RapF. Sequence analyses of the Rap proteins, guided by the RapF-PhrF structure, unveil the molecular basis of Phr recognition and discrimination, allowing us to relax the Phr specificity of RapF by a single residue change. PMID:23526880

Human Lsg1 defines a family of essential GTPases that correlates with the evolution of compartmentalization

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Scheffzek Klaus

2005-10-01

Full Text Available Abstract Background Compartmentalization is a key feature of eukaryotic cells, but its evolution remains poorly understood. GTPases are the oldest enzymes that use nucleotides as substrates and they participate in a wide range of cellular processes. Therefore, they are ideal tools for comparative genomic studies aimed at understanding how aspects of biological complexity such as cellular compartmentalization evolved. Results We describe the identification and characterization of a unique family of circularly permuted GTPases represented by the human orthologue of yeast Lsg1p. We placed the members of this family in the phylogenetic context of the YlqF Related GTPase (YRG family, which are present in Eukarya, Bacteria and Archea and include the stem cell regulator Nucleostemin. To extend the computational analysis, we showed that hLsg1 is an essential GTPase predominantly located in the endoplasmic reticulum and, in some cells, in Cajal bodies in the nucleus. Comparison of localization and siRNA datasets suggests that all members of the family are essential GTPases that have increased in number as the compartmentalization of the eukaryotic cell and the ribosome biogenesis pathway have evolved. Conclusion We propose a scenario, consistent with our data, for the evolution of this family: cytoplasmic components were first acquired, followed by nuclear components, and finally the mitochondrial and chloroplast elements were derived from different bacterial species, in parallel with the formation of the nucleolus and the specialization of nuclear components.

Rapid Refresh (RAP) [13 km

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National Oceanic and Atmospheric Administration, Department of Commerce — The Rapid Refresh (RAP) numerical weather model took the place of the Rapid Update Cycle (RUC) on May 1, 2012. Run by the National Centers for Environmental...

Rapid Refresh (RAP) [20 km

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — The Rapid Refresh (RAP) numerical weather model took the place of the Rapid Update Cycle (RUC) on May 1, 2012. Run by the National Centers for Environmental...

Overview of the remedial action priority system (RAPS)

International Nuclear Information System (INIS)

Whelan, G.; Steelman, B.L.; Strenge, D.L.; Droppo, J.G.

1986-01-01

To provide DOE with a better management tool for prioritizing funding allocations for further site investigations and possible remediations, Pacific Northwest Laboratory developed a more objective, physics-based risk assessment methodology called the Remedial Action Priority System (RAPS). This methodology uses empirically, analytically, and semianalytically based mathematical algorithms and a pathways analysis to predict the potential for contaminant transport from a hazardous waste disposal site to local populations. Four major pathways for contaminant migration are considered in the RAPS methodology: groundwater, overland, surface water, and atmospheric. Using the predications of contaminant transport, simplified exposure assessments are performed for important receptors. The risks associated with the sites can then be calculated relative to other sites for each pathway and for all pathways together. The RAPS methodology addresses many of the typical limitations associated with other ranking systems; it considers: (1) more site information and constituent characteristics associated with the transport pathways; (2) chemical and radioactive wastes; (3) the potential direction of contaminant movement; (4) contaminant retention (e.g., dispersion and decay/degradation), where applicable; (5) population distributions; (6) various routes of exposure (e.g., inhalations, ingestion, and external exposure); (7) contaminant toxicities; (8) duration of exposure of the surrounding population; and (9) contaminant arrival time to sensitive receptors. Because RAPS is based on more site information and constituent characteristics, the scoring system of the RAPS methodology also reduces the subjectivity associated with prioritizing hazardous waste sites. The RAPS methodology requires minimum user knowledge of risk assessment and a minimum amount of input data

Secretion of soluble vascular endothelial growth factor receptor 1 (sVEGFR1/sFlt1 requires Arf1, Arf6, and Rab11 GTPases.

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Jae-Joon Jung

Full Text Available The soluble form of vascular endothelial growth factor receptor 1 (sVEGFR-1/sFlt1 is generated by alternative splicing of the FLT1 gene. Secretion of sFlt1 from endothelial cells plays an important role in blood vessel sprouting and morphogenesis. However, excess sFlt1 secretion is associated with diseases such as preeclampsia and chronic kidney disease. To date, the secretory transport process involved in the secretion of sFlt1 is poorly understood. In the present study, we investigated the itinerary of sFlt1 trafficking along the secretory pathway. To understand the timecourse of sFlt1 secretion, endothelial cells stably expressing sFlt1 were metabolically radiolabeled with [(35S]-methionine and cysteine. Our results indicate that after initial synthesis the levels of secreted [(35S]-sFlt1 in the extracellular medium peaks at 8 hours. Treatment with brefeldin A (BFA, a drug which blocks trafficking between the endoplasmic reticulum (ER and the Golgi complex, inhibited extracellular release of sFlt1 suggesting that ER to Golgi and intra-Golgi trafficking of sFlt1 are essential for its secretion. Furthermore, we show that ectopic expression of dominant-negative mutant forms of Arf1, Arf6, and Rab11 as well as siRNA-mediated knockdown of these GTPases block secretion of sFlt1 during normoxic and hypoxic conditions suggesting role for these small GTPases. This work is the first to report role of regulatory proteins involved in sFlt1 trafficking along the secretory pathway and may provide insights and new molecular targets for the modulation of sFlt-1 release during physiological and pathological conditions.

Crosstalk between Bcl-2 family and Ras family small GTPases: potential cell fate regulation?

International Nuclear Information System (INIS)

Kang, Jia; Pervaiz, Shazib

2013-01-01

Cell fate regulation is a function of diverse cell signaling pathways that promote cell survival and or inhibit cell death execution. In this regard, the role of the Bcl-2 family in maintaining a tight balance between cell death and cell proliferation has been extensively studied. The conventional dogma links cell fate regulation by the Bcl-2 family to its effect on mitochondrial permeabilization and apoptosis amplification. However, recent evidence provide a novel mechanism for death regulation by the Bcl-2 family via modulating cellular redox metabolism. For example overexpression of Bcl-2 has been shown to contribute to a pro-oxidant intracellular milieu and down-regulation of cellular superoxide levels enhanced death sensitivity of Bcl-2 overexpressing cells. Interestingly, gene knockdown of the small GTPase Rac1 or pharmacological inhibition of its activity also reverted death phenotype in Bcl-2 expressing cells. This appears to be a function of an interaction between Bcl-2 and Rac1. Similar functional associations have been described between the Bcl-2 family and other members of the Ras superfamily. These interactions at the mitochondria provide novel opportunities for strategic therapeutic targeting of drug-resistant cancers.

Customized treatment in non-small-cell lung cancer based on EGFR mutations and BRCA1 mRNA expression.

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Rafael Rosell

Full Text Available BACKGROUND: Median survival is 10 months and 2-year survival is 20% in metastatic non-small-cell lung cancer (NSCLC treated with platinum-based chemotherapy. A small fraction of non-squamous cell lung cancers harbor EGFR mutations, with improved outcome to gefitinib and erlotinib. Experimental evidence suggests that BRCA1 overexpression enhances sensitivity to docetaxel and resistance to cisplatin. RAP80 and Abraxas are interacting proteins that form complexes with BRCA1 and could modulate the effect of BRCA1. In order to further examine the effect of EGFR mutations and BRCA1 mRNA levels on outcome in advanced NSCLC, we performed a prospective non-randomized phase II clinical trial, testing the hypothesis that customized therapy would confer improved outcome over non-customized therapy. In an exploratory analysis, we also examined the effect of RAP80 and Abraxas mRNA levels. METHODOLOGY/PRINCIPAL FINDINGS: We treated 123 metastatic non-squamous cell lung carcinoma patients using a customized approach. RNA and DNA were isolated from microdissected specimens from paraffin-embedded tumor tissue. Patients with EGFR mutations received erlotinib, and those without EGFR mutations received chemotherapy with or without cisplatin based on their BRCA1 mRNA levels: low, cisplatin plus gemcitabine; intermediate, cisplatin plus docetaxel; high, docetaxel alone. An exploratory analysis examined RAP80 and Abraxas expression. Median survival exceeded 28 months for 12 patients with EGFR mutations, and was 11 months for 38 patients with low BRCA1, 9 months for 40 patients with intermediate BRCA1, and 11 months for 33 patients with high BRCA1. Two-year survival was 73.3%, 41.2%, 15.6% and 0%, respectively. Median survival was influenced by RAP80 expression in the three BRCA1 groups. For example, for patients with both low BRCA1 and low RAP80, median survival exceeded 26 months. RAP80 was a significant factor for survival in patients treated according to BRCA1

The Remedial Action Priority System (RAPS): Mathematical formulations

International Nuclear Information System (INIS)

Whelan, G.; Strenge, D.L.; Droppo, J.G. Jr.; Steelman, B.L.; Buck, J.W.

1987-08-01

The Remedial Action Priority System (RAPS) represents a methodology that prioritizes inactive hazardous and radioactive mixed-waste disposal sites in a scientific and objective manner based on limited site information. This methodology is intended to bridge the technology gap between the initial site evaluation using the Hazard Ranking System (HRS) and the time-consuming process of actual field site characterization, assessment, and remediation efforts. The RAPS methodology provides the US Department of Energy with a management tool for assistance in prioritizing funding and human resource allocations for further investigations and possible remediations at its inactive waste sites. Use of RAPS will help DOE ensure that those sites posing the highest potential risk are addressed first. Chapters 1 through 10 were processed separately for the Energy Data Base

RAB10 Interacts with the Male Germ Cell-Specific GTPase-Activating Protein during Mammalian Spermiogenesis

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Ying-Hung Lin

2017-01-01

Full Text Available According to recent estimates, 2%–15% of couples are sterile, and approximately half of the infertility cases are attributed to male reproductive factors. However, the reasons remain undefined in approximately 25% of male infertility cases, and most infertility cases exhibit spermatogenic defects. Numerous genes involved in spermatogenesis still remain unknown. We previously identified Male Germ Cells Rab GTPase-Activating Proteins (MGCRABGAPs through cDNA microarray analysis of human testicular tissues with spermatogenic defects. MGCRABGAP contains a conserved RABGAP catalytic domain, TBC (Tre2/Bub2/Cdc16. RABGAP family proteins regulate cellular function (e.g., cytoskeletal remodeling, vesicular trafficking, and cell migration by inactivating RAB proteins. MGCRABGAP is a male germ cell-specific protein expressed in elongating and elongated spermatids during mammalian spermiogenesis. The purpose of this study was to identify proteins that interact with MGCRABGAP during mammalian spermiogenesis using a proteomic approach. We found that MGCRABGAP exhibited GTPase-activating bioability, and several MGCRABGAP interactors, possible substrates (e.g., RAB10, RAB5C, and RAP1, were identified using co-immunoprecipitation (co-IP and nano liquid chromatography-mass spectrometry/mass spectrometry (nano LC-MS/MS. We confirmed the binding ability between RAB10 and MGCRABGAP via co-IP. Additionally, MGCRABGAP–RAB10 complexes were specifically colocalized in the manchette structure, a critical structure for the formation of spermatid heads, and were slightly expressed at the midpiece of mature spermatozoa. Based on these results, we propose that MGCRABGAP is involved in mammalian spermiogenesis by modulating RAB10.

Rho GTPase expression in human myeloid cells.

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Suzanne F G van Helden

Full Text Available Myeloid cells are critical for innate immunity and the initiation of adaptive immunity. Strict regulation of the adhesive and migratory behavior is essential for proper functioning of these cells. Rho GTPases are important regulators of adhesion and migration; however, it is unknown which Rho GTPases are expressed in different myeloid cells. Here, we use a qPCR-based approach to investigate Rho GTPase expression in myeloid cells.We found that the mRNAs encoding Cdc42, RhoQ, Rac1, Rac2, RhoA and RhoC are the most abundant. In addition, RhoG, RhoB, RhoF and RhoV are expressed at low levels or only in specific cell types. More differentiated cells along the monocyte-lineage display lower levels of Cdc42 and RhoV, while RhoC mRNA is more abundant. In addition, the Rho GTPase expression profile changes during dendritic cell maturation with Rac1 being upregulated and Rac2 downregulated. Finally, GM-CSF stimulation, during macrophage and osteoclast differentiation, leads to high expression of Rac2, while M-CSF induces high levels of RhoA, showing that these cytokines induce a distinct pattern. Our data uncover cell type specific modulation of the Rho GTPase expression profile in hematopoietic stem cells and in more differentiated cells of the myeloid lineage.

Sevoflurane Inhalation Accelerates the Long-Term Memory Consolidation via Small GTPase Overexpression in the Hippocampus of Mice in Adolescence.

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Nakamura, Emi; Kinoshita, Hiroyuki; Feng, Guo-Gang; Hayashi, Hisaki; Satomoto, Maiko; Sato, Motohiko; Fujiwara, Yoshihiro

2016-01-01

Sevoflurane exposure impairs the long-term memory in neonates. Whether the exposure to animals in adolescence affects the memory, however, has been unclear. A small hydrolase enzyme of guanosine triphosphate (GTPase) rac1 plays a role in the F-actin dynamics related to the synaptic plasticity, as well as superoxide production via reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. The current study was designed to examine whether sevoflurane exposure to mice in early adolescence modifies the long-term learning ability concomitantly with the changes in F-actin constitution as well as superoxide production in the hippocampus according to the levels of rac1 protein expression. Four-week-old mice were subjected to the evaluation of long-term learning ability for three days. On day one, each mouse was allowed to enter a dark chamber for five min to acclimatization. On day two, the procedure was repeated with the addition of an electric shock as soon as a mouse entered the dark chamber. All mice subsequently inhaled 2 L/min air with (Sevoflurane group) and without (Control group) 2.5% sevoflurane for three hours. On day three, each mouse was placed on the platform and retention time, which is the latency to enter the dark chamber, was examined. The brain removed after the behavior test, was used for analyses of immunofluorescence, Western immunoblotting and intracellular levels of superoxide. Sevoflurane exposure significantly prolonged retention time, indicating the enhanced long-term memory. Sevoflurane inhalation augmented F-actin constitution coexisting with the rac1 protein overexpression in the hippocampus whereas it did not alter the levels of superoxide. Sevoflurane exposure to 4-week-old mice accelerates the long-term memory concomitantly with the enhanced F-actin constitution coexisting with the small GTPase rac1 overexpression in the hippocampus. These results suggest that sevoflurane inhalation may amplify long-term memory

Life extension and replacement management for RAPS type steam generators

International Nuclear Information System (INIS)

Arya, R.C.; Rastogi, A.K.

1996-01-01

The steam generating equipment in first four units of Indian PHWRs Rajasthan Atomic Power Station (RAPS) 1-2 and Madras Atomic Power Station (MAPS) 1-2 are hairpin type and comprise of eight boiler assemblies. Each assembly consists of identical, single pass, inverted and vertical hairpin heat exchangers (10 for RAPS and 11 for MAPS) containing 195 monel-400 U tubes of 12.7 mm dia x 1.242 mm thick. The hot heavy water flows through these tubes and imparts heat to feed, light demineralized water entering the shell at the bottom of preheat leg. The heat is generated on the outer surface of the tubes. Details of studies carried out for life extension and replacement management for RAPS type steam generators are given. 1 fig., 5 tabs

Identification of potential small molecule binding pockets on Rho family GTPases.

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Juan Manuel Ortiz-Sanchez

Full Text Available Rho GTPases are conformational switches that control a wide variety of signaling pathways critical for eukaryotic cell development and proliferation. They represent attractive targets for drug design as their aberrant function and deregulated activity is associated with many human diseases including cancer. Extensive high-resolution structures (>100 and recent mutagenesis studies have laid the foundation for the design of new structure-based chemotherapeutic strategies. Although the inhibition of Rho signaling with drug-like compounds is an active area of current research, very little attention has been devoted to directly inhibiting Rho by targeting potential allosteric non-nucleotide binding sites. By avoiding the nucleotide binding site, compounds may minimize the potential for undesirable off-target interactions with other ubiquitous GTP and ATP binding proteins. Here we describe the application of molecular dynamics simulations, principal component analysis, sequence conservation analysis, and ensemble small-molecule fragment mapping to provide an extensive mapping of potential small-molecule binding pockets on Rho family members. Characterized sites include novel pockets in the vicinity of the conformationaly responsive switch regions as well as distal sites that appear to be related to the conformations of the nucleotide binding region. Furthermore the use of accelerated molecular dynamics simulation, an advanced sampling method that extends the accessible time-scale of conventional simulations, is found to enhance the characterization of novel binding sites when conformational changes are important for the protein mechanism.

Translating Chicana Rap: Snow Tha Product

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Adriana Onita

2017-06-01

Full Text Available This project examines rap lyrics, interviews, and music videos by Chicana artist Snow Tha Product to show how rap has been culturally translated, performed, and appropriated by females in order to “flip the script,” or subvert the dichotomous model of female sexuality that has been imposed upon them. Weaving insights from three academic fields (cultural translation, Chican@ studies, and hip-hop feminism, this paper also aims to creatively expand the definition of translation by positioning rap music as a performative language in its own right, capable of encoding and translating complex cultural issues related to race, gender, and sexuality.

Polymorphisms of ST2-IL18R1-IL18RAP gene cluster: a new risk for autoimmune thyroid diseases.

Science.gov (United States)

Wang, X; Zhu, Y F; Li, D M; Qin, Q; Wang, Q; Muhali, F S; Jiang, W J; Zhang, J A

2016-02-01

Interleukin 33 (IL33) / ST2 pathway and ST2-interlukin18 receptor1-interlukin18 receptor accessory protein (ST2-IL18R1-IL18RAP) gene cluster have been involved in many autoimmune diseases but few report in autoimmune thyroid diseases (AITD). In this study, we investigated whether polymorphisms of IL33, ST2, IL18R1, and IL18RAP are associated with Graves' disease (GD) and Hashimoto's thyroiditis (HT), two major forms of AITD, among a Chinese population. A total of 11 SNPs were explored in a case-control study including 417 patients with GD, 250 HT patients and 301 controls, including rs1929992, rs10975519, rs10208293, rs6543116, rs1041973, rs3732127, rs11465597, rs1035130, rs2293225, rs1035127, rs917997 of IL 33, ST2-IL18R1-IL18RAP gene cluster. Genotyping of these SNPs was performed using matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer (MALDI-TOF-MS) platform from Sequenom. The frequencies of allele A and AA+AG genotype of rs6543116 (ST2) in HT patients were significantly increased compared with those of the controls (P = 0.029/0.021, OR = 1.31/1.62). And in another SNP rs917997, AA+AG genotype presented an increased frequency in HT subjects compared with controls (P = 0.046, OR = 1.53). Furthermore, the haplotype GAGCCCG from ST2-IL18R1-IL18RAP gene cluster (rs6543116, rs1041973, rs1035130, rs3732127, rs1035127, rs2293225, rs917997) was associated with increased susceptibility to GD with an OR of 2.03 (P = 0.022, 95% CI = 1.07-3.86). Some SNPs of ST2-IL18R1-IL18RAP gene cluster might increase the risk of susceptibility of HT and GD in Chinese Han population. © 2015 John Wiley & Sons Ltd.

Rap Music Genres and Deviant Behaviors in French-Canadian Adolescents

Science.gov (United States)

Miranda, Dave; Claes, Michel

2004-01-01

This study investigated the links between the preference for 4 rap music genres (American rap, French rap, hip hop/soul, and gangsta/hardcore rap) and 5 types of deviant behaviors in adolescence (violence, theft, street gangs, mild drug use, and hard drug use). The effects of peers' deviancy, violent media, and importance given to lyrics were…

Microstructure of shear-induced thixoformed Al- 4.5Cu-1.5Mg alloy via RAP and SSTT processes

Directory of Open Access Journals (Sweden)

Siamak Nikzad

2017-07-01

Full Text Available In this research, the effect of the thixoforming temperature on the microstructure and mechanical properties was investigated in the thixoforming of the feedstock produced by the RAP (recrystallization and partial melting and SSTT (semi-solid thermal transformation processes for Al-4.5Cu-1.5Mg alloy. In the RAP process, the percentage reduction in area was approximately 35%. Thixoforming was done at 610, 620, and 630 °C. Globular microstructure was observed at all temperatures and conditions. The minimum average globule size was 39 μm, and it was obtained in the thixoforming of the feedstock produced by the RAP process in the section of 4 mm in diameter at 620 ° C after applying shear. Its corresponding compressive strength was -877.44 MPa. The maximum average globule size was 136 μm, and it was obtained in the thixoforming of the feedstock produced by the SSTT process in the section of 10 mm in diameter at 630 °C before applying shear. Its corresponding compressive strength was -769.18 MPa. The finest and most spherical globules, as well as the highest compressive strength were obtained at 620 °C in both RAP and SSTT states.

Efficient transcription of the glycolytic gene ADH1 and three translational component genes requires the GCR1 product, which can act through TUF/GRF/RAP binding sites.

OpenAIRE

Santangelo, G M; Tornow, J

1990-01-01

Glycolytic gene expression in Saccharomyces cerevisiae is thought to be activated by the GCR and TUF proteins. We tested the hypothesis that GCR function is mediated by TUF/GRF/RAP binding sites (UASRPG elements). We found that UASRPG-dependent activation of a heterologous gene and transcription of ADH1, TEF1, TEF2, and RP59 were sensitive to GCR1 disruption. GCR is not required for TUF/GRF/RAP expression or in vitro DNA-binding activity.

Molas Baju Wara: Hybridity in Manggarai Rap Music

Directory of Open Access Journals (Sweden)

Ans. Prawati Yuliantari

2017-06-01

Full Text Available Rap music which has been popular since 2007 in Manggarai region, East Nusa Tenggara, Indonesia, gave rise to rap hybrid phenomenon. The mixture between American rap music formats and local elements of Manggarai attracted the attention of young people in the region. One of the local songs that feature hybridity in rap Manggarai is "Molas Baju Wara" created by Lipooz, one of the pioneers of rap in Ruteng, the capital city of Manggarai district. To discuss this phenomenon, the concept of hybridity in cultural territory proposed by James Lull is adopted. This concept is used particularly to analyze the forms of hybridity reflected in " Molas Baju Wara" and the ways they are used in showing the social and cultural conditions of Manggarai. "Molas Baju Wara" was selected as the object of study because the song is clearly showing the characteristics of hybridity in music. The study shows that hybridity could be perceived in Manggarai rap music specifically in the use of local musical instruments like drums, cajon, and tambourine as a substitute for percussive sounds of drums, boombox, or turn-table which are commonly used by rap musicians in their home country, the U.S.A. In addition, there are elements of local sound such as the sound of rain that represents Ruteng as the rain city. Hybridity characteristics can also be found in the use of Manggarai vernacular in the whole lyrics as well as the narration of local themes and certain sites that represent Ruteng.

Efficient transcription of the glycolytic gene ADH1 and three translational component genes requires the GCR1 product, which can act through TUF/GRF/RAP binding sites.

Science.gov (United States)

Santangelo, G M; Tornow, J

1990-01-01

Glycolytic gene expression in Saccharomyces cerevisiae is thought to be activated by the GCR and TUF proteins. We tested the hypothesis that GCR function is mediated by TUF/GRF/RAP binding sites (UASRPG elements). We found that UASRPG-dependent activation of a heterologous gene and transcription of ADH1, TEF1, TEF2, and RP59 were sensitive to GCR1 disruption. GCR is not required for TUF/GRF/RAP expression or in vitro DNA-binding activity. Images PMID:2405258

Reverse engineering GTPase programming languages with reconstituted signaling networks.

Science.gov (United States)

Coyle, Scott M

2016-07-02

The Ras superfamily GTPases represent one of the most prolific signaling currencies used in Eukaryotes. With these remarkable molecules, evolution has built GTPase networks that control diverse cellular processes such as growth, morphology, motility and trafficking. (1-4) Our knowledge of the individual players that underlie the function of these networks is deep; decades of biochemical and structural data has provided a mechanistic understanding of the molecules that turn GTPases ON and OFF, as well as how those GTPase states signal by controlling the assembly of downstream effectors. However, we know less about how these different activities work together as a system to specify complex dynamic signaling outcomes. Decoding this molecular "programming language" would help us understand how different species and cell types have used the same GTPase machinery in different ways to accomplish different tasks, and would also provide new insights as to how mutations to these networks can cause disease. We recently developed a bead-based microscopy assay to watch reconstituted H-Ras signaling systems at work under arbitrary configurations of regulators and effectors. (5) Here we highlight key observations and insights from this study and propose extensions to our method to further study this and other GTPase signaling systems.

miR-203a is involved in HBx-induced inflammation by targeting Rap1a

Energy Technology Data Exchange (ETDEWEB)

Wu, AiRong [Department of gastroenterology, The First affiliated Hospital of Soochow University, Suzhou 215006 (China); Chen, Huo [Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123 (China); Xu, ChunFang [Department of gastroenterology, The First affiliated Hospital of Soochow University, Suzhou 215006 (China); Zhou, Ji; Chen, Si [Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123 (China); Shi, YuQi [Department of gastroenterology, The First affiliated Hospital of Soochow University, Suzhou 215006 (China); Xu, Jie [Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123 (China); Gan, JianHe, E-mail: j_pzhang@suda.edu.cn [Department of gastroenterology, The First affiliated Hospital of Soochow University, Suzhou 215006 (China); Zhang, JinPing, E-mail: ganjianhe@aliyun.com [Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123 (China)

2016-11-15

Hepatitis B virus (HBV) causes acute and chronic hepatitis, and is one of the major causes of cirrhosis and hepatocellular carcinoma. Accumulating evidence suggests that inflammation is the key factor for liver cirrhosis and hepatocellular carcinoma. MicroRNAs play important roles in many biological processes. Here, we aim to explore the function of microRNAs in the HBX-induced inflammation. First, microarray experiment showed that HBV{sup +} liver samples expressed higher level of miR-203a compared to HBV{sup -} liver samples. To verify these alterations, HBx-coding plasmid was transfected into HepG2 cells to overexpress HBx protein. The real-time PCR results suggested that over-expression of HBx could induce up-regulation of miR-203a. To define how up-regulation of miR-203a can induce liver cells inflammation, we over-expressed miR-203a in HepG2 cells. Annexin V staining and BrdU staining suggested that overexpression of miR-203a significantly increased the cell apoptosis and proliferation, meanwhile, over-expression of miR-203a could lead to a decrease in G0/G1 phase cells and an increase in G2/M phase cells. Some cytokines production including IL-6 and IL-8 were significantly increased, but TGFβ and IFNγ were decreased in miR-203a over-expressed HepG2 cells. Luciferase reporter assay experiments, protein mass-spectrum assay and real-time PCR all together demonstrated that Rap1a was the target gene of miR-203a. Further experiments showed that these alterations were modulated through PI3K/ERK/p38/NFκB pathways. These data suggested that HBV-infection could up-regulate the expression of miR-203a, thus down regulated the expression of Rap1a and affected the PI3K/ERK/p38/NFκB pathways, finally induced the hepatitis inflammation. - Highlights: • HBX induces the over-expression of miR-203a in HepG2 cells. • miR-203a targets Rap1a to induce the inflammation in HepG2 cells. • miR-203a regulates the apoptosis and cell cycles of HepG2 cells. • miR-203a alters

2D-DIGE and MALDI TOF/TOF MS analysis reveal that small GTPase signaling pathways may play an important role in cadmium-induced colon cell malignant transformation

International Nuclear Information System (INIS)

Lu, Jian; Zhou, Zhongping; Zheng, Jianzhou; Zhang, Zhuyi; Lu, Rongzhu; Liu, Hanqing; Shi, Haifeng; Tu, Zhigang

2015-01-01

Cadmium is a toxic heavy metal present in the environment and in industrial materials. Cadmium has demonstrated carcinogenic activity that induces cell transformation, but how this occurs is unclear. We used 2D-DIGE and MALDI TOF/TOF MS combined with bioinformatics and immunoblotting to investigate the molecular mechanism of cadmium transformation. We found that small GTPases were critical for transformation. Additionally, proteins involved in mitochondrial transcription, DNA repair, and translation also had altered expression patterns in cadmium treated cells. Collectively, our results suggest that activation of small GTPases contributes to cadmium-induced transformation of colon cells. - Highlights: • Colon epithelial cell line is firstly successfully transformed by cadmium. • 2D-DIGE is applied to visualize the differentially expressed proteins. • RhoA plays an important role in cadmium induced malignant transformation. • Bioinformatic and experimental methods are combined to explore new mechanisms.

2D-DIGE and MALDI TOF/TOF MS analysis reveal that small GTPase signaling pathways may play an important role in cadmium-induced colon cell malignant transformation

Energy Technology Data Exchange (ETDEWEB)

Lu, Jian, E-mail: lujian@ujs.edu.cn [School of Medicine, Jiangsu University, Zhenjiang 212013 (China); Institute of Life Sciences, Jiangsu University, Zhenjiang 212013 (China); Zhou, Zhongping [School of Medicine, Jiangsu University, Zhenjiang 212013 (China); Institute of Life Sciences, Jiangsu University, Zhenjiang 212013 (China); Zheng, Jianzhou [Department of Respiration Medicine, Changzhou No.2 People' s Hospital, Changzhou 213003 (China); Zhang, Zhuyi [School of Medicine, Jiangsu University, Zhenjiang 212013 (China); Institute of Life Sciences, Jiangsu University, Zhenjiang 212013 (China); Lu, Rongzhu [School of Medicine, Jiangsu University, Zhenjiang 212013 (China); Liu, Hanqing [School of Pharmacy, Jiangsu University, Zhenjiang 212013 (China); Shi, Haifeng [Institute of Life Sciences, Jiangsu University, Zhenjiang 212013 (China); Tu, Zhigang, E-mail: tuzg_ujs@ujs.edu.cn [Institute of Life Sciences, Jiangsu University, Zhenjiang 212013 (China)

2015-10-01

Cadmium is a toxic heavy metal present in the environment and in industrial materials. Cadmium has demonstrated carcinogenic activity that induces cell transformation, but how this occurs is unclear. We used 2D-DIGE and MALDI TOF/TOF MS combined with bioinformatics and immunoblotting to investigate the molecular mechanism of cadmium transformation. We found that small GTPases were critical for transformation. Additionally, proteins involved in mitochondrial transcription, DNA repair, and translation also had altered expression patterns in cadmium treated cells. Collectively, our results suggest that activation of small GTPases contributes to cadmium-induced transformation of colon cells. - Highlights: • Colon epithelial cell line is firstly successfully transformed by cadmium. • 2D-DIGE is applied to visualize the differentially expressed proteins. • RhoA plays an important role in cadmium induced malignant transformation. • Bioinformatic and experimental methods are combined to explore new mechanisms.

The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation

Directory of Open Access Journals (Sweden)

Carolina Varela Chavez

2016-03-01

Full Text Available Clostridium sordellii lethal toxin (TcsL is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1–93 domain recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases.

Rapping dyslexia : learning rhythm, rhyme and flow in dyslectic children

NARCIS (Netherlands)

Tittarelli, M.; Marti, P.; Peppoloni, D.

2014-01-01

The paper presents a design case that draws inspiration from rap music as a way to tell stories rhythmically, with simple instruments for accompaniment. Rhythm, rhymes and flow are key features of rap music. In this study, we attempted to apply rap principles and dynamics to a very specific field of

The Drosophila small GTPase Rac2 is required for normal feeding and mating behaviour.

Science.gov (United States)

Goergen, Philip; Kasagiannis, Anna; Schiöth, Helgi B; Williams, Michael J

2014-03-01

All multicellular organisms require the ability to regulate bodily processes in order to maintain a stable condition, which necessitates fluctuations in internal metabolics, as well as modifications of outward behaviour. Understanding the genetics behind this modulation is important as a general model for the metabolic modification of behaviour. This study demonstrates that the activity of the small GTPase Rac2 is required in Drosophila for the proper regulation of lipid storage and feeding behaviour, as well as aggression and mating behaviours. Rac2 mutant males and females are susceptible to starvation and contain considerably less lipids than controls. Furthermore, Rac2 mutants also have disrupted feeding behaviour, eating fewer but larger meals than controls. Intriguingly, Rac2 mutant males rarely initiate aggressive behaviour and display significantly increased levels of courtship behaviour towards other males and mated females. From these results we conclude that Rac2 has a central role in regulating the Drosophila homeostatic system.

LHC rap: a global phenomenon

CERN Multimedia

2008-01-01

Do you think the LHC is super duper fly? Does it make you want to compose some slick rhymes and bust out some killer beats? It did for one CERN rapper, and the results have become a YouTube smash hit! Katie McAlpine will sing for the CMS party on 24 September, and for the ATLAS Fest on 4 October.The Large Hadron Rap, to give it its full name, is the brainchild of AlpineKat, AKA Katie McAlpine, who is currently working for ATLAS e-News and outreach. To date, the YouTube video rap has been viewed more than 2.5 million times, to say nothing of the media coverage. Featured in newspapers around the world, including the New York Times in the US, The Telegraph in the UK and Geneva’s very own Matin Bleu, the rap is officially a sensation! Katie wrote the inspired (and pretty accurate) physics lyrics during her commute on the number 56 bus between Geneva and CERN. After obtaining permission to film in the experiment caverns and tunnel,...

The Rac1 hypervariable region in targeting and signaling

Science.gov (United States)

Lam, B. Daniel; Hordijk, Peter L.

2013-01-01

Cellular signaling by small GTPases is critically dependent on proper spatio-temporal orchestration of activation and output. In addition to their core G (guanine nucleotide binding)-domain, small GTPases comprise a hypervariable region (HVR) and a lipid anchor that are generally accepted to control subcellullar localization. The HVR encodes in many small GTPases a polybasic region (PBR) that permits charge-mediated association to the inner leaflet of the plasma membrane or to intracellular organelles. Over the past 15–20 years, evidence has accumulated for specific protein–protein interactions, mediated by the HVR, that control both targeting and signaling specificity of small GTPases. Using the RhoGTPase Rac1 as a paradigm we here review a series of protein partners that require the Rac1 HVR for association and that control various aspects of localized Rac1 signaling. Some of these proteins represent Rac1 activators, whereas others mediate Rac1 inactivation and degradation and yet others potentiate Rac1 downstream signaling. Finally, evidence is discussed which shows that the HVR of Rac1 also contributes to effector interactions, co-operating with the N-terminal effector domain. The complexity of localized Rac1 signaling, reviewed here, is most likely exemplary for many other small GTPases as well, representing a challenge to identify and define similar mechanisms controlling the specific signaling induced by small GTPases. PMID:23354415

Volumetric Analysis and Performance of Hot Mix Asphalt with Variable Rap Content

Directory of Open Access Journals (Sweden)

Arshad Ahmad Kamil

2017-01-01

Full Text Available Incorporating Reclaimed Asphalt Pavement (RAP to the asphalt concrete mixture for highway construction offer many benefits including energy consumption, conservation of natural resources and preservation of the environment to associated emissions. This paper presents a study on performance of Hot Mix Asphalt with variable RAP content. The study is carried out to evaluate the Marshall Properties and Performance of RAP-Asphalt mixes using conventional asphaltic concrete mix AC14. Marshall Mix Design Method was used to produce control mix (0% RAP and RAP-Asphalt mixes samples which consist of 15% RAP, 25% RAP and 35% RAP in accordance with Specifications for Road Works of Public Works Department, Malaysia. The Marshall Properties analysis was performed to ensure compliance with Marshall Requirements, The resilient modulus test was performed to measure the stiffness of the mixes while Modified Lottman test was conducted to evaluate the moisture susceptibility of these mixes. The results obtained showed that there were no substantial difference in Marshall Properties, moisture susceptibility and indirect tensile strength between RAP-Asphalt mixes with the control mix. The test results indicated that recycled mixes performed as good as the performance of conventional HMA in terms of moisture susceptibility and resilient modulus. It is recommended that further research be carried out for asphalt mixes containing more than 35% of RAP material.

Product (P1) from project 0-6738 : performance studies and future directions for mixes containing RAP and RAS.

Science.gov (United States)

2013-01-01

In recent years both reclaimed asphalt pavement (RAP) and recycled asphalt shingles (RAS) have been widely used in asphalt mixes by the asphalt paving industry in Texas. The use of RAP and RAS can save tax payers money, and it is also good for the...

Synergistic regulation of competence development in Bacillus subtilis by two Rap-Phr systems.

Science.gov (United States)

Bongiorni, Cristina; Ishikawa, Shu; Stephenson, Sophie; Ogasawara, Naotake; Perego, Marta

2005-07-01

The 11 Rap proteins of Bacillus subtilis comprise a conserved family of tetratricopeptide (TPR)-containing regulatory proteins. Their activity is inhibited by specific Phr pentapeptides produced from the product of phr genes through an export-import maturation process. We found that one of the proteins, namely RapF, is involved in the regulation of competence to DNA transformation. The ComA response regulator and transcription factor for initiation of competence development is the target of RapF. Specific binding of RapF to the carboxy-terminal DNA-binding domain of ComA inhibits the response regulator's ability to bind its target DNA promoters. The PhrF C-terminal pentapeptide, QRGMI, inhibits RapF activity. The activity of RapF and PhrF in regulating competence development is analogous to the previously described activity of RapC and PhrC (L. J. Core and M. Perego, Mol. Microbiol. 49:1509-1522, 2003). In fact, the RapF and PhrF pair of proteins acts synergistically with RapC and PhrC in the overall regulation of the ComA transcription factor. Since the transcription of the RapC- and RapF-encoding genes is positively regulated by their own target ComA, an autoregulatory circuit must exist for the competence transcription factor in order to modulate its activity.

ATPase and GTPase Tangos Drive Intracellular Protein Transport.

Science.gov (United States)

Shan, Shu-Ou

2016-12-01

The GTPase superfamily of proteins provides molecular switches to regulate numerous cellular processes. The 'GTPase switch' paradigm, in which external regulatory factors control the switch of a GTPase between 'on' and 'off' states, has been used to interpret the regulatory mechanism of many GTPases. However, recent work unveiled a class of nucleotide hydrolases that do not adhere to this classical paradigm. Instead, they use nucleotide-dependent dimerization cycles to regulate key cellular processes. In this review article, recent studies of dimeric GTPases and ATPases involved in intracellular protein targeting are summarized. It is suggested that these proteins can use the conformational plasticity at their dimer interface to generate multiple points of regulation, thereby providing the driving force and spatiotemporal coordination of complex cellular pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

An Action Research Study on the Influence of Gangsta Rap on Academic and Behavioral Issues of 5th Grade African-American Males

Science.gov (United States)

Lewis, Shaun; Boes, Susan R.; Chibbaro, Julie S.

2015-01-01

This small action research study (ARS) began with a review of the literature examining the relationship of gangsta rap in regards to academic achievement, self-esteem, decision-making, identity issues and development of young African American males. The purpose of the ARS was to examine the correlation between gangsta rap and its influence on 5th…

Performance Evaluation of Hot Mix Asphalt with Different Proportions of RAP Content

Science.gov (United States)

Kamil Arshad, Ahmad; Awang, Haryati; Shaffie, Ekarizan; Hashim, Wardati; Rahman, Zanariah Abd

2018-03-01

Reclaimed Asphalt Pavement (RAP) is old asphalt pavement that has been removed from a road by milling or full depth removal. The use of RAP in hot mix asphalt (HMA) eliminates the need to dispose old asphalt pavements and conserves asphalt binders and aggregates, resulting in significant cost savings and benefits to society. This paper presents a study on HMA with different RAP proportions carried out to evaluate the volumetric properties and performance of asphalt mixes containing different proportions of RAP. Marshall Mix Design Method was used to produce control mix (0% RAP) and asphalt mixes containing 15% RAP, 25% RAP and 35% RAP in accordance with Specifications for Road Works of Public Works Department, Malaysia for AC14 dense graded asphalt gradation. Volumetric analysis was performed to ensure that the result is compliance with specification requirements. The resilient modulus test was performed to measure the stiffness of the mixes while the Modified Lottman test was conducted to evaluate the moisture susceptibility of these mixes. The Hamburg wheel tracking test was used to evaluate the rutting performance of these mixes. The results obtained showed that there were no substantial difference in Marshall Properties, moisture susceptibility, resilient modulus and rutting resistance between asphalt mixes with RAP and the control mix. The test results indicated that recycled mixes performed as good as the performance of conventional HMA in terms of moisture susceptibility and resilient modulus. It is recommended that further research be carried out for asphalt mixes containing more than 35% RAP material.

Performance Evaluation of Hot Mix Asphalt with Different Proportions of RAP Content

Directory of Open Access Journals (Sweden)

Kamil Arshad Ahmad

2018-01-01

Full Text Available Reclaimed Asphalt Pavement (RAP is old asphalt pavement that has been removed from a road by milling or full depth removal. The use of RAP in hot mix asphalt (HMA eliminates the need to dispose old asphalt pavements and conserves asphalt binders and aggregates, resulting in significant cost savings and benefits to society. This paper presents a study on HMA with different RAP proportions carried out to evaluate the volumetric properties and performance of asphalt mixes containing different proportions of RAP. Marshall Mix Design Method was used to produce control mix (0% RAP and asphalt mixes containing 15% RAP, 25% RAP and 35% RAP in accordance with Specifications for Road Works of Public Works Department, Malaysia for AC14 dense graded asphalt gradation. Volumetric analysis was performed to ensure that the result is compliance with specification requirements. The resilient modulus test was performed to measure the stiffness of the mixes while the Modified Lottman test was conducted to evaluate the moisture susceptibility of these mixes. The Hamburg wheel tracking test was used to evaluate the rutting performance of these mixes. The results obtained showed that there were no substantial difference in Marshall Properties, moisture susceptibility, resilient modulus and rutting resistance between asphalt mixes with RAP and the control mix. The test results indicated that recycled mixes performed as good as the performance of conventional HMA in terms of moisture susceptibility and resilient modulus. It is recommended that further research be carried out for asphalt mixes containing more than 35% RAP material.

Cep169, a Novel Microtubule Plus-End-Tracking Centrosomal Protein, Binds to CDK5RAP2 and Regulates Microtubule Stability.

Directory of Open Access Journals (Sweden)

Yusuke Mori

Full Text Available The centrosomal protein, CDK5RAP2, is a microcephaly protein that regulates centrosomal maturation by recruitment of a γ-tubulin ring complex (γ-TuRC onto centrosomes. In this report, we identified a novel human centrosomal protein, Cep169, as a binding partner of CDK5RAP2, a member of microtubule plus-end-tracking proteins (+TIPs. Cep169 interacts directly with CDK5RAP2 through CM1, an evolutionarily conserved domain, and colocalizes at the pericentriolar matrix (PCM around centrioles with CDK5RAP2. In addition, Cep169 interacts with EB1 through SxIP-motif responsible for EB1 binding, and colocalizes with CDK5RAP2 at the microtubule plus-end. EB1-binding-deficient Cep169 abolishes EB1 interaction and microtubule plus-end attachment, indicating Cep169 as a novel member of +TIPs. We further show that ectopic expression of either Cep169 or CDK5RAP2 induces microtubule bundling and acetylation in U2OS cells, and depletion of Cep169 induces microtubule depolymerization in HeLa cells, although Cep169 is not required for assembly of γ-tubulin onto centrosome by CDK5RAP2. These results show that Cep169 targets microtubule tips and regulates stability of microtubules with CDK5RAP2.

Rap van tong, scherp van pen. Literaire discussiecultuur in Nederlandse praatjespamfletten (circa 1600-1750)

NARCIS (Netherlands)

Dingemanse, C.W.

2008-01-01

In the early modern period pamphlets constituted the most important medium to influence public opinion in the Netherlands. The thesis Rap van tong, scherp van pen (Glib tongues, sharp pens) focuses on the literary and rhetorical aspects of a remarkable type of pamphlet called praatje (small-talk),

Assessment of transferring Sr-90 and Cs-137 in products of processing of seeds raps for getting raps' oil and biodiesel

International Nuclear Information System (INIS)

Yatsino, T.S.; Mironov, V.P.

2009-01-01

The objects of research are soil assays, seeds of raps, straw and the stalks selected on polluted radio nuclides of territory. The work purpose is to measure specific activity of strontium and cesium in assays, to calculate factors of transition Sr 90 and Sr 137 in products of processing of seeds of raps. (authors)

Synergistic Regulation of Competence Development in Bacillus subtilis by Two Rap-Phr Systems† ‡

Science.gov (United States)

Bongiorni, Cristina; Ishikawa, Shu; Stephenson, Sophie; Ogasawara, Naotake; Perego, Marta

2005-01-01

The 11 Rap proteins of Bacillus subtilis comprise a conserved family of tetratricopeptide (TPR)-containing regulatory proteins. Their activity is inhibited by specific Phr pentapeptides produced from the product of phr genes through an export-import maturation process. We found that one of the proteins, namely RapF, is involved in the regulation of competence to DNA transformation. The ComA response regulator and transcription factor for initiation of competence development is the target of RapF. Specific binding of RapF to the carboxy-terminal DNA-binding domain of ComA inhibits the response regulator's ability to bind its target DNA promoters. The PhrF C-terminal pentapeptide, QRGMI, inhibits RapF activity. The activity of RapF and PhrF in regulating competence development is analogous to the previously described activity of RapC and PhrC (L. J. Core and M. Perego, Mol. Microbiol. 49:1509-1522, 2003). In fact, the RapF and PhrF pair of proteins acts synergistically with RapC and PhrC in the overall regulation of the ComA transcription factor. Since the transcription of the RapC- and RapF-encoding genes is positively regulated by their own target ComA, an autoregulatory circuit must exist for the competence transcription factor in order to modulate its activity. PMID:15968044

Two biomarker-directed randomized trials in European and Chinese patients with nonsmall-cell lung cancer: the BRCA1-RAP80 Expression Customization (BREC) studies.

Science.gov (United States)

Moran, T; Wei, J; Cobo, M; Qian, X; Domine, M; Zou, Z; Bover, I; Wang, L; Provencio, M; Yu, L; Chaib, I; You, C; Massuti, B; Song, Y; Vergnenegre, A; Lu, H; Lopez-Vivanco, G; Hu, W; Robinet, G; Yan, J; Insa, A; Xu, X; Majem, M; Chen, X; de Las Peñas, R; Karachaliou, N; Sala, M A; Wu, Q; Isla, D; Zhou, Y; Baize, N; Zhang, F; Garde, J; Germonpre, P; Rauh, S; ALHusaini, H; Sanchez-Ronco, M; Drozdowskyj, A; Sanchez, J J; Camps, C; Liu, B; Rosell, R

2014-11-01

In a Spanish Lung Cancer Group (SLCG) phase II trial, the combination of BRCA1 and receptor-associated protein 80 (RAP80) expression was significantly associated with outcome in Caucasian patients with nonsmall-cell lung cancer (NSCLC). The SLCG therefore undertook an industry-independent collaborative randomized phase III trial comparing nonselected cisplatin-based chemotherapy with therapy customized according to BRCA1/RAP80 expression. An analogous randomized phase II trial was carried out in China under the auspices of the SLCG to evaluate the effect of BRCA1/RAP80 expression in Asian patients. Eligibility criteria included stage IIIB-IV NSCLC and sufficient tumor specimen for molecular analysis. Randomization to the control or experimental arm was 1 : 1 in the SLCG trial and 1 : 3 in the Chinese trial. In both trials, patients in the control arm received docetaxel/cisplatin; in the experimental arm, patients with low RAP80 expression received gemcitabine/cisplatin, those with intermediate/high RAP80 expression and low/intermediate BRCA1 expression received docetaxel/cisplatin, and those with intermediate/high RAP80 expression and high BRCA1 expression received docetaxel alone. The primary end point was progression-free survival (PFS). Two hundred and seventy-nine patients in the SLCG trial and 124 in the Chinese trial were assessable for PFS. PFS in the control and experimental arms in the SLCG trial was 5.49 and 4.38 months, respectively [log rank P = 0.07; hazard ratio (HR) 1.28; P = 0.03]. In the Chinese trial, PFS was 4.74 and 3.78 months, respectively (log rank P = 0.82; HR 0.95; P = 0.82). Accrual was prematurely closed on the SLCG trial due to the absence of clinical benefit in the experimental over the control arm. However, the BREC studies provide proof of concept that an international, nonindustry, biomarker-directed trial is feasible. Thanks to the groundwork laid by these studies, we expect that ongoing further research on alternative biomarkers to

Neuronal Rho GTPase Rac1 elimination confers neuroprotection in a mouse model of permanent ischemic stroke

DEFF Research Database (Denmark)

Karabiyik, Cansu; Fernandes, Rui; Figueiredo, Francisco Rosário

2018-01-01

The Rho GTPase Rac1 is a multifunctional protein involved in distinct pathways ranging from development to pathology. The aim of the present study was to unravel the contribution of neuronal Rac1 in regulating the response to brain injury induced by permanent focal cerebral ischemia (pMCAO). Our ...

Neuronal Rho GTPase Rac1 elimination confers neuroprotection in a mice model of permanent ischemic stroke

DEFF Research Database (Denmark)

Karabiyik, Cansu; Fernandes, Rui; Figueiredo, Francisci Rosário

2017-01-01

The Rho GTPase Rac1 is a multifunctional protein involved in distinct pathways ranging from development to pathology. The aim of the present study was to unravel the contribution of neuronal Rac1 in regulating the response to brain injury induced by permanent focal cerebral ischemia (pMCAO). Our ...

Palmitoylation of the immunity related GTPase, Irgm1: impact on membrane localization and ability to promote mitochondrial fission.

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Stanley C Henry

Full Text Available The Immunity-Related GTPases (IRG are a family of large GTPases that mediate innate immune responses. Irgm1 is particularly critical for immunity to bacteria and protozoa, and for inflammatory homeostasis in the intestine. Although precise functions for Irgm1 have not been identified, prior studies have suggested roles in autophagy/mitophagy, phagosome remodeling, cell motility, and regulating the activity of other IRG proteins. These functions ostensibly hinge on the ability of Irgm1 to localize to intracellular membranes, such as those of the Golgi apparatus and mitochondria. Previously, it has been shown that an amphipathic helix, the αK helix, in the C-terminal portion of the protein partially mediates membrane binding. However, in absence of αK, there is still substantial binding of Irgm1 to cellular membranes, suggesting the presence of other membrane binding motifs. In the current work, an additional membrane localization motif was found in the form of palmitoylation at a cluster of cysteines near the αK. An Irgm1 mutant possessing alanine to cysteine substitutions at these amino acids demonstrated little residual palmitoylation, yet it displayed only a small decrease in localization to the Golgi and mitochondria. In contrast, a mutant containing the palmitoylation mutations in combination with mutations disrupting the amphipathic character of the αK displayed a complete loss of apparent localization to the Golgi and mitochondria, as well as an overall loss of association with cellular membranes in general. Additionally, Irgm1 was found to promote mitochondrial fission, and this function was undermined in Irgm1 mutants lacking the palmitoylation domain, and to a greater extent in those lacking the αK, or the αK and palmitoylation domains combined. Our data suggest that palmitoylation together with the αK helix firmly anchor Irgm1 in the Golgi and mitochondria, thus facilitating function of the protein.

Changing images of violence in Rap music lyrics: 1979-1997.

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Herd, Denise

2009-12-01

Rap music has been at the center of concern about the potential harmful effects of violent media on youth social behavior. This article explores the role of changing images of violence in rap music lyrics from the 1970s to the 1990s. The results indicate that there has been a dramatic and sustained increase in the level of violence in rap music. The percentage of songs mentioning violence increased from 27 per cent during 1979-1984 to 60 per cent during 1994-1997. In addition, portrayals of violence in later songs are viewed in a more positive light as shown by their increased association with glamor, wealth, masculinity, and personal prowess. Additional analyses revealed that genre, specifically gangster rap, is the most powerful predictor of the increased number of violent references in songs. The discussion suggests that violence in rap music has increased in response to the complex interplay of changing social conditions such as the elevated levels of youth violence in the 1980s and changing commercial practices within the music industry.

Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus.

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Kai Xu

2016-10-01

Full Text Available Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs. The VRCs built by Tomato bushy stunt virus (TBSV are enriched with phosphatidylethanolamine (PE through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5-positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment.

Role of Arf GTPases in fungal morphogenesis and virulence.

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Hayet Labbaoui

2017-02-01

Full Text Available Virulence of the human fungal pathogen Candida albicans depends on the switch from budding to filamentous growth, which requires sustained membrane traffic and polarized growth. In many organisms, small GTPases of the Arf (ADP-ribosylation factor family regulate membrane/protein trafficking, yet little is known about their role in fungal filamentous growth. To investigate these GTPases in C. albicans, we generated loss of function mutants in all 3 Arf proteins, Arf1-Arf3, and 2 Arf-like proteins, Arl1 and Arl3. Our results indicate that of these proteins, Arf2 is required for viability and sensitivity to antifungal drugs. Repressible ARF2 expression results in defects in filamentous growth, cell wall integrity and virulence, likely due to alteration of the Golgi. Arl1 is also required for invasive filamentous growth and, although arl1/arl1 cells can initiate hyphal growth, hyphae are substantially shorter than that of the wild-type, due to the inability of this mutant to maintain hyphal growth at a single site. We show that this defect does not result from an alteration of phospholipid distribution and is unlikely to result from the sole Golgin Imh1 mislocalization, as Imh1 is not required for invasive filamentous growth. Rather, our results suggest that the arl1/arl1 hyphal growth defect results from increased secretion in this mutant. Strikingly, the arl1/arl1 mutant is drastically reduced in virulence during oropharyngeal candidiasis. Together, our results highlight the importance of Arl1 and Arf2 as key regulators of hyphal growth and virulence in C. albicans and identify a unique function of Arl1 in secretion.

Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

Energy Technology Data Exchange (ETDEWEB)

Kamada, Katsuhiko; De Angelis, Jacqueline; Roeder, Robert G.; Burley, Stephen K. (Rockefeller)

2012-12-13

The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-{angstrom} resolution. The {alpha}/{beta} structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3{gamma} (HNF-3{gamma}), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3{gamma} and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the {beta} subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

A signaling pathway contributing to platelet storage lesion development: targeting PI3-kinase–dependent Rap1 activation slows storage-induced platelet deterioration

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Schubert, Peter; Thon, Jonathan N.; Walsh, Geraldine M.; Chen, Cindy H.I.; Moore, Edwin D.; Devine, Dana V.; Kast, Juergen

2015-01-01

BACKGROUND The term platelet storage lesion (PSL) describes the structural and biochemical changes in platelets (PLTs) during storage. These are typified by alterations of morphologic features and PLT metabolism leading to reduced functionality and hence reduced viability for transfusion. While the manifestations of the storage lesion are well characterized, the biochemical pathways involved in the initiation of this process are unknown. STUDY DESIGN AND METHODS A complementary proteomic approach has recently been applied to analyze changes in the PLT proteome during storage. By employing stringent proteomic criteria, 12 proteins were identified as significantly and consistently changing in relative concentration over a 7-day storage period. Microscopy, Western blot analysis, flow cytometry, and PLT functionality analyses were used to unravel the involvement of a subset of these 12 proteins, which are connected through integrin signaling in one potential signaling pathway underlying storage lesion development. RESULTS Microscopic analysis revealed changes in localization of glycoprotein IIIa, Rap1, and talin during storage. Rap1 activation was observed to correlate with expression of the PLT activation marker CD62P. PLTs incubated for 7 days with the PI3-kinase inhibitor LY294002 showed diminished Rap1 activation as well as a moderate reduction in integrin αIIbβ3 activation and release of α-granules. Furthermore, this inhibitor seemed to improve PLT integrity and quality during storage as several in vitro probes showed a deceleration of PLT activation. CONCLUSION These results provide the first evidence for a signaling pathway mediating PSL in which PI3-kinase–dependent Rap1 activation leads to integrin αIIbβ3 activation and PLT degranulation. PMID:19497060

Radiological Assistance Program (RAP) Regions

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Department of Homeland Security — The U.S. Department of Energy (DOE) created the Radiological Assistance Program (RAP) in the 1950s to make DOE resources and expertise available to organizations...

Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

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Vallon, Mario, E-mail: m.vallon@arcor.de [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany); Rohde, Franziska; Janssen, Klaus-Peter [Chirurgische Klinik und Poliklinik, Technische Universitaet Muenchen, Munich (Germany); Essler, Markus [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany)

2010-02-01

Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

Rab GTPases in Immunity and Inflammation.

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Prashar, Akriti; Schnettger, Laura; Bernard, Elliott M; Gutierrez, Maximiliano G

2017-01-01

Strict spatiotemporal control of trafficking events between organelles is critical for maintaining homeostasis and directing cellular responses. This regulation is particularly important in immune cells for mounting specialized immune defenses. By controlling the formation, transport and fusion of intracellular organelles, Rab GTPases serve as master regulators of membrane trafficking. In this review, we discuss the cellular and molecular mechanisms by which Rab GTPases regulate immunity and inflammation.

Expression analysis and biological characterization of Babesia sp. BQ1 (Lintan) (Babesia motasi-like) rhoptry-associated protein 1 and its potential use in serodiagnosis via ELISA.

Science.gov (United States)

Niu, Qingli; Liu, Zhijie; Yang, Jifei; Yu, Peifa; Pan, Yuping; Zhai, Bintao; Luo, Jianxun; Moreau, Emmanuelle; Guan, Guiquan; Yin, Hong

2016-05-31

In China, ovine babesiosis is one of the most important tick-borne haemoparasitic diseases of small ruminants. It has a significant economic impact, and several Babesia motasi-like isolates have been recently shown to be responsible for ovine babesiosis in this country. Full-length and C-terminal-truncated forms of the rap-1a61-1 gene of Babesia sp. BQ1 (Lintan) were cloned into the pET-30a plasmid and subsequently expressed as His-fusion proteins. The resulting recombinant RAP-1a proteins (rRAP-1a61-1 and rRAP-1a61-1/CT) were purified and evaluated as diagnostic antigens using Western blot analysis and ELISA. The native Babesia sp. BQ1 (Lintan) RAP-1 protein was recognized using Western blots and IFAT by antibodies that were raised in rabbits against rRAP-1a61-1/CT. The specificity, sensitivity and positive threshold values for rRAP-1a61-1/CT in ELISA were evaluated. Cross-reactivity was observed between rRAP-1a61-1/CT and positive sera for Babesia sp. BQ1 (Lintan), Babesia sp. BQ1 (Ningxian) and Babesia sp. Tianzhu isolates obtained from infected sheep. At one week post-inoculation, a significant increase was observed in the amount of antibodies produced against RAP-1a, and high levels of antibodies against RAP-1a were observed for 3 months (at 84 days p.i.). A total of 3198 serum samples were collected from small ruminants in 54 different regions in 23 provinces of China. These samples were tested using ELISA based on the rRAP-1a61-1/CT protein. The results indicated that the average positive rate was 36.02 %. The present study suggests that rRAP-1a61-1/CT might be a potential diagnostic antigen for detecting several isolates of B. motasi-like parasites infection.

CDK5RAP2 gene and tau pathophysiology in late-onset sporadic Alzheimer's disease.

Science.gov (United States)

Miron, Justin; Picard, Cynthia; Nilsson, Nathalie; Frappier, Josée; Dea, Doris; Théroux, Louise; Poirier, Judes

2018-06-01

Because currently known Alzheimer's disease (AD) single-nucleotide polymorphisms only account for a small fraction of the genetic variance in this disease, there is a need to identify new variants associated with AD. Our team performed a genome-wide association study in the Quebec Founder Population isolate to identify novel protective or risk genetic factors for late-onset sporadic AD and examined the impact of these variants on gene expression and AD pathology. The rs10984186 variant is associated with an increased risk of developing AD and with a higher CDK5RAP2 mRNA prevalence in the hippocampus. On the other hand, the rs4837766 variant, which is among the best cis-expression quantitative trait loci in the CDK5RAP2 gene, is associated with lower mild cognitive impairment/AD risk and conversion rate. The rs10984186 risk and rs4837766 protective polymorphic variants of the CDK5RAP2 gene might act as potent genetic modifiers for AD risk and/or conversion by modulating the expression of this gene. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Chance Encounters: Rap Music as a Relational and Pedagogical Resource in Clinical Pastoral Education.

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Gilmore, Jeremy

2018-03-01

Music has long been regarded as a valuable tool for educators. Over the last three decades, rap music has grown to become a global phenomenon. However, due to historical and cultural factors, rap music is often underutilized in Clinical Pastoral Education. This article discusses the social significance of rap music, highlights how rap music informed my supervision of a clinical pastoral education student, and examines Chance the Rapper's mixtape Coloring Book as a case study on the utilization of rap music as a relational and pedagogical resource in spiritual education.

Rab GTPases in Immunity and Inflammation

Directory of Open Access Journals (Sweden)

Akriti Prashar

2017-09-01

Full Text Available Strict spatiotemporal control of trafficking events between organelles is critical for maintaining homeostasis and directing cellular responses. This regulation is particularly important in immune cells for mounting specialized immune defenses. By controlling the formation, transport and fusion of intracellular organelles, Rab GTPases serve as master regulators of membrane trafficking. In this review, we discuss the cellular and molecular mechanisms by which Rab GTPases regulate immunity and inflammation.

Ultrastructural and biochemical localization of N-RAP at the interface between myofibrils and intercalated disks in the mouse heart.

Science.gov (United States)

Zhang, J Q; Elzey, B; Williams, G; Lu, S; Law, D J; Horowits, R

2001-12-11

N-RAP is a recently discovered muscle-specific protein found at cardiac intercalated disks. Double immunogold labeling of mouse cardiac muscle reveals that vinculin is located immediately adjacent to the fascia adherens region of the intercalated disk membrane, while N-RAP extends approximately 100 nm further toward the interior of the cell. We partially purified cardiac intercalated disks using low- and high-salt extractions followed by density gradient centrifugation. Immunoblots show that this preparation is highly enriched in desmin and junctional proteins, including N-RAP, talin, vinculin, beta1-integrin, N-cadherin, and connexin 43. Electron microscopy and immunolabeling demonstrate that N-RAP and vinculin are associated with the large fragments of intercalated disks that are present in this preparation, which also contains numerous membrane vesicles. Detergent treatment of the partially purified intercalated disks removed the membrane vesicles and extracted vinculin and beta1-integrin. Further separation on a sucrose gradient removed residual actin and myosin and yielded a fraction morphologically similar to fasciae adherentes that was highly enriched in N-RAP, N-cadherin, connexin 43, talin, desmin, and alpha-actinin. The finding that N-RAP copurifies with detergent-extracted intercalated disk fragments even though beta-integrin and vinculin have been completely removed suggests that N-RAP association with the adherens junction region is mediated by the cadherin system. Consistent with this hypothesis, we found that recombinant N-RAP fragments bind alpha-actinin in a gel overlay assay. In addition, immunofluorescence shows that N-RAP remains bound at the ends of isolated, detergent-treated cardiac myofibrils. These results demonstrate that N-RAP remains tightly bound to myofibrils and fasciae adherentes during biochemical purification and may be a key constituent in the mechanical link between these two structures.

Literature review : performance of RAP/RAS mixes and new direction.

Science.gov (United States)

2014-04-01

In the last several years reclaimed asphalt pavement (RAP) and recycled asphalt shingles (RAS) have been : widely used in asphalt mixes in Texas. The use of RAP/RAS can significantly reduce the initial cost of : asphalt mixtures, conserve energy, and...

Fatigue Durability Analysis of Collecting Rapping System in Electrostatic Precipitators under Impact Loading

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Ali Akbar Lotfi Neyestanak

2014-01-01

Full Text Available Due to the importance of collecting rapping system in electrostatic precipitators (ESP and controlling the relevant damage under impact loading, fatigue durability of this system is analyzed in the present study based on the numerical and experimental results considering fatigue damage growth and vibration acceleration in the collecting system because of the successive impact of rapping hammers. By microscopic examination of the fracture surface of rapping hammer, beach marks obviously show typical fatigue failure in the rapping hammer arm. In addition, the microscopic examination of the cross section of the collecting plates indicates the corrosion voids which cause crack and eventually fatigue failure. The finite element method is applied to determine both the stress and concentration positions of dynamic stress on the rapping system under impact loading. The paper results can be utilized in system optimization and new material selection for the system by evaluating rapping system durability.

Rap Music Use, Perceived Peer Behavior, and Sexual Initiation Among Ethnic Minority Youth.

Science.gov (United States)

Johnson-Baker, Kimberly A; Markham, Christine; Baumler, Elizabeth; Swain, Honora; Emery, Susan

2016-03-01

Research shows that rap music use is associated with risky sexual behavior in ethnic minority youth; however, it is unknown whether rap music use impacts sexual initiation specifically and, if so, which factors mediate this impact. Thus, we investigated the longitudinal relationship between hours spent listening to rap music in seventh grade and sexual initiation in ninth grade. We also examined the role of perceived peer sexual behavior as a potential mediator of this relationship. We analyzed data from students (n = 443) enrolled in a school-based randomized controlled trial of a sexual health education curriculum collected at baseline and at 18-month follow-up. Rap music use and perceived peer sexual behavior were assessed in seventh grade, whereas sexual initiation was assessed in ninth grade. Univariate, multivariate, and mediation analyses were conducted. At baseline, rap music use was significantly associated with race/ethnicity, parental music rules, and sexual behavior, but not with gender or parental education. Rap music use was a significant predictor of sexual initiation on univariate analysis but not multivariate analysis. Mediation analysis showed that the association between hours spent listening to rap music and sexual initiation was significantly mediated by perceived peer sexual behavior. Rap music use in early adolescence significantly impacts sexual initiation in late adolescence, partially mediated by perceived peer sexual behavior. More research is needed to understand how rap music influences perceptions of peer sexual behavior, which, in turn, influence early sexual initiation. Copyright © 2016 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.

Ras-dva1 small GTPase regulates telencephalon development in Xenopus laevis embryos by controlling Fgf8 and Agr signaling at the anterior border of the neural plate

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Maria B. Tereshina

2014-07-01

Full Text Available We previously found that the small GTPase Ras-dva1 is essential for the telencephalic development in Xenopus laevis because Ras-dva1 controls the Fgf8-mediated induction of FoxG1 expression, a key telencephalic regulator. In this report, we show, however, that Ras-dva1 and FoxG1 are expressed in different groups of cells; whereas Ras-dva1 is expressed in the outer layer of the anterior neural fold, FoxG1 and Fgf8 are activated in the inner layer from which the telencephalon is derived. We resolve this paradox by demonstrating that Ras-dva1 is involved in the transduction of Fgf8 signal received by cells in the outer layer, which in turn send a feedback signal that stimulates FoxG1 expression in the inner layer. We show that this feedback signal is transmitted by secreted Agr proteins, the expression of which is activated in the outer layer by mediation of Ras-dva1 and the homeodomain transcription factor Otx2. In turn, Agrs are essential for maintaining Fgf8 and FoxG1 expression in cells at the anterior neural plate border. Our finding reveals a novel feedback loop mechanism based on the exchange of Fgf8 and Agr signaling between neural and non-neural compartments at the anterior margin of the neural plate and demonstrates a key role of Ras-dva1 in this mechanism.

The Rab-GTPase-activating protein TBC1D1 regulates skeletal muscle glucose metabolism

DEFF Research Database (Denmark)

Szekeres, Ferenc; Chadt, Alexandra; Tom, Robby Z

2012-01-01

The Rab-GTPase-activating protein TBC1D1 has emerged as a novel candidate involved in metabolic regulation. Our aim was to determine whether TBC1D1 is involved in insulin as well as energy-sensing signals controlling skeletal muscle metabolism. TBC1D1-deficient congenic B6.SJL-Nob1.10 (Nob1.10(SJL...... be explained partly by a 50% reduction in GLUT4 protein, since proximal signaling at the level of Akt, AMPK, and acetyl-CoA carboxylase (ACC) was unaltered. Paradoxically, in vivo insulin-stimulated 2-deoxyglucose uptake was increased in EDL and tibialis anterior muscle from TBC1D1-deficient mice......)) and wild-type littermates were studied. Glucose and insulin tolerance, glucose utilization, hepatic glucose production, and tissue-specific insulin-mediated glucose uptake were determined. The effect of insulin, AICAR, or contraction on glucose transport was studied in isolated skeletal muscle. Glucose...

MDA-9/Syntenin (SDCBP) modulates small GTPases RhoA and Cdc42 via transforming growth factor β1 to enhance epithelial-mesenchymal transition in breast cancer.

Science.gov (United States)

Menezes, Mitchell E; Shen, Xue-Ning; Das, Swadesh K; Emdad, Luni; Sarkar, Devanand; Fisher, Paul B

2016-12-06

Epithelial-mesenchymal transition (EMT) is one of the decisive steps regulating cancer invasion and metastasis. However, the molecular mechanisms underlying this transition require further clarification. MDA-9/syntenin (SDCBP) expression is elevated in breast cancer patient samples as well as cultured breast cancer cells. Silencing expression of MDA-9 in mesenchymal metastatic breast cancer cells triggered a change in cell morphology in both 2D- and 3D-cultures to a more epithelial-like phenotype, along with changes in EMT markers, cytoskeletal rearrangement and decreased invasion. Conversely, over expressing MDA-9 in epithelial non-metastatic breast cancer cells instigated a change in morphology to a more mesenchymal phenotype with corresponding changes in EMT markers, cytoskeletal rearrangement and an increase in invasion. We also found that MDA-9 upregulated active levels of known modulators of EMT, the small GTPases RhoA and Cdc42, via TGFβ1. Reintroducing TGFβ1 in MDA-9 silenced cells restored active RhoA and cdc42 levels, modulated cytoskeletal rearrangement and increased invasion. We further determined that MDA-9 interacts with TGFβ1 via its PDZ1 domain. Finally, in vivo studies demonstrated that silencing the expression of MDA-9 resulted in decreased lung metastasis and TGFβ1 re-expression partially restored lung metastases. Our findings provide evidence for the relevance of MDA-9 in mediating EMT in breast cancer and support the potential of MDA-9 as a therapeutic target against metastatic disease.

Differential regulation of the Rac1 GTPase-activating protein (GAP) BCR during oxygen/glucose deprivation in hippocampal and cortical neurons.

Science.gov (United States)

Smith, Katharine R; Rajgor, Dipen; Hanley, Jonathan G

2017-12-08

Brain ischemia causes oxygen and glucose deprivation (OGD) in neurons, triggering a cascade of events leading to synaptic accumulation of glutamate. Excessive activation of glutamate receptors causes excitotoxicity and delayed cell death in vulnerable neurons. Following global cerebral ischemia, hippocampal CA1 pyramidal neurons are more vulnerable to injury than their cortical counterparts, but the mechanisms that underlie this difference are unclear. Signaling via Rho-family small GTPases, their upstream guanine nucleotide exchange factors, and GTPase-activating proteins (GAPs) is differentially dysregulated in response to OGD/ischemia in hippocampal and cortical neurons. Increased Rac1 activity caused by OGD/ischemia contributes to neuronal death in hippocampal neurons via diverse effects on NADPH oxidase activity and dendritic spine morphology. The Rac1 guanine nucleotide exchange factor Tiam1 mediates an OGD-induced increase in Rac1 activity in hippocampal neurons; however, the identity of an antagonistic GAP remains elusive. Here we show that the Rac1 GAP breakpoint cluster region (BCR) associates with NMDA receptors (NMDARs) along with Tiam1 and that this protein complex is more abundant in hippocampal compared with cortical neurons. Although total BCR is similar in the two neuronal types, BCR is more active in hippocampal compared with cortical neurons. OGD causes an NMDAR- and Ca 2+ -permeable AMPAR-dependent deactivation of BCR in hippocampal but not cortical neurons. BCR knockdown occludes OGD-induced Rac1 activation in hippocampal neurons. Furthermore, disrupting the Tiam1-NMDAR interaction with a fragment of Tiam1 blocks OGD-induced Tiam1 activation but has no effect on the deactivation of BCR. This work identifies BCR as a critical player in Rac1 regulation during OGD in hippocampal neurons. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments.

Science.gov (United States)

Dodonova, Svetlana O; Aderhold, Patrick; Kopp, Juergen; Ganeva, Iva; Röhling, Simone; Hagen, Wim J H; Sinning, Irmgard; Wieland, Felix; Briggs, John A G

2017-06-16

COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of βδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.

Identification of the GTPase superfamily in Mycoplasma synoviae and Mycoplasma hyopneumoniae

Directory of Open Access Journals (Sweden)

Clayton Luiz Borges

2007-01-01

Full Text Available Mycoplasmas are the smallest known prokaryotes with self-replication ability. They are obligate parasites, taking up many molecules of their hosts and acting as pathogens in men, animals, birds and plants. Mycoplasma hyopneumoniae is the infective agent of swine mycoplasmosis and Mycoplasma synoviae is responsible for subclinical upper respiratory infections that may result in airsacculitis and synovitis in chickens and turkeys. These highly infectious organisms present a worldwide distribution and are responsible for major economic problems. Proteins of the GTPase superfamily occur in all domains of life, regulating functions such as protein synthesis, cell cycle and differentiation. Despite their functional diversity, all GTPases are believed to have evolved from a single common ancestor. In this work we have identified mycoplasma GTPases by searching the complete genome databases of Mycoplasma synoviae and Mycoplasma hyopneumoniae, J (non-pathogenic and 7448 (pathogenic strains. Fifteen ORFs encoding predicted GTPases were found in M. synoviae and in the two strains of M. hyopneumoniae. Searches for conserved G domains in GTPases were performed and the sequences were classified into families. The GTPase phylogenetic analysis showed that the subfamilies were well resolved into clades. The presence of GTPases in the three strains suggests the importance of GTPases in 'minimalist' genomes.

The Effect of Rap/Hip-Hop Music on Young Adult Smoking: An Experimental Study.

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Harakeh, Zeena; Bogt, Tom F M Ter

2018-02-16

Music may influence young people's behavior through its lyrics. Substance use references occur more frequently in rap/hip-hop than in other music genres. The aim was to examine whether the exposure to rap/hip-hop lyrics referring to substance use affected cigarette smoking. An experiment with a 3-group between subject design was conducted among 74 daily-smoking young adults ranging in age from 17 to 25 years old. Three conditions were tested in a mobile lab (camper vehicle) from May to December 2011, i.e., regular chart pop music (N = 28), rap/hip-hop with non-frequent references to substance use (N = 24), and rap/hip-hop with frequent references to substance use (N = 22). One-way ANOVA showed that participants listening to substance use infused rap/hip-hop songs felt significantly less pleasant, liked the songs less, and comprehended the songs less compared to participants listening to pop songs. Poisson loglinear analyses revealed that compared to the pop music condition, none of the two rap/hip-hop music conditions had a significant effect on acute smoking. Thus, contrary to expectations, the two different rap/hip-hop conditions did not have a significantly different effect on acute smoking. Listening to rap/hip-hop, even rap hip/hop with frequent referrals to substance use (primarily alcohol and drug use, and general smoking referrals), does not seem to encourage cigarette smoking among Dutch daily-smoking young adults, at least short term.

The interdependence of the Rho GTPases and apicobasal cell polarity.

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Mack, Natalie Ann; Georgiou, Marios

2014-01-01

Signaling via the Rho GTPases provides crucial regulation of numerous cell polarization events, including apicobasal (AB) polarity, polarized cell migration, polarized cell division and neuronal polarity. Here we review the relationships between the Rho family GTPases and epithelial AB polarization events, focusing on the 3 best-characterized members: Rho, Rac and Cdc42. We discuss a multitude of processes that are important for AB polarization, including lumen formation, apical membrane specification, cell-cell junction assembly and maintenance, as well as tissue polarity. Our discussions aim to highlight the immensely complex regulatory mechanisms that encompass Rho GTPase signaling during AB polarization. More specifically, in this review we discuss several emerging common themes, that include: 1) the need for Rho GTPase activities to be carefully balanced in both a spatial and temporal manner through a multitude of mechanisms; 2) the existence of signaling feedback loops and crosstalk to create robust cellular responses; and 3) the frequent multifunctionality that exists among AB polarity regulators. Regarding this latter theme, we provide further discussion of the potential plasticity of the cell polarity machinery and as a result the possible implications for human disease.

Coupling mechanical tension and GTPase signaling to generate cell and tissue dynamics

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Zmurchok, Cole; Bhaskar, Dhananjay; Edelstein-Keshet, Leah

2018-07-01

Regulators of the actin cytoskeleton such Rho GTPases can modulate forces developed in cells by promoting actomyosin contraction. At the same time, through mechanosensing, tension is known to affect the activity of Rho GTPases. What happens when these effects act in concert? Using a minimal model (1 GTPase coupled to a Kelvin–Voigt element), we show that two-way feedback between signaling (‘RhoA’) and mechanical tension (stretching) leads to a spectrum of cell behaviors, including contracted or relaxed cells, and cells that oscillate between these extremes. When such ‘model cells’ are connected to one another in a row or in a 2D sheet (‘epithelium’), we observe waves of contraction/relaxation and GTPase activity sweeping through the tissue. The minimal model lends itself to full bifurcation analysis, and suggests a mechanism that explains behavior observed in the context of development and collective cell behavior.

The Ras GTPase-activating protein Rasal3 supports survival of naive T cells.

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Ryunosuke Muro

Full Text Available The Ras-mitogen-activated protein kinase (MAPK pathway is crucial for T cell receptor (TCR signaling in the development and function of T cells. The significance of various modulators of the Ras-MAPK pathway in T cells, however, remains to be fully understood. Ras-activating protein-like 3 (Rasal3 is an uncharacterized member of the SynGAP family that contains a conserved Ras GTPase-activating protein (GAP domain, and is predominantly expressed in the T cell lineage. In the current study, we investigated the function and physiological roles of Rasal3. Our results showed that Rasal3 possesses RasGAP activity, but not Rap1GAP activity, and represses TCR-stimulated ERK phosphorylation in a T cell line. In systemic Rasal3-deficient mice, T cell development in the thymus including positive selection, negative selection, and β-selection was unaffected. However, the number of naive, but not effector memory CD4 and CD8 T cell in the periphery was significantly reduced in Rasal3-deficient mice, and associated with a marked increase in apoptosis of these cells. Indeed, survival of Rasal3 deficient naive CD4 T cells in vivo by adoptive transfer was significantly impaired, whereas IL-7-dependent survival of naive CD4 T cells in vitro was unaltered. Collectively, Rasal3 is required for in vivo survival of peripheral naive T cells, contributing to the maintenance of optimal T cell numbers.

The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

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Sato, Mai [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan); Kitaguchi, Tetsuya [Cell Signaling Group, Waseda Bioscience Research Institute in Singapore (WABOIS), Waseda University, 11 Biopolis Way, 05-01/02 Helios, Singapore 138667 (Singapore); Numano, Rika [The Electronics-Inspired Interdisciplinary Research Institute (EIIRIS), Toyohashi University of Technology, 1-1 Hibarigaoka, Tennpaku-cho, Toyohashi, Aichi 441-8580 (Japan); Ikematsu, Kazuya [Forensic Pathology and Science, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8523 (Japan); Kakeyama, Masaki [Laboratory of Environmental Health Sciences, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Murata, Masayuki; Sato, Ken [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan); Tsuboi, Takashi, E-mail: takatsuboi@bio.c.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan)

2012-04-06

Highlights: Black-Right-Pointing-Pointer Regulation of exocytosis by Rho GTPase Cdc42. Black-Right-Pointing-Pointer Cdc42 increases the number of fusion events from newly recruited vesicles. Black-Right-Pointing-Pointer Cdc42 increases the number of exocytosis-competent dense-core vesicles. -- Abstract: Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott-Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.

The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

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Sato, Mai; Kitaguchi, Tetsuya; Numano, Rika; Ikematsu, Kazuya; Kakeyama, Masaki; Murata, Masayuki; Sato, Ken; Tsuboi, Takashi

2012-01-01

Highlights: ► Regulation of exocytosis by Rho GTPase Cdc42. ► Cdc42 increases the number of fusion events from newly recruited vesicles. ► Cdc42 increases the number of exocytosis-competent dense-core vesicles. -- Abstract: Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott–Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.

3D printing with RepRap cookbook

CERN Document Server

Salinas, Richard

2014-01-01

A systematic guide consisting of over 100 recipes which focus on helping you understand the process of 3D printing using RepRap machines. The book aims at providing professionals with a series of working recipes to help make their fuzzy notions into real, saleable projects/objects using 3D printing technology. This book is for novice designers and artists who own a RepRap-based 3D printer, have fundamental knowledge of its working, and who desire to gain better mastery of the printing process. For the more experienced user, it will provide a handy visual resource, with side-by-side comparisons

Potential involvement of drought-induced Ran GTPase CLRan1 in root growth enhancement in a xerophyte wild watermelon.

Science.gov (United States)

Akashi, Kinya; Yoshimura, Kazuya; Kajikawa, Masataka; Hanada, Kouhei; Kosaka, Rina; Kato, Atsushi; Katoh, Akira; Nanasato, Yoshihiko; Tsujimoto, Hisashi; Yokota, Akiho

2016-10-01

Enhanced root growth is known as the survival strategy of plants under drought. Previous proteome analysis in drought-resistant wild watermelon has shown that Ran GTPase, an essential regulator of cell division and proliferation, was induced in the roots under drought. In this study, two cDNAs were isolated from wild watermelon, CLRan1 and CLRan2, which showed a high degree of structural similarity with those of other plant Ran GTPases. Quantitative RT-PCR and promoter-GUS assays suggested that CLRan1 was expressed mainly in the root apex and lateral root primordia, whereas CLRan2 was more broadly expressed in other part of the roots. Immunoblotting analysis confirmed that the abundance of CLRan proteins was elevated in the root apex region under drought stress. Transgenic Arabidopsis overexpressing CLRan1 showed enhanced primary root growth, and the growth was maintained under osmotic stress, indicating that CLRan1 functions as a positive factor for maintaining root growth under stress conditions.

A Small Ras-like protein Ray/Rab1c modulates the p53-regulating activity of PRPK

International Nuclear Information System (INIS)

Abe, Yasuhito; Takeuchi, Takashi; Imai, Yoshinori; Murase, Ryuichi; Kamei, Yoshiaki; Fujibuchi, Taketsugu; Matsumoto, Suguru; Ueda, Norifumi; Ogasawara, Masahito; Shigemoto, Kazuhiro; Kito, Katsumi

2006-01-01

PRPK phosphorylates serine-15 residue of p53 and enhances transcriptional activity. PRPK possesses a bipartite nuclear localization signal and localizes in nucleus when over-expressed in cells. However, intrinsic PRPK localizes mainly in the cytosol in situ. While studying the mechanisms in the distribution of intrinsic PRPK, we identified a PRPK binding protein, an ubiquitously expressed Small Ras-like GTPase, Rab1c, also named Ray or Rab35. The over-expressed Ray was distributed in the nucleus, cytosol, and cell membrane. Both Ray wild type and GTP-restrictively binding mutant Ray-Q67L, but not guanine nucleotide unstable binding mutant Ray-N120I, partially distributed the over-expressed PRPK to the cytosol and also suppressed the PRPK-induced p53-transcriptional activity profoundly. A Small Ras-like GTPase protein Ray was thus indicated to modulate p53 transcriptional activity of PRPK

Affiliation and Alienation: Hip-Hop, Rap, and Urban Science Education

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Emdin, Christopher

2010-01-01

The critiques of rap artists and other participants in hip-hop culture provide data for teachers and researchers to investigate the attitudes of US urban youth towards schooling. This study explores the complex relationships between hip-hop and science education by examining how rap lyrics project beliefs about schooling, the relevance of existing…

Rab27 GTPases Distribute Extracellular Nanomaps for Invasive Growth and Metastasis: Implications for Prognosis and Treatment

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Olivier De Wever

2013-05-01

Full Text Available The Rab27 family of small GTPases regulates exocytosis of distinct vesicle types including multivesicular endosomes, which results in the release of exosomes. Exosomes are nanometer-sized membrane vesicles that enclose soluble factors such as proteins and nucleic acids within a lipid bilayer and can travel toward distant tissues to influence multiple aspects of cell behavior. In our view that tumors are endocrine organs producing exosomes, Rab27 GTPases and their effector proteins are critical determinants for invasive growth and metastasis. Rab27 proteins and their effectors may serve as prognostic biomarkers or as targets for patient-tailored therapy.

Small GTPase R-Ras participates in neural tube formation in zebrafish embryonic spinal cord.

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Ohata, Shinya; Uga, Hideko; Okamoto, Hitoshi; Katada, Toshiaki

2018-06-27

Ras related (R-Ras), a small GTPase, is involved in the maintenance of apico-basal polarity in neuroepithelial cells of the zebrafish hindbrain, axonal collapse in cultured murine hippocampal neurons, and maturation of blood vessels in adult mice. However, the role of R-Ras in neural tube formation remains unknown. Using antisense morpholino oligonucleotides (AMOs), we found that in the spinal cord of zebrafish embryos, the lumen was formed bilaterally in rras morphants, whereas it was formed at the midline in control embryos. As AMO can cause off-target effects, we generated rras mutant zebrafish lines using CRISPR/Cas9 technology. Although these rras mutant embryos did not have a bilateral lumen in the spinal cord, the following findings suggest that the phenotype is unlikely due to an off-target effect of rras AMO: 1) The rras morphant phenotype was rescued by an injection of AMO-resistant rras mRNA, and 2) a bilaterally segregated spinal cord was not observed in rras mutant embryos injected with rras AMO. The results suggest that the function of other ras family genes may be redundant in rras mutants. Previous research reported a bilaterally formed lumen in the spinal cord of zebrafish embryos with a mutation in a planar cell polarity (PCP) gene, van gogh-like 2 (vangl2). In the present study, in cultured cells, R-Ras was co-immunoprecipitated with Vangl2 but not with another PCP regulator, Pricke1. Interestingly, the interaction between R-Ras and Vangl2 was stronger in guanine-nucleotide free point mutants of R-Ras than in wild-type or constitutively active (GTP-bound) forms of R-Ras. R-Ras may regulate neural tube formation in cooperation with Vangl2 in the developing zebrafish spinal cord. Copyright © 2018 Elsevier Inc. All rights reserved.

Expression of GIMAP1, a GTPase of the immunity-associated protein family, is not up-regulated in malaria

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Carter Christine

2009-04-01

Full Text Available Abstract Background GIMAP (GTPase of the immunity-associated protein family proteins are a family of putative GTPases believed to be regulators of cell death in lymphomyeloid cells. GIMAP1 was the first reported member of this gene family, identified as a gene up-regulated at the RNA level in the spleens of mice infected with the malarial parasite, Plasmodium chabaudi. Methods A monoclonal antibody against mouse GIMAP1 was developed and was used to analyse the expression of the endogenous protein in tissues of normal mice and in defined sub-populations of cells prepared from lymphoid tissues using flow cytometry. It was also used to assess the expression of GIMAP1 protein after infection and/or immunization of mice with P. chabaudi. Real-time PCR analysis was employed to measure the expression of GIMAP1 for comparison with the protein level analysis. Results GIMAP1 protein expression was detected in all lineages of lymphocytes (T, B, NK, in F4/80+ splenic macrophages and in some lymphoid cell lines. Additional evidence is presented suggesting that the strong expression by mature B cells of GIMAP1 and other GIMAP genes and proteins seen in mice may be a species-dependent characteristic. Unexpectedly, no increase was found in the expression of GIMAP1 in P. chabaudi infected mice at either the mRNA or protein level, and this remained so despite applying a number of variations to the protocol. Conclusion The model of up-regulation of GIMAP1 in response to infection/immunization with P. chabaudi is not a robustly reproducible experimental system. The GIMAP1 protein is widely expressed in lymphoid cells, with an interesting increase in expression in the later stages of B cell development. Alternative approaches will be required to define the functional role of this GTPase in immune cells.

Mortality in American Hip-Hop and Rap Recording Artists, 1987-2014.

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Lawson, Carl J

2015-12-01

The deaths of American hip-hop and rap recording artists often receive considerable media attention. However, these artists' deaths have not been examined as a distinct group like the deaths of rock, classical, jazz, and pop music artists. This is a seminal epidemiological analysis on the deaths of an understudied group, American hip-hop and rap music recording artists. Media reports were analyzed of the deaths of American hip-hop and rap music recording artists that occurred from January 1, 1987 to December 31, 2014. The decedents' age, sex, race, cause of death, stage names, and city and state of death were recorded for analysis. The most commonly reported cause of death was homicide. The 280 deaths were categorized as homicide (55%), unintentional injury (13%), cardiovascular (7%), undetermined/undisclosed (7%), cancer (6%), other (5%), suicide (4%), and infectious disease (3%). The mean reported age at death was 30 yrs (range 15-75) and the median was 29 yrs; 97% were male and 92% were black. All but one of the homicides were committed with firearms. Homicide was the most commonly reported cause of death. Public health focus and guidance for hip-hop and rap recording artists should mirror that for African-American men and adolescent males ages 15-54 yrs, for whom the leading causes of death are homicide, unintentional injury, and heart disease. Given the preponderance of homicide deaths in this analysis, premature mortality reduction efforts should focus on violence prevention and conflict mitigation.

Entre ritmo e poesia: rap e literatura oral urbana

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Marcus Rogerio Salgado

2015-11-01

Full Text Available O objetivo do presente artigo é um estudo do rap enquanto manifestação de literatura oral urbana e forma de oralidade tecnológica. Para tanto, o artigo passará em revista as relações entre literatura e palavra falada/cantada, assim como as possibilidades de interface estética entre a literatura e a música que estão em questão quando tratamos do rap.

Rac1 GTPase Promotes Interaction of Hematopoietic Stem/Progenitor Cell with Niche and Participates in Leukemia Initiation and Maintenance in Mouse.

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Chen, Shuying; Li, Huan; Li, Shouyun; Yu, Jing; Wang, Min; Xing, Haiyan; Tang, Kejing; Tian, Zheng; Rao, Qing; Wang, Jianxiang

2016-07-01

Interaction between hematopoietic stem/progenitor cells (HSPCs) with their niche is critical for HSPC function. The interaction also plays an important role in the multistep process of leukemogenesis. Rac1 GTPase has been found to be highly expressed and activated in leukemia patients. Here, by forced expression of constitutively active form of Rac1 (Rac1-V12) in HSPCs, we demonstrate that active Rac1 promotes interaction of HSPC with niche. We then established an active Rac1 associated acute myeloid leukemia (AML) model by expression of Rac1-V12 cooperated with AML1-ETO9a (AE9a) in mouse HSPCs. Compared with AE9a alone, Rac1-V12 cooperated with AE9a (AER) drives an AML with a short latency, demonstrating that activation of Rac1 GTPase in mice promotes AML development. The mechanism of this AML promotion is by a better homing and lodging of leukemia cells in niche, which further enhancing their colony formation, quiescence and preventing leukemia cells from apoptosis. Further study showed that an inhibitor targeting activated Rac1 can increase the efficacy of chemotherapeutic agents to leukemia cells. This study provides evidence that activation of Rac1 promotes leukemia development through enhancing leukemia cells' homing and retention in niche, and suggests that inhibition of Rac1 GTPase could be an effective way of eliminating AML cells. Stem Cells 2016;34:1730-1741. © 2016 AlphaMed Press.

Source localization using recursively applied and projected (RAP) MUSIC

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Mosher, J.C. [Los Alamos National Lab., NM (United States); Leahy, R.M. [Univ. of Southern California, Los Angeles, CA (United States). Signal and Image Processing Inst.

1998-03-01

A new method for source localization is described that is based on a modification of the well known multiple signal classification (MUSIC) algorithm. In classical MUSIC, the array manifold vector is projected onto an estimate of the signal subspace, but errors in the estimate can make location of multiple sources difficult. Recursively applied and projected (RAP) MUSIC uses each successively located source to form an intermediate array gain matrix, and projects both the array manifold and the signal subspace estimate into its orthogonal complement. The MUSIC projection is then performed in this reduced subspace. Using the metric of principal angles, the authors describe a general form of the RAP-MUSIC algorithm for the case of diversely polarized sources. Through a uniform linear array simulation, the authors demonstrate the improved Monte Carlo performance of RAP-MUSIC relative to MUSIC and two other sequential subspace methods, S and IES-MUSIC.

Interaction of the Small GTPase Cdc42 with Arginine Kinase Restricts White Spot Syndrome Virus in Shrimp.

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Xu, Ji-Dong; Jiang, Hai-Shan; Wei, Tian-Di; Zhang, Ke-Yi; Wang, Xian-Wei; Zhao, Xiao-Fan; Wang, Jin-Xing

2017-03-01

Many types of small GTPases are widely expressed in eukaryotes and have different functions. As a crucial member of the Rho GTPase family, Cdc42 serves a number of functions, such as regulating cell growth, migration, and cell movement. Several RNA viruses employ Cdc42-hijacking tactics in their target cell entry processes. However, the function of Cdc42 in shrimp antiviral immunity is not clear. In this study, we identified a Cdc42 protein in the kuruma shrimp ( Marsupenaeus japonicus ) and named it Mj Cdc42. Mj Cdc42 was upregulated in shrimp challenged by white spot syndrome virus (WSSV). The knockdown of Mj Cdc42 and injection of Cdc42 inhibitors increased the proliferation of WSSV. Further experiments determined that Mj Cdc42 interacted with an arginine kinase ( Mj AK). By analyzing the binding activity and enzyme activity of Mj AK and its mutant, Δ Mj AK, we found that Mj AK could enhance the replication of WSSV in shrimp. Mj AK interacted with the envelope protein VP26 of WSSV. An inhibitor of AK activity, quercetin, could impair the function of Mj AK in WSSV replication. Further study demonstrated that the binding of Mj Cdc42 and Mj AK depends on Cys 271 of Mj AK and suppresses the WSSV replication-promoting effect of Mj AK. By interacting with the active site of Mj AK and suppressing its enzyme activity, Mj Cdc42 inhibits WSSV replication in shrimp. Our results demonstrate a new function of Cdc42 in the cellular defense against viral infection in addition to the regulation of actin and phagocytosis, which has been reported in previous studies. IMPORTANCE The interaction of Cdc42 with arginine kinase plays a crucial role in the host defense against WSSV infection. This study identifies a new mechanism of Cdc42 in innate immunity and enriches the knowledge of the antiviral innate immunity of invertebrates. Copyright © 2017 American Society for Microbiology.

Activation of Rho GTPases by Cytotoxic Necrotizing Factor 1 Induces Macropinocytosis and Scavenging Activity in Epithelial Cells

Science.gov (United States)

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

Investigation on performances of asphalt mixtures made with Reclaimed Asphalt Pavement: Effects of interaction between virgin and RAP bitumen

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Luca Noferini

2017-07-01

Full Text Available According to most recent surveys, the European area produced 265 mil tonnes of asphalt for road applications in 2014. In the same year, the amount of available RAP was more than 50 mil tonnes. The use of RAP in new blended mixes reduces the need of neat bitumen, making RAP recycling economically attractive. Despite the economic and environmental benefits, road authorities tend to limit the use of RAP in asphalt mixes due to uncertainty about field performances. The present study focuses on the interaction between neat and RAP bitumen in asphalt mixes made with different RAP content. The effects of RAP on physical and rheological properties of the final bituminous blend were investigated. This study is part of a wider research, where a specific type of asphalt mixture was produced with different RAP contents being 10%, 20% and 30% by mass of the mix. Bitumen was extracted and recovered from asphalt mixes, then it was subjected to the following laboratory tests: standard characterization, dynamic viscosity and rheological analysis with DSR. Findings showed that the effects of RAP bitumen on the final blend varied in proportion to RAP content. A threshold value of RAP content was found, below which bitumen was not subjected to significant changes in physical and rheological properties. Practical implications on production methods and paving of RAP mixes are also proposed. Keywords: Reclaimed Asphalt Pavement (RAP, Recycling, Bitumen blending, Bitumen rheology

Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region*

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Jang, Hyunbum; Abraham, Sherwin J.; Chavan, Tanmay S.; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I.; Nussinov, Ruth; Gaponenko, Vadim

2015-01-01

K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. PMID:25713064

Specificity versus redundancy in the RAP2.4 transcription factor family of Arabidopsis thaliana: transcriptional regulation of genes for chloroplast peroxidases.

Science.gov (United States)

Rudnik, Radoslaw; Bulcha, Jote Tafese; Reifschneider, Elena; Ellersiek, Ulrike; Baier, Margarete

2017-08-23

The Arabidopsis ERFIb / RAP2.4 transcription factor family consists of eight members with highly conserved DNA binding domains. Selected members have been characterized individually, but a systematic comparison is pending. The redox-sensitive transcription factor RAP2.4a mediates chloroplast-to-nucleus redox signaling and controls induction of the three most prominent chloroplast peroxidases, namely 2-Cys peroxiredoxin A (2CPA) and thylakoid- and stromal ascorbate peroxidase (tAPx and sAPx). To test the specificity and redundancy of RAP2.4 transcription factors in the regulation of genes for chloroplast peroxidases, we compared the DNA-binding sites of the transcription factors in tertiary structure models, analyzed transcription factor and target gene regulation by qRT-PCR in RAP2.4, 2-Cys peroxiredoxin and ascorbate peroxidase T-DNA insertion lines and RAP2.4 overexpressing lines of Arabidopsis thaliana and performed promoter binding studies. All RAP2.4 proteins bound the tAPx promoter, but only the four RAP2.4 proteins with identical DNA contact sites, namely RAP2.4a, RAP2.4b, RAP2.4d and RAP2.4h, interacted stably with the redox-sensitive part of the 2CPA promoter. Gene expression analysis in RAP2.4 knockout lines revealed that RAP2.4a is the only one supporting 2CPA and chloroplast APx expression. Rap2.4h binds to the same promoter region as Rap2.4a and antagonizes 2CPA expression. Like the other six RAP2.4 proteins, Rap2.4 h promotes APx mRNA accumulation. Chloroplast ROS signals induced RAP2.4b and RAP2.4d expression, but these two transcription factor genes are (in contrast to RAP2.4a) insensitive to low 2CP availability, and their expression decreased in APx knockout lines. RAP2.4e and RAP2.4f gradually responded to chloroplast APx availability and activated specifically APx expression. These transcription factors bound, like RAP2.4c and RAP2.4g, the tAPx promoter, but hardly the 2CPA promoter. The RAP2.4 transcription factors form an environmentally and

BAR domain proteins regulate Rho GTPase signaling.

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Aspenström, Pontus

2014-01-01

BAR proteins comprise a heterogeneous group of multi-domain proteins with diverse biological functions. The common denominator is the Bin-Amphiphysin-Rvs (BAR) domain that not only confers targeting to lipid bilayers, but also provides scaffolding to mold lipid membranes into concave or convex surfaces. This function of BAR proteins is an important determinant in the dynamic reconstruction of membrane vesicles, as well as of the plasma membrane. Several BAR proteins function as linkers between cytoskeletal regulation and membrane dynamics. These links are provided by direct interactions between BAR proteins and actin-nucleation-promoting factors of the Wiskott-Aldrich syndrome protein family and the Diaphanous-related formins. The Rho GTPases are key factors for orchestration of this intricate interplay. This review describes how BAR proteins regulate the activity of Rho GTPases, as well as how Rho GTPases regulate the function of BAR proteins. This mutual collaboration is a central factor in the regulation of vital cellular processes, such as cell migration, cytokinesis, intracellular transport, endocytosis, and exocytosis.

Rac1 GTPase regulates 11β hydroxysteroid dehydrogenase type 2 and fibrotic remodeling.

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Lavall, Daniel; Schuster, Pia; Jacobs, Nadine; Kazakov, Andrey; Böhm, Michael; Laufs, Ulrich

2017-05-05

The aim of the study was to characterize the role of Rac1 GTPase for the mineralocorticoid receptor (MR)-mediated pro-fibrotic remodeling. Transgenic mice with cardiac overexpression of constitutively active Rac1 (RacET) develop an age-dependent phenotype with atrial dilatation, fibrosis, and atrial fibrillation. Expression of MR was similar in RacET and WT mice. The expression of 11β hydroxysteroid dehydrogenase type 2 (11β-HSD2) was age-dependently up-regulated in the atria and the left ventricles of RacET mice on mRNA and protein levels. Statin treatment inhibiting Rac1 geranylgeranylation reduced 11β-HSD2 up-regulation. Samples of human left atrial myocardium showed a positive correlation between Rac1 activity and 11β-HSD2 expression ( r = 0.7169). Immunoprecipitation showed enhanced Rac1-bound 11β-HSD2 relative to Rac1 expression in RacET mice that was diminished with statin treatment. Both basal and phorbol 12-myristate 13-acetate (PMA)-induced NADPH oxidase activity were increased in RacET and correlated positively with 11β-HSD2 expression ( r = 0.788 and r = 0.843, respectively). In cultured H9c2 cardiomyocytes, Rac1 activation with l-buthionine sulfoximine increased; Rac1 inhibition with NSC23766 decreased 11β-HSD2 mRNA and protein expression. Connective tissue growth factor (CTGF) up-regulation induced by aldosterone was prevented with NSC23766. Cardiomyocyte transfection with 11β-HSD2 siRNA abolished the aldosterone-induced CTGF up-regulation. Aldosterone-stimulated MR nuclear translocation was blocked by the 11β-HSD2 inhibitor carbenoxolone. In cardiac fibroblasts, nuclear MR translocation induced by aldosterone was inhibited with NSC23766 and spironolactone. NSC23766 prevented the aldosterone-induced proliferation and migration of cardiac fibroblasts and the up-regulation of CTGF and fibronectin. In conclusion, Rac1 GTPase regulates 11β-HSD2 expression, MR activation, and MR-mediated pro-fibrotic signaling. © 2017 by The American Society for

Neurotrophin Promotes Neurite Outgrowth by Inhibiting Rif GTPase Activation Downstream of MAPKs and PI3K Signaling.

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Tian, Xiaoxia; Yan, Huijuan; Li, Jiayi; Wu, Shuang; Wang, Junyu; Fan, Lifei

2017-01-13

Members of the well-known semaphorin family of proteins can induce both repulsive and attractive signaling in neural network formation and their cytoskeletal effects are mediated in part by small guanosine 5'-triphosphatase (GTPases). The aim of this study was to investigate the cellular role of Rif GTPase in the neurotrophin-induced neurite outgrowth. By using PC12 cells which are known to cease dividing and begin to show neurite outgrowth responding to nerve growth factor (NGF), we found that semaphorin 6A was as effective as nerve growth factor at stimulating neurite outgrowth in PC12 cells, and that its neurotrophic effect was transmitted through signaling by mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3K). We further found that neurotrophin-induced neurite formation in PC12 cells could be partially mediated by inhibition of Rif GTPase activity downstream of MAPKs and PI3K signaling. In conclusion, we newly identified Rif as a regulator of the cytoskeletal rearrangement mediated by semaphorins.

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

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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.

Development of low-cost open source 3D gel printer "RepRap SWIM-ER"

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Sato, Kei; Basher, Samiul; Ota, Takafumi; Tase, Taishi; Takamatsu, Kyuichiro; Saito, Azusa; Khosla, Ajit; Kawakami, Masaru; Furuawa, Hidemitsu

2017-04-01

Gels are soft and wet materials having low friction, good biocompatibility, and material permeability. It is expected that gel materials will be used as new kinds of industrial materials in the engineering and medical applications. But it cannot build a complicated shape. Soft & Wet Matter Engineering Laboratory developed a 3D gel Printer "SWIM-ER", has enabled modeling of complex shapes of the gel. However, this is expensive. Therefore not all of the gel researchers and the companies have such a device. To solve this problem, we manufacture a low-cost open-source 3D gel printer "RepRap SWIM-ER" from the RepRap. We made the components required to manufacture the "RepRap SWIM-ER" from the 3D printer and chose a light source. In addition, we produced the P-DN gel for RepRap SWIM-ER and conducted the molding test to confirm whether RepRap SWIM-ER can used it.

Implications of Rho GTPase signaling in glioma cell invasion and tumor progression

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Shannon Patricia Fortin Ensign

2013-10-01

Full Text Available Glioblastoma (GB is the most malignant of primary adult brain tumors, characterized by a highly locally-invasive cell population, as well as abundant proliferative cells, neoangiogenesis, and necrosis. Clinical intervention with chemotherapy or radiation may either promote or establish an environment for manifestation of invasive behavior. Understanding the molecular drivers of invasion in the context of glioma progression may be insightful in directing new treatments for patients with GB. Here, we review current knowledge on Rho family GTPases, their aberrant regulation in GB, and their effect on GB cell invasion and tumor progression. Rho GTPases are modulators of cell migration through effects on actin cytoskeleton rearrangement; in non-neoplastic tissue, expression and activation of Rho GTPases are normally under tight regulation. In GB, Rho GTPases are deregulated, often via hyperactivity or overexpression of their activators, Rho GEFs. Downstream effectors of Rho GTPases have been shown to promote invasiveness and, importantly, glioma cell survival. The study of aberrant Rho GTPase signaling in GB is thus an important investigation of cell invasion as well as treatment resistance and disease progression.

As mensagens sobre drogas no rap: como sobreviver na periferia Messages about drugs from rap: how to survive in the periferia

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Vinícius Gonçalves Bento da Silva

2004-12-01

Full Text Available O objetivo é analisar as mensagens sobre drogas nas letras de rap de grupos com representatividade e influência entre jovens da periferia de São Paulo. O rap é um gênero musical que faz parte de um movimento cultural - o hip hop -, que difunde uma visão social de mundo, principalmente nas periferias das grandes cidades. Por meio da Análise de Discurso estudaram-se 11 letras de 9 grupos. O tema marcante é a vida na periferia retratada pelo tráfico e consumo de drogas, pela violência e pela discriminação. O uso de drogas é compreendido por alguns grupos como conseqüência do modo de produção, e por outros, pelas características individuais, pela influência da família e dos amigos. As propostas para o enfrentamento e para a superação dos problemas estão voltadas à responsabilização do sujeito que, através de um esforço pessoal, não se envolveria com o tráfico e consumo de drogas consideradas perigosas e destrutivas como o crack e a cocaína. O fortalecimento de laços familiares e de amizade e a educação são também vistos como saídas para os problemas advindos do envolvimento com as drogas. As propostas invocam um discurso que, além de denunciar a situação dos jovens da periferia, propõe mecanismos de proteção para criar uma alternativa de "vida possível" - de convivência com a violência, com o tráfico e consumo de drogas.The objective was to analyze the messages about drugs of the rap lyrics from representative and influential groups among the youth living in the periferias of São Paulo. It understands that rap is a kind of music that is part of cultural movement - the hip hop - that disseminates a particular set of ideas, mainly in the periferias of the big cities. Through Discourse Analysis 11 lyrics of 9 rap groups were taken as a sample. The outstanding theme of the lyrics is the situation of the segregated areas of the city, including the presence of drug trafficking and consumption, as well as

Rap Music Literacy: A Case Study of Millennial Audience Reception to Rap Lyrics Depicting Independent Women

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Moody-Ramirez, Mia; Scott, Lakia M.

2015-01-01

Using a feminist lens and a constructivist approach as the theoretical framework, we used rap lyrics and videos to help college students explore mass media's representation of the "independent" Black woman and the concept of "independence" in general. Students must be able to formulate their own concept of independence to…

O rap radical e a "nova classe média"

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Ricardo Indig Teperman

2015-04-01

Full Text Available Este artigo discute a recente alteração na posição relativa do rap e dos rappers no campo da produção cultural no Brasil. O grupo Racionais MCs, tão central no campo do rap nacional que acaba por determinar a tendência hegemônica do gênero, vem se afastando do posicionamento revolucionário que marcou seus primeiros anos. Proponho que o aumento do poder de consumo e a democratização do acesso à tecnologia e à educação são aspectos que marcam a experiência da nova geração do rap (a chamada "nova escola", personificada em Emicida, e que provocaram o reposicionamento do Racionais. Recupero uma formulação de Antonio Candido para propor que essa nova posição pode ser considerada "radical".

Novel Alternative Splice Variants of Mouse Cdk5rap2.

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Nadine Kraemer

Full Text Available Autosomal recessive primary microcephaly (MCPH is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.

External radiation monitoring in TAPS and RAPS environs (1980-81) using TLD

International Nuclear Information System (INIS)

Basu, A.S.; Nambi, K.S.V.; Sunta, C.M.

1983-01-01

Results of environmental external radiation monitoring using quarterly integrated TLD measurements are presented for environments of the Tarapur Atomic Power Station (TAPS) and the Rajasthan Atomic Power Station (RAPS) for the two year monitoring period (1980-81). The data fit into the unimodal log-normal distribution except for locations where gaseous radioactivity escaping from the plant makes a significant contribution. The average natural radiation background in TAPS and RAPS environment is estimated to be 59.6 +- 4.7 mR yr -1 and 65.1 +- 9.8 mR yr -1 respectively. Contribution from the plant superimposed over the natural level leads frequently to bi-normal distribution. The effect of stack-released gaseous radioactivity is seen in locations within 1.6 km of TAPS: for example Ghivoli village registered an excess of 9.3 mR yr -1 over the natural background. The quarterly background values indicate minor temporal and spatial variations which can be attributed to changes in natural as well as stack released radioactivity. (author)

Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites

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Brautigam, Chad A; Xing, Wenmin; Yang, Sheng; Henry, Lisa; Doolittle, Lynda K; Walz, Thomas

2017-01-01

The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly. PMID:28949297

C3G knock-down enhances migration and invasion by increasing Rap1-mediated p38α activation, while it impairs tumor growth through p38α-independent mechanisms

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Priego, Neibla; Arechederra, María; Sequera, Celia; Bragado, Paloma; Vázquez-Carballo, Ana; Gutiérrez-Uzquiza, Álvaro; Martín-Granado, Víctor; Ventura, Juan José; Kazanietz, Marcelo G.; Guerrero, Carmen; Porras, Almudena

2016-01-01

C3G, a Guanine nucleotide Exchange Factor (GEF) for Rap1 and R-Ras, has been shown to play important roles in development and cancer. Previous studies determined that C3G regulates cell death through down-regulation of p38α MAPK activity. Here, we found that C3G knock-down in MEFs and HCT116 cells promotes migration and invasion through Rap1-mediated p38α hyper-activation. These effects of C3G were inhibited by Rap1 knock-down or inactivation. The enhanced migration observed in C3G depleted HCT116 cells was associated with reduction in E-cadherin expression, internalization of ZO-1, actin cytoskeleton reorganization and decreased adhesion. We also found that matrix metalloproteases MMP2 and MMP9 are involved in the pro-invasive effect of C3G down-regulation. Additionally, our studies revealed that both C3G and p38α collaborate to promote growth of HCT116 cells in vitro and in vivo, possibly by enhancing cell survival. In fact, knocking-down C3G or p38α individually or together promoted cell death in vitro, although only the double C3G-p38α silencing was able to increase cell death within tumors. Notably, we found that the pro-tumorigenic function of C3G does not depend on p38α or Rap1 activation. Altogether, our studies uncover novel mechanisms by which C3G controls key aspects of tumorigenesis. PMID:27286263

Mechanisms of membrane binding of small GTPase K-Ras4B farnesylated hypervariable region.

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Jang, Hyunbum; Abraham, Sherwin J; Chavan, Tanmay S; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I; Nussinov, Ruth; Gaponenko, Vadim

2015-04-10

K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Ras GTPases Modulate Morphogenesis, Sporulation and Cellulase Gene Expression in the Cellulolytic Fungus Trichoderma reesei

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Zhang, Jiwei; Zhang, Yanmei; Zhong, Yaohua; Qu, Yinbo; Wang, Tianhong

2012-01-01

Background The model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae, but their functions remain unknown. Methodology/Principal Findings Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2G16V allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway. Conclusions/Significance Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the

Changes in the prevalence of alcohol in rap music lyrics 1979-2009.

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Herd, Denise

2014-02-01

This study examines the prevalence and context of alcohol references in rap music lyrics from 1979 through 2009. Four hundred nine top-ranked rap music songs released were sampled from Billboard magazine rating charts. Songs were analyzed using systematic content analysis and were coded for alcohol beverage types and brand names, drinking behaviors, drinking contexts, attitudes towards alcohol, and consequences of drinking. Trends were analyzed using regression analyses. The results of the study reveal significant increases in the presence of alcohol in rap songs; a decline in negative attitudes towards alcohol; decreases in consequences attributed to alcohol; increases in the association of alcohol with glamour and wealth, drugs, and nightclubs; and increases in references to liquor and champagne.

Characterization of a Rab11-like GTPase, EhRab11, of Entamoeba histolytica.

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McGugan, Glen C; Temesvari, Lesly A

2003-07-01

The Entamoeba histolytica Rab11 family of small molecular weight GTPases consists of three members, EhRab11, EhRab11B, and EhRab11C. The functions of these Rabs in Entamoeba have not been determined. Therefore, as an approach to elucidate the role of the Rab11 family of GTPases in Entamoeba, immunofluorescence microscopy was undertaken to define the subcellular localization of one member of this family, EhRab11. Under conditions of growth, EhRab11 displayed a punctate pattern in the cytoplasm of trophozoites. EhRab11 did not colocalize with markers for the Golgi apparatus, endoplasmic reticulum, pinosomes, phagosomes, or compartments formed by receptor-mediated endocytosis, suggesting that this Rab may not play a role in vesicle trafficking between these organelles. Under conditions of iron and serum starvation, EhRab11 was translocated to the periphery of the cell. The altered cellular localization was accompanied by multinucleation of the cells as well as the acquisition of detergent resistance by the cells, features that are characteristic of Entamoeba cysts. The translocation of EhRab11 to the periphery of the cell during iron and serum starvation was specific as the subcellular localizations of two other Rab GTPases, EhRab7 and EhRabA, were not altered under the same conditions. In addition, the formation of multinucleated cells by inhibition of cytokinesis was not sufficient to induce the translocation of EhRab11 to the cell periphery. Taken together, the data suggest that iron and serum starvation may induce encystation in E. histolytica and that EhRab11 may play a role in this process. Moreover, these studies are the first to describe a putative role for a Rab GTPase in encystation in Entamoeba sp.

Sequential sentinel SNP Regional Association Plots (SSS-RAP): an approach for testing independence of SNP association signals using meta-analysis data.

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Zheng, Jie; Gaunt, Tom R; Day, Ian N M

2013-01-01

Genome-Wide Association Studies (GWAS) frequently incorporate meta-analysis within their framework. However, conditional analysis of individual-level data, which is an established approach for fine mapping of causal sites, is often precluded where only group-level summary data are available for analysis. Here, we present a numerical and graphical approach, "sequential sentinel SNP regional association plot" (SSS-RAP), which estimates regression coefficients (beta) with their standard errors using the meta-analysis summary results directly. Under an additive model, typical for genes with small effect, the effect for a sentinel SNP can be transformed to the predicted effect for a possibly dependent SNP through a 2×2 2-SNP haplotypes table. The approach assumes Hardy-Weinberg equilibrium for test SNPs. SSS-RAP is available as a Web-tool (http://apps.biocompute.org.uk/sssrap/sssrap.cgi). To develop and illustrate SSS-RAP we analyzed lipid and ECG traits data from the British Women's Heart and Health Study (BWHHS), evaluated a meta-analysis for ECG trait and presented several simulations. We compared results with existing approaches such as model selection methods and conditional analysis. Generally findings were consistent. SSS-RAP represents a tool for testing independence of SNP association signals using meta-analysis data, and is also a convenient approach based on biological principles for fine mapping in group level summary data. © 2012 Blackwell Publishing Ltd/University College London.

Regulation of cerebral cortex development by Rho GTPases: insights from in vivo studies

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Roberta eAzzarelli

2015-01-01

Full Text Available The cerebral cortex is the site of higher human cognitive and motor functions. Histologically, it is organized into six horizontal layers, each containing unique populations of molecularly and functionally distinct excitatory projection neurons and inhibitory interneurons. The stereotyped cellular distribution of cortical neurons is crucial for the formation of functional neural circuits and it is predominantly established during embryonic development. Cortical neuron development is a multiphasic process characterized by sequential steps of neural progenitor proliferation, cell cycle exit, neuroblast migration and neuronal differentiation. This series of events requires an extensive and dynamic remodeling of the cell cytoskeleton at each step of the process. As major regulators of the cytoskeleton, the family of small Rho GTPases has been shown to play essential functions in cerebral cortex development. Here we review in vivo findings that support the contribution of Rho GTPases to cortical projection neuron development and we address their involvement in the etiology of cerebral cortex malformations.

Characterization and Functional Analysis of the Calmodulin-Binding Domain of Rac1 GTPase

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Xu, Bing; Chelikani, Prashen; Bhullar, Rajinder P.

2012-01-01

Rac1, a member of the Rho family of small GTPases, has been shown to promote formation of lamellipodia at the leading edge of motile cells and affect cell migration. We previously demonstrated that calmodulin can bind to a region in the C-terminal of Rac1 and that this interaction is important in the activation of platelet Rac1. Now, we have analyzed amino acid residue(s) in the Rac1-calmodulin binding domain that are essential for the interaction and assessed their functional contribution in Rac1 activation. The results demonstrated that region 151–164 in Rac1 is essential for calmodulin binding. Within the 151–164 region, positively-charged amino acids K153 and R163 were mutated to alanine to study impact on calmodulin binding. Mutant form of Rac1 (K153A) demonstrated significantly reduced binding to calmodulin while the double mutant K153A/R163A demonstrated complete lack of binding to calmodulin. Thrombin or EGF resulted in activation of Rac1 in CHRF-288-11 or HeLa cells respectively and W7 inhibited this activation. Immunoprecipitation studies demonstrated that higher amount of CaM was associated with Rac1 during EGF dependent activation. In cells expressing mutant forms of Rac1 (K153A or K153A/R163A), activation induced by EGF was significantly decreased in comparison to wild type or the R163A forms of Rac1. The lack of Rac1 activation in mutant forms was not due to an inability of GDP-GTP exchange or a change in subcelllular distribution. Moreover, Rac1 activation was decreased in cells where endogenous level of calmodulin was reduced using shRNA knockdown and increased in cells where calmodulin was overexpressed. Docking analysis and modeling demonstrated that K153 in Rac1 interacts with Q41 in calmodulin. These results suggest an important role for calmodulin in the activation of Rac1 and thus, in cytoskeleton reorganization and cell migration. PMID:22905193

Characterization and functional analysis of the calmodulin-binding domain of Rac1 GTPase.

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Bing Xu

Full Text Available Rac1, a member of the Rho family of small GTPases, has been shown to promote formation of lamellipodia at the leading edge of motile cells and affect cell migration. We previously demonstrated that calmodulin can bind to a region in the C-terminal of Rac1 and that this interaction is important in the activation of platelet Rac1. Now, we have analyzed amino acid residue(s in the Rac1-calmodulin binding domain that are essential for the interaction and assessed their functional contribution in Rac1 activation. The results demonstrated that region 151-164 in Rac1 is essential for calmodulin binding. Within the 151-164 region, positively-charged amino acids K153 and R163 were mutated to alanine to study impact on calmodulin binding. Mutant form of Rac1 (K153A demonstrated significantly reduced binding to calmodulin while the double mutant K153A/R163A demonstrated complete lack of binding to calmodulin. Thrombin or EGF resulted in activation of Rac1 in CHRF-288-11 or HeLa cells respectively and W7 inhibited this activation. Immunoprecipitation studies demonstrated that higher amount of CaM was associated with Rac1 during EGF dependent activation. In cells expressing mutant forms of Rac1 (K153A or K153A/R163A, activation induced by EGF was significantly decreased in comparison to wild type or the R163A forms of Rac1. The lack of Rac1 activation in mutant forms was not due to an inability of GDP-GTP exchange or a change in subcelllular distribution. Moreover, Rac1 activation was decreased in cells where endogenous level of calmodulin was reduced using shRNA knockdown and increased in cells where calmodulin was overexpressed. Docking analysis and modeling demonstrated that K153 in Rac1 interacts with Q41 in calmodulin. These results suggest an important role for calmodulin in the activation of Rac1 and thus, in cytoskeleton reorganization and cell migration.

Analysis of the usage of rubberized asphalt in hot mix asphalt using Reclaimed Asphalt Pavement (RAP)

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Dwidarma Nataadmadja, Adelia; Prahara, Eduardi; Sumbung, Pierre Christian

2017-12-01

There has been an increasing demand in using more environmentally friendly materials in pavement construction. One of the alternative materials that have been widely used is the Reclaimed Asphalt Pavement (RAP) aggregates. The RAP aggregates are derived from the crushed and screened pavement materials that contain asphalt and aggregates. This material is usually combined with natural aggregates and virgin asphalt binder to construct a new pavement. There have been numerous positive feedbacks in using this material although RAP aggregates also have certain weaknesses, such as questionable interaction between virgin and recycled materials and increased stiffness of RAP binder. Moreover, there has been a push on using rubber as an additive to asphalt binder to improve the welfare of rubber farmers. This research combines the usage of both latex and RAP as the ingredients to design hot mix asphalt (HMA) as latex could help in improving the flexibility of HMA and the interaction between the virgin and recycled materials. The main objective of this research is to find a suitable percentage of RAP aggregates to be used in HMA with certain percentage of latex as the binder additive.

The Arf GTPase-activating protein family is exploited by Salmonella enterica serovar Typhimurium to invade nonphagocytic host cells.

Science.gov (United States)

Davidson, Anthony C; Humphreys, Daniel; Brooks, Andrew B E; Hume, Peter J; Koronakis, Vassilis

2015-02-10

To establish intracellular infections, Salmonella bacteria trigger host cell membrane ruffling and invasion by subverting cellular Arf guanine nucleotide exchange factors (GEFs) that activate Arf1 and Arf6 GTPases by promoting GTP binding. A family of cellular Arf GTPase-activating proteins (GAPs) can downregulate Arf signaling by stimulating GTP hydrolysis, but whether they do this during infection is unknown. Here, we uncovered a remarkable role for distinct Arf GAP family members in Salmonella invasion. The Arf6 GAPs ACAP1 and ADAP1 and the Arf1 GAP ASAP1 localized at Salmonella-induced ruffles, which was not the case for the plasma membrane-localized Arf6 GAPs ARAP3 and GIT1 or the Golgi-associated Arf1 GAP1. Surprisingly, we found that loss of ACAP1, ADAP1, or ASAP1 impaired Salmonella invasion, revealing that GAPs cannot be considered mere terminators of cytoskeleton remodeling. Salmonella invasion was restored in Arf GAP-depleted cells by expressing fast-cycling Arf derivatives, demonstrating that Arf GTP/GDP cycles facilitate Salmonella invasion. Consistent with this view, both constitutively active and dominant-negative Arf derivatives that cannot undergo GTP/GDP cycles inhibited invasion. Furthermore, we demonstrated that Arf GEFs and GAPs colocalize at invading Salmonella and collaborate to drive Arf1-dependent pathogen invasion. This study revealed that Salmonella bacteria exploit a remarkable interplay between Arf GEFs and GAPs to direct cycles of Arf GTPase activation and inactivation. These cycles drive Salmonella cytoskeleton remodeling and enable intracellular infections. To initiate infections, the Salmonella bacterial pathogen remodels the mammalian actin cytoskeleton and invades host cells by subverting host Arf GEFs that activate Arf1 and Arf6 GTPases. Cellular Arf GAPs deactivate Arf GTPases and negatively regulate cell processes, but whether they target Arfs during infection is unknown. Here, we uncovered an important role for the Arf GAP

Thermodynamics between RAP/RAS and virgin aggregates during asphalt concrete production : a literature review.

Science.gov (United States)

2015-09-01

In hot-mix asphalt (HMA) plants, virgin aggregates are heated and dried separately before being mixed with : RAP/RAS and virgin asphalt binder. RAP/RAS materials are not heated or dried directly by a burner to avoid : burning of aged binder coating o...

Uncoupling of dynamin polymerization and GTPase activity revealed by the conformation-specific nanobody dynab.

Science.gov (United States)

Galli, Valentina; Sebastian, Rafael; Moutel, Sandrine; Ecard, Jason; Perez, Franck; Roux, Aurélien

2017-10-12

Dynamin is a large GTPase that forms a helical collar at the neck of endocytic pits, and catalyzes membrane fission (Schmid and Frolov, 2011; Ferguson and De Camilli, 2012). Dynamin fission reaction is strictly dependent on GTP hydrolysis, but how fission is mediated is still debated (Antonny et al., 2016): GTP energy could be spent in membrane constriction required for fission, or in disassembly of the dynamin polymer to trigger fission. To follow dynamin GTP hydrolysis at endocytic pits, we generated a conformation-specific nanobody called dynab, that binds preferentially to the GTP hydrolytic state of dynamin-1. Dynab allowed us to follow the GTPase activity of dynamin-1 in real-time. We show that in fibroblasts, dynamin GTP hydrolysis occurs as stochastic bursts, which are randomly distributed relatively to the peak of dynamin assembly. Thus, dynamin disassembly is not coupled to GTPase activity, supporting that the GTP energy is primarily spent in constriction.

Unraveling the molecular mechanism of interactions of the Rho GTPases Cdc42 and Rac1 with the scaffolding protein IQGAP2.

Science.gov (United States)

Ozdemir, E Sila; Jang, Hyunbum; Gursoy, Attila; Keskin, Ozlem; Li, Zhigang; Sacks, David B; Nussinov, Ruth

2018-03-09

IQ motif-containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell-cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP-Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 "insert loop" was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis.

From Rage to Rap and Prison to Print:

Directory of Open Access Journals (Sweden)

Josephine Metcalf

2009-11-01

Full Text Available 1. IntroductionBy the late 1980s, while thousands of former gang members were either dead or incarcerated, their legacy and the gang subculture was being celebrated and commodified through gangsta rap music and videos, film and fashion. In the early 1990s this lucrative cultural trend sparked interest from potential authors and publishers. Such literary attention was partially fuelled by a public fascination with life in the ghettos following the 1992 Los Angeles (LA riots, resulting in a ne...

A class of dynamin-like GTPases involved in the generation of the tubular ER network

Science.gov (United States)

Hu, Junjie; Shibata, Yoko; Zhu, Peng-Peng; Voss, Christiane; Rismanchi, Neggy; Prinz, William A.; Rapoport, Tom A.; Blackstone, Craig

2009-01-01

The endoplasmic reticulum (ER) consists of tubules that are shaped by the reticulons and DP1/Yop1p, but how the tubules form an interconnected network is unknown. Here, we show that mammalian atlastins, which are dynamin-like, integral membrane GTPases, interact with the tubule-shaping proteins. The atlastins localize to the tubular ER and are required for proper network formation in vivo and in vitro. Depletion of the atlastins or overexpression of dominant-negative forms inhibits tubule interconnections. The Sey1p GTPase in S. cerevisiae is likely a functional ortholog of the atlastins; it shares the same signature motifs and membrane topology and interacts genetically and physically with the tubule-shaping proteins. Cells simultaneously lacking Sey1p and a tubule-shaping protein have ER morphology defects. These results indicate that formation of the tubular ER network depends on conserved dynamin-like GTPases. Since atlastin-1 mutations cause a common form of hereditary spastic paraplegia, we suggest ER shaping defects as a novel neuropathogenic mechanism. PMID:19665976

Gallic acid inhibits gastric cancer cells metastasis and invasive growth via increased expression of RhoB, downregulation of AKT/small GTPase signals and inhibition of NF-κB activity

Energy Technology Data Exchange (ETDEWEB)

Ho, Hsieh-Hsun [Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan (China); Chang, Chi-Sen [Department of Medicine, Chung Shan Medical University, Taichung 402, Taiwan (China); Division of Gastroenterology, Taichung Veterans General Hospital, Taichung 402, Taiwan (China); Ho, Wei-Chi [Division of Gastroenterology, Jen-Ai Hospital, Taichung 402, Taiwan (China); Liao, Sheng-You [Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan (China); Lin, Wea-Lung [Department of Pathology, School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan (China); Department of Pathology, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China); Wang, Chau-Jong, E-mail: wcj@csmu.edu.tw [Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan (China); Department of Medical Research, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China)

2013-01-01

Our previous study demonstrated the therapeutic potential of gallic acid (GA) for controlling tumor metastasis through its inhibitory effect on the motility of AGS cells. A noteworthy finding in our previous experiment was increased RhoB expression in GA-treated cells. The aim of this study was to evaluate the role of RhoB expression on the inhibitory effects of GA on AGS cells. By applying the transfection of RhoB siRNA into AGS cells and an animal model, we tested the effect of GA on inhibition of tumor growth and RhoB expression. The results confirmed that RhoB-siRNA transfection induced GA to inhibit AGS cells’ invasive growth involving blocking the AKT/small GTPase signals pathway and inhibition of NF-κB activity. Finally, we evaluated the effect of GA on AGS cell metastasis by colonization of tumor cells in nude mice. It showed GA inhibited tumor cells growth via the expression of RhoB. These data support the inhibitory effect of GA which was shown to inhibit gastric cancer cell metastasis and invasive growth via increased expression of RhoB, downregulation of AKT/small GTPase signals and inhibition of NF-κB activity. Thus, GA might be a potential agent in treating gastric cancer. Highlights: ► GA could downregulate AKT signal via increased expression of RhoB. ► GA inhibits metastasis in vitro in gastric carcinoma. ► GA inhibits tumor growth in nude mice model.

Changes in the prevalence of alcohol use in rap song lyrics, 1979-97.

Science.gov (United States)

Herd, Denise

2005-09-01

This paper explores the role of changing images of drinking and alcoholic beverage use in rap music from its beginnings in the United States in the late 1970s to the late 1990s. A sample of 341 rap music song lyrics released from 1979 to 1997 were selected using Billboard and Gavin rating charts. Song lyrics were coded for music genres, alcohol beverage types and brand names, drinking behaviors, drinking contexts, intoxication, attitudes towards alcohol and consequences of drinking. From 1979 to 1997, songs with references to alcohol increased fivefold (from 8 to 44%); those exhibiting positive attitudes rose from 43% to 73%; and brand name mentions increased from 46% to 71%. There were also significant increases in songs mentioning champagne and liquor (mainly expensive brand names) when comparing songs released after 1994 with those from previous years. In addition, there were significant increases in references to alcohol to signify glamour and wealth, and using alcohol with drugs and for recreational purposes. The findings also showed that alcohol use in rap music was much more likely to result in positive than negative consequences. Many of these findings are consistent with the idea that rap music has been profoundly affected by commercial forces and the marketing of alcoholic beverages. In addition, it is possible that the increase in references to alcoholic beverages in rap music, particularly spirits, is a reflection of a broader advertising culture which increasingly associates African Americans with alcohol use.

The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes

Science.gov (United States)

Khatter, Divya; Raina, Vivek B.; Dwivedi, Devashish; Sindhwani, Aastha; Bahl, Surbhi; Sharma, Mahak

2015-01-01

The homotypic fusion and protein sorting (HOPS) complex is a multi-subunit complex conserved from yeast to mammals that regulates late endosome and lysosome fusion. However, little is known about how the HOPS complex is recruited to lysosomes in mammalian cells. Here, we report that the small GTPase Arl8b, but not Rab7 (also known as RAB7A), is essential for membrane localization of the human (h)Vps41 subunit of the HOPS complex. Assembly of the core HOPS subunits to Arl8b- and hVps41-positive lysosomes is guided by their subunit–subunit interactions. RNA interference (RNAi)-mediated depletion of hVps41 resulted in the impaired degradation of EGFR that was rescued upon expression of wild-type but not an Arl8b-binding-defective mutant of hVps41, suggesting that Arl8b-dependent lysosomal localization of hVps41 is required for its endocytic function. Furthermore, we have also identified that the Arl8b effector SKIP (also known as PLEKHM2) interacts with and recruits HOPS subunits to Arl8b and kinesin-positive peripheral lysosomes. Accordingly, RNAi-mediated depletion of SKIP impaired lysosomal trafficking and degradation of EGFR. These findings reveal that Arl8b regulates the association of the human HOPS complex with lysosomal membranes, which is crucial for the function of this tethering complex in endocytic degradation. PMID:25908847

The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes.

Science.gov (United States)

Khatter, Divya; Raina, Vivek B; Dwivedi, Devashish; Sindhwani, Aastha; Bahl, Surbhi; Sharma, Mahak

2015-05-01

The homotypic fusion and protein sorting (HOPS) complex is a multi-subunit complex conserved from yeast to mammals that regulates late endosome and lysosome fusion. However, little is known about how the HOPS complex is recruited to lysosomes in mammalian cells. Here, we report that the small GTPase Arl8b, but not Rab7 (also known as RAB7A), is essential for membrane localization of the human (h)Vps41 subunit of the HOPS complex. Assembly of the core HOPS subunits to Arl8b- and hVps41-positive lysosomes is guided by their subunit-subunit interactions. RNA interference (RNAi)-mediated depletion of hVps41 resulted in the impaired degradation of EGFR that was rescued upon expression of wild-type but not an Arl8b-binding-defective mutant of hVps41, suggesting that Arl8b-dependent lysosomal localization of hVps41 is required for its endocytic function. Furthermore, we have also identified that the Arl8b effector SKIP (also known as PLEKHM2) interacts with and recruits HOPS subunits to Arl8b and kinesin-positive peripheral lysosomes. Accordingly, RNAi-mediated depletion of SKIP impaired lysosomal trafficking and degradation of EGFR. These findings reveal that Arl8b regulates the association of the human HOPS complex with lysosomal membranes, which is crucial for the function of this tethering complex in endocytic degradation. © 2015. Published by The Company of Biologists Ltd.

Metric Ambiguity and Flow in Rap Music: A Corpus-Assisted Study of Outkast's "Mainstream" (1996

Directory of Open Access Journals (Sweden)

Mitchell Ohriner

2017-01-01

Full Text Available Recent years have seen the rise of musical corpus studies, primarily detailing harmonic tendencies of tonal music. This article extends this scholarship by addressing a new genre (rap music and a new parameter of focus (rhythm. More specifically, I use corpus methods to investigate the relation between metric ambivalence in the instrumental parts of a rap track (i.e., the beat and an emcee's rap delivery (i.e., the flow. Unlike virtually every other rap track, the instrumental tracks of Outkast's "Mainstream" (1996 simultaneously afford hearing both a four-beat and a three-beat metric cycle. Because three-beat durations between rhymes, phrase endings, and reiterated rhythmic patterns are rare in rap music, an abundance of them within a verse of "Mainstream" suggests that an emcee highlights the three-beat cycle, especially if that emcee is not prone to such durations more generally. Through the construction of three corpora, one representative of the genre as a whole, and two that are artist specific, I show how the emcee T-Mo Goodie's expressive practice highlights the rare three-beat affordances of the track.

Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts

International Nuclear Information System (INIS)

Wan, Qiaoqiao; Cho, Eunhye; Yokota, Hiroki; Na, Sungsoo

2013-01-01

Highlights: •Shear stress increased TCF/LEF activity and stimulated β-catenin nuclear localization. •Rac1, Cdc42, and RhoA displayed distinct dynamic activity patterns under flow. •Rac1 and Cdc42, but not RhoA, regulate shear stress-driven TCF/LEF activation. •Cytoskeleton did not significantly affect shear stress-induced TCF/LEF activation. -- Abstract: Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm 2 ) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate

Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts

Energy Technology Data Exchange (ETDEWEB)

Wan, Qiaoqiao; Cho, Eunhye [Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 (United States); Yokota, Hiroki [Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 (United States); Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Na, Sungsoo, E-mail: sungna@iupui.edu [Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 (United States)

2013-04-19

Highlights: •Shear stress increased TCF/LEF activity and stimulated β-catenin nuclear localization. •Rac1, Cdc42, and RhoA displayed distinct dynamic activity patterns under flow. •Rac1 and Cdc42, but not RhoA, regulate shear stress-driven TCF/LEF activation. •Cytoskeleton did not significantly affect shear stress-induced TCF/LEF activation. -- Abstract: Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm{sup 2}) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate

Determination of usable residual asphalt binder in RAP.

Science.gov (United States)

2009-01-01

For current recycled mix designs, the Illinois Department of Transportation (IDOT) assumes 100% contribution of : working binder from Recycled Asphalt Pavement (RAP) materials when added to Hot Mix Asphalt (HMA). However, it is : unclear if this assu...

RAP2.4a Is Transported through the Phloem to Regulate Cold and Heat Tolerance in Papaya Tree (Carica papaya cv. Maradol: Implications for Protection Against Abiotic Stress.

Directory of Open Access Journals (Sweden)

Luis Figueroa-Yañez

Full Text Available Plants respond to stress through metabolic and morphological changes that increase their ability to survive and grow. To this end, several transcription factor families are responsible for transmitting the signals that are required for these changes. Here, we studied the transcription factor superfamily AP2/ERF, particularly, RAP2.4 from Carica papaya cv. Maradol. We isolated four genes (CpRap2.4a, CpRAap2.4b, CpRap2.1 and CpRap2.10, and an in silico analysis showed that the four genes encode proteins that contain a conserved APETALA2 (AP2 domain located within group I and II transcription factors of the AP2/ERF superfamily. Semiquantitative PCR experiments indicated that each CpRap2 gene is differentially expressed under stress conditions, such as extreme temperatures. Moreover, genetic transformants of tobacco plants overexpressing CpRap2.4a and CpRap2.4b genes show a high level of tolerance to cold and heat stress compared to non-transformed plants. Confocal microscopy analysis of tobacco transgenic plants showed that CpRAP2.4a and CpRAP2.4b proteins were mainly localized to the nuclei of cells from the leaves and roots and also in the sieve elements. Moreover, the movement of CpRap2.4a RNA in tobacco grafting was analyzed. Our results indicate that CpRap2.4a and CpRap2.4b RNA in the papaya tree have a functional role in the response to stress conditions such as exposure to extreme temperatures via direct translation outside the parental RNA cell.

O rap dos Racionais MC\\'s em sala de aula como via de emancipação de jovens na periferia de São Paulo: análises de oficinas musicais com ênfase no rap

OpenAIRE

Raquel Mendonça Martins

2015-01-01

A presente dissertação de mestrado teve o propósito de pesquisar em que medida a estética multifacetada do rap pode ser utilizada nos processos de formação de adolescentes pobres e moradores das periferias, em sua maioria, afrodescendentes. Ao identificar no rap um potencial de ruptura e de resistência frente às formas atuais de discriminação racial - tanto em seus aspectos musicais, quanto narrativos - foram realizadas oficinas de música com ênfase no rap, envolvendo jovens entre treze e qui...

Analysis of the finescale timing of repeated signals: does shell rapping in hermit crabs signal stamina?

Science.gov (United States)

Briffa; Elwood

2000-01-01

Hermit crabs, Pagurus bernhardus, sometimes exchange shells after a period of shell rapping, when the initiating or attacking crab brings its shell rapidly and repeatedly into contact with the shell of the noninitiator or defender in a series of bouts. Bouts are separated by pauses, and raps within bouts are separated by very short periods called 'gaps'. Since within-contest variation is missed when signals are studied by averaging performance rates over entire contests, we analysed the fine within-bout structure of this repeated, aggressive signal. We found that the pattern is consistent with high levels of fatigue in initiators. The duration of the gaps between individual raps increased both within bouts and from bout to bout, and we conclude that this activity is costly to perform. Furthermore, long pauses between bouts is correlated with increased vigour of rapping in the subsequent bout, which suggests that the pause allows for recovery from fatigue induced by rapping. These between-bout pauses may be assessed by noninitiators and provide a signal of stamina. Copyright 2000 The Association for the Study of Animal Behaviour.

Ras and relatives--job sharing and networking keep an old family together.

Science.gov (United States)

Ehrhardt, Annette; Ehrhardt, Götz R A; Guo, Xuecui; Schrader, John W

2002-10-01

Many members of the Ras superfamily of GTPases have been implicated in the regulation of hematopoietic cells, with roles in growth, survival, differentiation, cytokine production, chemotaxis, vesicle-trafficking, and phagocytosis. The well-known p21 Ras proteins H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B are also frequently mutated in human cancer and leukemia. Besides the four p21 Ras proteins, the Ras subfamily of the Ras superfamily includes R-Ras, TC21 (R-Ras2), M-Ras (R-Ras3), Rap1A, Rap1B, Rap2A, Rap2B, RalA, and RalB. They exhibit remarkable overall amino acid identities, especially in the regions interacting with the guanine nucleotide exchange factors that catalyze their activation. In addition, there is considerable sharing of various downstream effectors through which they transmit signals and of GTPase activating proteins that downregulate their activity, resulting in overlap in their regulation and effector function. Relatively little is known about the physiological functions of individual Ras family members, although the presence of well-conserved orthologs in Caenorhabditis elegans suggests that their individual roles are both specific and vital. The structural and functional similarities have meant that commonly used research tools fail to discriminate between the different family members, and functions previously attributed to one family member may be shared with other members of the Ras family. Here we discuss similarities and differences in activation, effector usage, and functions of different members of the Ras subfamily. We also review the possibility that the differential localization of Ras proteins in different parts of the cell membrane may govern their responses to activation of cell surface receptors.

The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

Energy Technology Data Exchange (ETDEWEB)

Tsukamoto, Yuta; Katayama, Chisako [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Shinohara, Miki; Shinohara, Akira [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Maekawa, Shohei [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan)

2013-11-29

Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.

The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

International Nuclear Information System (INIS)

Tsukamoto, Yuta; Katayama, Chisako; Shinohara, Miki; Shinohara, Akira; Maekawa, Shohei; Miyamoto, Masaaki

2013-01-01

Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions

Addiction and Lifestyles in Contemporary Europe: Reframing Addictions Project (ALICE RAP). Final Evaluation Report Deliverable 21.1, Work Package 21

OpenAIRE

Mittelmark, Maurice B.

2016-01-01

At the onset of the ALICE RAP project, the following objectives, description of work, and main tasks were agreed for Work Package 21: 1. To evaluate the overall functioning of the collaborative research project, using a state-of-art systems model of partnership functioning. 2. To document the interactions and linkages between project inputs, throughputs and outputs as the project unfolds over five years. 3. To facilitate structured discussions involving all pa...

Plant Rho-type (Rop) GTPase-dependent activation of receptor-like cytoplasmic kinases in vitro.

Science.gov (United States)

Dorjgotov, Dulguun; Jurca, Manuela E; Fodor-Dunai, Csilla; Szucs, Attila; Otvös, Krisztina; Klement, Eva; Bíró, Judit; Fehér, Attila

2009-04-02

Plants have evolved distinct mechanisms to link Rho-type (Rop) GTPases to downstream signaling pathways as compared to other eukaryotes. Here, experimental data are provided that members of the Medicago, as well as Arabidopsis, receptor-like cytoplasmic kinase family (RLCK Class VI) were strongly and specifically activated by GTP-bound Rop GTPases in vitro. Deletion analysis indicated that the residues implicated in the interaction might be distributed on various parts of the kinases. Using a chimaeric Rop GTPase protein, the importance of the Rho-insert region in kinase activation could also be verified. These data strengthen the possibility that RLCKs may serve as Rop GTPase effectors in planta.

Rap-rimas afetivas da periferia: reflexões na perspectiva sócio-histórica Rap-affective rhymes of the periphery: reflections in the social-historical perspective

Directory of Open Access Journals (Sweden)

Jaison Hinkel

2007-01-01

Full Text Available Considerando a dimensão afetiva como constitutiva do agir e do pensar humano, e reconhecendo que sua presença é uma constante no Rap, este artigo busca investigar como a afetividade é expressa nas músicas de quatro grupos de Rap nacional. A partir da análise das músicas, pode-se considerar que estas expressam as vivências advindas de uma ordem social baseada na inclusão social perversa. Há instantes em que a tônica está no sentimento da vergonha, culpa, humilhação, tristeza, revolta e medo que assola os moradores da periferia. Em contrapartida, há propostas de enfrentamento desta condição, expressando a importância da união, irmandade, humildade, esperança, amor, alegria e solidariedade. Assim, nestas canções, a afetividade expressa tanto a denúncia do sofrimento ético-político, como a possibilidade de aumentar a potência de ação do sujeito para a superação da condição de padecimento humano, indicando a música, especialmente o Rap, como temática importante na compreensão psicossocial do sujeito em contextos de exclusão social.Considering the affectionate dimension as the constituent of act and of human thought, and recognizing that his presence is a constant one in the Rap, this article seeks to investigate like the affection is espress in the music of four groups of Rap national. Starting from the analysis of the music, we can consider that these express the resulting experiences of a social order based on the perverse social inclusion. There are instants in that to tonic is in the feeling of the shame, blame, humiliation, sorrow, revolt and fear that devastates the inhabitants of the periphery. In compensation, there is proposals to face of this condition, expressing the importance of the union, fraternity, humility, hope, love, joy and solidarity. Like this, in these songs, the so much express affection the denunciation of the ethical-political suffering, as the possibility of potency the action of the

Whats the Rap about Ecstasy? Popular Music Lyrics and Drug Trends among American Youth

Science.gov (United States)

Diamond, Sarah; Bermudez, Rey; Schensul, Jean

2006-01-01

Trends in ecstasy use in America during the past decade were reflected in mainstream, American rap-music lyrics between 1996 and 2003. Drawing on communication and cultural studies theory, this article provides a content analysis of 69 rap songs mentioning the club drug ecstasy. The songs are coded according to whether they contain positive, mixed…

Expression of sheep pathogen Babesia sp. Xinjiang rhoptry-associated protein 1 and evaluation of its diagnostic potential by enzyme-linked immunosorbent assay.

Science.gov (United States)

Niu, Qingli; Liu, Zhijie; Yang, Jifei; Yu, Peifa; Pan, Yuping; Zhai, Bintao; Luo, Jianxun; Guan, Guiquan; Yin, Hong

2016-12-01

Ovine babesiosis is one of the most important tick-borne haemoparasitic diseases of small ruminants. The ovine parasite Babesia sp. Xinjiang is widespread in China. In this study, recombinant full-length XJrRAP-1aα2 (rhoptry-associated protein 1aα2) and C-terminal XJrRAP-1aα2 CT of Babesia sp. Xinjiang were expressed and used to evaluate their diagnostic potential for Babesia sp. Xinjiang infections by indirect enzyme-linked immunosorbent assay (ELISA). Purified XJrRAP-1aα2 was tested for reactivity with sera from animals experimentally infected with Babesia sp. Xinjiang and other haemoparasites using Western blotting and ELISA. The results showed no cross-reactivities between XJrRAP-1aα2 CT and sera from animals infected by other pathogens. High level of antibodies against RAP-1a usually lasted 10 weeks post-infection (wpi). A total of 3690 serum samples from small ruminants in 23 provinces located in 59 different regions of China were tested by ELISA. The results indicated that the average positive rate was 30·43%, and the infections were found in all of the investigated provinces. This is the first report on the expression and potential use of a recombinant XJrRAP-1aα2 CT antigen for the development of serological assays for the diagnosis of ovine babesiosis, caused by Babesia sp. Xinjiang.

Feeling the beat: the meaning of rap music for ethnically diverse Midwestern college students--a phenomenological study.

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Iwamoto, Derek K; Creswell, John; Caldwell, Leon

2007-01-01

Despite its national and international appeal, rap is considered one of the most controversial of music genres. Given the political charge it generates, rap music has spawned research across the social and health sciences. The majority of the research has investigated its impact on African Americans. Further, the research has tended to focus on negative aspects of the music; there has been a dearth of in-depth qualitative studies that explore how rap impacts the listener. Our phenomenological study explores that impact on ethnically diverse college students. Results indicate a profound psychological and educational effect and the discussion goes on to highlight the potential and innovative ways rap music can be utilized with adolescents in fields such as education, risk reduction programs, and counseling psychology.

Binding of the GTPase Sar1 to a Lipid Membrane Monolayer: Insertion and Orientation Studied by Infrared Reflection–Absorption Spectroscopy

Directory of Open Access Journals (Sweden)

Christian Schwieger

2017-11-01

Full Text Available Membrane-interacting proteins are polyphilic polymers that engage in dynamic protein–protein and protein–lipid interactions while undergoing changes in conformation, orientation and binding interfaces. Predicting the sites of interactions between such polypeptides and phospholipid membranes is still a challenge. One example is the small eukaryotic GTPase Sar1, which functions in phospholipid bilayer remodeling and vesicle formation as part of the multimeric coat protein complex (COPII. The membrane interaction of Sar1 is strongly dependent on its N-terminal 23 amino acids. By monolayer adsorption experiments and infrared reflection-absorption spectroscopy (IRRAS, we elucidate the role of lipids in inducing the amphipathicity of this N-terminal stretch, which inserts into the monolayer as an amphipathic helix (AH. The AH inserting angle is determined and is consistent with the philicities and spatial distribution of the amino acid monomers. Using an advanced method of IRRAS data evaluation, the orientation of Sar1 with respect to the lipid layer prior to the recruitment of further COPII proteins is determined. The result indicates that only a slight reorientation of the membrane-bound Sar1 is needed to allow coat assembly. The time-course of the IRRAS analysis corroborates a role of slow GTP hydrolysis in Sar1 desorption from the membrane.

Inhibition of hippocampal synaptic transmission by impairment of Ral function

DEFF Research Database (Denmark)

Owe-Larsson, Björn; Chaves-Olarte, Esteban; Chauhan, Ashok

2005-01-01

Large clostridial cytotoxins and protein overexpression were used to probe for involvement of Ras-related GTPases (guanosine triphosphate) in synaptic transmission in cultured rat hippocampal neurons. The toxins TcdA-10463 (inactivates Rho, Rac, Cdc42, Rap) and TcsL-1522 (inactivates Ral, Rac, Ras......, R-Ras, Rap) both inhibited autaptic responses. In a proportion of the neurons (25%, TcdA-10463; 54%, TcsL-1522), the inhibition was associated with a shift from activity-dependent depression to facilitation, indicating that the synaptic release probability was reduced. Overexpression of a dominant...... negative Ral mutant, Ral A28N, caused a strong inhibition of autaptic responses, which was associated with a shift to facilitation in a majority (80%) of the neurons. These results indicate that Ral, along with at least one other non-Rab GTPase, participates in presynaptic regulation in hippocampal neurons....

Hepatic trans-Golgi action coordinated by the GTPase ARFRP1 is crucial for lipoprotein lipidation and assembly[S

Science.gov (United States)

Hesse, Deike; Radloff, Katrin; Jaschke, Alexander; Lagerpusch, Merit; Chung, Bomee; Tailleux, Anne; Staels, Bart; Schürmann, Annette

2014-01-01

The liver is a major organ in whole body lipid metabolism and malfunctioning can lead to various diseases including dyslipidemia, fatty liver disease, and type 2 diabetes. Triglycerides and cholesteryl esters are packed in the liver as very low density lipoproteins (VLDLs). Generation of these lipoproteins is initiated in the endoplasmic reticulum and further maturation likely occurs in the Golgi. ADP-ribosylation factor-related protein 1 (ARFRP1) is a small trans-Golgi-associated guanosine triphosphatase (GTPase) that regulates protein sorting and is required for chylomicron lipidation and assembly in the intestine. Here we show that the hepatocyte-specific deletion of Arfrp1 (Arfrp1liv−/−) results in impaired VLDL lipidation leading to reduced plasma triglyceride levels in the fasted state as well as after inhibition of lipoprotein lipase activity by Triton WR-1339. In addition, the concentration of ApoC3 that comprises 40% of protein mass of secreted VLDLs is markedly reduced in the plasma of Arfrp1liv−/− mice but accumulates in the liver accompanied by elevated triglycerides. Fractionation of Arfrp1liv−/− liver homogenates reveals more ApoB48 and a lower concentration of triglycerides in the Golgi compartments than in the corresponding fractions from control livers. In conclusion, ARFRP1 and the Golgi apparatus play an important role in lipoprotein maturation in the liver by influencing lipidation and assembly of proteins to the lipid particles. PMID:24186947

Sprouty regulates cell migration by inhibiting the activation of Rac1 GTPase

International Nuclear Information System (INIS)

Poppleton, Helen M.; Edwin, Francis; Jaggar, Laura; Ray, Ramesh; Johnson, Leonard R.; Patel, Tarun B.

2004-01-01

Sprouty (SPRY) protein negatively modulates fibroblast growth factor and epidermal growth factor actions. We showed that human SPRY2 inhibits cell growth and migration in response to serum and several growth factors. Using rat intestinal epithelial (IEC-6) cells, we investigated the involvement of the Rho family of GTPases, RhoA, Rac1, and cdc42 in SPRY2-mediated inhibition of cell migration and proliferation. The ability of TAT-tagged SPRY2 to inhibit proliferation and migration of IEC-6 cells transfected with constitutively active mutants of RhoA(G14V), Rac1(G12V), and cdc42 (F28L) was determined. Constitutively active RhoA(G14V), Rac1(G12V), or cdc42(F28L) did not protect cells from the anti-proliferative actions of TAT-SPRY2. The ability of TAT-hSPRY2 to inhibit migration was not altered by of RhoA(G14V) and cdc42(F28L). However, Rac1(G12V) obliterated the ability of SPRY2 to inhibit cell autonomous or serum-induced migration. Also, the activation of endogenous Rac1 was attenuated by TAT-SPRY2. Thus, SPRY2 mediates its anti-migratory actions by inhibiting Rac1 activation

Molecular characterization and chromosomal assignment of equine cartilage derived retinoic acid sensitive protein (CD-RAP)/melanoma inhibitory activity (MIA)

DEFF Research Database (Denmark)

Berg, Lise Charlotte; Mata, Xavier; Thomsen, Preben Dybdahl

2008-01-01

Cartilage-derived retinoic acid sensitive protein (CD-RAP) also known as melanoma inhibitory activity (MIA) has already been established as a marker for chondrocyte differentiation and a number of cancerous condition sin humans. Studies have also shown that CD-RAP/MIA is a potential marker of joint......RNA in articular cartilage and chondrocytes from horses with no signs of joint disease. The expression decreased as the cells dedifferentiated in monolayer culture. We also identified an equine CD-RAP/MIA splioce variant similar to that reported in humans. The CD_RAP/MIA protein was detected in equine synovial...... fluid, serum and culture medium from chondrocyte cultures. In conclusion, CD-RAP/MIA is expressed in equine cartilage and chondrocytes, and the protein can be detected in equine serum, synovial fluid and in culture medium from chondrocyte cultures. The equine gene and resulting protein share great...

Human Mammary Epithelial Cell Transformation by Rho GTPase Through a Novel Mechanism

Science.gov (United States)

2009-08-01

87: 635-44. 18. Burbelo P, Wellstein A, Pestell RG. Altered Rho GTPase signaling pathways in breast cancer cells. Breast Cancer Res Treat 2004; 84...Burbelo P, Wellstein A, Pestell RG. Altered Rho GTPase signaling pathways in breast cancer cells. Breast Cancer Res Treat 2004;84:43–8. 19. Band V

Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein

DEFF Research Database (Denmark)

Järås, Marcus; Johnels, Petra; Hansen, Nils Gunder

2010-01-01

stem cells could be prospectively separated. In addition, by generating an anti-IL1RAP antibody, we provide proof of concept that IL1RAP can be used as a target on CML CD34(+)CD38(-) cells to induce antibody-dependent cell-mediated cytotoxicity. This study thus identifies IL1RAP as a unique cell...... will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test...... whether IL1RAP expression distinguishes normal (Ph(-)) and leukemic (Ph(+)) cells within the CML CD34(+)CD38(-) cell compartment, we established a unique protocol for conducting FISH on small numbers of sorted cells. By using this method, we sorted cells directly into drops on slides to investigate...

Characterization of mitochondrion-targeted GTPases in Plasmodium falciparum.

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Gupta, Kirti; Gupta, Ankit; Haider, Afreen; Habib, Saman

2018-04-12

Ribosome assembly is critical for translation and regulating the response to cellular events and requires a complex interplay of ribosomal RNA and proteins with assembly factors. We investigated putative participants in the biogenesis of the reduced organellar ribosomes of Plasmodium falciparum and identified homologues of two assembly GTPases - EngA and Obg that were found in mitochondria. Both are indispensable in bacteria and P. berghei EngA is among the 'essential' parasite blood stage proteins identified recently. PfEngA and PfObg1 interacted with parasite mitoribosomes in vivo. GTP stimulated PfEngA interaction with the 50S subunit of Escherichia coli surrogate ribosomes. Although PfObg1-ribosome interaction was independent of nucleotide binding, GTP hydrolysis by PfObg1 was enhanced upon ribosomal association. An additional function for PfObg1 in mitochondrial DNA transactions was suggested by its specific interaction with the parasite mitochondrial genome in vivo. Deletion analysis revealed that the positively-charged OBG (spoOB-associated GTP-binding protein) domain mediates DNA-binding. A role for PfEngA in mitochondrial genotoxic stress response was indicated by its over-expression upon methyl methanesulfonate-induced DNA damage. PfEngA had lower sensitivity to an E. coli EngA inhibitor suggesting differences with bacterial counterparts. Our results show the involvement of two important GTPases in P. falciparum mitochondrial function, with the first confirmed localization of an EngA homologue in eukaryotic mitochondria.

Evaluation of the RapID-ANA system for identification of anaerobic bacteria of veterinary origin.

Science.gov (United States)

Adney, W S; Jones, R L

1985-12-01

This study evaluated the ability of the RapID-ANA system (Innovative Diagnostic Systems, Inc., Atlanta, Ga.) to accurately identify a spectrum of freshly isolated veterinary anaerobes. A total of 183 isolates were tested and included 7 Actinomyces spp., 53 Bacteroides spp., 32 Clostridium spp., 2 Eubacterium spp., 65 Fusobacterium spp., 1 Peptococcus spp., 22 Peptostreptococcus spp., and 1 Propionibacterium spp. All isolates were initially identified by conventional biochemical testing and gas-liquid chromatography of short-chain fatty acid metabolites. Additional tests were performed as required by the RapID-ANA system. Of these isolates, 81.4% were correctly identified to the genus level, including 59.6% to the species level, 14.2% were incorrectly identified at the genus level, and 4.4% were not identified. Initially, 20.2% of the strains were not identified because the microcodes were not in the code book. The majority of the incorrect identifications were caused by the misidentification of Fusobacterium spp. as Bacteroides spp. Errors also occurred when veterinary anaerobes not included in the data base were assigned an identification from the existing data base. The RapID-ANA system appears to be a promising new method for rapid identification of veterinary anaerobes; however, further evaluation with an extended data base is needed before the system can accurately identify all clinically significant anaerobes.

Hybrid Texts: Fifth Graders, Rap Music, and Writing

Science.gov (United States)

Christianakis, Mary

2011-01-01

Consistent with a sociocritical frame and the analytic tools of hybridity theory, this article explicates how urban fifth-grade children made language hybrids using rap and poetry to participate in classroom literacy. Ethnographic data from a yearlong study illustrate two key findings. First, standards-based and canon-driven writing models…

DNA topoisomerase IIβ stimulates neurite outgrowth in neural differentiated human mesenchymal stem cells through regulation of Rho-GTPases (RhoA/Rock2 pathway) and Nurr1 expression.

Science.gov (United States)

Zaim, Merve; Isik, Sevim

2018-04-25

DNA topoisomerase IIβ (topo IIβ) is known to regulate neural differentiation by inducing the neuronal genes responsible for critical neural differentiation events such as neurite outgrowth and axon guidance. However, the pathways of axon growth controlled by topo IIβ have not been clarified yet. Microarray results of our previous study have shown that topo IIβ silencing in neural differentiated primary human mesenchymal stem cells (hMSCs) significantly alters the expression pattern of genes involved in neural polarity, axonal growth, and guidance, including Rho-GTPases. This study aims to further analyze the regulatory role of topo IIβ on the process of axon growth via regulation of Rho-GTPases. For this purpose, topo IIβ was silenced in neurally differentiated hMSCs. Cells lost their morphology because of topo IIβ deficiency, becoming enlarged and flattened. Additionally, a reduction in both neural differentiation efficiency and neurite length, upregulation in RhoA and Rock2, downregulation in Cdc42 gene expression were detected. On the other hand, cells were transfected with topo IIβ gene to elucidate the possible neuroprotective effect of topo IIβ overexpression on neural-induced hMSCs. Topo IIβ overexpression prompted all the cells to exhibit neural cell morphology as characterized by longer neurites. RhoA and Rock2 expressions were downregulated, whereas Cdc42 expression was upregulated. Nurr1 expression level correlated with topo IIβ in both topo IIβ-overexpressed and -silenced cells. Furthermore, differential translocation of Rho-GTPases was detected by immunostaining in response to topo IIβ. Our results suggest that topo IIβ deficiency could give rise to neurodegeneration through dysregulation of Rho-GTPases. However, further in-vivo research is needed to demonstrate if re-regulation of Rho GTPases by topo IIβ overexpression could be a neuroprotective treatment in the case of neurodegenerative diseases.

Re-active Passive (RAP) Devices for Control of Noise Transmission through a Panel

Science.gov (United States)

Carneal, James P.; Giovanardi, Marco; Fuller, Chris R.; Palumbo, Daniel L.

2008-01-01

Re-Active Passive (RAP) devices have been developed to control low frequency (transmission through a panel. These devices use a combination of active, re-active, and passive technologies packaged into a single unit to control a broad frequency range utilizing the strength of each technology over its best suited frequency range. The RAP device uses passive constrained layer damping to cover the relatively high frequency range (>200 Hz), reactive distributed vibration absorber) to cover the medium frequency range (75 to 250 Hz), and active control for controlling low frequencies (transmission through a panel mounted in a transmission loss test facility. Experimental results are presented for the bare panel, and combinations of passive treatment, reactive treatment, and active control. Results indicate that three RAP devices were able to increase the overall broadband (15-1000 Hz) transmission loss by 9.4 dB. These three devices added a total of 285 grams to the panel mass of 6.0 kg, or approximately 5%, not including control electronics.

Definition of molecular determinants of prostate cancer cell bone extravasation.

Science.gov (United States)

Barthel, Steven R; Hays, Danielle L; Yazawa, Erika M; Opperman, Matthew; Walley, Kempland C; Nimrichter, Leonardo; Burdick, Monica M; Gillard, Bryan M; Moser, Michael T; Pantel, Klaus; Foster, Barbara A; Pienta, Kenneth J; Dimitroff, Charles J

2013-01-15

Advanced prostate cancer commonly metastasizes to bone, but transit of malignant cells across the bone marrow endothelium (BMEC) remains a poorly understood step in metastasis. Prostate cancer cells roll on E-selectin(+) BMEC through E-selectin ligand-binding interactions under shear flow, and prostate cancer cells exhibit firm adhesion to BMEC via β1, β4, and αVβ3 integrins in static assays. However, whether these discrete prostate cancer cell-BMEC adhesive contacts culminate in cooperative, step-wise transendothelial migration into bone is not known. Here, we describe how metastatic prostate cancer cells breach BMEC monolayers in a step-wise fashion under physiologic hemodynamic flow. Prostate cancer cells tethered and rolled on BMEC and then firmly adhered to and traversed BMEC via sequential dependence on E-selectin ligands and β1 and αVβ3 integrins. Expression analysis in human metastatic prostate cancer tissue revealed that β1 was markedly upregulated compared with expression of other β subunits. Prostate cancer cell breaching was regulated by Rac1 and Rap1 GTPases and, notably, did not require exogenous chemokines as β1, αVβ3, Rac1, and Rap1 were constitutively active. In homing studies, prostate cancer cell trafficking to murine femurs was dependent on E-selectin ligand, β1 integrin, and Rac1. Moreover, eliminating E-selectin ligand-synthesizing α1,3 fucosyltransferases in transgenic adenoma of mouse prostate mice dramatically reduced prostate cancer incidence. These results unify the requirement for E-selectin ligands, α1,3 fucosyltransferases, β1 and αVβ3 integrins, and Rac/Rap1 GTPases in mediating prostate cancer cell homing and entry into bone and offer new insight into the role of α1,3 fucosylation in prostate cancer development.

Rho GTPases, their post-translational modifications, disease-associated mutations and pharmacological inhibitors.

Science.gov (United States)

Olson, Michael F

2018-05-04

The 20 members of the Rho GTPase family are key regulators of a wide-variety of biological activities. In response to activation, they signal via downstream effector proteins to induce dynamic alterations in the organization of the actomyosin cytoskeleton. In this review, post-translational modifications, mechanisms of dysregulation identified in human pathological conditions, and the ways that Rho GTPases might be targeted for chemotherapy will be discussed.

Inhibition of RhoA GTPase and the subsequent activation of PTP1B protects cultured hippocampal neurons against amyloid β toxicity

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Rodriguez-Tebar Alfredo

2011-02-01

Full Text Available Abstract Background Amyloid beta (Aβ is the main agent responsible for the advent and progression of Alzheimer's disease. This peptide can at least partially antagonize nerve growth factor (NGF signalling in neurons, which may be responsible for some of the effects produced by Aβ. Accordingly, better understanding the NGF signalling pathway may provide clues as to how to protect neurons from the toxic effects of Aβ. Results We show here that Aβ activates the RhoA GTPase by binding to p75NTR, thereby preventing the NGF-induced activation of protein tyrosine phosphatase 1B (PTP1B that is required for neuron survival. We also show that the inactivation of RhoA GTPase and the activation of PTP1B protect cultured hippocampal neurons against the noxious effects of Aβ. Indeed, either pharmacological inhibition of RhoA with C3 ADP ribosyl transferase or the transfection of cultured neurons with a dominant negative form of RhoA protects cultured hippocampal neurons from the effects of Aβ. In addition, over-expression of PTP1B also prevents the deleterious effects of Aβ on cultured hippocampal neurons. Conclusion Our findings indicate that potentiating the activity of NGF at the level of RhoA inactivation and PTP1B activation may represent a new means to combat the noxious effects of Aβ in Alzheimer's disease.

Cortactin deficiency is associated with reduced neutrophil recruitment but increased vascular permeability in vivo.

Science.gov (United States)

Schnoor, Michael; Lai, Frank P L; Zarbock, Alexander; Kläver, Ruth; Polaschegg, Christian; Schulte, Dörte; Weich, Herbert A; Oelkers, J Margit; Rottner, Klemens; Vestweber, Dietmar

2011-08-01

Neutrophil extravasation and the regulation of vascular permeability require dynamic actin rearrangements in the endothelium. In this study, we analyzed in vivo whether these processes require the function of the actin nucleation-promoting factor cortactin. Basal vascular permeability for high molecular weight substances was enhanced in cortactin-deficient mice. Despite this leakiness, neutrophil extravasation in the tumor necrosis factor-stimulated cremaster was inhibited by the loss of cortactin. The permeability defect was caused by reduced levels of activated Rap1 (Ras-related protein 1) in endothelial cells and could be rescued by activating Rap1 via the guanosine triphosphatase (GTPase) exchange factor EPAC (exchange protein directly activated by cAMP). The defect in neutrophil extravasation was caused by enhanced rolling velocity and reduced adhesion in postcapillary venules. Impaired rolling interactions were linked to contributions of β(2)-integrin ligands, and firm adhesion was compromised by reduced ICAM-1 (intercellular adhesion molecule 1) clustering around neutrophils. A signaling process known to be critical for the formation of ICAM-1-enriched contact areas and for transendothelial migration, the ICAM-1-mediated activation of the GTPase RhoG was blocked in cortactin-deficient endothelial cells. Our results represent the first physiological evidence that cortactin is crucial for orchestrating the molecular events leading to proper endothelial barrier function and leukocyte recruitment in vivo.

The Small GTPase Rac1 Contributes to Extinction of Aversive Memories of Drug Withdrawal by Facilitating GABAA Receptor Endocytosis in the vmPFC.

Science.gov (United States)

Wang, Weisheng; Ju, Yun-Yue; Zhou, Qi-Xin; Tang, Jian-Xin; Li, Meng; Zhang, Lei; Kang, Shuo; Chen, Zhong-Guo; Wang, Yu-Jun; Ji, Hui; Ding, Yu-Qiang; Xu, Lin; Liu, Jing-Gen

2017-07-26

Extinction of aversive memories has been a major concern in neuropsychiatric disorders, such as anxiety disorders and drug addiction. However, the mechanisms underlying extinction of aversive memories are not fully understood. Here, we report that extinction of conditioned place aversion (CPA) to naloxone-precipitated opiate withdrawal in male rats activates Rho GTPase Rac1 in the ventromedial prefrontal cortex (vmPFC) in a BDNF-dependent manner, which determines GABA A receptor (GABA A R) endocytosis via triggering synaptic translocation of activity-regulated cytoskeleton-associated protein (Arc) through facilitating actin polymerization. Active Rac1 is essential and sufficient for GABA A R endocytosis and CPA extinction. Knockdown of Rac1 expression within the vmPFC of rats using Rac1-shRNA suppressed GABA A R endocytosis and CPA extinction, whereas expression of a constitutively active form of Rac1 accelerated GABA A R endocytosis and CPA extinction. The crucial role of GABA A R endocytosis in the LTP induction and CPA extinction is evinced by the findings that blockade of GABA A R endocytosis by a dynamin function-blocking peptide (Myr-P4) abolishes LTP induction and CPA extinction. Thus, the present study provides first evidence that Rac1-dependent GABA A R endocytosis plays a crucial role in extinction of aversive memories and reveals the sequence of molecular events that contribute to learning experience modulation of synaptic GABA A R endocytosis. SIGNIFICANCE STATEMENT This study reveals that Rac1-dependent GABA A R endocytosis plays a crucial role in extinction of aversive memories associated with drug withdrawal and identifies Arc as a downstream effector of Rac1 regulations of synaptic plasticity as well as learning and memory, thereby suggesting therapeutic targets to promote extinction of the unwanted memories. Copyright © 2017 the authors 0270-6474/17/377096-15$15.00/0.

The adaptor molecule RIAM integrates signaling events critical for integrin-mediated control of immune function and cancer progression.

Science.gov (United States)

Patsoukis, Nikolaos; Bardhan, Kankana; Weaver, Jessica D; Sari, Duygu; Torres-Gomez, Alvaro; Li, Lequn; Strauss, Laura; Lafuente, Esther M; Boussiotis, Vassiliki A

2017-08-22

Lymphocyte activation requires adhesion to antigen-presenting cells. This is a critical event linking innate and adaptive immunity. Lymphocyte adhesion is accomplished through LFA-1, which must be activated by a process referred to as inside-out integrin signaling. Among the few signaling molecules that have been implicated in inside-out integrin activation in hematopoietic cells are the small guanosine triphosphatase (GTPase) Rap1 and its downstream effector Rap1-interacting molecule (RIAM), a multidomain protein that defined the Mig10-RIAM-lamellipodin (MRL) class of adaptor molecules. Through its various domains, RIAM is a critical node of signal integration for activation of T cells, recruits monomeric and polymerized actin to drive actin remodeling and cytoskeletal reorganization, and promotes inside-out integrin signaling in T cells. As a regulator of inside-out integrin activation, RIAM affects multiple functions of innate and adaptive immunity. The effects of RIAM on cytoskeletal reorganization and integrin activation have implications in cell migration and trafficking of cancer cells. We provide an overview of the structure and interactions of RIAM, and we discuss the implications of RIAM functions in innate and adaptive immunity and cancer. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Performance evaluation of reactor operated zircaloy-2 pressure tubes of RAPS-1

International Nuclear Information System (INIS)

Chatterjee, S.; Ramadasan, E.; Balakrishnan, K.S.; Bahl, J.K.

1992-01-01

Detailed post irradiation examination was carried out on pressure tube sections from E-10, F-9 and F-10 locations of RAPS-1 after an in-reactor residence equivalent to 3.6 effective full power years. The F-10 pressure tube was studied in detail on sections obtained from one end to the other, whereas in the case of E-9 and F-9 pressure tubes only the end sections were examined. The studies carried out were visual examination, metallography, hydrogen i.e. H(D) analysis and mechanical testing at 300 C. Microstructural observations revealed uniform and random hydride/deuteride platelet distribution and absence of blisters or hydride segregation. The H(D) content in the F-10 pressure tube was found to vary in the range 6-12 ppm. The typical H(D) content in the three tubes was around 1 ppm. The H(D) pick-up evaluated from the observed oxide layer thickness was 8 ppm. Longitudinal tensile specimens fabricated from the F-10 pressure tube section and tested at 300 C exhibited increase in yield strength and tensile strength of 39% and 30% respectively. The residual uniform elongation was typically 1.8%. The observed changes in the tensile properties were found to be lower than those reported on unstressed specimens irradiated to similar neutron fluences. The observed hydrogen content and tensile properties obtained in F-10 pressure tube would not be detrimental under normal reactor operating conditions. (author). 10 refs., 4 figs., 2 tabs., 1 annexure

PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells

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Halayko Andrew J

2009-09-01

Full Text Available Abstract Background Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8. IL-8 production is in part regulated via activation of Gq-and Gs-coupled receptors. Here we study the role of the cyclic AMP (cAMP effectors protein kinase A (PKA and exchange proteins directly activated by cAMP (Epac1 and Epac2 in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response. Methods IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors Clostridium difficile toxin B-1470 (GTPases, U0126 (extracellular signal-regulated kinases ERK1/2 and Rp-8-CPT-cAMPS (PKA. The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used. Results The β2-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-O-Me-cAMPS mimicked the effects of 8-pCPT-2'-O-Me-cAMP, whereas the negative control 8-pCPT-2'-O-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP induced GTP

Rac1 recruits the adapter protein CMS/CD2AP to cell-cell contacts

NARCIS (Netherlands)

van Duijn, Trynette J.; Anthony, Eloise C.; Hensbergen, Paul J.; Deelder, André M.; Hordijk, Peter L.

2010-01-01

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in

Open-Source Wax RepRap 3-D Printer for Rapid Prototyping Paper-Based Microfluidics.

Science.gov (United States)

Pearce, J M; Anzalone, N C; Heldt, C L

2016-08-01

The open-source release of self-replicating rapid prototypers (RepRaps) has created a rich opportunity for low-cost distributed digital fabrication of complex 3-D objects such as scientific equipment. For example, 3-D printable reactionware devices offer the opportunity to combine open hardware microfluidic handling with lab-on-a-chip reactionware to radically reduce costs and increase the number and complexity of microfluidic applications. To further drive down the cost while improving the performance of lab-on-a-chip paper-based microfluidic prototyping, this study reports on the development of a RepRap upgrade capable of converting a Prusa Mendel RepRap into a wax 3-D printer for paper-based microfluidic applications. An open-source hardware approach is used to demonstrate a 3-D printable upgrade for the 3-D printer, which combines a heated syringe pump with the RepRap/Arduino 3-D control. The bill of materials, designs, basic assembly, and use instructions are provided, along with a completely free and open-source software tool chain. The open-source hardware device described here accelerates the potential of the nascent field of electrochemical detection combined with paper-based microfluidics by dropping the marginal cost of prototyping to nearly zero while accelerating the turnover between paper-based microfluidic designs. © 2016 Society for Laboratory Automation and Screening.

Rho GTPases: Novel Players in the Regulation of the DNA Damage Response?

Directory of Open Access Journals (Sweden)

Gerhard Fritz

2015-09-01

Full Text Available The Ras-related C3 botulinum toxin substrate 1 (Rac1 belongs to the family of Ras-homologous small GTPases. It is well characterized as a membrane-bound signal transducing molecule that is involved in the regulation of cell motility and adhesion as well as cell cycle progression, mitosis, cell death and gene expression. Rac1 also adjusts cellular responses to genotoxic stress by regulating the activity of stress kinases, including c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK and p38 kinases as well as related transcription factors. Apart from being found on the inner side of the outer cell membrane and in the cytosol, Rac1 has also been detected inside the nucleus. Different lines of evidence indicate that genotoxin-induced DNA damage is able to activate nuclear Rac1. The exact mechanisms involved and the biological consequences, however, are unclear. The data available so far indicate that Rac1 might integrate DNA damage independent and DNA damage dependent cellular stress responses following genotoxin treatment, thereby coordinating mechanisms of the DNA damage response (DDR that are related to DNA repair, survival and cell death.

Rap as the language of conflict in hip-hop subculture

Directory of Open Access Journals (Sweden)

Т М Кожелупенко

2008-12-01

Full Text Available This article is devoted to such notions of sociolinguistics as subculture and the language of conflict. These aspects are studied by the example of the rapidly developing hip-hop subculture and rap-texts as its main component.

Acoustic detection of particles, the RAP experiment: present status and results

International Nuclear Information System (INIS)

Bassan, M; Buonomo, B; Coccia, E; Blair, D; D'Antonio, S; Monache, G Delle; Gioacchino, D Di; Fafone, V; Ligi, C; Marini, A; Mazzitelli, G; Modestino, G; Pizzella, G; Quintieri, L; Roccella, S; Rocchi, A; Ronga, F; Tripodi, P; Valente, P

2006-01-01

Cosmic ray events with rate and energy much higher than expected were detected by the ultracryogenic gravitational antenna Nautilus located at the Laboratori Nazionali di Frascati, when it was operating in superconducting state. Mechanisms related to the superconductivity state of the material could be involved in such a way to enhance the conversion efficiency of the particle energy into vibrational energy of the detector. The RAP experiment has the aim to study the mechanical response of a small metallic resonant bar to short pulses of high energy electron beam, investigating the response of the bar both in normal and in superconducting state. The results obtained for an Al5056 bar down to a temperature of 4 K are reported and the preliminary results for a niobium bar at temperature below and above the superconducting-normal transition are also discussed

Triptolide disrupts the actin-based Sertoli-germ cells adherens junctions by inhibiting Rho GTPases expression

Energy Technology Data Exchange (ETDEWEB)

Wang, Xiang; Zhao, Fang [Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009 (China); Lv, Zhong-ming; Shi, Wei-qin [Jiangsu Provincial Center for Disease Control and Prevention, Nanjing (China); Zhang, Lu-yong, E-mail: lyzhang@cpu.edu.cn [Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009 (China); Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Nanjing (China); State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009 (China); Yan, Ming, E-mail: brookming@cpu.edu.cn [Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009 (China)

2016-11-01

Triptolide (TP), derived from the medicinal plant Triterygium wilfordii Hook. f. (TWHF), is a diterpene triepoxide with variety biological and pharmacological activities. However, TP has been restricted in clinical application due to its narrow therapeutic window especially in reproductive system. During spermatogenesis, Sertoli cell cytoskeleton plays an essential role in facilitating germ cell movement and cell-cell actin-based adherens junctions (AJ). At Sertoli cell-spermatid interface, the anchoring device is a kind of AJ, known as ectoplasmic specializations (ES). In this study, we demonstrate that β-actin, an important component of cytoskeleton, has been significantly down-regulated after TP treatment. TP can inhibit the expression of Rho GTPase such as, RhoA, RhoB, Cdc42 and Rac1. Downstream of Rho GTPase, Rho-associated protein kinase (ROCKs) gene expressions were also suppressed by TP. F-actin immunofluorescence proved that TP disrupts Sertoli cells cytoskeleton network. As a result of β-actin down-regulation, TP treatment increased expression of testin, which indicating ES has been disassembled. In summary, this report illustrates that TP induces cytoskeleton dysfunction and disrupts cell-cell adherens junctions via inhibition of Rho GTPases. - Highlights: • Triptolide induced the disruption of Sertoli-germ cell adherens junction. • Rho GTPases expression and actin dynamics have been suppressed by triptolide. • Actin-based adherens junction is a potential antifertility target of triptolide. • Rho-Rock is involved in the regulation of actin dynamics.

Triptolide disrupts the actin-based Sertoli-germ cells adherens junctions by inhibiting Rho GTPases expression

International Nuclear Information System (INIS)

Wang, Xiang; Zhao, Fang; Lv, Zhong-ming; Shi, Wei-qin; Zhang, Lu-yong; Yan, Ming

2016-01-01

Triptolide (TP), derived from the medicinal plant Triterygium wilfordii Hook. f. (TWHF), is a diterpene triepoxide with variety biological and pharmacological activities. However, TP has been restricted in clinical application due to its narrow therapeutic window especially in reproductive system. During spermatogenesis, Sertoli cell cytoskeleton plays an essential role in facilitating germ cell movement and cell-cell actin-based adherens junctions (AJ). At Sertoli cell-spermatid interface, the anchoring device is a kind of AJ, known as ectoplasmic specializations (ES). In this study, we demonstrate that β-actin, an important component of cytoskeleton, has been significantly down-regulated after TP treatment. TP can inhibit the expression of Rho GTPase such as, RhoA, RhoB, Cdc42 and Rac1. Downstream of Rho GTPase, Rho-associated protein kinase (ROCKs) gene expressions were also suppressed by TP. F-actin immunofluorescence proved that TP disrupts Sertoli cells cytoskeleton network. As a result of β-actin down-regulation, TP treatment increased expression of testin, which indicating ES has been disassembled. In summary, this report illustrates that TP induces cytoskeleton dysfunction and disrupts cell-cell adherens junctions via inhibition of Rho GTPases. - Highlights: • Triptolide induced the disruption of Sertoli-germ cell adherens junction. • Rho GTPases expression and actin dynamics have been suppressed by triptolide. • Actin-based adherens junction is a potential antifertility target of triptolide. • Rho-Rock is involved in the regulation of actin dynamics.

Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide.

Science.gov (United States)

Diaz, Alejandra R; Core, Leighton J; Jiang, Min; Morelli, Michela; Chiang, Christina H; Szurmant, Hendrik; Perego, Marta

2012-03-01

Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0F∼P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0F∼P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.

Feeling the Beat: The Meaning of Rap Music for Ethnically Diverse Midwestern College Students--A Phenomenological Study

Science.gov (United States)

Iwamoto, Derek K.; Creswell, John; Caldwell, Leon

2007-01-01

Despite its national and international appeal, rap is considered one of the most controversial of music genres. Given the political charge it generates, rap music has spawned research across the social and health sciences. The majority of the research has investigated its impact on African Americans. Further, the research has tended to focus on…

An extracellular-matrix-specific GEF-GAP interaction regulates Rho GTPase crosstalk for 3D collagen migration.

Science.gov (United States)

Kutys, Matthew L; Yamada, Kenneth M

2014-09-01

Rho-family GTPases govern distinct types of cell migration on different extracellular matrix proteins in tissue culture or three-dimensional (3D) matrices. We searched for mechanisms selectively regulating 3D cell migration in different matrix environments and discovered a form of Cdc42-RhoA crosstalk governing cell migration through a specific pair of GTPase activator and inhibitor molecules. We first identified βPix, a guanine nucleotide exchange factor (GEF), as a specific regulator of migration in 3D collagen using an affinity-precipitation-based GEF screen. Knockdown of βPix specifically blocks cell migration in fibrillar collagen microenvironments, leading to hyperactive cellular protrusion accompanied by increased collagen matrix contraction. Live FRET imaging and RNAi knockdown linked this βPix knockdown phenotype to loss of polarized Cdc42 but not Rac1 activity, accompanied by enhanced, de-localized RhoA activity. Mechanistically, collagen phospho-regulates βPix, leading to its association with srGAP1, a GTPase-activating protein (GAP), needed to suppress RhoA activity. Our results reveal a matrix-specific pathway controlling migration involving a GEF-GAP interaction of βPix with srGAP1 that is critical for maintaining suppressive crosstalk between Cdc42 and RhoA during 3D collagen migration.

O negro drama do rap: entre a lei do cão e a lei da selva

Directory of Open Access Journals (Sweden)

Bruno Zeni

2004-04-01

Full Text Available NESTE texto são feitas algumas considerações que partem de interesses e preocupações pessoais do autor a respeito do rap, tais como a elaboração das letras, a relação entre essa manifestação e o universo da pobreza e da prisão, o diálogo do ritmo com a tradição da música e da literatura brasileiras. São discutidas ainda algumas das questões estéticas e éticas relacionadas ao rap feito em São Paulo.IN THIS text I set out some considerations that stem from keen personal interests and concerns regarding rap - e.g., the production of lyrics, the relationship between such manifestations and the universe of poverty and imprisonment, and the dialogue between this rhythm and the broader tradition of Brazilian music and literature. I discuss some aesthetic and ethical issues related to the variety of rap that I am most intimately involved with, namely, the one made in São Paulo.

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

Energy Technology Data Exchange (ETDEWEB)

Malaby, Andrew W.; Das, Sanchaita; Chakravarthy, Srinivas; Irving, Thomas C.; Bilsel, Osman; Lambright, David G.

2018-01-01

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 and 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.

Opposing roles for RhoH GTPase during T-cell migration and activation

Science.gov (United States)

Baker, Christina M.; Comrie, William A.; Hyun, Young-Min; Chung, Hung-Li; Fedorchuk, Christine A.; Lim, Kihong; Brakebusch, Cord; McGrath, James L.; Waugh, Richard E.; Meier-Schellersheim, Martin; Kim, Minsoo

2012-01-01

T cells spend the majority of their time perusing lymphoid organs in search of cognate antigen presented by antigen presenting cells (APCs) and then quickly recirculate through the bloodstream to another lymph node. Therefore, regulation of a T-cell response is dependent upon the ability of cells to arrive in the correct location following chemokine gradients (“go” signal) as well as to receive appropriate T-cell receptor (TCR) activation signals upon cognate antigen recognition (“stop” signal). However, the mechanisms by which T cells regulate these go and stop signals remain unclear. We found that overexpression of the hematopoietic-specific RhoH protein in the presence of chemokine signals resulted in decreased Rap1–GTP and LFA-1 adhesiveness to ICAM-1, thus impairing T-cell chemotaxis; while in the presence of TCR signals, there were enhanced and sustained Rap1–GTP and LFA-1 activation as well as prolonged T:APC conjugates. RT-PCR analyses of activated CD4+ T cells and live images of T-cell migration and immunological synapse (IS) formation revealed that functions of RhoH took place primarily at the levels of transcription and intracellular distribution. Thus, we conclude that RhoH expression provides a key molecular determinant that allows T cells to switch between sensing chemokine-mediated go signals and TCR-dependent stop signals. PMID:22689994

RAP: RNA-Seq Analysis Pipeline, a new cloud-based NGS web application.

Science.gov (United States)

D'Antonio, Mattia; D'Onorio De Meo, Paolo; Pallocca, Matteo; Picardi, Ernesto; D'Erchia, Anna Maria; Calogero, Raffaele A; Castrignanò, Tiziana; Pesole, Graziano

2015-01-01

The study of RNA has been dramatically improved by the introduction of Next Generation Sequencing platforms allowing massive and cheap sequencing of selected RNA fractions, also providing information on strand orientation (RNA-Seq). The complexity of transcriptomes and of their regulative pathways make RNA-Seq one of most complex field of NGS applications, addressing several aspects of the expression process (e.g. identification and quantification of expressed genes and transcripts, alternative splicing and polyadenylation, fusion genes and trans-splicing, post-transcriptional events, etc.). In order to provide researchers with an effective and friendly resource for analyzing RNA-Seq data, we present here RAP (RNA-Seq Analysis Pipeline), a cloud computing web application implementing a complete but modular analysis workflow. This pipeline integrates both state-of-the-art bioinformatics tools for RNA-Seq analysis and in-house developed scripts to offer to the user a comprehensive strategy for data analysis. RAP is able to perform quality checks (adopting FastQC and NGS QC Toolkit), identify and quantify expressed genes and transcripts (with Tophat, Cufflinks and HTSeq), detect alternative splicing events (using SpliceTrap) and chimeric transcripts (with ChimeraScan). This pipeline is also able to identify splicing junctions and constitutive or alternative polyadenylation sites (implementing custom analysis modules) and call for statistically significant differences in genes and transcripts expression, splicing pattern and polyadenylation site usage (using Cuffdiff2 and DESeq). Through a user friendly web interface, the RAP workflow can be suitably customized by the user and it is automatically executed on our cloud computing environment. This strategy allows to access to bioinformatics tools and computational resources without specific bioinformatics and IT skills. RAP provides a set of tabular and graphical results that can be helpful to browse, filter and export

Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

International Nuclear Information System (INIS)

Huang, Xionggao; Wei, Yantao; Ma, Haizhi; Zhang, Shaochong

2012-01-01

Highlights: ► Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. ► Rac1 is activated in vitreous-transformed RPE cells. ► Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. ► Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. ► The vitreous-induced effects are mediated by a Rac1 GTPase/LIMK1/cofilin pathway. -- Abstract: Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous-transformed human RPE cells undergo cytoskeletal rearrangements via Rac1 GTPase-dependent pathways that modulate LIMK1 and

Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Huang, Xionggao [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China); Department of Ophthalmology, Hainan Medical College, Haikou (China); Wei, Yantao; Ma, Haizhi [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China); Zhang, Shaochong, E-mail: zhshaochong@163.com [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China)

2012-03-09

Highlights: Black-Right-Pointing-Pointer Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. Black-Right-Pointing-Pointer Rac1 is activated in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer The vitreous-induced effects are mediated by a Rac1 GTPase/LIMK1/cofilin pathway. -- Abstract: Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous

GTPase activity plays a key role in the pathobiology of LRRK2.

Directory of Open Access Journals (Sweden)

Yulan Xiong

2010-04-01

Full Text Available Mutations in the leucine-rich repeat kinase 2 (LRRK2 gene are associated with late-onset, autosomal-dominant, familial Parkinson's disease (PD and also contribute to sporadic disease. The LRRK2 gene encodes a large protein with multiple domains, including functional Roc GTPase and protein kinase domains. Mutations in LRRK2 most likely cause disease through a toxic gain-of-function mechanism. The expression of human LRRK2 variants in cultured primary neurons induces toxicity that is dependent on intact GTP binding or kinase activities. However, the mechanism(s underlying LRRK2-induced neuronal toxicity is poorly understood, and the contribution of GTPase and/or kinase activity to LRRK2 pathobiology is not well defined. To explore the pathobiology of LRRK2, we have developed a model of LRRK2 cytotoxicity in the baker's yeast Saccharomyces cerevisiae. Protein domain analysis in this model reveals that expression of GTPase domain-containing fragments of human LRRK2 are toxic. LRRK2 toxicity in yeast can be modulated by altering GTPase activity and is closely associated with defects in endocytic vesicular trafficking and autophagy. These truncated LRRK2 variants induce similar toxicity in both yeast and primary neuronal models and cause similar vesicular defects in yeast as full-length LRRK2 causes in primary neurons. The toxicity induced by truncated LRRK2 variants in yeast acts through a mechanism distinct from toxicity induced by human alpha-synuclein. A genome-wide genetic screen identified modifiers of LRRK2-induced toxicity in yeast including components of vesicular trafficking pathways, which can also modulate the trafficking defects caused by expression of truncated LRRK2 variants. Our results provide insight into the basic pathobiology of LRRK2 and suggest that the GTPase domain may contribute to the toxicity of LRRK2. These findings may guide future therapeutic strategies aimed at attenuating LRRK2-mediated neurodegeneration.

End-joining inhibition at telomeres requires the translocase and polySUMO-dependent ubiquitin ligase Uls1.

Science.gov (United States)

Lescasse, Rachel; Pobiega, Sabrina; Callebaut, Isabelle; Marcand, Stéphane

2013-03-20

In eukaryotes, permanent inhibition of the non-homologous end joining (NHEJ) repair pathway at telomeres ensures that chromosome ends do not fuse. In budding yeast, binding of Rap1 to telomere repeats establishes NHEJ inhibition. Here, we show that the Uls1 protein is required for the maintenance of NHEJ inhibition at telomeres. Uls1 protein is a non-essential Swi2/Snf2-related translocase and a Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) with unknown targets. Loss of Uls1 results in telomere-telomere fusions. Uls1 requirement is alleviated by the absence of poly-SUMO chains and by rap1 alleles lacking SUMOylation sites. Furthermore, Uls1 limits the accumulation of Rap1 poly-SUMO conjugates. We propose that one of Uls1 functions is to clear non-functional poly-SUMOylated Rap1 molecules from telomeres to ensure the continuous efficiency of NHEJ inhibition. Since Uls1 is the only known STUbL with a translocase activity, it can be the general molecular sweeper for the clearance of poly-SUMOylated proteins on DNA in eukaryotes.

‘When i rap, i feel more like myself’

DEFF Research Database (Denmark)

Vitus, Kathrine

2016-01-01

This article analyzes the relationship between subjectivity and ideology in a short film, Rapper Girl, produced by young women living in multiethnic Copenhagen, and develops the concept of the ‘RapX fantasy’. Through Jacques Rancière’s and Slavoj Žižek’s theoretical lenses, the article explores how...

RAPS: an innovative active pixel for particle detection integrated in CMOS technology

International Nuclear Information System (INIS)

Passeri, Daniele; Placidi, Pisana; Verducci, Leonardo; Ciampolini, Paolo; Matrella, Guido; Marras, Alessandro; Bilei, G.M.

2004-01-01

In this paper we discuss some design, implementation and test issues, with respect to the development of the RAPS01 chip in the framework of the Radiation Active Pixel Sensors (RAPS) INFN project. The project aimed at verifying feasibility of smart, high-resolution pixel arrays with a fully standard, submicron CMOS technology for particle detection purposes. Layout optimization of the pixel, including sensitive element and local read and amplification circuits has been carried out. Different basic pixel schemes and read-out options have been proposed and devised. Chip fabrication has been completed and test phase is now under way: to this purpose a suitable test environment has been devised and test strategies have been planned

Diaspora portoricaine et musique rap à New-York : entre latinité et culture africaine américaine Puerto Rican diaspora and rap music in New-York city: between “latininad” and African American culture

Directory of Open Access Journals (Sweden)

Stéphane Partel

2012-05-01

Full Text Available Le rap a atteint un succès sans précédent auprès des jeunes urbains aux Etats-Unis et dans les grandes mégalopoles du monde entier. Ce genre musical, figure de proue du hip-hop, s’est affirmé dès les années 1970 comme un moyen d’expression diasporique reflétant les expériences, les rapports complexes et les interactions culturelles entre Africains Américains, Jamaïcains et Portoricains de la diaspora vivant à New York. Cependant, le positionnement des artistes issus de la diaspora portoricaine a été peu étudié. Nourri de séjours d’observation participante et de recherches réalisés entre 2005 et 2008, ainsi que de la fréquentation assidue des concerts du collectif artistique portoricain The Terror Squad auxquels l’auteur a pu assister, cet article se propose doncd’analyser le rôle central des artistes issus de la diaspora portoricaine de New York à travers l’étude du rap, élément le plus médiatisé du hip-hop. Il devient ainsi possible de mieux entrevoir les tensions, convergences et interactions culturelles qui régissent les rapports complexes qui s’établissent entre la latinité des Portoricains et la culture africaine-américaine urbaine depuis plus d’une trentaine d’années dans le milieu du rap.Rap music has achieved unprecedented success among urban youth in the United States and in big cities around the world. As a key element of hip-hop this musical style has been asserting its strength since the 1970s as a reflection of the diasporic experience and the complex cultural interactions between African Americans, Jamaicans and Puerto Ricans of the diaspora living in New York. However, scant research has been conducted on the role of Puerto Rican artists of the diaspora living in New York. Therefore, based on field trips and participant observation undertaken between 2005 and 2008 and the concerts of the Puerto Rican rap group The Terror Squad that the author was able to attend frequently, the

RAC1 GTP-ase signals Wnt-beta-catenin pathway mediated integrin-directed metastasis-associated tumor cell phenotypes in triple negative breast cancers.

Science.gov (United States)

De, Pradip; Carlson, Jennifer H; Jepperson, Tyler; Willis, Scooter; Leyland-Jones, Brian; Dey, Nandini

2017-01-10

The acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes by Triple-Negative Breast Cancer (TNBC) cells is caused by an upregulation of the Wnt-beta-catenin pathway (WP). We reported that WP is one of the salient genetic features of TNBC. RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. The collective percentage of alteration(s) in RAC1 in ER+ve BC was lower as compared to ER-ve BC (35% vs 57%) (brca/tcga/pub2015). High expression of RAC1 was associated with poor outcome for RFS with HR=1.48 [CI: 1.15-1.9] p=0.0019 in the Hungarian ER-veBC cohort. Here we examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC. Using pharmacological agents (sulindac sulfide), genetic tools (beta-catenin siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed (1) migration, (2) matrigel invasion, (3) RAC1 and Cdc42 activation, (4) actin dynamics (confocal microscopy) and (5) podia-parameters. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) decreased invasion, (d) altered actin dynamics and (e) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation by LWnt3ACM as well as Wnt3A recombinant protein. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by

Laboratory testing and economic analysis of high RAP warm mixed asphalt.

Science.gov (United States)

2009-03-24

This report contains laboratory testing, economic analysis, literature review, and information obtained from multiple producers throughout the state of Mississippi regarding the use of high RAP (50 % to 100%) mixtures containing warm mix additives. T...

Migrant Rap in the Periphery: Performing Politics of Belonging

Science.gov (United States)

Leppänen, Sirpa; Westinen, Elina

2017-01-01

Focusing on a YouTube performance by an emergent Finnish Somali rapper and the audience responses it has generated, this paper looks at ways in which rap music engages with the issue of belonging. Drawing on recent theorizations of belonging as a multi-dimensional, contingent and fluid process, along with sociolinguistic work on globalization and…

Vesicular Trafficking Defects, Developmental Abnormalities, and Alterations in the Cellular Death Process Occur in Cell Lines that Over-Express Dictyostelium GTPase, Rab2, and Rab2 Mutants

Directory of Open Access Journals (Sweden)

Katherine Maringer

2014-08-01

Full Text Available Small molecular weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and required for protein transport from the ER to the Golgi complex, however, the function of Rab2 in Dictyostelium has yet to be fully characterized. Using cell lines that over-express DdRab2, as well as cell lines over-expressing constitutively active (CA, and dominant negative (DN forms of the GTPase, we report a functional role in vesicular transport specifically phagocytosis, and endocytosis. Furthermore, Rab2 like other GTPases cycles between an active GTP-bound and an inactive GDP-bound state. We found that this GTP/GDP cycle for DdRab2 is crucial for normal Dictyostelium development and cell–cell adhesion. Similar to Rab5 and Rab7 in C. elegans, we found that DdRab2 plays a role in programmed cell death, possibly in the phagocytic removal of apoptotic corpses.

Rap Therapy? An Innovative Approach to Groupwork with Urban Adolescents.

Science.gov (United States)

DeCarlo, Alonzo

2001-01-01

Describes a study in which young, urban African American adolescents with behavior problems participated in weekly group sessions that used rap music to promote the development of appropriate social skills related to morality, identity, judgement, decision making, anger management, impulse control, and crime and punishment. Overall, student…

Physics evaluation for testino. of RAPS and TAPS fuel pins in CIRUS pressurised water loop

International Nuclear Information System (INIS)

John, Benjamin; Paul, O.P.K.

1976-01-01

Relevant calculations carried out to assess the reactivity effect, heat generation and other parameters for testing of RAPS and TAPS fuel pins in the Cirus pressurised water loop are summarised. The Cirus neutron flux level being low, in order to simulate the RAPS design heat rating of ∫ Kdtheta = 40 w/cm, the required plutonium enrichment in mixed plutonium uranium oxide fuel pin was worked out. The results showed that a PuO 2 enrichment of 1.5 wt percent would be necessary to meet the above requirement. The analysis for the TAPS pin indicated that the desired heat flux of 115w/cm 2 cannot be obtained in the Cirus loop with either a 7 pin cluster geometry, or with a single pin with the enrichment level as used in TAPS pin. Lattice code DUMLAC and the core simulation code AECLHEX were used for these studies. (author)

RAP-2A Computer code for transients analysis in fast reactors

International Nuclear Information System (INIS)

Iftode, I.; Popescu, C.; Turcu, I.; Biro, L.

1975-10-01

The RAP-2A computer code is designed for analyzing thermohydraulic transients and/or steady state problems for large LMFBR cores. Physical and mathematical models, main input-output data, the flow chart of the code and a sample problem are given. RAP-2A calculates the power and the thermoydraulic transients initiated by a flow or reactivity changes, from a normal operating state of the reactor up to core disassembly. In this analysis a representative fuel pin is considered: a one-group space-independent (point) kinetics model to describe the neutron kinetics and a one-dimensional model describing the heat transfer (radial in the fuel and axial in the coolant) are used. Mechanical deformations due to temperature gradient, pressure losses, fuel melting, etc., are also calculated. The code is written in FORTRAN-4 language and is running on a IBM-370/135 computer

Nanofibrillar scaffolds induce preferential activation of Rho GTPases in cerebral cortical astrocytes

Science.gov (United States)

Tiryaki, Volkan Mujdat; Ayres, Virginia M; Khan, Adeel A; Ahmed, Ijaz; Shreiber, David I; Meiners, Sally

2012-01-01

Cerebral cortical astrocyte responses to polyamide nanofibrillar scaffolds versus poly-L-lysine (PLL)-functionalized planar glass, unfunctionalized planar Aclar coverslips, and PLL-functionalized planar Aclar surfaces were investigated by atomic force microscopy and immunocytochemistry. The physical properties of the cell culture environments were evaluated using contact angle and surface roughness measurements and compared. Astrocyte morphological responses, including filopodia, lamellipodia, and stress fiber formation, and stellation were imaged using atomic force microscopy and phalloidin staining for F-actin. Activation of the corresponding Rho GTPase regulators was investigated using immunolabeling with Cdc42, Rac1, and RhoA. Astrocytes cultured on the nanofibrillar scaffolds showed a unique response that included stellation, cell–cell interactions by stellate processes, and evidence of depression of RhoA. The results support the hypothesis that the extracellular environment can trigger preferential activation of members of the Rho GTPase family, with demonstrable morphological consequences for cerebral cortical astrocytes. PMID:22915841

Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

International Nuclear Information System (INIS)

Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.; Lee, Benhur; Moncman, Carole L.; McCann, Richard O.; Dutch, Rebecca Ellis

2006-01-01

The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1 V12 or Cdc42 V12 could increase cell-cell fusion promoted by the Hendra or SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA L63 decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia

Discovery and characterization of small molecule Rac1 inhibitors.

Science.gov (United States)

Arnst, Jamie L; Hein, Ashley L; Taylor, Margaret A; Palermo, Nick Y; Contreras, Jacob I; Sonawane, Yogesh A; Wahl, Andrew O; Ouellette, Michel M; Natarajan, Amarnath; Yan, Ying

2017-05-23

Aberrant activation of Rho GTPase Rac1 has been observed in various tumor types, including pancreatic cancer. Rac1 activates multiple signaling pathways that lead to uncontrolled proliferation, invasion and metastasis. Thus, inhibition of Rac1 activity is a viable therapeutic strategy for proliferative disorders such as cancer. Here we identified small molecule inhibitors that target the nucleotide-binding site of Rac1 through in silico screening. Follow up in vitro studies demonstrated that two compounds blocked active Rac1 from binding to its effector PAK1. Fluorescence polarization studies indicate that these compounds target the nucleotide-binding site of Rac1. In cells, both compounds blocked Rac1 binding to its effector PAK1 following EGF-induced Rac1 activation in a dose-dependent manner, while showing no inhibition of the closely related Cdc42 and RhoA activity. Furthermore, functional studies indicate that both compounds reduced cell proliferation and migration in a dose-dependent manner in multiple pancreatic cancer cell lines. Additionally, the two compounds suppressed the clonogenic survival of pancreatic cancer cells, while they had no effect on the survival of normal pancreatic ductal cells. These compounds do not share the core structure of the known Rac1 inhibitors and could serve as additional lead compounds to target pancreatic cancers with high Rac1 activity.

Rap como identidade cultural negra e periférica: a aversão de rappers brasileiros a Rede Globo

OpenAIRE

Júnior, Francisco Carlos Guerra de Mendonça

2014-01-01

Dissertação de Mestrado em Comunicação e Jornalismo, apresentada à Faculdade de Letras da Universidade de Coimbra. O rap (rhythm and poetry – ritmo e poesia) é a vertente musical do movimento hip hop, que surgiu nos Estados Unidos na década de 1960. Além do rap, o hip hop conta com MC´s (Mestres de Cerimônia), os DJ´s (disc-joqueys), a dança (break dance) e a pintura (grafith). O rap passou a ser um método utilizado para conscientizar a população sobre os problemas vivenciados pelos negros...

Remote ultrasonic characterisation of an irradiated pressure tube from RAPS-II

Energy Technology Data Exchange (ETDEWEB)

Gangotra, S; Muralidhar, S; Raut, S D; Ouseph, P M; Ghosh, J K; Sahoo, K C [Bhabha Atomic Research Centre, Bombay (India). Radiometallurgy Div.

1994-12-31

The Rajasthan Atomic Power Station Unit-2 (RAPS-2) has reached a stage of operation where the contacting pressure tubes are suspect to failure as a result of irradiation creep and displacement of the garter springs, the hot pressure tube coming in contact with the cold calandria tube. To study and assess the safety of these pressure tubes, two channels believed to be in contact with the calandria tubes, have been removed from the reactor for detailed full length post irradiation examination. Some of the test results are presented. 2 refs., 3 figs., 1 tab.

Targeting GTPases in Parkinson’s disease: comparison to the historic path of kinase drug discovery and perspectives

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LIN eHONG

2014-06-01

Full Text Available Neurological diseases have placed heavy social and financial burdens on modern society. As the life expectancy of humans is extended, neurological diseases, such as Parkinson’s disease, have become increasingly common among senior populations. Although the enigmas of Parkinson’s diseases await resolution, more vivid pictures on the cause, progression and control of the illness are emerging after years of research. On the molecular level, GTPases are implicated in the etiology of Parkinson’s disease and are rational pharmaceutical targets for their control. However, targeting individual GTPases, which belong to a superfamily of proteins containing multiple members with a conserved guanine nucleotide binding domain, has proven to be challenging. In contrast, pharmaceutical pursuit of inhibition of kinases, which constitute another superfamily of proteins with more than 500 members, has been fairly successful. We reviewed the breakthroughs in the history of kinase drug discovery to provide guidance for the GTPase field. We summarize recent progress made in the regulation of GTPase activity. We also present an efficient and cost effective approach to drug screening, which uses multiplex flow cytometry and mixture-based positional scanning libraries. These methods allow simultaneous measurements of both the activity and the selectivity of the screened library. Several GTPase activator clusters were identified which showed selectivity against different GTPase subfamilies. While the clusters need to be further deconvoluted to identify individual active compounds, the method described here and the structure information gathered create a foundation for further developments to build upon.

The Arabidopsis gene DIG6 encodes a large 60S subunit nuclear export GTPase 1 that is involved in ribosome biogenesis and affects multiple auxin-regulated development processes

KAUST Repository

Zhao, Huayan

2015-08-13

The circularly permuted GTPase large subunit GTPase 1 (LSG1) is involved in the maturation step of the 60S ribosome and is essential for cell viability in yeast. Here, an Arabidopsis mutant dig6 (drought inhibited growth of lateral roots) was isolated. The mutant exhibited multiple auxin-related phenotypes, which included reduced lateral root number, altered leaf veins, and shorter roots. Genetic mapping combined with next-generation DNA sequencing identified that the mutation occurred in AtLSG1-2. This gene was highly expressed in regions of auxin accumulation. Ribosome profiling revealed that a loss of function of AtLSG1-2 led to decreased levels of monosomes, further demonstrating its role in ribosome biogenesis. Quantitative proteomics showed that the expression of certain proteins involved in ribosome biogenesis was differentially regulated, indicating that ribosome biogenesis processes were impaired in the mutant. Further investigations showed that an AtLSG1-2 deficiency caused the alteration of auxin distribution, response, and transport in plants. It is concluded that AtLSG1-2 is integral to ribosome biogenesis, consequently affecting auxin homeostasis and plant development.

The Arabidopsis gene DIG6 encodes a large 60S subunit nuclear export GTPase 1 that is involved in ribosome biogenesis and affects multiple auxin-regulated development processes

KAUST Repository

Zhao, Huayan; Lü , Shiyou; Li, Ruixi; Chen, Tao; Zhang, Huoming; Cui, Peng; Ding, Feng; Liu, Pei; Wang, Guangchao; Xia, Yiji; Running, Mark P.; Xiong, Liming

2015-01-01

The circularly permuted GTPase large subunit GTPase 1 (LSG1) is involved in the maturation step of the 60S ribosome and is essential for cell viability in yeast. Here, an Arabidopsis mutant dig6 (drought inhibited growth of lateral roots) was isolated. The mutant exhibited multiple auxin-related phenotypes, which included reduced lateral root number, altered leaf veins, and shorter roots. Genetic mapping combined with next-generation DNA sequencing identified that the mutation occurred in AtLSG1-2. This gene was highly expressed in regions of auxin accumulation. Ribosome profiling revealed that a loss of function of AtLSG1-2 led to decreased levels of monosomes, further demonstrating its role in ribosome biogenesis. Quantitative proteomics showed that the expression of certain proteins involved in ribosome biogenesis was differentially regulated, indicating that ribosome biogenesis processes were impaired in the mutant. Further investigations showed that an AtLSG1-2 deficiency caused the alteration of auxin distribution, response, and transport in plants. It is concluded that AtLSG1-2 is integral to ribosome biogenesis, consequently affecting auxin homeostasis and plant development.

Vitek 2 ANC card versus BBL Crystal Anaerobe and RapID ANA II for identification of clinical anaerobic bacteria.

Science.gov (United States)

Blairon, Laurent; Maza, Mengi L; Wybo, Ingrid; Piérard, Denis; Dediste, Anne; Vandenberg, Olivier

2010-08-01

The Vitek 2 Anaerobe and Corynebacterium Identification Card (ANC) was recently evaluated in a multicentre study. In the present work, this system was compared with the BBL Crystal Anaerobe and RapID ANA II panels. These kits were tested using 196 strains of anaerobes that had been previously identified by gas-liquid chromatography. Identification to the species or to the genus level was 75.0%, 81.1% and 70.9% for Crystal, RapID and Vitek, respectively. Vitek ANC failed to provide any identification in 20.4% of the strains, but it had fewer misidentifications than RapID. The confidence factors provided on the results report of each kit were not always correlated with a lower risk of major errors, with the exception of Vitek 2 in which a confidence factor higher than 0.86 excluded the risk of misidentification in more than 87% of isolates. The lower rate of identification by the Vitek and Crystal panels is mostly due the lower ability of these systems to identify the Clostridia. Overall, the three panels are comparable but need improvement to a better accuracy. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Constitutively active signaling by the G protein βγ-subunit mediates intrinsically increased phosphodiesterase-4 activity in human asthmatic airway smooth muscle cells.

Directory of Open Access Journals (Sweden)

Aihua Hu

Full Text Available Signaling by the Gβγ subunit of Gi protein, leading to downstream c-Src-induced activation of the Ras/c-Raf1/MEK-ERK1/2 signaling pathway and its upregulation of phosphodiesterase-4 (PDE4 activity, was recently shown to mediate the heightened contractility in proasthmatic sensitized isolated airway smooth muscle (ASM, as well as allergen-induced airway hyperresponsiveness and inflammation in an in vivo animal model of allergic asthma. This study investigated whether cultured human ASM (HASM cells derived from asthmatic donor lungs exhibit constitutively increased PDE activity that is attributed to intrinsically upregulated Gβγ signaling coupled to c-Src activation of the Ras/MEK/ERK1/2 cascade. We show that, relative to normal cells, asthmatic HASM cells constitutively exhibit markedly increased intrinsic PDE4 activity coupled to heightened Gβγ-regulated phosphorylation of c-Src and ERK1/2, and direct co-localization of the latter with the PDE4D isoform. These signaling events and their induction of heightened PDE activity are acutely suppressed by treating asthmatic HASM cells with a Gβγ inhibitor. Importantly, along with increased Gβγ activation, asthmatic HASM cells also exhibit constitutively increased direct binding of the small Rap1 GTPase-activating protein, Rap1GAP, to the α-subunit of Gi protein, which serves to cooperatively facilitate Ras activation and, thereby, enable enhanced Gβγ-regulated ERK1/2-stimulated PDE activity. Collectively, these data are the first to identify that intrinsically increased signaling via the Gβγ subunit, facilitated by Rap1GAP recruitment to the α-subunit, mediates the constitutively increased PDE4 activity detected in asthmatic HASM cells. These new findings support the notion that interventions targeted at suppressing Gβγ signaling may lead to novel approaches to treat asthma.

RhoA GTPase regulates radiation-induced alterations in endothelial cell adhesion and migration

International Nuclear Information System (INIS)

Rousseau, Matthieu; Gaugler, Marie-Hélène; Rodallec, Audrey; Bonnaud, Stéphanie; Paris, François; Corre, Isabelle

2011-01-01

Highlights: ► We explore the role of RhoA in endothelial cell response to ionizing radiation. ► RhoA is rapidly activated by single high-dose of radiation. ► Radiation leads to RhoA/ROCK-dependent actin cytoskeleton remodeling. ► Radiation-induced apoptosis does not require the RhoA/ROCK pathway. ► Radiation-induced alteration of endothelial adhesion and migration requires RhoA/ROCK. -- Abstract: Endothelial cells of the microvasculature are major target of ionizing radiation, responsible of the radiation-induced vascular early dysfunctions. Molecular signaling pathways involved in endothelial responses to ionizing radiation, despite being increasingly investigated, still need precise characterization. Small GTPase RhoA and its effector ROCK are crucial signaling molecules involved in many endothelial cellular functions. Recent studies identified implication of RhoA/ROCK in radiation-induced increase in endothelial permeability but other endothelial functions altered by radiation might also require RhoA proteins. Human microvascular endothelial cells HMEC-1, either treated with Y-27632 (inhibitor of ROCK) or invalidated for RhoA by RNA interference were exposed to 15 Gy. We showed a rapid radiation-induced activation of RhoA, leading to a deep reorganisation of actin cytoskeleton with rapid formation of stress fibers. Endothelial early apoptosis induced by ionizing radiation was not affected by Y-27632 pre-treatment or RhoA depletion. Endothelial adhesion to fibronectin and formation of focal adhesions increased in response to radiation in a RhoA/ROCK-dependent manner. Consistent with its pro-adhesive role, ionizing radiation also decreased endothelial cells migration and RhoA was required for this inhibition. These results highlight the role of RhoA GTPase in ionizing radiation-induced deregulation of essential endothelial functions linked to actin cytoskeleton.

Extensive in silico analysis of Mimivirus coded Rab GTPase homolog suggests a possible role in virion membrane biogenesis

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Amrutraj eZade

2015-09-01

Full Text Available Rab GTPases are the key regulators of intracellular membrane trafficking in eukaryotes. Many viruses and intracellular bacterial pathogens have evolved to hijack the host Rab GTPase functions, mainly through activators and effector proteins, for their benefit. Acanthamoeba polyphaga mimivirus (APMV is one of the largest viruses and belongs to the monophyletic clade of nucleo-cytoplasmic large DNA viruses (NCLDV. The inner membrane lining is integral to the APMV virion structure. APMV assembly involves extensive host membrane modifications, like vesicle budding and fusion, leading to the formation of a membrane sheet that is incorporated into the virion. Intriguingly, APMV and all group I members of the Mimiviridae family code for a putative Rab GTPase protein. APMV is the first reported virus to code for a Rab GTPase (encoded by R214 gene. Our thorough in silico analysis of the subfamily specific (SF region of Mimiviridae Rab GTPase sequences suggests that they are related to Rab5, a member of the group II Rab GTPases, of lower eukaryotes. Because of their high divergence from the existing three isoforms, A, B and C of the Rab5-family, we suggest that Mimiviridae Rabs constitute a new isoform, Rab5D. Phylogenetic analysis indicated probable horizontal acquisition from a lower eukaryotic ancestor followed by selection and divergence. Furthermore, interaction network analysis suggests that vps34 (a Class III P13K homolog, coded by APMV L615, Atg-8 and dynamin (host proteins are recruited by APMV Rab GTPase during capsid assembly. Based on these observations, we hypothesize that APMV Rab plays a role in the acquisition of inner membrane during virion assembly.

The Rho GTPase Effector ROCK Regulates Cyclin A, Cyclin D1, and p27Kip1 Levels by Distinct Mechanisms

OpenAIRE

Croft, Daniel R.; Olson, Michael F.

2006-01-01

The members of the Rho GTPase family are well known for their regulation of actin cytoskeletal structures. In addition, they influence progression through the cell cycle. The RhoA and RhoC proteins regulate numerous effector proteins, with a central and vital signaling role mediated by the ROCK I and ROCK II serine/threonine kinases. The requirement for ROCK function in the proliferation of numerous cell types has been revealed by studies utilizing ROCK-selective inhibitors such as Y-27632. H...

Fundamental evaluation of the interaction between RAS/RAP and virgin asphalt binders.

Science.gov (United States)

2017-08-01

A comprehensive laboratory testing program was conducted in this research project to examine the blending between reclaimed asphalt pavement (RAP)/recycled asphalt shingles (RAS) and virgin asphalt binders and to evaluate the factors that may affect ...

The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion.

Science.gov (United States)

Schulte, Kathrin; Pawlowski, Nikolaus; Faelber, Katja; Fröhlich, Chris; Howard, Jonathan; Daumke, Oliver

2016-03-02

The immunity-related GTPases (IRGs) constitute a powerful cell-autonomous resistance system against several intracellular pathogens. Irga6 is a dynamin-like protein that oligomerizes at the parasitophorous vacuolar membrane (PVM) of Toxoplasma gondii leading to its vesiculation. Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization. We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. Contrary to the previous model, the structure shows that the GTPase domains dimerize in a parallel fashion. The nucleotides in the center of the interface participate in dimerization by forming symmetric contacts with each other and with the switch I region of the opposing Irga6 molecule. The latter contact appears to activate GTP hydrolysis by stabilizing the position of the catalytic glutamate 106 in switch I close to the active site. Further dimerization contacts involve switch II, the G4 helix and the trans stabilizing loop. The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. This study contributes important insights into the assembly and catalytic mechanisms of IRG proteins as prerequisite to understand their anti-microbial action.

Drosophila-Cdh1 (Rap/Fzr) a regulatory subunit of APC/C is required for synaptic morphology, synaptic transmission and locomotion.

Science.gov (United States)

Wise, Alexandria; Schatoff, Emma; Flores, Julian; Hua, Shao-Ying; Ueda, Atsushi; Wu, Chun-Fang; Venkatesh, Tadmiri

2013-11-01

The assembly of functional synapses requires the orchestration of the synthesis and degradation of a multitude of proteins. Protein degradation and modification by the conserved ubiquitination pathway has emerged as a key cellular regulatory mechanism during nervous system development and function (Kwabe and Brose, 2011). The anaphase promoting complex/cyclosome (APC/C) is a multi-subunit ubiquitin ligase complex primarily characterized for its role in the regulation of mitosis (Peters, 2002). In recent years, a role for APC/C in nervous system development and function has been rapidly emerging (Stegmuller and Bonni, 2005; Li et al., 2008). In the mammalian central nervous system the activator subunit, APC/C-Cdh1, has been shown to be a regulator of axon growth and dendrite morphogenesis (Konishi et al., 2004). In the Drosophila peripheral nervous system (PNS), APC2, a ligase subunit of the APC/C complex has been shown to regulate synaptic bouton size and activity (van Roessel et al., 2004). To investigate the role of APC/C-Cdh1 at the synapse we examined loss-of-function mutants of Rap/Fzr (Retina aberrant in pattern/Fizzy related), a Drosophila homolog of the mammalian Cdh1 during the development of the larval neuromuscular junction in Drosophila. Our cell biological, ultrastructural, electrophysiological, and behavioral data showed that rap/fzr loss-of-function mutations lead to changes in synaptic structure and function as well as locomotion defects. Data presented here show changes in size and morphology of synaptic boutons, and, muscle tissue organization. Electrophysiological experiments show that loss-of-function mutants exhibit increased frequency of spontaneous miniature synaptic potentials, indicating a higher rate of spontaneous synaptic vesicle fusion events. In addition, larval locomotion and peristaltic movement were also impaired. These findings suggest a role for Drosophila APC/C-Cdh1 mediated ubiquitination in regulating synaptic morphology

Composite mechanisms for improving Bubble Rap in delay tolerant networks

Directory of Open Access Journals (Sweden)

Sweta Jain

2014-01-01

Full Text Available Delay tolerant networks (DTNs are a subset of mobile ad hoc networks where connections are sparse and intermittent. This often results in a network graph which is rarely connected which introduces a challenge in message forwarding because of a lack of end-to-end connectivity towards the destination. Recently, social-based forwarding algorithms are gaining popularity because of the social nature displayed by the node movements in a DTN, especially in application areas like the pocket switched networks. The social-based metrics like community, similarity, centrality etc. are used to determine the carrier to which a node has to forward its message. Composite methods are used to improve the performance of Bubble Rap social-based forwarding algorithm. In the proposed mechanism, a new social metric termed ‘friendship’ has been introduced along with a time-to-live (TTL-based ‘threshold’ and acknowledgement (ACK IDs. Real trace data and working day movement models are used for simulations in the opportunistic network environment simulator to demonstrate that the proposed algorithm gives better delivery ratio than the original Bubble Rap algorithm.

Inter-cellular transport of ran GTPase.

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Deepak Khuperkar

Full Text Available Ran, a member of the Ras-GTPase superfamily, has a well-established role in regulating the transport of macromolecules across the nuclear envelope (NE. Ran has also been implicated in mitosis, cell cycle progression, and NE formation. Over-expression of Ran is associated with various cancers, although the molecular mechanism underlying this phenomenon is unclear. Serendipitously, we found that Ran possesses the ability to move from cell-to-cell when transiently expressed in mammalian cells. Moreover, we show that the inter-cellular transport of Ran is GTP-dependent. Importantly, Ran displays a similar distribution pattern in the recipient cells as that in the donor cell and co-localizes with the Ran binding protein Nup358 (also called RanBP2. Interestingly, leptomycin B, an inhibitor of CRM1-mediated export, or siRNA mediated depletion of CRM1, significantly impaired the inter-cellular transport of Ran, suggesting a function for CRM1 in this process. These novel findings indicate a possible role for Ran beyond nucleo-cytoplasmic transport, with potential implications in inter-cellular communication and cancers.

Inhibition of Cdc42 and Rac1 activities in pheochromocytoma, the adrenal medulla tumor.

Science.gov (United States)

Croisé, Pauline; Brunaud, Laurent; Tóth, Petra; Gasman, Stéphane; Ory, Stéphane

2017-04-03

Altered Rho GTPase signaling has been linked to many types of cancer. As many small G proteins, Rho GTPases cycle between an active and inactive state thanks to specific regulators that catalyze exchange of GDP into GTP (Rho-GEF) or hydrolysis of GTP into GDP (Rho-GAP). Recent studies have shown that alteration takes place either at the level of Rho proteins themselves (expression levels, point mutations) or at the level of their regulators, mostly RhoGEFs and RhoGAPs. Most reports describe Rho GTPases gain of function that may participate to the tumorigenesis processes. In contrast, we have recently reported that decreased activities of Cdc42 and Rac1 as well as decreased expression of 2 Rho-GEFs, FARP1 and ARHGEF1, correlate with pheochromocytomas, a tumor developing in the medulla of the adrenal gland (Croisé et al., Endocrine Related Cancer, 2016). Here we highlight the major evidence and further study the correlation between Rho GTPases activities and expression levels of ARHGEF1 and FARP1. Finally we also discuss how the decrease of Cdc42 and Rac1 activities may help human pheochromocytomas to develop and comment the possible relationship between FARP1, ARHGEF1 and the 2 Rho GTPases Cdc42 and Rac1 in tumorigenesis.

The Rab2A GTPase Promotes Breast Cancer Stem Cells and Tumorigenesis via Erk Signaling Activation

Directory of Open Access Journals (Sweden)

Man-Li Luo

2015-04-01

Full Text Available Proline-directed phosphorylation is regulated by the prolyl isomerase Pin1, which plays a fundamental role in driving breast cancer stem-like cells (BCSCs. Rab2A is a small GTPase critical for vesicle trafficking. Here, we show that Pin1 increases Rab2A transcription to promote BCSC expansion and tumorigenesis in vitro and in vivo. Mechanistically, Rab2A directly interacts with and prevents dephosphorylation/inactivation of Erk1/2 by the MKP3 phosphatase, resulting in Zeb1 upregulation and β-catenin nuclear translocation. In cancer cells, Rab2A is activated via gene amplification, mutation or Pin1 overexpression. Rab2A overexpression or mutation endows BCSC traits to primary normal human breast epithelial cells, whereas silencing Rab2A potently inhibits the expansion and tumorigenesis of freshly isolated BCSCs. Finally, Rab2A overexpression correlates with poor clinical outcome in breast cancer patients. Thus, Pin1/Rab2A/Erk drives BCSC expansion and tumorigenicity, suggesting potential drug targets.

Rap and Resistance: A Social Movement of the Wu-Tang Clan.

Science.gov (United States)

Chasteen, Amy L.; Shriver, Thomas

1998-01-01

Examines specific collective identity and political expression of the rap group the Wu-Tang Clan. Reveals a multi-layered political strategy that has been conscientiously designed and implemented to instigate a social movement. Prioritizes the voices of marginalized Black peoples and provides raw narratives about oppression. (MMU)

RAP-IA code for calculus thermodinamic of the fast reactors

International Nuclear Information System (INIS)

Popescu, C.; Turcu, I.; Boeriu, S.; Biro, L.

1975-01-01

The RAP-IA code is developed in order to perform a complete calculation for a thermal channel of a Na-cooled fast reactor. Calculation may be effected for both stationary state and dynamic regime following modification of some in-put data: total thermal power, multiplication coefficient, flow-rate and in-put temperature of the thermal agent, pressure level

Effect of Bio based rejuvenator on mix design, Energy consumption and GHG Emission of High RAP Mixture

Science.gov (United States)

Abdullahi Ahmad, Kabiru; Ezree Abdullah, Mohd; Hassan, Norhidayah Abdul; Usman, Nura; Hassan, Mohd Rosli Mohd; Bilema, Munder A. M.; Modibbo Saeed, Saeed; Batari, Ahmad

2018-04-01

Concerns about the cost, availability, and environmental impact of using petroleum-based materials have led to increased usage of reclaimed asphalt pavement (RAP). Mean-while, the demand of the road industry to decrease the energy consumptions and reduce the release of greenhouse gases as well as other harmful gases, which cause serious air pollution has increased due to the amount of energy consumed is a major component of pavement construction that significantly contributes to the total cost. This paper evaluates the effects of Biobased rejuvenator known as JCO on the required heat energy and the amount of CO2 produced to increase the temperature of RAP and virgin aggregates and one binder from 25C to the point of mixing. The results showed that incorporating Biobased rejuvenator (JCO) can potentially reduce the required heat energy and amount of greenhouse gas produced by RAP and virgin, respectively.

Suppressed invasive and migratory behaviors of SW1353 chondrosarcoma cells through the regulation of Src, Rac1 GTPase, and MMP13.

Science.gov (United States)

Xu, Wenxiao; Wan, Qiaoqiao; Na, Sungsoo; Yokota, Hiroki; Yan, Jing-Long; Hamamura, Kazunori

2015-12-01

Chondrosarcoma is the second frequent type of primary bone cancer. In response to stress to the endoplasmic reticulum, activation of eIF2α-mediated signaling is reported to induce apoptosis. However, its effects on invasive and migratory behaviors of chondrosarcoma have not been understood. Focusing on potential roles of Src kinase, Rac1 GTPase, and MMP13, we investigated eIF2α-driven regulation of SW1353 chondrosarcoma cells. In particular, we employed two chemical agents (salubrinal, Sal; and guanabenz, Gu) that elevate the level of eIF2α phosphorylation. The result revealed that both Sal and Gu reduced invasion and motility of SW1353 chondrosarcoma cells in a dose dependent manner. Live imaging using a fluorescent resonance energy transfer (FRET) technique showed that Sal and Gu downregulated activities of Src kinase as well as Rac1 GTPase in an eIF2α dependent manner. RNA interference experiments supported an eIF2α-mediated regulatory network in the inhibitory role of Sal and Gu. Partial silencing of MMP13 also suppressed malignant phenotypes of SW1353 chondrosarcoma cells. However, MMP13 was not regulated via eIF2α since administration of Sal but not Gu reduced expression of MMP13. In summary, we demonstrate that eIF2α dependent and independent pathways regulate invasion and motility of SW1353 chondrosarcoma cells, and inactivation of Src, Rac1, and MMP13 by Sal could provide a potential adjuvant therapy for combating metastatic chondrosarcoma cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Rapping in Catalan in Class and the Empowerment of the Learner

Science.gov (United States)

Aliagas, Cristina; Fernández, Júlia-Alba; Llonch, Pau

2016-01-01

Despite the well-known educational possibilities afforded by "Rhythm And Poetry" (RAP) for the development of musical, lyrical and critical skills [Morrell, E., & Duncan-Andrade, J. M. R. (2002). Promoting Academic Literacy with Urban Youth through Engaging Hip-hop Culture. "The English Journal," 91(6), 88-92. Retrieved…

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania*

Science.gov (United States)

Bahl, Surbhi; Parashar, Smriti; Malhotra, Himanshu; Raje, Manoj; Mukhopadhyay, Amitabha

2015-01-01

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania. PMID:26499792

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania.

Science.gov (United States)

Bahl, Surbhi; Parashar, Smriti; Malhotra, Himanshu; Raje, Manoj; Mukhopadhyay, Amitabha

2015-12-11

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Rac1 hypervariable region in targeting and signaling: a tail of many stories

NARCIS (Netherlands)

Lam, B. Daniel; Hordijk, Peter L.

2013-01-01

Cellular signaling by small GTPases is critically dependent on proper spatio-temporal orchestration of activation and output. In addition to their core G (guanine nucleotide binding)-domain, small GTPases comprise a hypervariable region (HVR) and a lipid anchor that are generally accepted to control

Involvement of rho-gtpases in fibroblast adhesion and fibronectine fibrillogenesis under stretch

Science.gov (United States)

Guignandon, A.; Lambert, C.; Rattner, A.; Servotte, S.; Lapiere, C.; Nusgens, B.; Vico, L.

The Rho family small GTPases play a crucial role in mediating cellular adaptation to mechanical stimulation (MS), and possibly to microgravity (μg), through effects on the cytoskeleton and cell adhesion which is, in turn, mainly regulated by fibronectin fibrillogenesis (FnF). It remains unclear how mechanical stimulation is transduced to the Rho signaling pathways and how it impacts on fibronectin (fbn) fibrillogenesis (FnF). μg (2 days, mission STS-095) led to de-adhesion of fibroblasts and modification of the underlying extracellular matrix. To determine whether GTPases modulated FnF, we generated stable cell lines expressing high level of activated RhoA and Rac1 (QL) as compared to wild type (WI26-WT). After MS application [8% deformation, 1Hz, 15 min., 3 times/day for 1-2 days], we quantified focal adhesion (vinculin, paxillin, FAKY397), f-actin stress fibers (Sf) and FnF with home-developed softwares. We reported that after MS, Sf are more rapidly (30min) formed under the nucleus in Wi26-WT (+100%) and Rac1 (+200%) than in RhoA (+20%). Vinculin & paxillin were only restricted to the cell edge in static conditions and homogeneously distributed after MS in WT and Rac1. The relative area of contacts (vinculin & paxillin) was more dramatically enhanced by MS in Rac1 (+80%) than in WT (+40%) and RhoA (+25%) indicating that new focal contacts are formed under MS and supported the presence of Sf. MS Activation of FAK (FAKY397) was clear in WT and Rac1 and reduced in RhoA. FnF was restricted to cell-cell contacts zone without any change in the relative area of fbn after a 2-days MS. However we found more numerous spots of fbn at the cell center in Rac1 as compared with RhoA & WT suggesting that these fibrillar contacts will grow upon maturation and modulate FnF. The results indicate that MS induces formation of Sf and focal adhesions and enhances FF. RhoA has been shown to induce the formation of Sf and focal adhesions, and Rac1 activation decreases Rho activity in

Tandem duplications of a degenerated GTP-binding domain at the origin of GTPase receptors Toc159 and thylakoidal SRP

International Nuclear Information System (INIS)

Hernandez Torres, Jorge; Maldonado, Monica Alexandra Arias; Chomilier, Jacques

2007-01-01

The evolutionary origin of some nuclear encoded proteins that translocate proteins across the chloroplast envelope remains unknown. Therefore, sequences of GTPase proteins constituting the Arabidopsis thaliana translocon at the outer membrane of chloroplast (atToc) complexes were analyzed by means of HCA. In particular, atToc159 and related proteins (atToc132, atToc120, and atToc90) do not have proven homologues of prokaryotic or eukaryotic ancestry. We established that the three domains commonly referred to as A, G, and M originate from the GTPase G domain, tandemly repeated, and probably evolving toward an unstructured conformation in the case of the A domain. It resulted from this study a putative common ancestor for these proteins and a new domain definition, in particular the splitting of A into three domains (A1, A2, and A3), has been proposed. The family of Toc159, previously containing A. thaliana and Pisum sativum, has been extended to Medicago truncatula and Populus trichocarpa and it has been revised for Oryza sativa. They have also been compared to GTPase subunits involved in the cpSRP system. A distant homology has been revealed among Toc and cpSRP GTP-hydrolyzing proteins of A. thaliana, and repetitions of a GTPase domain were also found in cpSRP protein receptors, by means of HCA analysis

Illuminating Chaucer through Poetry, Manuscript Illuminations, and a Critical Rap Album

Science.gov (United States)

Lynch, Tom Liam

2007-01-01

Drawing connections between Chaucer, Eminem, and social issues, New York City high school teacher Tom Liam Lynch helped students become familiar with "The Canterbury Tales." Students wrote poems of rhymed couplets about today's social and political issues, created illuminated manuscripts, and recorded a rap CD. A book and album were…

A educação informal e o rap como agente educativo

Directory of Open Access Journals (Sweden)

Alexandre Felipe Fiuza

2013-01-01

Full Text Available In this text are broached the relationship between rap music and the educational processes. To achieve this goal, the study deals with the conceptualization of the modalities of education, namely formal, non-formal and informal education, based on national and foreign bibliography. Considering the object of study, this paper is based on interdisciplinary reflections, consisting of theoretical and methodological approaches from the fields of Education, Music, Communication and Sociology of Culture. These theoretical frameworks contribute to the accuracy of concepts and the observation of the multiple dimensions that hip hop, and specifically rap, holds as a social and cultural phenomenon. By focusing on the particularity of informal education, which is even more prevalent in the so-called knowledge or media society, this study aims to contribute to the discussion of educational processes intrinsic to the culture industry and the media, and their significant influence on the audience.

Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes.

Science.gov (United States)

Fu, Yulong; Zhu, Jun-Yi; Zhang, Fujian; Richman, Adam; Zhao, Zhanzheng; Han, Zhe

2017-06-01

The Drosophila nephrocyte is a critical component of the fly renal system and bears structural and functional homology to podocytes and proximal tubule cells of the mammalian kidney. Investigations of nephrocyte cell biological processes are fundamental to understanding the insect renal system. Nephrocytes are highly active in endocytosis and vesicle trafficking. Rab GTPases regulate endocytosis and trafficking but specific functions of nephrocyte Rabs remain undefined. We analyzed Rab GTPase expression and function in Drosophila nephrocytes and found that 11 out of 27 Drosophila Rabs were required for normal activity. Rabs 1, 5, 7, 11 and 35 were most important. Gene silencing of the nephrocyte-specific Rab5 eliminated all intracellular vesicles and the specialized plasma membrane structures essential for nephrocyte function. Rab7 silencing dramatically increased clear vacuoles and reduced lysosomes. Rab11 silencing increased lysosomes and reduced clear vacuoles. Our results suggest that Rab5 mediates endocytosis that is essential for the maintenance of functionally critical nephrocyte plasma membrane structures and that Rabs 7 and 11 mediate alternative downstream vesicle trafficking pathways leading to protein degradation and membrane recycling, respectively. Elucidating molecular pathways underlying nephrocyte function has the potential to yield important insights into human kidney cell physiology and mechanisms of cell injury that lead to disease. The Drosophila nephrocyte is emerging as a useful in vivo model system for molecular target identification and initial testing of therapeutic approaches in humans.

Anti-inflammatory and analgesic activity of r.a.p . ( Radix Angelicae ...

African Journals Online (AJOL)

The objective of this paper was to study the anti-inflammatory and analgesic effects of Radix Angelicae Pubescentis (R.A.P) ethanol extracts. Three classic anti-inflammatory models and two analgesic models were used in this research. In anti-inflammatory tests, all the extracts have a certain inhibition on the acute ...

The GTPase Rab37 Participates in the Control of Insulin Exocytosis.

Directory of Open Access Journals (Sweden)

Sanda Ljubicic

Full Text Available Rab37 belongs to a subclass of Rab GTPases regulating exocytosis, including also Rab3a and Rab27a. Proteomic studies indicate that Rab37 is associated with insulin-containing large dense core granules of pancreatic β-cells. In agreement with these observations, we detected Rab37 in extracts of β-cell lines and human pancreatic islets and confirmed by confocal microscopy the localization of the GTPase on insulin-containing secretory granules. We found that, as is the case for Rab3a and Rab27a, reduction of Rab37 levels by RNA interference leads to impairment in glucose-induced insulin secretion and to a decrease in the number of granules in close apposition to the plasma membrane. Pull-down experiments revealed that, despite similar functional effects, Rab37 does not interact with known Rab3a or Rab27a effectors and is likely to operate through a different mechanism. Exposure of insulin-secreting cells to proinflammatory cytokines, fatty acids or oxidized low-density lipoproteins, mimicking physiopathological conditions that favor the development of diabetes, resulted in a decrease in Rab37 expression. Our data identify Rab37 as an additional component of the machinery governing exocytosis of β-cells and suggest that impaired expression of this GTPase may contribute to defective insulin release in pre-diabetic and diabetic conditions.

Loss of γ-tubulin, GCP-WD/NEDD1 and CDK5RAP2 from the Centrosome of Neurons in Developing Mouse Cerebral and Cerebellar Cortex

International Nuclear Information System (INIS)

Yonezawa, Satoshi; Shigematsu, Momoko; Hirata, Kazuto; Hayashi, Kensuke

2015-01-01

It has been recently reported that the centrosome of neurons does not have microtubule nucleating activity. Microtubule nucleation requires γ-tubulin as well as its recruiting proteins, GCP-WD/NEDD1 and CDK5RAP2 that anchor γ-tubulin to the centrosome. Change in the localization of these proteins during in vivo development of brain, however, has not been well examined. In this study we investigate the localization of γ-tubulin, GCP-WD and CDK5RAP2 in developing cerebral and cerebellar cortex with immunofluorescence. We found that γ-tubulin and its recruiting proteins were localized at centrosomes of immature neurons, while they were lost at centrosomes in mature neurons. This indicated that the loss of microtubule nucleating activity at the centrosome of neurons is due to the loss of γ-tubulin-recruiting proteins from the centrosome. RT-PCR analysis revealed that these proteins are still expressed after birth, suggesting that they have a role in microtubule generation in cell body and dendrites of mature neurons. Microtubule regrowth experiments on cultured mature neurons showed that microtubules are nucleated not at the centrosome but within dendrites. These data indicated the translocation of microtubule-organizing activity from the centrosome to dendrites during maturation of neurons, which would explain the mixed polarity of microtubules in dendrites

Activation of G protein-coupled estrogen receptor 1 induces coronary artery relaxation via Epac/Rap1-mediated inhibition of RhoA/Rho kinase pathway in parallel with PKA.

Directory of Open Access Journals (Sweden)

Xuan Yu

Full Text Available Previously, we reported that cAMP/PKA signaling is involved in GPER-mediated coronary relaxation by activating MLCP via inhibition of RhoA pathway. In the current study, we tested the hypothesis that activation of GPER induces coronary artery relaxation via inhibition of RhoA/Rho kinase pathway by cAMP downstream targets, exchange proteins directly activated by cAMP (Epac as well as PKA. Our results show that Epac inhibitors, brefeldin A (BFA, 50 μM, or ESI-09 (20 μM, or CE3F4 (100 μM, all partially inhibited porcine coronary artery relaxation response to the selective GPER agonist, G-1 (0.3-3 μM; while concurrent administration of BFA and PKI (5 μM, a PKA inhibitor, almost completely blocked the relaxation effect of G-1. The Epac specific agonist, 8-CPT-2Me-cAMP (007, 1-100 μM, induced a concentration-dependent relaxation response. Furthermore, the activity of Ras-related protein 1 (Rap1 was up regulated by G-1 (1 μM treatment of porcine coronary artery smooth muscle cells (CASMCs. Phosphorylation of vasodilator-stimulated phosphoprotein (p-VASP was elevated by G-1 (1 μM treatment, but not by 007 (50 μM; and the effect of G-1 on p-VASP was blocked by PKI, but not by ESI-09, an Epac antagonist. RhoA activity was similarly down regulated by G-1 and 007, whereas ESI-09 restored most of the reduced RhoA activity by G-1 treatment. Furthermore, G-1 decreased PGF2α-induced p-MYPT1, which was partially reversed with either ESI-09 or PKI; whereas, concurrent administration of ESI-09 and PKI totally prevented the inhibitory effect of G-1. The inhibitory effects of G-1 on p- MLC levels in CASMCs were mostly restored by either ESI-09 or PKI. These results demonstrate that activation of GPER induces coronary artery relaxation via concurrent inhibition of RhoA/Rho kinase by Epac/Rap1 and PKA. GPER could be a potential drug target for preventing and treating cardiovascular diseases.

Frequency of helicobacter pylori (hp) infection in children with recurrent abdominal pain (rap)

International Nuclear Information System (INIS)

Mahmud, S.; Ali, S.

2015-01-01

To study the frequency of Helicobacter Pylori (HP) infection among children with recurrent abdominal pain (RAP) Study Design: Cross-sectional comparative study. Place and Duration of Study: Military Hospital (MH), Rawalpindi from December 2011 to February 2012. Patients and Methods: One hundred children of either gender aged 2 to 12 years presenting with RAP were tested for HP at Paediatric OPD MH, Rawalpindi who consented to participate in the study. Those children who tested positive for Helicobacter Pylori Stool Antigen Test (HPSAT) were labeled as those having Hp infection. The stool assay was performed using the HpSAT kit and the socio-demographic and clinical profiles of children were associated. Results: Out of 100 children included in the study HpSAT was positive in 38% children. Frequency of Hp infection was significantly associated with source of drinking water (p = 0.014), socioeconomic status (p = 0.001) and positive family history of yspepsia (p= 0.023). While age and gender have no significant association with HP infection. Conclusion: Hp infection is very common in children presenting with RAP in our Paediatric OPD. Children with family history of dyspepsia, from low socioeconomic class and those drinking filtered water are at greater risk for HP infection. It is recommended that children from other populations in our country should also be tested in their medical health facilities in order to have a wider analysis of this problem in our setup. (author)

Truncated RAP-MUSIC (TRAP-MUSIC) for MEG and EEG source localization.

Science.gov (United States)

Mäkelä, Niko; Stenroos, Matti; Sarvas, Jukka; Ilmoniemi, Risto J

2018-02-15

Electrically active brain regions can be located applying MUltiple SIgnal Classification (MUSIC) on magneto- or electroencephalographic (MEG; EEG) data. We introduce a new MUSIC method, called truncated recursively-applied-and-projected MUSIC (TRAP-MUSIC). It corrects a hidden deficiency of the conventional RAP-MUSIC algorithm, which prevents estimation of the true number of brain-signal sources accurately. The correction is done by applying a sequential dimension reduction to the signal-subspace projection. We show that TRAP-MUSIC significantly improves the performance of MUSIC-type localization; in particular, it successfully and robustly locates active brain regions and estimates their number. We compare TRAP-MUSIC and RAP-MUSIC in simulations with varying key parameters, e.g., signal-to-noise ratio, correlation between source time-courses, and initial estimate for the dimension of the signal space. In addition, we validate TRAP-MUSIC with measured MEG data. We suggest that with the proposed TRAP-MUSIC method, MUSIC-type localization could become more reliable and suitable for various online and offline MEG and EEG applications. Copyright © 2017 Elsevier Inc. All rights reserved.

NADPH oxidase complex-derived reactive oxygen species, the actin cytoskeleton, and rho GTPases in cell migration

DEFF Research Database (Denmark)

Stanley, Alanna; Thompson, Kerry; Hynes, Ailish

2014-01-01

Abstract Significance: Rho GTPases are historically known to be central regulators of actin cytoskeleton reorganization. This affects many processes including cell migration. In addition, members of the Rac subfamily are known to be involved in reactive oxygen species (ROS) production through...... mediating cytoskeletal reorganization. Critical Issues: The role of the actin cytoskeleton in providing a scaffold for components of the Nox complex needs to be examined in the light of these new advances. During cell migration, Rho GTPases, ROS, and cytoskeletal organization appear to function as a complex...... compartments. This in conjunction with the analysis of tissues lacking specific Rho GTPases, and Nox components will facilitate a detailed examination of the interactions of these structures with the actin cytoskeleton. In combination with the analysis of ROS production, including its subcellular location...

Rab4GTPase modulates CFTR function by impairing channel expression at plasma membrane

International Nuclear Information System (INIS)

Saxena, Sunil K.; Kaur, Simarna; George, Constantine

2006-01-01

Cystic fibrosis (CF), an autosomal recessive disorder, is caused by the disruption of biosynthesis or the function of a membrane cAMP-activated chloride channel, CFTR. CFTR regulatory mechanisms include recruitment of channel proteins to the cell surface from intracellular pools and by protein-protein interactions. Rab proteins are small GTPases involved in regulated trafficking controlling vesicle docking and fusion. Rab4 controls recycling events from endosome to the plasma membrane, fusion, and degradation. The colorectal cell line HT-29 natively expresses CFTR and responds to cAMP stimulation with an increase in CFTR-mediated currents. Rab4 over-expression in HT-29 cells inhibits both basal and cAMP-stimulated CFTR-mediated currents. GTPase-deficient Rab4Q67L and GDP locked Rab4S22N both inhibit channel activity, which appears characteristically different. Active status of Rab4 was confirmed by GTP overlay assay, while its expression was verified by Western blotting. The pull-down and immunoprecipitation experiments suggest that Rab4 physically interacts with CFTR through protein-protein interaction. Biotinylation with cell impermeant NHS-Sulfo-SS-Biotin implies that Rab4 impairs CFTR expression at cell surface. The enhanced cytosolic CFTR indicates that Rab4 expression restrains CFTR appearance at the cell membrane. The study suggests that Rab4 regulates the channel through multiple mechanisms that include protein-protein interaction, GTP/GDP exchange, and channel protein trafficking. We propose that Rab4 is a dynamic molecule with a significant role in CFTR function

Structural basis for the interaction of the adaptor protein grb14 with activated ras.

Directory of Open Access Journals (Sweden)

Rohini Qamra

Full Text Available Grb14, a member of the Grb7-10-14 family of cytoplasmic adaptor proteins, is a tissue-specific negative regulator of insulin signaling. Grb7-10-14 contain several signaling modules, including a Ras-associating (RA domain, a pleckstrin-homology (PH domain, a family-specific BPS (between PH and SH2 region, and a C-terminal Src-homology-2 (SH2 domain. We showed previously that the RA and PH domains, along with the BPS region and SH2 domain, are necessary for downregulation of insulin signaling. Here, we report the crystal structure at 2.4-Å resolution of the Grb14 RA and PH domains in complex with GTP-loaded H-Ras (G12V. The structure reveals that the Grb14 RA and PH domains form an integrated structural unit capable of binding simultaneously to small GTPases and phosphoinositide lipids. The overall mode of binding of the Grb14 RA domain to activated H-Ras is similar to that of the RA domains of RalGDS and Raf1 but with important distinctions. The integrated RA-PH structural unit in Grb7-10-14 is also found in a second adaptor family that includes Rap1-interacting adaptor molecule (RIAM and lamellipodin, proteins involved in actin-cytoskeleton rearrangement. The structure of Grb14 RA-PH in complex with H-Ras represents the first detailed molecular characterization of tandem RA-PH domains bound to a small GTPase and provides insights into the molecular basis for specificity.

Induction of Non-Apoptotic Cell Death by Activated Ras Requires Inverse Regulation of Rac1 and Arf6

OpenAIRE

Bhanot, Haymanti; Young, Ashley M.; Overmeyer, Jean H.; Maltese, William A.

2010-01-01

Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating-protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to...

Improving Quality Control of Asphalt Pavement with RAP Using a Portable Infrared Spectroscopy Device

Science.gov (United States)

2016-04-01

This project has investigated the effectiveness of a Portable Infrared Spectrometer (PIRS) device in estimating percent of Reclaimed Asphalt Pavement (RAP) and its contribution into oxidative aging of a new asphalt mixture immediately after productio...

Investigation on the Combined Effect of Fibers and Cement on the Mechanical Performance of Foamed Bitumen Mixtures Containing 100% RAP

OpenAIRE

Ehsan Ashouri Taziani; Emanuele Toraldo; Filippo Giustozzi; Maurizio Crispino

2016-01-01

Concerns about virgin aggregate sources and increasing demands for construction materials of transport infrastructures as the key parameters in development are the most important reasons, which convinced pavement engineers to develop new methods in order to use higher amount of recycled asphalt pavement (RAP). One of the common methodologies to produce mixtures containing RAP is foamed bitumen mix (FBM). In addition, according to previous research studies, incorporating various types of fiber...

Structure of the ribosomal interacting GTPase YjeQ from the enterobacterial species Salmonella typhimurium

International Nuclear Information System (INIS)

Nichols, C. E.; Johnson, C.; Lamb, H. K.; Lockyer, M.; Charles, I. G.; Hawkins, A. R.; Stammers, D. K.

2007-01-01

The X-ray crystal structure of the GTPase YjeQ from S. typhimurium is presented and compared with those of orthologues from T. maritima and B. subtilis. The YjeQ class of P-loop GTPases assist in ribosome biogenesis and also bind to the 30S subunit of mature ribosomes. YjeQ ribosomal binding is GTP-dependent and thought to specifically direct protein synthesis, although the nature of the upstream signal causing this event in vivo is as yet unknown. The attenuating effect of YjeQ mutants on bacterial growth in Escherichia coli makes it a potential target for novel antimicrobial agents. In order to further explore the structure and function of YjeQ, the isolation, crystallization and structure determination of YjeQ from the enterobacterial species Salmonella typhimurium (StYjeQ) is reported. Whilst the overall StYjeQ fold is similar to those of the previously reported Thematoga maritima and Bacillus subtilis orthologues, particularly the GTPase domain, there are larger differences in the three OB folds. Although the zinc-finger secondary structure is conserved, significant sequence differences alter the nature of the external surface in each case and may reflect varying signalling pathways. Therefore, it may be easier to develop YjeQ-specific inhibitors that target the N- and C-terminal regions, disrupting the metabolic connectivity rather than the GTPase activity. The availability of coordinates for StYjeQ will provide a significantly improved basis for threading Gram-negative orthologue sequences and in silico compound-screening studies, with the potential for the development of species-selective drugs

Structure of the ribosomal interacting GTPase YjeQ from the enterobacterial species Salmonella typhimurium

Energy Technology Data Exchange (ETDEWEB)

Nichols, C. E. [Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Johnson, C.; Lamb, H. K. [Institute of Cell and Molecular Biosciences, Catherine Cookson Building, Medical School, Framlington Place, Newcastle University, Newcastle-upon-Tyne NE2 4HH (United Kingdom); Lockyer, M. [Arrow Therapeutics Ltd, Britannia House, Trinity Street, Borough, London SE1 1DA (United Kingdom); Charles, I. G. [The Wolfson Institute for Biomedical Research, The Cruciform Building, University College London, Gower Street, London WC1E 6BT (United Kingdom); Hawkins, A. R. [Institute of Cell and Molecular Biosciences, Catherine Cookson Building, Medical School, Framlington Place, Newcastle University, Newcastle-upon-Tyne NE2 4HH (United Kingdom); Stammers, D. K., E-mail: daves@strubi.ox.ac.uk [Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

2007-11-01

The X-ray crystal structure of the GTPase YjeQ from S. typhimurium is presented and compared with those of orthologues from T. maritima and B. subtilis. The YjeQ class of P-loop GTPases assist in ribosome biogenesis and also bind to the 30S subunit of mature ribosomes. YjeQ ribosomal binding is GTP-dependent and thought to specifically direct protein synthesis, although the nature of the upstream signal causing this event in vivo is as yet unknown. The attenuating effect of YjeQ mutants on bacterial growth in Escherichia coli makes it a potential target for novel antimicrobial agents. In order to further explore the structure and function of YjeQ, the isolation, crystallization and structure determination of YjeQ from the enterobacterial species Salmonella typhimurium (StYjeQ) is reported. Whilst the overall StYjeQ fold is similar to those of the previously reported Thematoga maritima and Bacillus subtilis orthologues, particularly the GTPase domain, there are larger differences in the three OB folds. Although the zinc-finger secondary structure is conserved, significant sequence differences alter the nature of the external surface in each case and may reflect varying signalling pathways. Therefore, it may be easier to develop YjeQ-specific inhibitors that target the N- and C-terminal regions, disrupting the metabolic connectivity rather than the GTPase activity. The availability of coordinates for StYjeQ will provide a significantly improved basis for threading Gram-negative orthologue sequences and in silico compound-screening studies, with the potential for the development of species-selective drugs.

Regulation of Rac1 GTPase activity by quinine through G-protein and bitter taste receptor T2R4.

Science.gov (United States)

Sidhu, Crystal; Jaggupilli, Appalaraju; Chelikani, Prashen; Bhullar, Rajinder P

2017-02-01

Rac1 belongs to the Rho family of small GTPases and regulates actin cytoskeleton reorganization. T2R4 is a bitter taste receptor belonging to the G protein-coupled receptor family of proteins. In addition to mediating bitter taste perception from the tongue, T2R4s are found in extra-oral tissues, e.g., nasal epithelium, airways, brain, testis suggesting a much broader physiological function for these receptors. Anti-malarial drug and a bitter tasting compound, quinine, is a known agonist for T2R4, whereas BCML (Nα,Nα-Bis(carboxymethyl)-L-lysine) acts as an inverse agonist. Using western blot and Ca ++ mobilization assays, the effects of quinine on Rac1 activity in HEK293T cells stably expressing T2R4/Gα 16/44 , T2R4, or Gα 16/44 and transiently transfected with HA-Rac1 were investigated. Quinine treatment caused a significant reduction in the amount of active Rac1, whereas in the presence of BCML, quinine failed to cause any significant change in active Rac1. No significant change in Rac1 activity was observed in BAPTA-AM plus quinine-treated Gα 16/44 cells, suggesting possibility of a pathway in addition to the canonical Ca ++ -dependent pathway. A noticeable role for Gα 16/44 independent of T2R4 is observed in quinine-mediated Rac1 inactivation. Further, a significant difference in quinine-induced Ca ++ response in T2R4/Gα 16/44 or T2R4 cells was observed validating the partial role of calcium and importance of Gα 16/44 . This study is the first to show an inhibitory downstream action of a T2R4 agonist on Rac1 function. Further investigation will help in better understanding the downstream signal transduction network of T2R4 and its extra-oral physiological roles.

Apropriação musical: a arte de ouvir Rap

Directory of Open Access Journals (Sweden)

Jaison Hinkel

2011-09-01

Full Text Available Esta pesquisa teve por objetivo investigar se há e como se processa a relação estética entre sujeitos ouvintes e a música Rap, no processo de apropriação musical. Partimos da perspectiva sócio-histórica, segundo a qual nas reflexões metodológicas propostas por Vigotski estiveram tecidas as ideias de Bakhtin, como também as contribuições de Sartre, em um movimento dialógico com alguns leitores que se fazem interlocutores destes autores. Realizamos entrevistas individuais e abertas com cinco jovens moradores de periferia da região de Blumenau - SC. As análises indicaram a apropriação musical dos sujeitos investigados como um complexo processo que envolve aspectos referentes às propriedades físico-perceptuais do objeto estético Rap e a biografia de cada sujeito/ouvinte. A apropriação musical se mostrou como um complexo processo de conversão do coletivo em singular, fenômeno que exige um lugar cocriador do sujeito-ouvinte que se apropria dos significados expressos nas músicas e produz, a partir destes, novas zonas de sentido.

Investigation on the Combined Effect of Fibers and Cement on the Mechanical Performance of Foamed Bitumen Mixtures Containing 100% RAP

Directory of Open Access Journals (Sweden)

Ehsan Ashouri Taziani

2016-01-01

Full Text Available Concerns about virgin aggregate sources and increasing demands for construction materials of transport infrastructures as the key parameters in development are the most important reasons, which convinced pavement engineers to develop new methods in order to use higher amount of recycled asphalt pavement (RAP. One of the common methodologies to produce mixtures containing RAP is foamed bitumen mix (FBM. In addition, according to previous research studies, incorporating various types of fibers and hydraulic binders such as cement could significantly improve the mechanical performance of mixtures. The present research study evaluated FBM containing 100% RAP and two types of fiber and Portland cement. Dynamic modulus, unconfined dynamic creep compression, and indirect tensile strength were evaluated in the laboratory at optimum moisture content, which was investigated in this research. Both types of fiber and cement proved to enhance specific properties of mixtures.

The Small Rho GTPases Rac1 and Rac2 Are Important for T-Cell Independent Antigen Responses and for Suppressing Switching to IgG2b in Mice.

Science.gov (United States)

Gerasimčik, Natalija; He, Minghui; Dahlberg, Carin I M; Kuznetsov, Nikolai V; Severinson, Eva; Westerberg, Lisa S

2017-01-01

The Rho GTPases Cdc42, Rac1, and Rac2 coordinate receptor signaling to cell adhesion, migration, and proliferation. Deletion of Rac1 and Rac2 early during B cell development leads to failure in B cell entry into the splenic white pulp. Here, we sought to understand the role of Rac1 and Rac2 in B cell functionality and during the humoral antibody response. To circumvent the migratory deficiency of B cells lacking both Rac1 and Rac2, we took the approach to inducibly delete Rac1 in Rac2 -/- B cells in the spleen (Rac1 B Rac2 -/- B cells). Rac1 B Rac2 -/- mice had normal differentiation of splenic B cell populations, except for a reduction in marginal zone B cells. Rac1 B Rac2 -/- B cells showed normal spreading response on antibody-coated layers, while both Rac2 -/- and Rac1 B Rac2 -/- B cells had reduced homotypic adhesion and decreased proliferative response when compared to wild-type B cells. Upon challenge with the T-cell-independent antigen TNP-conjugated lipopolysaccharide, Rac1 B Rac2 -/- mice showed reduced antibody response. In contrast, in response to the T-cell-dependent antigen sheep red blood cells, Rac1 B Rac2 -/- mice had increased serum titers of IgG1 and IgG2b. During in vitro Ig class switching, Rac1 B Rac2 -/- B cells had elevated germline γ2b transcripts leading to increased Ig class switching to IgG2b. Our data suggest that Rac1 and Rac2 serve an important role in regulation of the B cell humoral immune response and in suppressing Ig class switching to IgG2b.

THERAPIE - THErmix-RAps-Plot-InterfacE. A graphic software for representation of THERMIX-2D results with the interactive plot program RAPS

International Nuclear Information System (INIS)

Duensing, P.; Jahn, W.; Rehm, W.

1986-09-01

The performance of safety analyses for gas-cooled high temperature reactor power plants requires efficient plot codes for the evaluation and representation of computer results. The report describes the coupling between the thermodynamic simulation code THERMIX and the graphic plot code RAPS via the interface program THERAPIE. Especially the structure and the handling of the interface program are explained as well as the dialogue with the plot code. Further options of the colour graphic system are demonstrated for the representation of temperature distributions in components of HTR concepts (HTR-500). (orig.) [de

BRAF and RAS oncogenes regulate Rho GTPase pathways to mediate migration and invasion properties in human colon cancer cells: a comparative study

Directory of Open Access Journals (Sweden)

Shirasawa Senji

2011-09-01

Full Text Available Abstract Background Colorectal cancer is a common disease that involves genetic alterations, such as inactivation of tumour suppressor genes and activation of oncogenes. Among them are RAS and BRAF mutations, which rarely coexist in the same tumour. Individual members of the Rho (Ras homology GTPases contribute with distinct roles in tumour cell morphology, invasion and metastasis. The aim of this study is to dissect cell migration and invasion pathways that are utilised by BRAFV600E as compared to KRASG12V and HRASG12V oncoproteins. In particular, the role of RhoA (Ras homolog gene family, member A, Rac1 (Ras-related C3 botulinum toxin substrate 1 and Cdc42 (cell division cycle 42 in cancer progression induced by each of the three oncogenes is described. Methods Colon adenocarcinoma cells with endogenous as well as ectopically expressed or silenced oncogenic mutations of BRAFV600E, KRASG12V and HRASG12V were employed. Signalling pathways and Rho GTPases were inhibited with specific kinase inhibitors and siRNAs. Cell motility and invasion properties were correlated with cytoskeletal properties and Rho GTPase activities. Results Evidence presented here indicate that BRAFV600E significantly induces cell migration and invasion properties in vitro in colon cancer cells, at least in part through activation of RhoA GTPase. The relationship established between BRAFV600E and RhoA activation is mediated by the MEK-ERK pathway. In parallel, KRASG12V enhances the ability of colon adenocarcinoma cells Caco-2 to migrate and invade through filopodia formation and PI3K-dependent Cdc42 activation. Ultimately increased cell migration and invasion, mediated by Rac1, along with the mesenchymal morphology obtained through the Epithelial-Mesenchymal Transition (EMT were the main characteristics rendered by HRASG12V in Caco-2 cells. Moreover, BRAF and KRAS oncogenes are shown to cooperate with the TGFβ-1 pathway to provide cells with additional transforming

C9orf72’s interaction with Rab GTPases - modulation of membrane traffic and autophagy

Directory of Open Access Journals (Sweden)

Bor Luen Tang

2016-10-01

Full Text Available Hexanucleotide repeat expansion in an intron of Chromosome 9 open reading frame 72 (C9orf72 is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS and Frontotemporal Dementia (FTD. While functional haploinsufficiency of C9orf72 resulting from the mutation may play a role in ALS/FTD, the actual cellular role of the protein has been unclear. Recent findings have now shown that C9orf72 physically and functionally interacts with multiple members of the Rab small GTPases family, consequently exerting important influences on cellular membrane traffic and the process of autophagy. Loss of C9orf72 impairs endocytosis in neuronal cell lines, and attenuated autophagosome formation. Interestingly, C9orf72 could influence autophagy both as part of a Guanine nucleotide exchange factor (GEF complex, or as a Rab effector that facilitates transport of the Unc-51-like Autophagy Activating Kinase 1 (Ulk1 autophagy initiation complex. The cellular function of C9orf72 is discussed in the light of these recent findings

Defect in the GTPase activating protein (GAP) function of eIF5 causes repression of GCN4 translation.

Science.gov (United States)

Antony A, Charles; Alone, Pankaj V

2017-05-13

In eukaryotes, the eIF5 protein plays an important role in translation start site selection by providing the GAP (GTPase activating protein) function. However, in yeast translation initiation fidelity defective eIF5 G31R mutant causes preferential utilization of UUG as initiation codon and is termed as Suppressor of initiation codon (Sui - ) phenotype due to its hyper GTPase activity. The eIF5 G31R mutant dominantly represses GCN4 expression and confers sensitivity to 3-Amino-1,2,4-Trizole (3AT) induced starvation. The down-regulation of the GCN4 expression (Gcn - phenotype) in the eIF5 G31R mutant was not because of leaky scanning defects; rather was due to the utilization of upUUG initiation codons at the 5' regulatory region present between uORF1 and the main GCN4 ORF. Copyright © 2017 Elsevier Inc. All rights reserved.

Visualization of the Activity of Rac1 Small GTPase in a Cell

International Nuclear Information System (INIS)

Higashi, Morihiro; Yu, Jianyong; Tsuchiya, Hiroshi; Saito, Teruyoshi; Oyama, Toshinao; Kawana, Hidetada; Kitagawa, Motoo; Tamaru, Jun-ichi; Harigaya, Kenichi

2010-01-01

Rho family G proteins including Rac regulate a variety of cellular functions, such as morphology, motility, and gene expression. Here we developed a fluorescence resonance energy transfer-based analysis in which we could monitor the activity of Rac1. To detect fluorescence resonance energy transfer, yellow fluorescent protein fused Rac1 and cyan fluorescent protein fused Cdc42-Rac1-interaction-binding domain of Pak1 protein were used as intermolecular probes of FRET. The fluorophores were separated with linear unmixing method. The fluorescence resonance energy transfer efficiency was measured by acceptor photobleaching assisted assay. With these methods, the Rac1 activity was visualized in a cell. The present findings indicate that this approach is sensitive enough to achieve results similar to those from ratiometric fluorescence resonance energy transfer analysis

Økologisk risikovurdering af en genmodificeret glyfosat-tolerant raps GT73 i anmeldelse til godkendelse vedr. import til markedsføring under forordning 1829/2003/EF

DEFF Research Database (Denmark)

Kjellsson, Gøsta; Sørensen, Jesper Givskov; Damgaard, Christian

2012-01-01

"BIOSCIENCE konklusioner vedr. den økolo-giske risikovurdering af den genmodifice-rede GT73-raps i Danmark Den genmodificerede raps, GT73, adskiller sig fra konventionel raps ved at have indsat gener der gør planten tolerant over for herbicidet glyfosat. Rapsen søges kun godkendt til import til f...... to monitor such dispersal of the GMO-plant into cultivated fields and surroundings. Although, not directly an environmental issue, dispersal of the GT73 could result in problems for co-existence with non-GMO-cultivation. "...

Økologisk risikovurdering af en genmodificeret glyfosat-tolerant raps GT73 i anmeldelse til godkendelse vedr. import til markedsføring under Forordning 1829/2003/EF

DEFF Research Database (Denmark)

Damgaard, Christian; Sørensen, Jesper Givskov; Kjellsson, Gøsta

2012-01-01

BIOSCIENCE konklusioner vedr. den økolo-giske risikovurdering af den genmodifice-rede GT73-raps i Danmark Den genmodificerede raps, GT73, adskiller sig fra konventionel raps ved at have indsat gener der gør planten tolerant over for herbicidet glyfosat. Rapsen søges kun godkendt til import til fo...... to monitor such dispersal of the GMO-plant into cultivated fields and surroundings. Although, not directly an environmental issue, dispersal of the GT73 could result in problems for co-existence with non-GMO-cultivation....

Neariminio žemės dirbimo poveikis vasarinių rapsų agroekosistemos komponentams

OpenAIRE

Petrauskas, Tomas

2014-01-01

Tikslas – įvertinti tiesioginės sėjos, supaprastinto žemės dirbimo ir augalinių liekanų įtaką kai kuriems vasarinių rapsų agroekosistemos komponentams. Stacionarus dviejų veiksnių lauko eksperimentas įrengtas 1999 m. Aleksandro Stulginskio universiteto Bandymų stotyje. A veiksnys − šiaudų panaudojimas: šiaudai pašalinti (-Š); šiaudai susmulkinti ir paskleisti (+Š). B veiksnys − žemės dirbimos sistemos: įprastas gilus arimas 23–25 cm gyliu rudenį (GA), kontrolinis variantas; seklus arimas 10–1...

Influence of bacterial toxins on the GTPase activity of transducin from bovine retinal rod outer segments

International Nuclear Information System (INIS)

Rybin, V.O.; Gureeva, A.A.

1986-01-01

The action of cholera toxin, capable of ADP-ribosylation of the activator N/sub s/ protein, and pertussis toxin, capable of ADP-ribosylation of the inhibitor N/sub i/ protein of the adenylate cyclase complex, on transducin, the GTP-binding protein of the rod outer segments of the retina, was investigated. It was shown that under the action of pertussis and cholera toxins, the GTPase activity of transducin is inhibited. Pertussin toxin inhibits the GTPase of native retinal rod outer segments by 30-40%, while GTPase of homogeneous transducin produces a 70-80% inhibition. The action of toxins on transducin depends on the presence and nature of the guanylic nucleotide with which incubation is performed. On the basis of the data obtained it is suggested that pertussis toxin interacts with pretransducin and with the transducin-GDP complex, while cholera toxin ADP-ribosylates the transducin-GTP complex and does not act on transducin lacking GTP

Increased diacylglycerol kinase ζ expression in human metastatic colon cancer cells augments Rho GTPase activity and contributes to enhanced invasion

International Nuclear Information System (INIS)

Cai, Kun; Mulatz, Kirk; Ard, Ryan; Nguyen, Thanh; Gee, Stephen H

2014-01-01

Unraveling the signaling pathways responsible for the establishment of a metastatic phenotype in carcinoma cells is critically important for understanding the pathology of cancer. The acquisition of cell motility is a key property of metastatic tumor cells and is a prerequisite for invasion. Rho GTPases regulate actin cytoskeleton reorganization and the cellular responses required for cell motility and invasion. Diacylglycerol kinase ζ (DGKζ), an enzyme that phosphorylates diacylglycerol to yield phosphatidic acid, regulates the activity of the Rho GTPases Rac1 and RhoA. DGKζ mRNA is highly expressed in several different colon cancer cell lines, as well as in colon cancer tissue relative to normal colonic epithelium, and thus may contribute to the metastatic process. To investigate potential roles of DGKζ in cancer metastasis, a cellular, isogenic model of human colorectal cancer metastatic transition was used. DGKζ protein levels, Rac1 and RhoA activity, and PAK phosphorylation were measured in the non-metastatic SW480 adenocarcinoma cell line and its highly metastatic variant, the SW620 line. The effect of DGKζ silencing on Rho GTPase activity and invasion through Matrigel-coated Transwell inserts was studied in SW620 cells. Invasiveness was also measured in PC-3 prostate cancer and MDA-MB-231 breast cancer cells depleted of DGKζ. DGKζ protein levels were elevated approximately 3-fold in SW620 cells compared to SW480 cells. There was a concomitant increase in active Rac1 in SW620 cells, as well as substantial increases in the expression and phosphorylation of the Rac1 effector PAK1. Similarly, RhoA activity and expression were increased in SW620 cells. Knockdown of DGKζ expression in SW620 cells by shRNA-mediated silencing significantly reduced Rac1 and RhoA activity and attenuated the invasiveness of SW620 cells in vitro. DGKζ silencing in highly metastatic MDA-MB-231 breast cancer cells and PC-3 prostate cancer cells also significantly attenuated

El vídeo, el cómic y el rap: una forma diferente de desbloquear al candidato del DELE

Directory of Open Access Journals (Sweden)

Florencia Battagliero Bocco

2017-11-01

Full Text Available Resumen: El objetivo del presente artículo es trabajar y reforzar las destrezas y competencias necesarias para aprobar el Diploma de Español como Lengua Extranjera. Para ello, se planteará una serie de recursos que buscan desbloquear al estudiante en un ambiente distendido, sobre todo los días previos al examen. La propuesta se centrará en la Prueba de Expresión e Interacción Orales, concretamente en la tarea 3 del nivel B1 y en la tarea 2 del nivel B2, que corresponden a la descripción de una fotografía. Se empleará, por una parte, un vídeo de carácter estático (donde el interlocutor no expresa movimientos corporales pronunciados y por otra, cómics de Argentina que a su vez servirán para ampliar el léxico en la variante rioplatense. Por último, se tratará el uso del rap como herramienta para mejorar la pronunciación, entonación y el empleo del lenguaje no verbal. Palabras claves: DELE, vídeo, cómic, rap.   Abstract: The purpose of the following essay is to train and to strengthen the skills and the competences required to pass the “Spanish as a Foreign Language Certificate”. In order to do so, we will first consider various resources which aim to release students from any kind of stress by setting up a relaxed atmosphere – especially a few days before the exam. The focus of the present essay has to be linked to the Expression and Oral Interaction tests and above all to the assignments 3 (B1 level and 2 (B2 level, and their picture descriptions. On the one hand, we will use a static video and, on the other hand, we will tackle Argentinian comics in order to expand students’ knowledge of the Río de la Plata lexicon. Finally, we will deal with the use of rap as a tool to improve pronunciation, intonation, and non-verbal communication. Key words: DELE, video, comic, rap.

A meta-analysis of genome-wide association scans identifies IL18RAP, PTPN2, TAGAP, and PUS10 as shared risk loci for Crohn's disease and celiac disease.

Directory of Open Access Journals (Sweden)

Eleonora A M Festen

2011-01-01

Full Text Available Crohn's disease (CD and celiac disease (CelD are chronic intestinal inflammatory diseases, involving genetic and environmental factors in their pathogenesis. The two diseases can co-occur within families, and studies suggest that CelD patients have a higher risk to develop CD than the general population. These observations suggest that CD and CelD may share common genetic risk loci. Two such shared loci, IL18RAP and PTPN2, have already been identified independently in these two diseases. The aim of our study was to explicitly identify shared risk loci for these diseases by combining results from genome-wide association study (GWAS datasets of CD and CelD. Specifically, GWAS results from CelD (768 cases, 1,422 controls and CD (3,230 cases, 4,829 controls were combined in a meta-analysis. Nine independent regions had nominal association p-value <1.0 x 10⁻⁵ in this meta-analysis and showed evidence of association to the individual diseases in the original scans (p-value < 1 x 10⁻² in CelD and < 1 x 10⁻³ in CD. These include the two previously reported shared loci, IL18RAP and PTPN2, with p-values of 3.37 x 10⁻⁸ and 6.39 x 10⁻⁹, respectively, in the meta-analysis. The other seven had not been reported as shared loci and thus were tested in additional CelD (3,149 cases and 4,714 controls and CD (1,835 cases and 1,669 controls cohorts. Two of these loci, TAGAP and PUS10, showed significant evidence of replication (Bonferroni corrected p-values <0.0071 in the combined CelD and CD replication cohorts and were firmly established as shared risk loci of genome-wide significance, with overall combined p-values of 1.55 x 10⁻¹⁰ and 1.38 x 10⁻¹¹ respectively. Through a meta-analysis of GWAS data from CD and CelD, we have identified four shared risk loci: PTPN2, IL18RAP, TAGAP, and PUS10. The combined analysis of the two datasets provided the power, lacking in the individual GWAS for single diseases, to detect shared loci with a

JMJD1C demethylates MDC1 to regulate the RNF8 and BRCA1-mediated chromatin response to DNA breaks

DEFF Research Database (Denmark)

Watanabe, Sugiko; Watanabe, Kenji; Akimov, Vyacheslav

2013-01-01

Chromatin ubiquitylation flanking DNA double-strand breaks (DSBs), mediated by RNF8 and RNF168 ubiquitin ligases, orchestrates a two-branch pathway, recruiting repair factors 53BP1 or the RAP80-BRCA1 complex. We report that human demethylase JMJD1C regulates the RAP80-BRCA1 branch of this DNA...

Neuronal Rac1 is required for learning-evoked neurogenesis

DEFF Research Database (Denmark)

Haditsch, Ursula; Anderson, Matthew P; Freewoman, Julia

2013-01-01

Hippocampus-dependent learning and memory relies on synaptic plasticity as well as network adaptations provided by the addition of adult-born neurons. We have previously shown that activity-induced intracellular signaling through the Rho family small GTPase Rac1 is necessary in forebrain projection...

O circuito rap “indé” em Paris: dinâmicas socioterritoriais e mensagem ultramar

Directory of Open Access Journals (Sweden)

Cristiano Nunes Alves

2016-05-01

Full Text Available Aborda-se o circuito rap independente em Paris, o chamado “rap indé”, produção musical da cultura hip hop, constituída por materialidades e fluxos dinamizados por agentes cujas raízes estão em territórios ultramarinos. Lançando mão de um levantamento documental e bibliográfico, e, de uma série de entrevistas e visitas técnicas, problematiza-se a relação do hip hop com o lugar, e propõe-se uma análise do rap indé a partir da teoria dos circuitos da economia urbana nos países subdesenvolvidos. Observa-se que o circuito indé, fortalecido na Île-de-France, sobretudo desde meados dos anos 1990, mobiliza toda a região, tendo em Clignancourt, importante lugar de encontro e articulação.  Sua produção dá-se em estúdios e selos de menor porte, caracterizando-se ainda por pequenas espessuras ligadas aos eventos musicais, e, divulgação e comercialização alternativas aos grandes circuitos da economia. Trata-se de um estudo buscando alternativas para pensar os modos de se analisar, a partir da música, as dinâmicas socioterritoriais na cidade contemporânea.

Rab GTPases Regulate Endothelial Cell Protein C Receptor-Mediated Endocytosis and Trafficking of Factor VIIa

Science.gov (United States)

Nayak, Ramesh C.; Keshava, Shiva; Esmon, Charles T.; Pendurthi, Usha R.; Rao, L. Vijaya Mohan

2013-01-01

Recent studies have established that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR may promote the endocytosis of this receptor/ligand complex. Rab GTPases are known to play a crucial role in the endocytic and exocytic pathways of receptors or receptor/ligand complexes. The present study was undertaken to investigate the role of Rab GTPases in the intracellular trafficking of EPCR and FVIIa. CHO-EPCR cells and human umbilical vein endothelial cells (HUVEC) were transduced with recombinant adenoviral vectors to express wild-type, constitutively active, or dominant negative mutant of various Rab GTPases. Cells were exposed to FVIIa conjugated with AF488 fluorescent probe (AF488-FVIIa), and intracellular trafficking of FVIIa, EPCR, and Rab proteins was evaluated by immunofluorescence confocal microscopy. In cells expressing wild-type or constitutively active Rab4A, internalized AF488-FVIIa accumulated in early/sorting endosomes and its entry into the recycling endosomal compartment (REC) was inhibited. Expression of constitutively active Rab5A induced large endosomal structures beneath the plasma membrane where EPCR and FVIIa accumulated. Dominant negative Rab5A inhibited the endocytosis of EPCR-FVIIa. Expression of constitutively active Rab11 resulted in retention of accumulated AF488-FVIIa in the REC, whereas expression of a dominant negative form of Rab11 led to accumulation of internalized FVIIa in the cytoplasm and prevented entry of internalized FVIIa into the REC. Expression of dominant negative Rab11 also inhibited the transport of FVIIa across the endothelium. Overall our data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR-FVIIa. PMID:23555015

Structural integrity test of Indian PHWR containment of Kaiga- 1 and 2 and RAPS- 3 and 4 atomic power stations

International Nuclear Information System (INIS)

Samota, Arun; Verma, U.S.P.; Warudkar, A.S.; Mohan, Nalini; Bhawal, R.N.; Bajaj, S.S.

2002-01-01

Full text: The structural integrity tests of Kaiga 1, 2 and RAPS 3, 4 primary containment were carried out as per requirements specified in RCC-G code by pressurizing the containment with oil free air at design pressure of 1.73 Kg/cm 2 (g). Dilations were monitored in vertical and radial directions by installing dial gauges at various locations on the outer surface of primary containment. The dilations were generally observed to be linear with the pressure and the residual displacements were within the acceptable limit. Strains were measured in meridional and circumferential directions at different pressure levels by using a large number of different types of strain gauge. While the behaviour of embedded type vibrating wire strain gauges was excellent, the behaviour of surface mounted electrical resistance (SMER) was satisfactory. Other parameters monitored were crack, differential and absolute settlements. No crack was noticed in any of the containment. No absolute and differential settlements were noticed. The results of the tests have demonstrated elastic behaviour of the containment. The acceptance criteria of the tests were met with good margin

A Novel Plasma Membrane-Anchored Protein Regulates Xylem Cell-Wall Deposition through Microtubule-Dependent Lateral Inhibition of Rho GTPase Domains.

Science.gov (United States)

Sugiyama, Yuki; Wakazaki, Mayumi; Toyooka, Kiminori; Fukuda, Hiroo; Oda, Yoshihisa

2017-08-21

Spatial control of cell-wall deposition is essential for determining plant cell shape [1]. Rho-type GTPases, together with the cortical cytoskeleton, play central roles in regulating cell-wall patterning [2]. In metaxylem vessel cells, which are the major components of xylem tissues, active ROP11 Rho GTPases form oval plasma membrane domains that locally disrupt cortical microtubules, thereby directing the formation of oval pits in secondary cell walls [3-5]. However, the regulatory mechanism that determines the planar shape of active Rho of Plants (ROP) domains is still unknown. Here we show that IQD13 associates with cortical microtubules and the plasma membrane to laterally restrict the localization of ROP GTPase domains, thereby directing the formation of oval secondary cell-wall pits. Loss and overexpression of IQD13 led to the formation of abnormally round and narrow secondary cell-wall pits, respectively. Ectopically expressed IQD13 increased the presence of parallel cortical microtubules by promoting microtubule rescue. A reconstructive approach revealed that IQD13 confines the area of active ROP domains within the lattice of the cortical microtubules, causing narrow ROP domains to form. This activity required the interaction of IQD13 with the plasma membrane. These findings suggest that IQD13 positively regulates microtubule dynamics as well as their linkage to the plasma membrane, which synergistically confines the area of active ROP domains, leading to the formation of oval secondary cell-wall pits. This finding sheds light on the role of microtubule-plasma membrane linkage as a lateral fence that determines the planar shape of Rho GTPase domains. Copyright © 2017 Elsevier Ltd. All rights reserved.

Rab GTPases and the Autophagy Pathway: Bacterial Targets for a Suitable Biogenesis and Trafficking of Their Own Vacuoles

Directory of Open Access Journals (Sweden)

María Milagros López de Armentia

2016-03-01

Full Text Available Autophagy is an intracellular process that comprises degradation of damaged organelles, protein aggregates and intracellular pathogens, having an important role in controlling the fate of invading microorganisms. Intracellular pathogens are internalized by professional and non-professional phagocytes, localizing in compartments called phagosomes. To degrade the internalized microorganism, the microbial phagosome matures by fusion events with early and late endosomal compartments and lysosomes, a process that is regulated by Rab GTPases. Interestingly, in order to survive and replicate in the phagosome, some pathogens employ different strategies to manipulate vesicular traffic, inhibiting phagolysosomal biogenesis (e.g., Staphylococcus aureus and Mycobacterium tuberculosis or surviving in acidic compartments and forming replicative vacuoles (e.g., Coxiella burnetti and Legionella pneumophila. The bacteria described in this review often use secretion systems to control the host’s response and thus disseminate. To date, eight types of secretion systems (Type I to Type VIII are known. Some of these systems are used by bacteria to translocate pathogenic proteins into the host cell and regulate replicative vacuole formation, apoptosis, cytokine responses, and autophagy. Herein, we have focused on how bacteria manipulate small Rab GTPases to control many of these processes. The growing knowledge in this field may facilitate the development of new treatments or contribute to the prevention of these types of bacterial infections.

Specific T-cell recognition of the merozoite proteins rhoptry-associated protein 1 and erythrocyte-binding antigen 1 of Plasmodium falciparum

DEFF Research Database (Denmark)

Jakobsen, P H; Hviid, L; Theander, T G

1993-01-01

The merozoite proteins merozoite surface protein 1 (MSP-1) and rhoptry-associated protein 1 (RAP-1) and synthetic peptides containing sequences of MSP-1, RAP-1, and erythrocyte-binding antigen 1, induced in vitro proliferative responses of lymphocytes collected from Ghanaian blood donors living i...... by individuals living in an area with a high transmission rate of malaria. Most of the donor plasma samples tested contained immunoglobulin G (IgG) and IgM antibodies recognizing the merozoite proteins, while only a minority showed high IgG reactivity to the synthetic peptides.......The merozoite proteins merozoite surface protein 1 (MSP-1) and rhoptry-associated protein 1 (RAP-1) and synthetic peptides containing sequences of MSP-1, RAP-1, and erythrocyte-binding antigen 1, induced in vitro proliferative responses of lymphocytes collected from Ghanaian blood donors living...

A demonstration of the applicability of implementing the enhanced Remedial Action Priority System (RAPS) for environmental releases

Energy Technology Data Exchange (ETDEWEB)

Whelan, G.; Droppo, J.G. Jr.; Strenge, D.L.; Walter, M.B.; Buck, J.W.

1989-12-01

The Remedial Action Priority System (RAPS) and the Multimedia Environmental Pollutant Assessment System (MEPAS) were developed to prioritize problems associated with potential releases of hazardous chemical and radioactive materials in a scientific and objective manner based on limited site information. This report documents the model testing efforts of the RAPS/MEPAS methodology for the atmospheric, surface water, groundwater, and exposure components. Comparisons are given of model outputs with measured data at three sites: the US Department of Energy's Mound facility in Ohio and Hanford facility in Washington, and a chromium-cadmium plating site in New York. The results show that the simulated magnitudes, spacial and temporal trends, and distributions of contaminants corresponded well with the measured data. 25 refs., 86 figs., 26 tabs.

Genetic characterization and molecular survey of Babesia sp. Xinjiang infection in small ruminants and ixodid ticks in China.

Science.gov (United States)

Niu, Qingli; Liu, Zhijie; Yang, Jifei; Gao, Shandian; Pan, Yuping; Guan, Guiquan; Luo, Jianxun; Yin, Hong

2017-04-01

Babesia sp. Xinjiang is a large ovine Babesia species that was recently isolated in China. Compared with other ovine Babesia species, it has different morphological features, pathogenicity and vector tick species. The known transmitting vector is Hyalomma anatolicum. In this study, the distribution and the presence of Babesia sp. Xinjiang in small ruminants and ixodid ticks in China were assessed by specific nested-PCR assay based on the rap-1a gene. A total of 978 blood samples from sheep or goats from 15 provinces and 797 tick specimens from vegetation from 10 provinces were collected and analysed for the presence of the Babesia sp. Xinjiang. Full-length and partial rap-1a of Babesia sp. Xinjiang were amplified from field samples. The PCR results were further confirmed by DNA sequencing. Overall, 38 (3.89%) blood samples and 51 (6.4%) tick samples were positive for Babesia sp. Xinjiang infection. The highest presence (26.92%) was found in blood samples from Yunnan province, while H. qinghaiensis ticks with the highest presence of infection (21.3%) were from Gansu province. This study identified for the first time Babesia sp. Xinjiang infection in H. longicornis tick species. The rap-1a sequences of Babesia sp. Xinjiang from field blood and tick samples indicated 100% identity. The presence of Babesia sp. Xinjiang infection may increase in China. Novel potential transmitting vectors might be more extensive than previously thought. Copyright © 2017. Published by Elsevier B.V.

From Violent Rap to Lovely Blues: The Transformation of Aggressive Behavior through Vocal Music Therapy.

NARCIS (Netherlands)

Meadows, Tony; Uhlig, S.

2011-01-01

The voice as a primary therapeutic instrument will be addressed in this chapter. Through vocal expression, chaos can be transformed into order – crying into singing, aggressive shouting into the structure of a rap song. This transformation of emotions demonstrates the ability to change behavior and

An Interview with Cathy Fowler about Sharing a Love of Reading through Book Raps.

Science.gov (United States)

Strangman, Nicole

2002-01-01

Includes an interview with Cathy Fowler, a Year 7 teacher at Kawungan State School in Queensland, Australia. Explains that Cathy is a participant and coordinator of the extremely popular Harry Potter Book Rap, a guided Internet book discussion among students all over the world. Discusses how this activity fueled her students' love for reading. (PM)

Neuronal Rho GTPase Rac1 elimination confers neuroprotection in a mouse model of permanent ischemic stroke.

Science.gov (United States)

Karabiyik, Cansu; Fernandes, Rui; Figueiredo, Francisco Rosário; Socodato, Renato; Brakebusch, Cord; Lambertsen, Kate Lykke; Relvas, João Bettencourt; Santos, Sofia Duque

2017-09-28

The Rho GTPase Rac1 is a multifunctional protein involved in distinct pathways ranging from development to pathology. The aim of the present study was to unravel the contribution of neuronal Rac1 in regulating the response to brain injury induced by permanent focal cerebral ischemia (pMCAO). Our results show that pMCAO significantly increased total Rac1 levels in wild type mice, mainly through rising nuclear Rac1, while a reduction in Rac1 activation was observed. Such changes preceded cell death induced by excitotoxic stress. Pharmacological inhibition of Rac1 in primary neuronal cortical cells prevented the increase in oxidative stress induced after overactivation of glutamate receptors. However, this was not sufficient to prevent the associated neuronal cell death. In contrast, RNAi-mediated knock down of Rac1 in primary cortical neurons prevented cell death elicited by glutamate excitotoxicity and decreased the activity of NADPH oxidase. To test whether in vivo down regulation of neuronal Rac1 was neuroprotective after pMCAO, we used tamoxifen-inducible neuron-specific conditional Rac1-knockout mice. We observed a significant 50% decrease in brain infarct volume of knockout mice and a concomitant increase in HIF-1α expression compared to littermate control mice, demonstrating that ablation of Rac1 in neurons is neuroprotective. Transmission electron microscopy performed in the ischemic brain showed that lysosomes in the infarct of Rac1- knockout mice were preserved at similar levels to those of non-infarcted tissue, while littermate mice displayed a decrease in the number of lysosomes, further corroborating the notion that Rac1 ablation in neurons is neuroprotective. Our results demonstrate that Rac1 plays important roles in the ischemic pathological cascade and that modulation of its levels is of therapeutic interest. © 2017 International Society of Neuropathology.

Quantification of local morphodynamics and local GTPase activity by edge evolution tracking.

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Yuki Tsukada

2008-11-01

Full Text Available Advances in time-lapse fluorescence microscopy have enabled us to directly observe dynamic cellular phenomena. Although the techniques themselves have promoted the understanding of dynamic cellular functions, the vast number of images acquired has generated a need for automated processing tools to extract statistical information. A problem underlying the analysis of time-lapse cell images is the lack of rigorous methods to extract morphodynamic properties. Here, we propose an algorithm called edge evolution tracking (EET to quantify the relationship between local morphological changes and local fluorescence intensities around a cell edge using time-lapse microscopy images. This algorithm enables us to trace the local edge extension and contraction by defining subdivided edges and their corresponding positions in successive frames. Thus, this algorithm enables the investigation of cross-correlations between local morphological changes and local intensity of fluorescent signals by considering the time shifts. By applying EET to fluorescence resonance energy transfer images of the Rho-family GTPases Rac1, Cdc42, and RhoA, we examined the cross-correlation between the local area difference and GTPase activity. The calculated correlations changed with time-shifts as expected, but surprisingly, the peak of the correlation coefficients appeared with a 6-8 min time shift of morphological changes and preceded the Rac1 or Cdc42 activities. Our method enables the quantification of the dynamics of local morphological change and local protein activity and statistical investigation of the relationship between them by considering time shifts in the relationship. Thus, this algorithm extends the value of time-lapse imaging data to better understand dynamics of cellular function.

Interaction between the p21ras GTPase activating protein and the insulin receptor

NARCIS (Netherlands)

Pronk, G.J.; Medema, R.H.; Burgering, B.M.T.; Clark, R.; McCormick, F.; Bos, J.L.

1992-01-01

We investigated the involvement of the p21ras-GTPase activating protein (GAP) in insulin-induced signal transduction. In cells overexpressing the insulin receptor, we did not observe association between GAP and the insulin receptor after insulin treatment nor the phosphorylation of GAP on tyrosine

Operating experience and radiation protection in RAPS-3 and 4 operations

International Nuclear Information System (INIS)

Khandelwal, Narendra; Dhakar, P.C.; Singh, G.K.; Gupta, Ashok

2008-01-01

Rajasthan Atomic Power Station (RAPS)-3 and 4 was designed and constructed using latest technological advancements in the field of nuclear energy. Operating experience of the station have taught many lessons and provided opportunities to take proactive corrective actions. Design modifications, effective implementation of radiological surveillance program and improvements in work culture have helped in achieving continual reduction in radiation exposures and effluent releases at the station. This paper discusses some of the modifications carried out at the station along with their radiological impacts. (author)

The monomeric GTPase RabA2 is required for progression and maintenance of membrane integrity of infection threads during root nodule symbiosis.

Science.gov (United States)

Dalla Via, Virginia; Traubenik, Soledad; Rivero, Claudio; Aguilar, O Mario; Zanetti, María Eugenia; Blanco, Flavio Antonio

2017-04-01

Progression of the infection canal that conducts rhizobia to the nodule primordium requires a functional Rab GTPase located in Golgi/trans-Golgi that also participate in root hair polar growth. Common bean (Phaseolus vulgaris) symbiotically associates with its partner Rhizobium etli, resulting in the formation of root nitrogen-fixing nodules. Compatible bacteria can reach cortical cells in a tightly regulated infection process, in which the specific recognition of signal molecules is a key step to select the symbiotic partner. In this work, we show that RabA2, a monomeric GTPase from common bean, is required for the progression of the infection canal, referred to as the infection thread (IT), toward the cortical cells. Expression of miss-regulated mutant variants of RabA2 resulted in an increased number of abortive infection events, including bursting of ITs and a reduction in the number of nodules. Nodules formed in these plants were small and contained infected cells with disrupted symbiosome membranes, indicating either early senescence of these cells or defects in the formation of the symbiosome membrane during bacterial release. RabA2 localized to mobile vesicles around the IT, but mutations that affect GTP hydrolysis or GTP/GDP exchange modified this localization. Colocalization of RabA2 with ArfA1 and a Golgi marker indicates that RabA2 localizes in Golgi stacks and the trans-Golgi network. Our results suggest that RabA2 is part of the vesicle transport events required to maintain the integrity of the membrane during IT progression.

Ran GTPase-activating protein 1 is a therapeutic target in diffuse large B-cell lymphoma.

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Kung-Chao Chang

Full Text Available Lymphoma-specific biomarkers contribute to therapeutic strategies and the study of tumorigenesis. Diffuse large B-cell lymphoma (DLBCL is the most common type of malignant lymphoma. However, only 50% of patients experience long-term survival after current treatment; therefore, developing novel therapeutic strategies is warranted. Comparative proteomic analysis of two DLBCL lines with a B-lymphoblastoid cell line (LCL showed differential expression of Ran GTPase-activating protein 1 (RanGAP1 between them, which was confirmed using immunoblotting. Immunostaining showed that the majority of DLBCLs (92%, 46/50 were RanGAP1(+, while reactive lymphoid hyperplasia (n = 12 was RanGAP1(+ predominantly in germinal centers. RanGAP1 was also highly expressed in other B-cell lymphomas (BCL, n = 180 with brisk mitotic activity (B-lymphoblastic lymphoma/leukemia: 93%, and Burkitt lymphoma: 95% or cell-cycle dysregulation (mantle cell lymphoma: 83%, and Hodgkin's lymphoma 91%. Interestingly, serum RanGAP1 level was higher in patients with high-grade BCL (1.71 ± 2.28 ng/mL, n = 62 than in low-grade BCL (0.75 ± 2.12 ng/mL, n = 52 and healthy controls (0.55 ± 1.58 ng/mL, n = 75 (high-grade BCL vs. low-grade BCL, p = 0.002; high-grade BCL vs. control, p < 0.001, Mann-Whitney U test. In vitro, RNA interference of RanGAP1 showed no effect on LCL but enhanced DLBCL cell death (41% vs. 60%; p = 0.035 and cell-cycle arrest (G0/G1: 39% vs. 49%, G2/M: 19.0% vs. 7.5%; p = 0.030 along with decreased expression of TPX2 and Aurora kinases, the central regulators of mitotic cell division. Furthermore, ON 01910.Na (Estybon, a multikinase inhibitor induced cell death, mitotic cell arrest, and hyperphosphorylation of RanGAP1 in DLBCL cell lines but no effects in normal B and T cells. Therefore, RanGAP1 is a promising marker and therapeutic target for aggressive B-cell lymphoma, especially DLBCL.

RAPS. A threedimensional plotprogram for testing of the model and for plotting of the results of finite-element calculations

International Nuclear Information System (INIS)

Koschmieder, D.; Altes, J.

1979-06-01

Usually in Finite-Element calculations a large amount of data is produced and because individual results have no meaning, graphic representation is bestsuited. It is convenient to link the F E Software-System with pre- and postprocessors. The plotting system RAPS, presented on the following pages, offers many possibilities for testing and description of two- or threedimensional structures, as well as for interpretation of results of static and dynamic calculations. The programm was developed for the F E System ASKA but it is possible to fit it to other F E Systems. At present the program is laid out for batchoperation. However it is planned to develop an interactive version of RAPS and to enlarge the postprocessor. (orig.) [de

Rac1 is dispensable for oocyte maturation and female fertility in vivo.

Science.gov (United States)

Hao, Jian-Xiu; Meng, Tie-Gang; Fan, Li-Hua; Yao, Yuan-Qing

2017-01-01

Oocyte maturation, the important process to produce female haploid gamete, accompanies with polarity establishment and highly asymmetric cell division to emit minor polar body within little cytoplasm. Microfilaments play central roles in polarity establishment and asymmetric cell division. Several actin regulators like WASP protein family as well as small GTPases function in microfilament dynamics, involving the process. Rac1, one member of RhoGTPases, has been reported to regulate the polarity and asymmetric cell division in mouse oocytes in vitro. The physiological role of Rac1 in mouse oocyte remains unknown. By conditional knockout technology, we specifically deleted Rac1 gene in mouse oocyte, and found that Rac1 deletion exerted little effect on mouse oocyte maturation including polarity establishment and asymmetric division, and the mutant mice showed normal fertility.

Vasoactive intestinal peptide-induced neurite remodeling in human neuroblastoma SH-SY5Y cells implicates the Cdc42 GTPase and is independent of Ras-ERK pathway

International Nuclear Information System (INIS)

Alleaume, Celine; Eychene, Alain; Harnois, Thomas; Bourmeyster, Nicolas; Constantin, Bruno; Caigneaux, Evelyne; Muller, Jean-Marc; Philippe, Michel

2004-01-01

Vasoactive intestinal peptide (VIP) is known to regulate proliferation or differentiation in normal and tumoral cells. SH-SY5Y is a differentiated cell subclone derived from the SK-N-SH human neuroblastoma cell line and possess all the components for an autocrine action of VIP. In the present study, we investigated the morphological changes and intracellular signaling pathways occurring upon VIP treatment of SH-SY5Y cells. VIP induced an early remodeling of cell projections: a branched neurite network spread out and prominent varicosities developed along neurites. Although activated by VIP, the Ras/ERK pathway was not required for the remodeling process. In contrast, pull-down experiments revealed a strong Cdc42 activation by VIP while expression of a dominant-negative Cdc42 prevented the VIP-induced neurite changes, suggesting an important role for this small GTPase in the process. These data provide the first evidence for a regulation of the activity of Rho family GTPases by VIP and bring new insights in the signaling pathways implicated in neurite remodeling process induced by VIP in neuroblastoma cells

The dual action of poly(ADP-ribose polymerase -1 (PARP-1 inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity

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Slava eRom

2015-08-01

Full Text Available The transcription of HIV-1 (HIV is regulated by complex mechanisms involving various cellular factors and virus-encoded transactivators. Poly(ADP-ribose polymerase 1 (PARP-1 inhibition has emerged recently as a potent anti-inflammatory tool, since PARP-1 is involved in the regulation of some genes through its interaction with various transcription factors. We propose a novel approach to diminish HIV replication via PARP-1 inhibition using human primary monocyte-derived macrophages (MDM as an in vitro model system. PARP-1 inhibitors were able to reduce HIV replication in MDM by 60-80% after 7 days infection. Long Terminal Repeat (LTR acts as a switch in virus replication and can be triggered by several agents such as: Tat, tumor necrosis factor α (TNFα, and phorbol 12-myristate 13-acetate (PMA. Overexpression of Tat in MDM transfected with an LTR reporter plasmid led to a 4.2-fold increase in LTR activation; PARP inhibition resulted in 70% reduction of LTR activity. LTR activity, which increased 3-fold after PMA or TNFα treatment, was reduced by PARP inhibition (by 85-95%. MDM treated with PARP inhibitors showed 90% reduction in NFκB activity (known to mediate PMA- and TNFα-induced HIV LTR activation. Cytoskeleton rearrangements are important in effective HIV-1 infection. PARP inactivation reduced actin cytoskeleton rearrangements by affecting Rho GTPase machinery. These findings suggest that HIV replication in MDM could be suppressed by PARP inhibition via NFκB suppression, diminution of LTR activation and its effects on the cytoskeleton. PARP appears to be essential for HIV replication and its inhibition may provide a potent approach to treatment of HIV infection.

Understanding cAMP-dependent allostery by NMR spectroscopy: comparative analysis of the EPAC1 cAMP-binding domain in its apo and cAMP-bound states.

Science.gov (United States)

Mazhab-Jafari, Mohammad T; Das, Rahul; Fotheringham, Steven A; SilDas, Soumita; Chowdhury, Somenath; Melacini, Giuseppe

2007-11-21

cAMP (adenosine 3',5'-cyclic monophosphate) is a ubiquitous second messenger that activates a multitude of essential cellular responses. Two key receptors for cAMP in eukaryotes are protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC), which is a recently discovered guanine nucleotide exchange factor (GEF) for the small GTPases Rap1 and Rap2. Previous attempts to investigate the mechanism of allosteric activation of eukaryotic cAMP-binding domains (CBDs) at atomic or residue resolution have been hampered by the instability of the apo form, which requires the use of mixed apo/holo systems, that have provided only a partial picture of the CBD apo state and of the allosteric networks controlled by cAMP. Here, we show that, unlike other eukaryotic CBDs, both apo and cAMP-bound states of the EPAC1 CBD are stable under our experimental conditions, providing a unique opportunity to define at an unprecedented level of detail the allosteric interactions linking two critical functional sites of this CBD. These are the phosphate binding cassette (PBC), where cAMP binds, and the N-terminal helical bundle (NTHB), which is the site of the inhibitory interactions between the regulatory and catalytic regions of EPAC. Specifically, the combined analysis of the cAMP-dependent changes in chemical shifts, 2 degrees structure probabilities, hydrogen/hydrogen exchange (H/H) and hydrogen/deuterium exchange (H/D) protection factors reveals that the long-range communication between the PBC and the NTHB is implemented by two distinct intramolecular cAMP-signaling pathways, respectively, mediated by the beta2-beta3 loop and the alpha6 helix. Docking of cAMP into the PBC perturbs the NTHB inner core packing and the helical probabilities of selected NTHB residues. The proposed model is consistent with the allosteric role previously hypothesized for L273 and F300 based on site-directed mutagenesis; however, our data show that such a contact is part of a

The 'invisible hand': regulation of RHO GTPases by RHOGDIs.

Science.gov (United States)

Garcia-Mata, Rafael; Boulter, Etienne; Burridge, Keith

2011-07-22

The 'invisible hand' is a term originally coined by Adam Smith in The Theory of Moral Sentiments to describe the forces of self-interest, competition and supply and demand that regulate the resources in society. This metaphor continues to be used by economists to describe the self-regulating nature of a market economy. The same metaphor can be used to describe the RHO-specific guanine nucleotide dissociation inhibitor (RHOGDI) family, which operates in the background, as an invisible hand, using similar forces to regulate the RHO GTPase cycle.

The invisible hand: regulation of RHO GTPases by RHOGDIs

Science.gov (United States)

Garcia-Mata, Rafael; Boulter, Etienne; Burridge, Keith

2011-01-01

Preface The 'invisible hand' is a term originally coined by Adam Smith in the Theory of Moral Sentiments to describe the forces of self-interest, competition, and supply and demand that regulate the resources in society. This metaphor continues to be used by economists to describe the self-regulating nature of a market economy. The same metaphor can be used to describe the RHO-specific guanine nucleotide dissociation inhibitor (RHOGDI) family, which operates in the background, as an invisible hand, using similar forces to regulate the RHO GTPase cycle. PMID:21779026

The GTPase Rab43 Controls the Anterograde ER-Golgi Trafficking and Sorting of GPCRs

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Chunman Li

2017-10-01

Full Text Available G-protein-coupled receptors (GPCRs constitute the largest superfamily of cell-surface signaling proteins. However, mechanisms underlying their surface targeting and sorting are poorly understood. Here, we screen the Rab family of small GTPases in the surface transport of multiple GPCRs. We find that manipulation of Rab43 function significantly alters the surface presentation and signaling of all GPCRs studied without affecting non-GPCR membrane proteins. Rab43 specifically regulates the transport of nascent GPCRs from the endoplasmic reticulum (ER to the Golgi. More interestingly, Rab43 directly interacts with GPCRs in an activation-dependent fashion. The Rab43-binding domain identified in the receptors effectively converts non-GPCR membrane protein transport into a Rab43-dependent pathway. These data reveal a crucial role for Rab43 in anterograde ER-Golgi transport of nascent GPCRs, as well as the ER sorting of GPCR members by virtue of its ability to interact directly.

Radiosensitivity of the swiss-rap mouse as a function of its growth rate

International Nuclear Information System (INIS)

Legeay, G.; Glas, J.F.

1969-01-01

The results of an exhaustive study of the age dependence of the radiosensitivity of female Swiss-Rap mice are given. A close relationship of radiosensitivity versus age could not be brought out, whereas the weekly growth rate could be accurately related to radiosensitivity. Thus, the latter should be studied when a strain is to be used for biological experiments, as the rates of growth are different with the strains. (author) [fr

Degrees of Intersectionality: Male Rap Artists in Sweden Negotiating Class, Race and Gender

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Kalle Berggren

2013-06-01

Full Text Available “Intersectionality” has become a highly influential concept in gender research over the last 25 years. Debates have focused on differences and power asymmetries between women, in terms of race but also addressing class, age, sexuality, ability and nation. However, intersectional paradigms have been used to a much lesser extent in gender studies on men. This article seeks to contribute to an emerging discussion about intersectionality and masculinity by analyzing rap lyrics in Swe-dish songs. The data consists of a broad sample of rap lyrics by male artists 1991-2011, which is analyzed through poststructuralist discourse analysis and queer phenomenology. The analysis shows how classed discourses can be described in terms of orientation and flow, how racialization is articulated in terms of place, and the role of normative notions of gender and sexuality in anti-racist discourses. It is argued that this interconnectedness – class being related to race, which in turn is profoundly gendered – is neither well captured by the prevailing notion of “masculinities” in gender studies on men, nor by the “constitution” vs. “addition” dichotomy in intersectionality debates. Instead, it is suggested that degrees of in-tersectionality might be a more fruitful way of theorizing intersectionality in rela-tion to men.

University-Community Collaboration to Promote Healthy Mothers and Infants: The Relationships and Parenting Support (RAPS) Program

Science.gov (United States)

Williams, Patricia Hrusa; Oravecz, Linda M.

2016-01-01

Research highlights the vulnerability of Black mothers and their infants, who experience higher rates of stress, preterm birth, low birth weight, and infant mortality than other racial groups. This article describes the development and implementation of the Relationships and Parenting Support (RAPS) Program, a community-based, family-focused…

Saccharomyces cerevisiae GTPase complex: Gtr1p-Gtr2p regulates cell-proliferation through Saccharomyces cerevisiae Ran-binding protein, Yrb2p

International Nuclear Information System (INIS)

Wang Yonggang; Nakashima, Nobutaka; Sekiguchi, Takeshi; Nishimoto, Takeharu

2005-01-01

A Gtr1p GTPase, the GDP mutant of which suppresses both temperature-sensitive mutants of Saccharomyces cerevisiae RanGEF/Prp20p and RanGAP/Rna1p, was presently found to interact with Yrb2p, the S. cerevisiae homologue of mammalian Ran-binding protein 3. Gtr1p bound the Ran-binding domain of Yrb2p. In contrast, Gtr2p, a partner of Gtr1p, did not bind Yrb2p, although it bound Gtr1p. A triple mutant: yrb2Δ gtr1Δ gtr2Δ was lethal, while a double mutant: gtr1Δ gtr2Δ survived well, indicating that Yrb2p protected cells from the killing effect of gtr1Δ gtr2Δ. Recombinant Gtr1p and Gtr2p were purified as a complex from Escherichia coli. The resulting Gtr1p-Gtr2p complex was comprised of an equal amount of Gtr1p and Gtr2p, which inhibited the Rna1p/Yrb2 dependent RanGAP activity. Thus, the Gtr1p-Gtr2p cycle was suggested to regulate the Ran cycle through Yrb2p

El rap español en el ámbito de los discursos de especialidad

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Francisco Jiménez Calderón

2012-11-01

Full Text Available Normal 0 21 false false false ES X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;} El presente artículo tiene como objetivo definir el rap español como un discurso de especialidad. Para ello, se revisa el concepto de ‘discurso de especialidad’, al que se alude con expresiones a menudo imprecisas como ‘lenguaje especial’, ‘lengua de especialidad’ o ‘jerga’. Ninguna, por distintas razones, parece adecuada; debe hablarse, en efecto, de ‘discurso de especialidad’, aunque una denominación como ‘discurso de actividad’ sería aún más precisa, pues nombraría el producto de comunicación que resulta de utilizar la lengua en un ámbito determinado, no necesariamente profesional, sino relativo a cualquier actividad concreta. Para situar al rap español en tal ámbito, se ha analizado un corpus de más de cien textos, que revela que, efectivamente, el rap presenta unos rasgos definitorios que le otorgan singularidad como discurso especial: terminología, desarrollo entre la escritura y la oralidad, reflexión metalingüística y acercamiento al género poético mediante el recurso a figuras retóricas consolidadas.

Escherichia coli α-hemolysin counteracts the anti-virulence innate immune response triggered by the Rho GTPase activating toxin CNF1 during bacteremia.

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Mamady Diabate

2015-03-01

Full Text Available The detection of the activities of pathogen-encoded virulence factors by the innate immune system has emerged as a new paradigm of pathogen recognition. Much remains to be determined with regard to the molecular and cellular components contributing to this defense mechanism in mammals and importance during infection. Here, we reveal the central role of the IL-1β signaling axis and Gr1+ cells in controlling the Escherichia coli burden in the blood in response to the sensing of the Rho GTPase-activating toxin CNF1. Consistently, this innate immune response is abrogated in caspase-1/11-impaired mice or following the treatment of infected mice with an IL-1β antagonist. In vitro experiments further revealed the synergistic effects of CNF1 and LPS in promoting the maturation/secretion of IL-1β and establishing the roles of Rac, ASC and caspase-1 in this pathway. Furthermore, we found that the α-hemolysin toxin inhibits IL-1β secretion without affecting the recruitment of Gr1+ cells. Here, we report the first example of anti-virulence-triggered immunity counteracted by a pore-forming toxin during bacteremia.

A Rac1--GDP trimer complex binds zinc with tetrahedral and octahedral coordination, displacing magnesium

Energy Technology Data Exchange (ETDEWEB)

Prehna, G.; Stebbins, C

2007-01-01

The Rho family of small GTPases represent well characterized signaling molecules that regulate many cellular functions such as actin cytoskeletal arrangement and the cell cycle by acting as molecular switches. A Rac1-GDP-Zn complex has been crystallized in space group P3221 and its crystal structure has been solved at 1.9 {angstrom} resolution. These trigonal crystals reveal the unexpected ability of Rac1 to coordinate Zn atoms in a tetrahedral fashion by use of its biologically relevant switch I and switch II regions. Upon coordination of zinc, the switch I region is stabilized in the GDP-bound conformation and contributes to a Rac1 trimer in the asymmetric unit. Zinc coordination causes switch II to adopt a novel conformation with a symmetry-related molecule. Additionally, zinc was found to displace magnesium from its octahedral coordination at switch I, although GDP binding remained stable. This structure represents the first reported Rac1-GDP-Zn complex, which further underscores the conformational flexibility and versatility of the small GTPase switch regions.

A Rac1-GDP Trimer Complex Binds Zinc with Tetrahedral and Octahedral Coordination, Displacing Magnesium

Energy Technology Data Exchange (ETDEWEB)

Prehna,G.; Stebbins, E.

2007-01-01

The Rho family of small GTPases represent well characterized signaling molecules that regulate many cellular functions such as actin cytoskeletal arrangement and the cell cycle by acting as molecular switches. A Rac1-GDP-Zn complex has been crystallized in space group P3{sub 2}21 and its crystal structure has been solved at 1.9 {angstrom} resolution. These trigonal crystals reveal the unexpected ability of Rac1 to coordinate Zn atoms in a tetrahedral fashion by use of its biologically relevant switch I and switch II regions. Upon coordination of zinc, the switch I region is stabilized in the GDP-bound conformation and contributes to a Rac1 trimer in the asymmetric unit. Zinc coordination causes switch II to adopt a novel conformation with a symmetry-related molecule. Additionally, zinc was found to displace magnesium from its octahedral coordination at switch I, although GDP binding remained stable. This structure represents the first reported Rac1-GDP-Zn complex, which further underscores the conformational flexibility and versatility of the small GTPase switch regions.

Degrees of Intersectionality: Male Rap Artists in Sweden Negotiating Class, Race and Gender

OpenAIRE

Kalle Berggren

2013-01-01

“Intersectionality” has become a highly influential concept in gender research over the last 25 years. Debates have focused on differences and power asymmetries between women, in terms of race but also addressing class, age, sexuality, ability and nation. However, intersectional paradigms have been used to a much lesser extent in gender studies on men. This article seeks to contribute to an emerging discussion about intersectionality and masculinity by analyzing rap lyrics in Swe-dish songs. ...

Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

International Nuclear Information System (INIS)

Tsukamoto, Yuta; Kagiwada, Satoshi; Shimazu, Sayuri; Takegawa, Kaoru; Noguchi, Tetsuko; Miyamoto, Masaaki

2015-01-01

The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed

Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

Energy Technology Data Exchange (ETDEWEB)

Tsukamoto, Yuta [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Kagiwada, Satoshi [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Shimazu, Sayuri [Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Takegawa, Kaoru [Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Noguchi, Tetsuko [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan)

2015-03-20

The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed.

Rap jako muzyka tożsamościowa: od czarnego getta do polskiego pop-nacjonalizmu

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Piotr Majewski

2016-01-01

Full Text Available Rap as identity music: from the black ghetto to Polish pop-nationalism In today’s world, cultural products, technologies, information and ideologies more and more permeate from one society to another, crossing all kinds of borders in the least expected way. Rap career is an illustration of this process. It was created in the late seventies and eighties of the twentieth century in New York ghettos and today it represents one of the most popular genres on the global scale. Rap is not only a symbol of revolution and the domination of Western capitalist business practices but also a cultural tool by which different groups, often marginalized or considering themselves as such, express their own identity. I am analyzing the indicated above phenomenon using two, extreme at first glance, examples. First, I present the story of the emergence and development of hip-hop culture in the United States. I try to show how rap music, which is an important element of this culture, allowed a marginalized part of American society for manifesting and communicating their views, beliefs and values, becoming a “transmission channel” for various ideologies, including the ideology of black nationalism. In the second part of the text, I am additionally analyzing the artwork and public appearances by Tadeusz “Tadek“ Polkowski, a Polish rapper, whom I consider a representative of an expanding hip-hop society relating to or sympathizing with the national movement. The music he creates is designed not only to restore the Poles’ “memory”, and therefore also the pride in their heroic and admirable past, but also to open their eyes to what is happening in their country that he believes is being colonized by the occupants.   Rap jako muzyka tożsamościowa: od czarnego getta do polskiego pop-nacjonalizmu We współczesnym świecie wytwory kultury, technologie, informacje i ideologie coraz częściej przenikają z jednego społeczeństwa do drugiego, przekraczaj

A Conserved Role for Atlastin GTPases in Regulating Lipid Droplet Size

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Robin W. Klemm

2013-05-01

Full Text Available Lipid droplets (LDs are the major fat storage organelles in eukaryotic cells, but how their size is regulated is unknown. Using genetic screens in C. elegans for LD morphology defects in intestinal cells, we found that mutations in atlastin, a GTPase required for homotypic fusion of endoplasmic reticulum (ER membranes, cause not only ER morphology defects, but also a reduction in LD size. Similar results were obtained after depletion of atlastin or expression of a dominant-negative mutant, whereas overexpression of atlastin had the opposite effect. Atlastin depletion in Drosophila fat bodies also reduced LD size and decreased triglycerides in whole animals, sensitizing them to starvation. In mammalian cells, co-overexpression of atlastin-1 and REEP1, a paralog of the ER tubule-shaping protein DP1/REEP5, generates large LDs. The effect of atlastin-1 on LD size correlates with its activity to promote membrane fusion in vitro. Our results indicate that atlastin-mediated fusion of ER membranes is important for LD size regulation.

RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

Energy Technology Data Exchange (ETDEWEB)

Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); James, John; Prescott, Alan [Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Haga, Ismar R. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); Beard, Philippa M., E-mail: pip.beard@roslin.ed.ac.uk [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom)

2015-01-15

Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.

NMR derived model of GTPase effector domain (GED self association: relevance to dynamin assembly.

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Swagata Chakraborty

Full Text Available Self-association of dynamin to form spiral structures around lipidic vesicles during endocytosis is largely mediated by its 'coiled coil' GTPase Effector Domain (GED, which, in vitro, self-associates into huge helical assemblies. Residue-level structural characterizations of these assemblies and understanding the process of association have remained a challenge. It is also impossible to get folded monomers in the solution phase. In this context, we have developed here a strategy to probe the self-association of GED by first dissociating the assembly using Dimethyl Sulfoxide (DMSO and then systematically monitoring the refolding into helix and concomitant re-association using NMR spectroscopy, as DMSO concentration is progressively reduced. The short segment, Arg109 - Met116, acts as the nucleation site for helix formation and self-association. Hydrophobic and complementary charge interactions on the surfaces drive self-association, as the helices elongate in both the directions resulting in an antiparallel stack. A small N-terminal segment remains floppy in the assembly. Following these and other published results on inter-domain interactions, we have proposed a plausible mode of dynamin self assembly.

La influencia de la Web 2.0 y sus condicionantes técnicos en la producción del videoclip de rap español

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Olga HEREDERO-DÍAZ

2017-07-01

Full Text Available El videoclip es un producto audiovisual definido a partir de sus fines, generalmente comerciales, pero también un ejercicio de expresión y experimentación artística. Tomando como punto de partida la transformación del modo de distribución y consumo del videoclip por la democratización del acceso a Internet del público juvenil, el objetivo principal de esta investigación será describir los cambios que han tenido lugar en la producción del videoclip de rap español de los últimos cinco años debido a la influencia de la Web 2.0 y sus condicionantes técnicos. A partir del análisis de los videoclips difundidos a través de la sección de rap del programa Ritmo Urbano de La 2 de RTVE durante sus cinco temporadas en antena (desde 2011 hasta 2016, se concluye que a día de hoy el vídeo musical de rap es un género que se produce mayoritariamente para Internet, lo que en el caso del videoclip de rap español ha supuesto una mayor autonomía y libertad creativa para los artistas, al tiempo que una merma en el estándar de calidad de la imagen y del sonido de las piezas finales que su público objetivo consume.

Yersinia outer protein YopE affects the actin cytoskeleton in Dictyostelium discoideum through targeting of multiple Rho family GTPases

LENUS (Irish Health Repository)

Vlahou, Georgia

2009-07-14

Abstract Background All human pathogenic Yersinia species share a virulence-associated type III secretion system that translocates Yersinia effector proteins into host cells to counteract infection-induced signaling responses and prevent phagocytosis. Dictyostelium discoideum has been recently used to study the effects of bacterial virulence factors produced by internalized pathogens. In this study we explored the potential of Dictyostelium as model organism for analyzing the effects of ectopically expressed Yersinia outer proteins (Yops). Results The Yersinia pseudotuberculosis virulence factors YopE, YopH, YopM and YopJ were expressed de novo within Dictyostelium and their effects on growth in axenic medium and on bacterial lawns were analyzed. No severe effect was observed for YopH, YopJ and YopM, but expression of YopE, which is a GTPase activating protein for Rho GTPases, was found to be highly detrimental. GFP-tagged YopE expressing cells had less conspicuous cortical actin accumulation and decreased amounts of F-actin. The actin polymerization response upon cAMP stimulation was impaired, although chemotaxis was unaffected. YopE also caused reduced uptake of yeast particles. These alterations are probably due to impaired Rac1 activation. We also found that YopE predominantly associates with intracellular membranes including the Golgi apparatus and inhibits the function of moderately overexpressed RacH. Conclusion The phenotype elicited by YopE in Dictyostelium can be explained, at least in part, by inactivation of one or more Rho family GTPases. It further demonstrates that the social amoeba Dictyostelium discoideum can be used as an efficient and easy-to-handle model organism in order to analyze the function of a translocated GAP protein of a human pathogen.

Development of a Novel NMR-based Rheb GTPase Assay and Molecular Characterization of TSC2 GAP Activity

Science.gov (United States)

2010-05-01

GTPase) that belongs to the Ras superfamily and has homologs in yeast, fungi , slime mold, fruit fly, zebra fish, and mammals (1–3). Ge- netic and...characterization of TSC2 disease mutations affecting its GAP activity (months 9-12) While the final aspects of this task are yet to be completed, we have...domain mutants of TSC2 that we examined affected its enzymatic activ- ity. This method can now be applied to study the function and regulation of other

Chlamydia abortus YhbZ, a truncated Obg family GTPase, associates with the Escherichia coli large ribosomal subunit.

Science.gov (United States)

Polkinghorne, Adam; Vaughan, Lloyd

2011-01-01

The stringent stress response is vital for bacterial survival under adverse environmental conditions. Obligate intracellular Chlamydia lack key stringent response proteins, but nevertheless can interrupt the cell cycle and enter stasis or persistence upon amino acid starvation. A possible key protein retained is YhbZ, a homologue of the ObgE guanosine triphosphatase (GTPase) superfamily connecting the stringent stress response to ribosome maturation. Curiously, chlamydial YhbZ lacks the ObgE C-terminal domain thought to be essential for binding the large ribosomal subunit. We expressed recombinant Chlamydia abortus YhbZ and showed it to be a functional GTPase, with similar activity to other Obg GTPase family members. As Chlamydia are resistant to genetic manipulation, we performed heterologous expression and gradient centrifugation experiments in Escherichia coli and found that, despite the missing C-terminal domain, C. abortus YhbZ co-fractionates with the E. coli 50S large ribosomal subunit. In addition, overexpression of chlamydial YhbZ in E. coli leads to growth defects and elongation, as reported for other Obg members. YhbZ did not complement an E. coli obgE temperature-sensitive mutant, indicating the C-terminal acidic domain may have an additional role. This data supports a role for YhbZ linking the chlamydial stress response to ribosome function and cellular growth. Copyright © 2010 Elsevier Ltd. All rights reserved.

O rap e o funk na socialização da juventude

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Dayrell Juarez

2002-01-01

Full Text Available O texto se propõe a discutir a importância dos grupos musicais juvenis nos processos de socialização vivenciados por jovens pobres na periferia de Belo Horizonte, problematizando o peso e o significado de ser membro de um grupo musical no conjunto da vida de cada um. Tem como foco os integrantes de três grupos de rap e três duplas de funk, procurando analisar as suas experiências culturais e o sentido que tais práticas adquirem no conjunto dos processos sociais que os constituem como sujeitos. Significa compreender como eles elaboram as suas vivências em torno do estilo, e os significados que atribuem a elas, no contexto social onde se inserem como jovens pobres. A discussão aponta que os jovens rappers e funkeiros encontram poucos espaços nas instituições do mundo adulto para construir referências e valores por meio dos quais possam se construir como sujeitos. Os estilos rap e funk assumem uma centralidade na vida desses jovens por intermédio das formas de sociabilidade que constroem, da música que criam, e dos eventos culturais que promovem. Esses estilos possibilitaram e vem possibilitando a esses jovens práticas, relações e símbolos por meio dos quais criam espaços próprios, significando uma referência na elaboração e vivência da sua condição juvenil, além de proporcionar a construção de uma auto-estima e identidades positivas.

Multivalent adhesion molecule 7 clusters act as signaling platform for host cellular GTPase activation and facilitate epithelial barrier dysfunction.

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Jenson Lim

2014-09-01

Full Text Available Vibrio parahaemolyticus is an emerging bacterial pathogen which colonizes the gastrointestinal tract and can cause severe enteritis and bacteraemia. During infection, V. parahaemolyticus primarily attaches to the small intestine, where it causes extensive tissue damage and compromises epithelial barrier integrity. We have previously described that Multivalent Adhesion Molecule (MAM 7 contributes to initial attachment of V. parahaemolyticus to epithelial cells. Here we show that the bacterial adhesin, through multivalent interactions between surface-induced adhesin clusters and phosphatidic acid lipids in the host cell membrane, induces activation of the small GTPase RhoA and actin rearrangements in host cells. In infection studies with V. parahaemolyticus we further demonstrate that adhesin-triggered activation of the ROCK/LIMK signaling axis is sufficient to redistribute tight junction proteins, leading to a loss of epithelial barrier function. Taken together, these findings show an unprecedented mechanism by which an adhesin acts as assembly platform for a host cellular signaling pathway, which ultimately facilitates breaching of the epithelial barrier by a bacterial pathogen.

Slit-Robo GTPase-Activating Protein 2 as a metastasis suppressor in osteosarcoma

OpenAIRE

Marko, Tracy A.; Shamsan, Ghaidan A.; Edwards, Elizabeth N.; Hazelton, Paige E.; Rathe, Susan K.; Cornax, Ingrid; Overn, Paula R.; Varshney, Jyotika; Diessner, Brandon J.; Moriarity, Branden S.; O?Sullivan, M. Gerard; Odde, David J.; Largaespada, David A.

2016-01-01

Osteosarcoma is the most common primary bone tumor, with metastatic disease responsible for most treatment failure and patient death. A forward genetic screen utilizing Sleeping Beauty mutagenesis in mice previously identified potential genetic drivers of osteosarcoma metastasis, including Slit-Robo GTPase-Activating Protein 2 (Srgap2). This study evaluates the potential role of SRGAP2 in metastases-associated properties of osteosarcoma cell lines through Srgap2 knockout via the CRISPR/Cas9 n...

Consideration on application of RAP to G-II NPP

International Nuclear Information System (INIS)

Shen Shen

2010-01-01

The design (D-RAP) for nuclear power plant has been adopted internationally in new-build advanced nuclear power plant design, which increases the ability of the risk-significant SSCs to carry out its functions during the accident circumstances especially in the severe accident situation, and reduces the risk of a nuclear power plant. The concept of reliability assurance program for advanced nuclear power plant can also be applied to nuclear power plants which second-generation NPP technology is used. Through analysis and research, the risk-significant SSCs in actual NPP can also be screened, and these SSCs can be managed appropriately, so that can improve the overall plant ability to against the severe accident, reduce the risk, and improve their safety and economy. Also such technology used does not exist any insurmountable technical difficulties and a lot of money input. (authors)