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.

Genome analysis of urease positive Serratia marcescens, co-producing SRT-2 and AAC(6')-Ic with multidrug efflux pumps for antimicrobial resistance.

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Srinivasan, Vijaya Bharathi; Rajamohan, Govindan

2018-04-05

In this study, we present the genome sequence of Serratia marcescens SM03, recovered from a human gut in India. The final assembly consists of 26 scaffolds (4620 coding DNA sequences, 5.08 Mb, 59.6% G + C ratio) and 79 tRNA genes. Analysis identified novel genes associated with lactose utilization, virulence, P-loop GTPases involved in urease production, CFA/I fimbriae apparatus and Yersinia - type CRISPR proteins. Antibiotic susceptibility testing indicated drug tolerant phenotype and inhibition assays demonstrated involvement of extrusion in resistance. Presence of enzymes SRT-2, AAC(6')-Ic, with additional Ybh transporter and EamA-like efflux pumps signifies the genetic plasticity observed in S. marcescens SM03. Copyright © 2018 Elsevier Inc. All rights reserved.

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.

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

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

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.

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.

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.

Concept and set-up of an IR-gas sensor construction kit

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Sieber, Ingo; Perner, Gernot; Gengenbach, Ulrich

2015-10-01

The paper presents an approach to a cost-efficient modularly built non-dispersive optical IR-gas sensor (NDIR) based on a construction kit. The modularity of the approach offers several advantages: First of all it allows for an adaptation of the performance of the gas sensor to individual specifications by choosing the suitable modular components. The sensitivity of the sensor e.g. can be altered by selecting a source which emits a favorable wavelength spectrum with respect to the absorption spectrum of the gas to be measured or by tuning the measuring distance (ray path inside the medium to be measured). Furthermore the developed approach is very well suited to be used in teaching. Together with students a construction kit on basis of an optical free space system was developed and partly implemented to be further used as a teaching and training aid for bachelor and master students at our institute. The components of the construction kit are interchangeable and freely fixable on a base plate. The components are classified into five groups: sources, reflectors, detectors, gas feed, and analysis cell. Source, detector, and the positions of the components are fundamental to experiment and test different configurations and beam paths. The reflectors are implemented by an aluminum coated adhesive foil, mounted onto a support structure fabricated by additive manufacturing. This approach allows derivation of the reflecting surface geometry from the optical design tool and generating the 3D-printing files by applying related design rules. The rapid fabrication process and the adjustment of the modules on the base plate allow rapid, almost LEGO®-like, experimental assessment of design ideas. Subject of this paper is modeling, design, and optimization of the reflective optical components, as well as of the optical subsystem. The realization of a sample set-up used as a teaching aid and the optical measurement of the beam path in comparison to the simulation results are

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.

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

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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.

Chlamydia abortus YhbZ, a truncated Obg family GTPase, associates with the Escherichia coli large ribosomal subunit.

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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.

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.

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.

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

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

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

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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.

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.

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.

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.

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,

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

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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.

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).

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

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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.

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.

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.

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.

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.

Small GTPases and Stress Responses of vvran1 in the Straw Mushroom Volvariella volvacea

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Jun-Jie Yan

2016-09-01

Full Text Available Small GTPases play important roles in the growth, development and environmental responses of eukaryotes. Based on the genomic sequence of the straw mushroom Volvariella volvacea, 44 small GTPases were identified. A clustering analysis using human small GTPases as the references revealed that V. volvacea small GTPases can be grouped into five families: nine are in the Ras family, 10 are in the Rho family, 15 are in the Rab family, one is in the Ran family and nine are in the Arf family. The transcription of vvran1 was up-regulated upon hydrogen peroxide (H2O2 stress, and could be repressed by diphenyleneiodonium chloride (DPI, a NADPH oxidase-specific inhibitor. The number of vvran1 transcripts also increased upon cold stress. Diphenyleneiodonium chloride, but not the superoxide dismutase (SOD inhibitor diethy dithiocarbamate (DDC, could suppress the up-regulation of vvran1 gene expression to cold stress. These results combined with the high correlations between gene expression and superoxide anion (O2− generation indicated that vvran1 could be one of the candidate genes in the downstream of O2− mediated pathways that are generated by NADPH oxidase under low temperature and oxidative stresses.

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....

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.

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.

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

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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.

Induction of nonapoptotic cell death by activated Ras requires inverse regulation of Rac1 and Arf6.

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

2010-10-01

Methuosis is a unique form of nonapoptotic 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 which graded expression of active H-Ras(G12V) triggers cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state. Blocking Rac1 activation with the specific Rac inhibitor, EHT 1864, or coexpression of dominant-negative Rac1(T17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activation, H-Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in the recycling of clathrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicating that the decrease in Arf6-GTP is directly linked to the activation of Rac1. Constitutively active Rac1(G12V) interacts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by short hairpin RNA prevents the decrease in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1(G12V). Together, the results suggest that perturbations of endosome morphology associated with Ras-induced methuosis are due to downstream activation of Rac1 combined with reciprocal inactivation of Arf6. The latter seems to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could suggest opportunities for the induction of methuosis in cancers that are resistant to apoptotic cell death.

The GTPase Rab37 Participates in the Control of Insulin Exocytosis.

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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.

Starch Digestibility and Physicochemical and Cooking Properties of Irradiated Rice Grains

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Luís Fernando Polesi

2017-01-01

Full Text Available This study evaluated the starch digestibility, physicochemical properties and cooking characteristics of polished rice (varieties IRGA417 and IAC202 subjected to the doses of 0 (as the control, 1, 2 and 5 kGy of gamma radiation. The highest dose decreased the apparent amylose content, peak viscosity, water absorption and volume expansion. Irradiation increased the solid loss by 119% and 187% for IAC202 and IRGA417, respectively, when comparing the higher dose with the control. For IAC202, irradiation decreased the rapidly digestible starch and increased the slowly digestible starch (SDS and resistant starch (RS. IRGA417 showed an elevation of SDS and a reduction in RS. And 1 kGy dose of gamma radiation generated the highest level of RS for both the two varieties and presented the smallest changes in other physicochemical and cooking properties.

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

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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 which graded expression of active H-Ras(G12V) triggers cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state. Blocking Rac1 activation with the specific Rac inhibitor, EHT 1864, or co-expression of dominant-negative Rac1(T17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activation, H-Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in recycling of clathrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicating that the decrease in Arf6-GTP is directly linked to activation of Rac1. Constitutively active Rac1(G12V) interacts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by shRNA prevents the decrease in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1(G12V). Together the results suggest that perturbations of endosome morphology associated with Ras-induced methuosis are due to downstream activation of Rac1, combined with reciprocal inactivation of Arf6. The latter appears to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could suggest opportunities for induction of methuosis in cancers that are resistant to apoptotic cell death. PMID:20713492

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...

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

FtsZ from radiation resistant bacterium Deinococcus radiodurans is different from its characterized homologues

International Nuclear Information System (INIS)

Mehta, Kruti P.; Misra, H.S.

2012-01-01

Polymerization/depolymerization dynamics of FtsZ and its GTPase activity are interdependent and the regulation of these processes determines the growth rate in a bacterium. Deinococcus radiodurans R1 that is best known for its extraordinary radiation resistance and efficient DNA double strand break repair is a comparatively slow growing bacterium and its growth gets arrested in response to gamma radiation. Mechanisms of cell division and its regulation under gamma stressed growth condition would be worth investigating. Genome of this bacterium encodes at least all the known components of divisome. Recombinant FtsZ of D. radiodurans (drFtsZ) preferred Mg 2+ for its GTPase activity. Relatively a very low GTPase activity was observed in presence of Mn 2+ , Co 2+ and Ni 2+ while release of inorganic phosphate could not be detected in presence of other divalent ions including Ca 2+ . GTPase activity of drFtsZ was lower than E. coli but higher than Mycobacterium and it required both Mg 2+ and GTP for its polymerization. Its GTPase activity did not increase with increasing concentration of Mg 2+ and correlates with the bundling of protofilaments. Results obtained from transmission electron microscopy and sedimentation analysis supported the reciprocal correlation of polymerization/depolymerization with the levels of GTPase activity. Dynamic light scattering in presence of 5mM or higher concentration of Mg 2+ and Mn 2 showed a characteristic cyclic change in light scattering without addition of extra metal ion or GTP

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.

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.

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

Science.gov (United States)

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

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

Directory of Open Access Journals (Sweden)

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.

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

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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

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

[Establishment of human multidrug-resistant lung carcinoma cell line (D6/MVP)].

Science.gov (United States)

Ma, Sheng-lin; Feng, Jian-guo; Gu, Lin-hui; Ling, Yu-tian

2003-03-01

To establish human multidrug-resistant lung carcinoma cell line (D6/MVP) with its characteristics studied. Intermittent administration of high-dose MMC, VDS and DDP (MVP) was used to induce human lung carcinoma cell line (D6) to a multidrug-resistant variety (D6/MVP). MTT assay was used to study the multidrug resistance of D6/MVP to multianticarcinogen. Flow cytometry was used to study the cell cycle distribution and the expression of P-gp, multidrug resistance-associated protein (MRP) and GSH/GST. 1. D6/MVP was resistant to many anti-tumor agents, with the IC(50) 13.3 times higher and the drug resistance 2 - 6 times higher than D6, 2. The multiplication time of D6/MVP was prolonged and the cell number of S-phase decreased while that of G1- and G(2)-phase increased and 3. The expression of P-gp and MRP was enhanced significantly (96.2% vs 51.7%), but the expression of GSH/GST kept stable. D6/MVP is a multidrug-resistant cell line possessing the basic characteristics of drug-resistance.

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.

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

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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

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

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.

Nucleophosmin1 is a negative regulator of the small GTPase Rac1

NARCIS (Netherlands)

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

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.

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.

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

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

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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

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.

Overexpression of Rad in muscle worsens diet-induced insulin resistance and glucose intolerance and lowers plasma triglyceride level

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Ilany, Jacob; Bilan, Philip J.; Kapur, Sonia; Caldwell, James S.; Patti, Mary-Elizabeth; Marette, Andre; Kahn, C. Ronald

2006-03-01

Rad is a low molecular weight GTPase that is overexpressed in skeletal muscle of some patients with type 2 diabetes mellitus and/or obesity. Overexpression of Rad in adipocytes and muscle cells in culture results in diminished insulin-stimulated glucose uptake. To further elucidate the potential role of Rad in vivo, we have generated transgenic (tg) mice that overexpress Rad in muscle using the muscle creatine kinase (MCK) promoter-enhancer. Rad tg mice have a 6- to 12-fold increase in Rad expression in muscle as compared to wild-type littermates. Rad tg mice grow normally and have normal glucose tolerance and insulin sensitivity, but have reduced plasma triglyceride levels. On a high-fat diet, Rad tg mice develop more severe glucose intolerance than the wild-type mice; this is due to increased insulin resistance in muscle, as exemplified by a rightward shift in the dose-response curve for insulin stimulated 2-deoxyglucose uptake. There is also a unexpected further reduction of the plasma triglyceride levels that is associated with increased levels of lipoprotein lipase in the Rad tg mice. These results demonstrate a potential synergistic interaction between increased expression of Rad and high-fat diet in creation of insulin resistance and altered lipid metabolism present in type 2 diabetes. diabetes mellitus | glucose transport | RGK GTPase | transgenic mouse

EFFECT OF NITROGEN-FIXING BACTERIA ON GRAIN YIELD AND DEVELOPMENT OF FLOODED IRRIGATED RICE

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AMAURI NELSON BEUTLER

2016-01-01

Full Text Available This study aimed at evaluating the effect of Azospirillum brasilense , a nitrogen - fixing bacterium, on flooded irrigated rice yield. Evaluations were carried out in a shaded nursery, with seedlings grown on an Alfisol. Were performed two sets of experiments. In the first, were carried out four experiments using the flooded rice cultivars INIA Olimar, Puitá Inta - CL, Br Irga 409 and Irga 424; these trials were set up as completely randomized design in a 5x4 factorial scheme, with four replications. Treatments consisted of five nitrogen rates (0, 40, 80, 120 and 160 kg ha - 1 and four levels of liquid inoculant Ab - V5 and Ab - V6 - A. brasilense (0, 1, 2 and 4 times the manufacturer's recommendation without seed treatment. In second set, were performed two experiments using the cultivars Puitá Inta - CL and Br Irga 409, arranged in the same design, but using a 4x2 factorial. In this set, treatments were composed of four levels of Ab - V5 and Ab - V6 - A. brasilense liquid inoculant (0, 1, 2 and 4 times the recommendation of 100 mL ha - 1 , using rice seeds with and without insecticide and fungicide treatment. Shoot dry matter, number of panicles, and rice grain yield per pot were the assessed variables. The results showed that rice seed inoculation with A. brasilense had no effects on rice grain yield of the cultivars INIA Olimar, Puitá Inta - CL, Br Irga 409 and Irga 424.

The Ins and Outs of Small GTPase Rac1 in the Vasculature

NARCIS (Netherlands)

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

Chemical composition and structural changes of irrigated rice during storageComposição química e alterações estruturais do arroz irrigado durante o armazenamento

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Tiago André Kaminski

2013-06-01

Full Text Available The effect storage time at different temperatures (0.5, 20 and 35 °C was periodically evaluated in regard to the chemical composition, elongation capacity and viscoamylographic properties of three irrigated rice varieties (BR-IRGA 410, IRGA 416 and IRGA 417 during 180 days. The alterations in the components of the polished grains were significant (P O efeito do tempo de armazenamento em diferentes temperaturas (0,5; 20 e 35°C foi avaliado sobre a composição química, capacidade de elongação e propriedades viscoamilográficas de três variedades de arroz irrigado (BR-IRGA 410, IRGA 416 e IRGA 417, durante 180 dias. As alterações nos componentes dos grãos polidos foram significativas (P<0,05 para o teor de matéria mineral, compostos fenólicos, amilose e amido resistente, com incrementos nos valores encontrados, principalmente em maiores períodos e temperaturas de armazenamento. As variáveis do experimento (variedade do arroz irrigado, tempo e temperatura do armazenamento influenciaram significativamente (P<0,01 nos parâmetros de elongação dos grãos, com aumentos expressivos durante o armazenamento a 35°C e nas propriedades viscoamilográficas, ou seja, observou-se maior viscosidade, resistência à quebra e retrogradação para as amostras com maior teor de amilose e mantidas a 35°C durante o armazenamento. As alterações nos componentes dos grãos, quando significativas (P<0,05, foram bastante discretas e não diretamente relacionadas às significativas alterações (P<0,01 de elongação e viscosidade. A temperatura se mostrou uma variável de grande influência no processo de envelhecimento do arroz, sugerindo a possibilidade do armazenamento deste cereal em maiores temperaturas para conferir qualidade culinária em menor tempo de armazenamento.

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

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

Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes.

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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.

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.

The 'invisible hand': regulation of RHO GTPases by RHOGDIs.

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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

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

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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.

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.

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.

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

Science.gov (United States)

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.

Molecular mapping of a sunflower rust resistance gene from HAR6.

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Bulos, Mariano; Ramos, María L; Altieri, Emiliano; Sala, Carlos A

2013-03-01

Sunflower rust, caused by Puccinia helianthi Schw., can result in significant yield losses in cultivated sunflower (Helianthus annuus L. var. macrocarpus Ckll.). HAR6 is a germplasm population resistant to most predominant rust races. The objectives of this study were to map the resistance factor present in HAR6 (R HAR6 ), and to provide and validate molecular tools for the identification of this gene for marker assisted selection purposes. Virulence reaction of seedlings for the F2 population and F2:3 families suggested that a single dominant gene confers rust resistance in HAR6-1, a selected rust resistance line from the original population. Genetic mapping with eight markers covered 97.4 cM of genetic distance on linkage group 13 of the sunflower consensus map. A co-dominant marker ZVG61 is the closest marker distal to R HAR6 at a genetic distance of 0.7 cM, while ORS581, a dominant marker linked in the coupling phase, is proximal to R HAR6 at a genetic distance of 1.5 cM. Validation of these markers was assessed by converting a susceptible line into a rust resistant isoline by means of marker assisted backcrossing. The application of these results to assist the breeding process and to design new strategies for rust control in sunflower is discussed.

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...

Racl Signaling Is Required for Insulin-Stimulated Glucose Uptake and Is Dysregulated in Insulin-Resistant Murine and Human Skeletal Muscle

DEFF Research Database (Denmark)

Sylow, L.; Jensen, T. E.; Kleinert, M.

2013-01-01

The actin cytoskeleton-regulating GTPase Racl is required for insulin-stimulated GLUT4 translocation in cultured muscle cells. However, involvement of Racl and its downstream signaling in glucose transport in insulin-sensitive and insulin-resistant mature skeletal muscle has not previously been i...

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.

MSH6 mutations arise in glioblastomas during temozolomide therapy and mediate temozolomide resistance

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Yip, Stephen; Miao, Jiangyong; Cahill, Daniel P.; Iafrate, A. John; Aldape, Ken; Nutt, Catherine L.; Louis, David N.

2009-01-01

Purpose Over the past few years, the alkylating agent temozolomide (TMZ) has become the standard-of-care therapy for patients with glioblastoma, the most common brain tumor. Recently, large-scale cancer genome sequencing efforts have identified a hypermutation phenotype and inactivating MSH6 mismatch repair gene mutations in recurrent, post-TMZ glioblastomas, particularly those growing more rapidly during TMZ treatment. This study aimed to clarify the timing and role of MSH6 mutations in mediating glioblastoma TMZ resistance. Experimental Design MSH6 sequence and microsatellite instability (MSI) status were determined in matched pre- and post-chemotherapy glioblastomas identified by The Cancer Genome Atlas (TCGA) as having post-treatment MSH6 mutations. TMZ-resistant lines were derived in vitro via selective growth under TMZ and the MSH6 gene was sequenced in resistant clones. The role of MSH6 inactivation in mediating resistance was explored using lentiviral shRNA knockdown and MSH6 reconstitution. Results MSH6 mutations were confirmed in post-treatment TCGA glioblastomas but absent in matched pre-treatment tumors. The post-treatment hypermutation phenotype displayed a signature bias toward CpC transitions and was not associated with MSI. In vitro modeling via exposure of an MSH6-wildtype glioblastoma line to TMZ resulted in resistant clones; one clone showed an MSH6 mutation, Thr1219Ile, that had been independently noted in two treated TCGA glioblastomas. Knockdown of MSH6 in the glioblastoma line U251 increased resistance to TMZ cytotoxicity and reconstitution restored cytotoxicity in MSH6-null glioma cells. Conclusions MSH6 mutations are selected for in glioblastomas during TMZ therapy both in vitro and in vivo, and are causally associated with TMZ resistance. PMID:19584161

Suscetibilidade de biótipos de arroz-vermelho e de cultivares de arroz irrigado ao herbicida imazethapyr Susceptibility of red rice biotypes and commercial rice cultivars to imazethapyr

Directory of Open Access Journals (Sweden)

S.H.B. Dornelles

2010-01-01

417. The methodology of dose-response curves proposed by Seefeldt et al. (1995 was applied. The methodology of response curves was generated from the parameters of the logistic model and the values of I50. The red rice biotypes and cultivars were subjected to six doses of the herbicide imazethapyr (0, 33.12, 66.25, 132.5, 265.0 and 530.0 g a.i. ha-1. Rice plants were counted and collected on the 20th day after treatment application. Damage percentage analysis was performed by visual assessment of phytotoxicity (%, plant green mass, and plant dry mass. Through the curves and results of the analysis of variance, it could be inferred that the cultivars Irga Clearfield CL 422 and CL Puit INTA were significantly equal to red rice biotype Catuçaba 1, resisting to imazethapyr doses higher than those recommended to the Match Clearfield ® system in the field. Biotypes Manoel Viana 2, Santa Maria 5, and Pelotas 3 grouped with the conventional cultivar IRGA 417, and were susceptible to the commercial dose of the herbicide. Rio Pardo 1 biotype is also resistant to the herbicide imazethapyr, but less than biotype Catuçaba 1.

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.

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.

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.

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...

Yb{sub 6}Ir{sub 5}Ga{sub 7} - a MgZn{sub 2} superstructure

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Seidel, Stefan; Poettgen, Rainer [Institut fuer Anorganische und Analytische Chemie, Universitaet Muenster, Corrensstrasse 30, 48149, Muenster (Germany)

2017-02-15

The gallide Yb{sub 6}Ir{sub 5}Ga{sub 7} was synthesized by high-frequency melting of the elements in a sealed niobium ampoule. The structure was refined from single-crystal X-ray diffractometer data: Nb{sub 6.4}Ir{sub 4}Al{sub 7.6} type, P6{sub 3}/mcm, a = 930.4(1), c = 843.0(1) pm, wR{sub 2} = 0.0597, 379 F{sup 2} values and 22 variables. Yb{sub 6}Ir{sub 5}Ga{sub 7} adopts a superstructure of the MgZn{sub 2} Laves phase by a complete ordering of the iridium and gallium atoms on the zinc substructure, i.e. the network consists of ordered and condensed Ir{sub 3}Ga and IrGa{sub 3} tetrahedra with Ir-Ga distances ranging from 260 to 265 pm. The crystal chemical details and the underlying group-subgroup scheme are discussed. (copyright 2017 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

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...

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

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.

Comprehensive behavioral analysis of mice deficient in Rapgef2 and Rapgef6, a subfamily of guanine nucleotide exchange factors for Rap small GTPases possessing the Ras/Rap-associating domain.

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Maeta, Kazuhiro; Hattori, Satoko; Ikutomo, Junji; Edamatsu, Hironori; Bilasy, Shymaa E; Miyakawa, Tsuyoshi; Kataoka, Tohru

2018-05-10

Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap small GTPases, characterized by the possession of the Ras/Rap-associating domain. Previous genomic analyses suggested their possible involvement in the etiology of schizophrenia. We recently demonstrated the development of an ectopic cortical mass (ECM), which resembles the human subcortical band heterotopia, in the dorsal telencephalon-specific Rapgef2 conditional knockout (Rapgef2-cKO) brains. Additional knockout of Rapgef6 in Rapgef2-cKO mice resulted in gross enlargement of the ECM whereas knockout of Rapgef6 alone (Rapgef6-KO) had no discernible effect on the brain morphology. Here, we performed a battery of behavioral tests to examine the effects of Rapgef2 or Rapgef6 deficiency on higher brain functions. Rapgef2-cKO mice exhibited hyperlocomotion phenotypes. They showed decreased anxiety-like behavior in the elevated plus maze and the open-field tests as well as increased depression-like behavior in the Porsolt forced swim and tail suspension tests. They also exhibited increased sociability especially in novel environments. They showed defects in cognitive function as evidenced by reduced learning ability in the Barnes circular maze test and by impaired working memory in the T maze tests. In contrast, although Rapgef6 and Rapgef2 share similarities in biochemical roles, Rapgef6-KO mice exhibited mild behavioral abnormalities detected with a number of behavioral tests, such as hyperlocomotion phenotype in the open-field test and the social interaction test with a novel environment and working-memory defects in the T-maze test. In conclusion, although there were differences in their brain morphology and the magnitude of the behavioral abnormalities, Rapgef2-cKO mice and Rapgef6-KO mice exhibited hyperlocomotion phenotype and working-memory defect, both of which could be recognized as schizophrenia-like behavior.

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...

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

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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.

Abnormally banded chromosomal regions in doxorubicin-resistant B16-BL6 murine melanoma cells.

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Slovak, M L; Hoeltge, G A; Ganapathi, R

1986-08-01

B16-BL6 murine melanoma cells were selected for cytogenetic evaluation during the stepwise development of increasing resistance in vitro to the antitumor antibiotic, doxorubicin (DOX). Karyotypic studies demonstrated extensive heteroploidy with both numerical and structural abnormalities which were not present in the parental DOX-sensitive B16-BL6 cells. Trypsin-Giemsa banding revealed the presence of several marker chromosomes containing abnormally banding regions (ABRs) in the 44-fold B16-BL6 DOX-resistant subline. These ABRs appeared to be more homogeneously staining at the higher DOX concentrations. Length measurements (ABR index) in seven banded metaphases indicated a direct correlation with increasing DOX concentration. When the DOX-resistant cells were grown in drug-free medium for 1 yr, the drug-resistant phenotype gradually declined in parallel with the level of resistance and the ABR index. DOX-induced cytogenetic damage examined by sister chromatid exchange methodology in parental B16-BL6 cells indicated a linear sister chromatid exchange:DOX dose-response relationship. However, after continuous treatment of parental B16-BL6 cells with DOX (0.01 microgram/ml) for 30 days, sister chromatid exchange scores were found to return to base-line values. The B16-BL6 resistant cells demonstrated a cross-resistant phenotype with N-trifluoroacetyladriamycin-14-valerate, actinomycin D, and the Vinca alkaloids but not with 1-beta-D-arabinofuranosylcytosine. The results suggest that ABR-containing chromosomes in DOX-resistant sublines may represent cytogenetic alterations of specific amplified genes involved in the expression of DOX resistance. Further studies are required to identify and define the possible gene products and to correlate their relationship to the cytotoxic action of doxorubicin.

Tetrahymena dynamin-related protein 6 self-assembles ...

Indian Academy of Sciences (India)

Usha P Kar

2017-12-30

Dec 30, 2017 ... multi-domain proteins, and share similar domain architecture. Classical dynamins ... domains: a GTPase domain, middle domain (MD), GTPase ..... influenced by bacterial environment, we have expressed human dynamin in ...

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...

The cytochrome P450 CYP6P4 is responsible for the high pyrethroid resistance in knockdown resistance-free Anopheles arabiensis.

Science.gov (United States)

Ibrahim, Sulaiman S; Riveron, Jacob M; Stott, Robert; Irving, Helen; Wondji, Charles S

2016-01-01

Pyrethroid insecticides are the front line vector control tools used in bed nets to reduce malaria transmission and its burden. However, resistance in major vectors such as Anopheles arabiensis is posing a serious challenge to the success of malaria control. Herein, we elucidated the molecular and biochemical basis of pyrethroid resistance in a knockdown resistance-free Anopheles arabiensis population from Chad, Central Africa. Using heterologous expression of P450s in Escherichia coli coupled with metabolism assays we established that the over-expressed P450 CYP6P4, located in the major pyrethroid resistance (rp1) quantitative trait locus (QTL), is responsible for resistance to Type I and Type II pyrethroid insecticides, with the exception of deltamethrin, in correlation with field resistance profile. However, CYP6P4 exhibited no metabolic activity towards non-pyrethroid insecticides, including DDT, bendiocarb, propoxur and malathion. Combining fluorescent probes inhibition assays with molecular docking simulation, we established that CYP6P4 can bind deltamethrin but cannot metabolise it. This is possibly due to steric hindrance because of the large vdW radius of bromine atoms of the dihalovinyl group of deltamethrin which docks into the heme catalytic centre. The establishment of CYP6P4 as a partial pyrethroid resistance gene explained the observed field resistance to permethrin, and its inability to metabolise deltamethrin probably explained the high mortality from deltamethrin exposure in the field populations of this Sudano-Sahelian An. arabiensis. These findings describe the heterogeneity in resistance towards insecticides, even from the same class, highlighting the need to thoroughly understand the molecular basis of resistance before implementing resistance management/control tools. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

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 ...

Mutation spectrum in the large GTPase dynamin 2, and genotype-phenotype correlation in autosomal dominant centronuclear myopathy

DEFF Research Database (Denmark)

Böhm, Johann; Biancalana, Valérie; Dechene, Elizabeth T

2012-01-01

Centronuclear myopathy (CNM) is a genetically heterogeneous disorder associated with general skeletal muscle weakness, type I fiber predominance and atrophy, and abnormally centralized nuclei. Autosomal dominant CNM is due to mutations in the large GTPase dynamin 2 (DNM2), a mechanochemical enzym...

Spontaneous focal activation of invariant natural killer T (iNKT cells in mouse liver and kidney

Directory of Open Access Journals (Sweden)

Zeng Jia

2010-11-01

Full Text Available Abstract Background Invariant natural killer T (iNKT cells differ from other T cells by their hyperactive effector T-cell status, in addition to the expression of NK lineage receptors and semi-invariant T-cell receptors. It is generally agreed that the immune phenotype of iNKT cells is maintained by repeated activation in peripheral tissues although no explicit evidence for such iNKT cell activity in vivo has so far been reported. Results We used an interferon (IFN-γ-inducible cytoplasmic protein, Irga6, as a histological marker for local IFN-γ production. Irga6 was intensely expressed in small foci of liver parenchymal cells and kidney tubular epithelium. Focal Irga6 expression was unaffected by germ-free status or loss of TLR signalling and was totally dependent on IFN-γ secreted by T cells in the centres of expression foci. These were shown to be iNKT cells by diagnostic T cell receptor usage and their activity was lost in both CD1 d and Jα-deficient mice. Conclusions This is the first report that supplies direct evidence for explicit activation events of NKT cells in vivo and raises issues about the triggering mechanism and consequences for immune functions in liver and kidney.

The effects of 6-mercaptopurine nucleotide derivatives on the growth and survival of 6-mercaptopurine-sensitive and -resistant cell culture lines.

OpenAIRE

Johnston, H. P.; Hawley, P.; White, S. E.; Gibson, I.; Tidd, D. M.

1985-01-01

6-Mercaptopurine (MP)-sensitive and -resistant cell culture lines were used to further characterize the apparent ability of MP nucleotide derivatives to overcome resistance to the parent drug. 6-Mercaptopurine-9-beta-D-ribofuranoside 5'-monophosphate [MPRP], bis(6-mercaptopurine-9-beta-D-ribofuranoside)-5', 5"'-monophosphate [bis(MPR)P], bis(O2',O3'-dibutyryl-6-mercaptopurine-9-beta-D-ribofuranoside)-5', 5"'-monophosphate [bis(dibut.MPR)P], and O2',O3'-dibutyryl-6-mercaptopurine-9-beta-D-ribo...

Nonuniformities of electrical resistivity in undoped 6H-SiC wafers

International Nuclear Information System (INIS)

Li, Q.; Polyakov, A.Y.; Skowronski, M.; Sanchez, E.K.; Loboda, M.J.; Fanton, M.A.; Bogart, T.; Gamble, R.D.

2005-01-01

Chemical elemental analysis, temperature-dependent Hall measurements, deep-level transient spectroscopy, and contactless resistivity mapping were performed on undoped semi-insulating (SI) and lightly nitrogen-doped conducting 6H-SiC crystals grown by physical vapor transport (PVT). Resistivity maps of commercial semi-insulating SiC wafers revealed resistivity variations across the wafers between one and two orders of magnitude. Two major types of variations were identified. First is the U-shape distribution with low resistivity in the center and high in the periphery of the wafer. The second type had an inverted U-shape distribution. Secondary-ion-mass spectrometry measurements of the distribution of nitrogen concentration along the growth axis and across the wafers sliced from different locations of lightly nitrogen-doped 6H-SiC boules were conducted. The measured nitrogen concentration gradually decreased along the growth direction and from the center to the periphery of the wafers. This change gives rise to the U-like distribution of resistivity in wafers of undoped SI-SiC. The concentrations of deep electron traps exhibited similar dependence. Compensation of nitrogen donors by these traps can result in the inverted U-like distribution of resistivity. Possible reasons for the observed nonuniformities include formation of a (0001) facet in PVT growth coupled with orientation-dependent nitrogen incorporation, systematic changes of the gas phase composition, and increase of the deposition temperature during boule growth

RhoGDI: multiple functions in the regulation of Rho family GTPase activities

DEFF Research Database (Denmark)

Dovas, Athanassios; Couchman, John R

2005-01-01

necessary for the correct targeting and regulation of Rho activities by conferring cues for spatial restriction, guidance and availability to effectors. These potential functions are discussed in the context of RhoGDI-associated multimolecular complexes, the newly emerged shuttling capability...... insight as to how RhoGDI exerts its effects on nucleotide binding, the membrane association-dissociation cycling of the GTPase and how these activities are controlled. Despite the initial negative roles attributed to RhoGDI, recent evidence has come to suggest that it may also act as a positive regulator...... of activities....

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

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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

Cisgenic Rvi6 scab-resistant apple lines show no differences in Rvi6 transcription when compared with conventionally bred cultivars.

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Chizzali, Cornelia; Gusberti, Michele; Schouten, Henk J; Gessler, Cesare; Broggini, Giovanni A L

2016-03-01

The expression of the apple scab resistance gene Rvi6 in different apple cultivars and lines is not modulated by biotic or abiotic factors. All commercially important apple cultivars are susceptible to Venturia inaequalis, the causal organism of apple scab. A limited number of apple cultivars were bred to express the resistance gene Vf from the wild apple genotype Malus floribunda 821. Positional cloning of the Vf locus allowed the identification of the Rvi6 (formerly HcrVf2) scab resistance gene that was subsequently used to generate cisgenic apple lines. It is important to understand and compare how this resistance gene is transcribed and modulated during infection in conventionally bred cultivars and in cisgenic lines. The aim of this work was to study the transcription pattern of Rvi6 in three classically bred apple cultivars and six lines of 'Gala' genetically modified to express Rvi6. Rvi6 transcription was analyzed at two time points using quantitative real-time PCR (RT-qPCR) following inoculation with V. inaequalis conidia or water. Rvi6 transcription was assessed in relation to five reference genes. β-Actin, RNAPol, and UBC were the most suited to performing RT-qPCR experiments on Malus × domestica. Inoculation with V. inaequalis conidia under conditions conducive to scab infection failed to produce any significant changes to the transcription level of Rvi6. Rvi6 expression levels were inconsistent in response to external treatments in the different apple cultivars, and transgenic, intragenic or cisgenic lines.

Enhanced accumulation of atropine in Atropa belladonna transformed by Rac GTPase gene isolated from Scoparia dulcis.

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Asano, Kyouhei; Lee, Jung-Bum; Yamamura, Yoshimi; Kurosaki, Fumiya

2013-12-01

Leaf tissues of Atropa belladonna were transformed by Sdrac2, a Rac GTPase gene, that is isolated from Scoparia dulcis, and the change in atropine concentration of the transformants was examined. Re-differentiated A. belladonna overexpressing Sdrac2 accumulated considerable concentration of atropine in the leaf tissues, whereas the leaves of plants transformed by an empty vector accumulated only a very low concentration of the compound. A. belladonna transformed by CASdrac2, a modified Sdrac2 of which translate was expected to bind guanosine triphosphate (GTP) permanently, accumulated very high concentrations of atropine (approximately 2.4-fold excess to those found in the wild-type plant in its natural habitat). In sharp contrast, the atropine concentration in transformed A. belladonna prepared with negatively modified Sdrac2, DNSdrac2, expected to bind guanosine diphosphate instead of GTP, was very low. These results suggested that Rac GTPases play an important role in the regulation of secondary metabolism in plant cells and that overexpression of the gene(s) may be capable of enhancing the production of natural products accumulated in higher plant cells.

The Rho-GTPase binding protein IQGAP2 is required for the glomerular filtration barrier.

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Sugano, Yuya; Lindenmeyer, Maja T; Auberger, Ines; Ziegler, Urs; Segerer, Stephan; Cohen, Clemens D; Neuhauss, Stephan C F; Loffing, Johannes

2015-11-01

Podocyte dysfunction impairs the size selectivity of the glomerular filter, leading to proteinuria, hypoalbuminuria, and edema, clinically defined as nephrotic syndrome. Hereditary forms of nephrotic syndrome are linked to mutations in podocyte-specific genes. To identify genes contributing to podocyte dysfunction in acquired nephrotic syndrome, we studied human glomerular gene expression data sets for glomerular-enriched gene transcripts differentially regulated between pretransplant biopsy samples and biopsies from patients with nephrotic syndrome. Candidate genes were screened by in situ hybridization for expression in the zebrafish pronephros, an easy-to-use in vivo assay system to assess podocyte function. One glomerulus-enriched product was the Rho-GTPase binding protein, IQGAP2. Immunohistochemistry found a strong presence of IQGAP2 in normal human and zebrafish podocytes. In zebrafish larvae, morpholino-based knockdown of iqgap2 caused a mild foot process effacement of zebrafish podocytes and a cystic dilation of the urinary space of Bowman's capsule upon onset of urinary filtration. Moreover, the glomerulus of zebrafish morphants showed a glomerular permeability for injected high-molecular-weight dextrans, indicating an impaired size selectivity of the glomerular filter. Thus, IQGAP2 is a Rho-GTPase binding protein, highly abundant in human and zebrafish podocytes, which controls normal podocyte structure and function as evidenced in the zebrafish pronephros.

Resistance monitoring and cross-resistance role of CYP6CW1 between buprofezin and pymetrozine in field populations of Laodelphax striatellus (Fallén).

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Zhang, Yueliang; Han, Yangchun; Liu, Baosheng; Yang, Qiong; Guo, Huifang; Liu, Zewen; Wang, Lihua; Fang, Jichao

2017-11-07

Monitoring resistance and investigating insecticide resistance mechanisms are necessary for controlling the small brown planthopper, Laodelphax striatellus. The susceptibility to four common insecticides of L. striatellus collected from Jiangsu, Anhui, Zhejiang and Jilin provinces of China in 2015 was monitored. The results showed that all field populations remained susceptible to chlorpyrifos and thiamethoxam with resistance ratios (RRs) of 2.3- to 9.5 and 1.6- to 3.3, respectively, while the insects had developed moderate pymetrozine resistance with RRs of 18.7 to 34.5. Resistance against buprofezin had developed to an alarmingly high level in three southeastern provinces of China with RRs of 108.8 to 156.1, but in Jilin it had an RR of only 26.6. Moreover, in line with both the buprofezin and pymetrozine resistance levels, we found LsCYP6CW1 to be over-expressed in all field L. striatellus populations, which indicated that it might be important for cross-resistance between buprofezin and pymetrozine. RNA interference (RNAi) ingestion resulted in the effective suppression of LsCYP6CW1 expression, and significantly increased susceptibility to both buprofezin and pymetrozine compared with the control, which further confirmed that overexpression of LsCYP6CW1 was involved in the cross-resistance to buprofezin and pymetrozine in field L. Striatellus populations.

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

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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.

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

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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

PlexinA2 Forward Signaling through Rap1 GTPases Regulates Dentate Gyrus Development and Schizophrenia-like Behaviors

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Xiao-Feng Zhao

2018-01-01

Full Text Available Summary: Dentate gyrus (DG development requires specification of granule cell (GC progenitors in the hippocampal neuroepithelium, as well as their proliferation and migration into the primordial DG. We identify the Plexin family members Plxna2 and Plxna4 as important regulators of DG development. Distribution of immature GCs is regulated by Sema5A signaling through PlxnA2 and requires a functional PlxnA2 GTPase-activating protein (GAP domain and Rap1 small GTPases. In adult Plxna2−/− but not Plxna2-GAP-deficient mice, the dentate GC layer is severely malformed, neurogenesis is compromised, and mossy fibers form aberrant synaptic boutons within CA3. Behavioral studies with Plxna2−/− mice revealed deficits in associative learning, sociability, and sensorimotor gating—traits commonly observed in neuropsychiatric disorder. Remarkably, while morphological defects are minimal in Plxna2-GAP-deficient brains, defects in fear memory and sensorimotor gating persist. Since allelic variants of human PLXNA2 and RAP1 associate with schizophrenia, our studies identify a biochemical pathway important for brain development and mental health. : Zhao et al. find that Sema5A-PlexinA2 forward signaling through Rap1 GTPases is required for progenitor distribution in the developing mouse dentate gyrus. Adult Plxna2−/−, but not Plxna2-GAP-deficient, mice show defects in dentate morphology, neurogenesis, and mossy fiber connectivity. Plxna2−/− and Plxna2-GAP mice exhibit behavioral defects suggestive of neuropsychiatric illness. Keywords: PlexinA2, semaphoring, Rap1, GAP, dentate gyrus, adult neurogenesis, mossy fiber, fear memory, sensorimotor gating, schizophrenia

A GTPase chimera illustrates an uncoupled nucleotide affinity and release rate, Providing insight into the activation mechanism

DEFF Research Database (Denmark)

Guilfoyle, Amy P.; Deshpande, Chandrika N.; Font Sadurni, Josep

2014-01-01

, biophysical studies on both the eukaryotic Gα proteins and the GTPase domain (NFeoB) of prokaryotic FeoB proteins have revealed conformational changes in the G5 loop that accompany nucleotide binding and release. However, it is unclear whether this conformational change in the G5 loop is a prerequisite...

IL6 induces TAM resistance via kinase-specific phosphorylation of ERα in OVCA cells.

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Wang, Yue; Niu, Xiu Long; Guo, Xiao Qin; Yang, Jing; Li, Ling; Qu, Ye; Xiu Hu, Cun; Mao, Li Qun; Wang, Dan

2015-06-01

About 40-60% of ovarian cancer (OVCA) cases express ERα, but only a small proportion of patients respond clinically to anti-estrogen treatment with estrogen receptor (ER) antagonist tamoxifen (TAM). The mechanism of TAM resistance in the course of OVCA progression remains unclear. However, IL6 plays a critical role in the development and progression of OVCA. Our recent results indicated that IL6 secreted by OVCA cells may promote the resistance of these cells to TAM via ER isoforms and steroid hormone receptor coactivator-1. Here we demonstrate that both exogenous (a relatively short period of treatment with recombinant IL6) and endogenous IL6 (generated as a result of transfection with a plasmid encoding sense IL6) increases expression of pERα-Ser118 and pERα-Ser167 in non-IL6-expressing A2780 cells, while deleting endogenous IL6 expression in IL6-overexpressing CAOV-3 cells (by transfection with a plasmid encoding antisense IL6) reduces expression of pERα-Ser118 and pERα-Ser167, indicating that IL6-induced TAM resistance may also be associated with increased expression of pERα-Ser118 and pERα-Ser167 in OVCA cells. Results of further investigation indicate that IL6 phosphorylates ERα at Ser118 and Ser167 by triggering activation of MEK/ERK and phosphotidylinositol 3 kinase/Akt signaling, respectively, to activate the ER pathway and thereby induce OVCA cells resistance to TAM. These results indicate that IL6 secreted by OVCA cells may also contribute to the refractoriness of these cells to TAM via the crosstalk between ER and IL6-mediated intracellular signal transduction cascades. Overexpression of IL6 not only plays an important role in OVCA progression but also promotes TAM resistance. Our results indicate that TAM-IL6-targeted adjunctive therapy may lead to a more effective intervention than TAM alone. © 2015 Society for Endocrinology.

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

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

The roles of CYP6AY1 and CYP6ER1 in imidacloprid resistance in the brown planthopper: Expression levels and detoxification efficiency.

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Bao, Haibo; Gao, Hongli; Zhang, Yixi; Fan, Dongzhe; Fang, Jichao; Liu, Zewen

2016-05-01

Two P450 monooxygenase genes, CYP6AY1 and CYP6ER1, were reported to contribute importantly to imidacloprid resistance in the brown planthopper, Nilaparvata lugens. Although recombinant CYP6AY1 could metabolize imidacloprid efficiently, the expression levels of CYP6ER1 gene were higher in most resistant populations. In the present study, three field populations were collected from different countries, and the bioassay, RNAi and imidacloprid metabolism were performed to evaluate the importance of two P450s in imidacloprid resistance. All three populations, DOT (Dongtai) from China, CNA (Chainat) from Thailand and HCM (Ho Chi Minh) from Vietnam, showed high resistance to imidacloprid (57.0-, 102.9- and 89.0-fold). CYP6AY1 and CYP6ER1 were both over expressed in three populations, with highest ratio of 13.2-fold for CYP6ER1 in HCM population. Synergism test and RNAi analysis confirmed the roles of both P450 genes in imidacloprid resistance. However, CYP6AY1 was indicated more important in CNA population, and CYP6AY1 and CYP6ER1 were equal in HCM population, although the expression level of CYP6ER1 (13.2-fold) was much higher than that of CYP6AY1 (4.11-fold) in HCM population. Although the recombinant proteins of both P450 genes could metabolize imidacloprid efficiently, the catalytic activity of CYP6AY1 (Kcat=3.627 pmol/min/pmol P450) was significantly higher than that of CYP6ER1 (Kcat=2.785 pmol/min/pmol P450). It was supposed that both P450 proteins were important for imidacloprid resistance, in which CYP6AY1 metabolized imidacloprid more efficiently and CYP6ER1 gene could be regulated by imidacloprid to a higher level. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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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.

Resistivity switching properties of Li-doped ZnO films deposited on LaB_6 electrode

International Nuclear Information System (INIS)

Igityan, A.; Kafadaryan, Y.; Aghamalyan, N.; Petrosyan, S.; Badalyan, G.; Vardanyan, V.; Nersisyan, M.; Hovsepyan, R.; Palagushkin, A.; Kryzhanovsky, B.

2015-01-01

Current–voltage (I–V) characteristics of Al/p-ZnO:Li/LaB_6 device, measured in voltage sweep mode, show unipolar resistive switching and monostable threshold switching (URS and MTS) for different bias voltage polarities. URS could be transformed to MTS by application of reverse bias voltage. With increasing number of cycles, URS is converted to bipolar resistive switching mode which is lost after certain number of cycles, and device turns into an ordinary resistor. Analysis of linear fitting I–V curves suggests that ohmic and space charge limited current laws are responsible for conductivity mechanism of Al/p-ZnO:Li/LaB_6 device. - Highlights: • Al/p-ZnO:Li/LaB_6 memristive device is fabricated using an e-beam evaporation technique. • Current–voltage (I–V) characteristics are studied. • Type of resistive switching mode depends on the bias voltage polarity and number of switching cycles. • Resistive switching in Al/ZnO:Li/LaB_6 has an interfacial effect. • Ohmic and SCLC laws are responsible for conductivity mechanism of resistive states.

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.

Therapeutic efficacy of interleukin-2 activated killer cells against adriamycin resistant mouse B16-BL6 melanoma.

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Gautam, S C; Chikkala, N F; Lewis, I; Grabowski, D R; Finke, J H; Ganapathi, R

1992-01-01

Development of multidrug-resistance (MDR) remains a major cause of failure in the treatment of cancer with chemotherapeutic agents. In our efforts to explore alternative treatment regimens for multidrug-resistant tumors we have examined the sensitivity of MDR tumor cell lines to lymphokine activated killer (LAK) cells. Adriamycin (ADM) resistant B16-BL6 melanoma, L1210 and P388 leukemic cell lines were tested for sensitivity to lysis by LAK cells in vitro. While ADM-resistant B16-BL6 and L1210 sublines were found to exhibit at least 2-fold greater susceptibility to lysis by LAK cells, sensitivity of ADM-resistant P388 cell was similar to that of parental cells. Since ADM-resistant B16-BL6 cells were efficiently lysed by LAK cells in vitro, the efficacy of therapy with LAK cells against the ADM-resistant B16-BL6 subline in vivo was evaluated. Compared to mice bearing parental B16-BL6 tumor cells, the adoptive transfer of LAK cells and rIL2 significantly reduced formation of experimental metastases (P less than 0.009) and extended median survival time (P less than 0.001) of mice bearing ADM-resistant B16-BL6 tumor cells. Results suggest that immunotherapy with LAK cells and rIL2 may be a useful modality in the treatment of cancers with the MDR phenotype.

The establishment of insulin resistance model in FL83B and L6 cell

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Liu, Lanlan; Han, Jizhong; Li, Haoran; Liu, Mengmeng; Zeng, Bin

2017-10-01

The insulin resistance models of mouse liver epithelial and rat myoblasts cells were induced by three kinds of inducers: dexamethasone, high insulin and high glucose. The purpose is to select the optimal insulin resistance model, to provide a simple and reliable TR cell model for the study of the pathogenesis of TR and the improvement of TR drugs and functional foods. The MTT method is used for toxicity screening of three compounds, selecting security and suitable concentration. We performed a Glucose oxidase peroxidase (GOD-POD) method involving FL83B and L6 cell with dexamethasone, high insulin and high glucose-induced insulin resistance. Results suggested that FL83B cells with dexamethasone-induced (0.25uM) were established insulin resistance and L6 cells with high-glucose (30mM) and dexamethasone-induced (0.25uM) were established insulin resistance.

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

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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.

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

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

Characterization in vitro and in vivo of progressively adriamycin-resistant B16-BL6 mouse melanoma cells.

Science.gov (United States)

Ganapathi, R; Grabowski, D; Schmidt, H; Bell, D; Melia, M

1987-07-01

Adriamycin (ADR)-resistant sublines of B16-BL6 mouse melanoma selected by exposure to increasing concentrations of ADR were characterized in vitro for growth properties and in vivo for tumorigenicity and pulmonary metastases. The progressively resistant sublines adapted to grow in the presence of 0.025, 0.05, 0.1, and 0.25 microgram/ml ADR in monolayer culture were found to be 5-, 10-, 20-, and 40-fold ADR-resistant, respectively, compared to the parental sensitive cells, using a soft-agar colony assay and continuous ADR treatment for 7 days. The doubling time in monolayer culture of the parent sensitive and progressively ADR-resistant sublines of B16-BL6 melanoma cells was approximately 16-18 h. Although the colony-forming efficiency in soft agar of parental sensitive cells was only 0.5-4%, the 5-, 10-, 20-, and 40-fold ADR-resistant sublines had colony-forming efficiencies of 15, 20, 30, and 77%, respectively. Tumorigenicity in C57BL/6 mice of progressively ADR-resistant sublines was similar to parental sensitive cells following s.c. and i.p. implantation of 10(5)-10(6) tumor cells. Experimental pulmonary metastases were significantly lower in ADR-resistant sublines with progressive resistance. Additionally, unlike the parental sensitive and 5-fold ADR-resistant B16-BL6 cells, the 10-, 20-, and 40-fold ADR-resistant sublines were spontaneously nonmetastatic. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical detection of P-glycoprotein revealed the presence of a Mr 170,000 plasma membrane glycoprotein in the 40-fold ADR-resistant subline and its counterpart maintained for 1 year in ADR-free medium. Results from this study suggest that progressively ADR-resistant B16-BL6 mouse melanoma cells selected in vitro demonstrate a marked increase in colony formation in soft agar and a decrease in the ability to produce pulmonary metastases, without alterations in tumorigenicity.

ADP-ribosylation factor arf6p may function as a molecular switch of new end take off in fission yeast

International Nuclear Information System (INIS)

Fujita, Atsushi

2008-01-01

Small GTPases act as molecular switches in a wide variety of cellular processes. In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from a monopolar manner to a bipolar manner, which is known as 'New End Take Off' (NETO). Here I report the identification of a gene, arf6 + , encoding an ADP-ribosylation factor small GTPase, that may be essential for NETO. arf6Δ cells completely fail to undergo NETO. arf6p localizes at both cell ends and presumptive septa in a cell-cycle dependent manner. And its polarized localization is not dependent on microtubules, actin cytoskeletons and some NETO factors (bud6p, for3p, tea1p, tea3p, and tea4p). Notably, overexpression of a fast GDP/GTP-cycling mutant of arf6p can advance the timing of NETO. These findings suggest that arf6p functions as a molecular switch for the activation of NETO in fission yeast

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

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.

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

Science.gov (United States)

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.

Assessment of insulin resistance in fructose-fed rats with 125I-6-deoxy-6-iodo-D-glucose, a new tracer of glucose transport

International Nuclear Information System (INIS)

Perret, Pascale; Slimani, Lotfi; Briat, Arnaud; Villemain, Daniele; Fagret, Daniel; Ghezzi, Catherine; Halimi, Serge; Demongeot, Jacques

2007-01-01

Insulin resistance, characterised by an insulin-stimulated glucose transport defect, is an important feature of the pre-diabetic state that has been observed in numerous pathological disorders. The purpose of this study was to assess variations in glucose transport in rats using 125 I-6-deoxy-6-iodo-D-glucose (6DIG), a new tracer of glucose transport proposed as an imaging tool to assess insulin resistance in vivo. Two protocols were performed, a hyperinsulinaemic-euglycaemic clamp and a normoinsulinaemic-normoglycaemic protocol, in awake control and insulin-resistant fructose-fed rats. The tracer was injected at steady state, and activity in 11 tissues and the blood was assessed ex vivo at several time points. A multicompartmental mathematical model was developed to obtain fractional transfer coefficients of 6DIG from the blood to the organs. Insulin sensitivity of fructose-fed rats, estimated by the glucose infusion rate, was reduced by 40% compared with control rats. At steady state, 6DIG uptake was significantly stimulated by insulin in insulin-sensitive tissues of control rats (basal versus insulin: diaphragm, p < 0.01; muscle, p < 0.05; heart, p < 0.001), whereas insulin did not stimulate 6DIG uptake in insulin-resistant fructose-fed rats. Moreover, in these tissues, the fractional transfer coefficients of entrance were significantly increased with insulin in control rats (basal vs insulin: diaphragm, p < 0.001; muscle, p < 0.001; heart, p < 0.01) whereas no significant changes were observed in fructose-fed rats. This study sets the stage for the future use of 6DIG as a non-invasive means for the evaluation of insulin resistance by nuclear imaging. (orig.)

Assessment of insulin resistance in fructose-fed rats with 125I-6-deoxy-6-iodo-D-glucose, a new tracer of glucose transport

Science.gov (United States)

Perret, Pascale; Slimani, Lotfi; Briat, Arnaud; Villemain, Danièle; Halimi, Serge; Demongeot, Jacques; Fagret, Daniel; Ghezzi, Catherine

2007-01-01

Purpose Insulin resistance, characterised by an insulin-stimulated glucose transport defect, is an important feature of the pre-diabetic state and it has been observed in numerous pathological disorders. The purpose of this study was to assess variations in glucose transport in rats with 125I-6-Deoxy-6-Iodo-D-glucose (6DIG), a new tracer of glucose transport proposed as an imaging tool to assess insulin resistance in vivo. Methods Two protocols were performed, a hyperinsulinaemic-euglycaemic clamp and a normoinsulinaemic normoglycaemic protocol, in awake control and insulin-resistant fructose-fed rats. The tracer was injected at steady state, and activity in 11 tissues and the blood were assessed ex vivo at several time points. A multicompartmental mathematical model was developed to obtain fractional transfer coefficients of 6DIG from the blood to the organs. Results Insulin sensitivity of fructose-fed rats, estimated by the glucose infusion rate, was reduced by 40% compared with control rats. At steady-state, 6DIG uptake was significantly stimulated by insulin in insulin-sensitive tissues of control rats (basal versus insulin: diaphragm, p<0.01; muscle, p<0.05; heart, p<0.001), whereas insulin did not stimulate 6DIG uptake in insulin-resistant fructose-fed rats. Moreover, in these tissues, the fractional transfer coefficients of entrance were significantly increased with insulin in control rats (basal vs insulin: diaphragm, p<0.001; muscle, p<0.001; heart, p<0.01) and whereas no significant changes were observed in fructose-fed rats. Conclusion This study sets the stage for the future use of 6DIG as a non-invasive means for the evaluation of insulin resistance by nuclear imaging. PMID:17171359

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.

High-resolution mapping of the brown planthopper resistance gene Bph6 in rice and characterizing its resistance in the 9311 and Nipponbare near isogenic backgrounds.

Science.gov (United States)

Qiu, Yongfu; Guo, Jianping; Jing, Shengli; Zhu, Lili; He, Guangcun

2010-11-01

Brown planthopper (Nilaparvata lugens Stål, BPH) is one of the most destructive insect pests of rice. Exploring resistance genes from diverse germplasms and incorporating them into cultivated varieties are critical for controlling this insect. The rice variety Swarnalata was reported to carry a resistance gene (designated Bph6), which has not yet been assigned to a chromosome location and the resistance mechanism is still unknown. In this study, we identified and mapped this gene using the F(2) and backcrossing populations and characterized its resistance in indica 9311 and japonica Nipponbare using near isogenic lines (NILs). In analysis of 9311/Swarnalata F(2) population, the Bph6 gene was located on the long arm of chromosome 4 between the SSR markers RM6997 and RM5742. The gene was further mapped precisely to a 25-kb region delimited between the STS markers Y19 and Y9; and the distance between these markers is 25-kb in Nipponbare genome. The Bph6 explained 77.5% of the phenotypic variance of BPH resistance in F(2) population and 84.9% in BC(2)F(2) population. Allele from Swarnalata significantly increased resistance to the BPH, resulted in a reduced damage score. In characterization of Bph6-mediated resistance, the BPH insects showed significant preference between NIL-9311 and 9311 in 3 h and between NIL-NIP and Nipponbare in 120 h after release. BPH growth and development were inhibited, and the insect's survival rates were lower on Bph6-NIL plants, compared with the parents 9311 and Nipponbare. The results indicate that the Bph6 exerted prolonged antixenotic and antibiotic effects in Bph6-NIL plants, and NIL-9311 plants showed a quicker and stronger effect toward BPH than NIL-NIP plants.

qnrA6 genetic environment and quinolone resistance conferred on Proteus mirabilis.

Science.gov (United States)

Jayol, Aurélie; Janvier, Frédéric; Guillard, Thomas; Chau, Françoise; Mérens, Audrey; Robert, Jérôme; Fantin, Bruno; Berçot, Béatrice; Cambau, Emmanuelle

2016-04-01

To determine the genetic location and environment of the qnrA6 gene in Proteus mirabilis PS16 where it was first described and to characterize the quinolone resistance qnrA6 confers. Transformation experiments and Southern blotting were performed for plasmid and genomic DNA of P. mirabilis PS16 to determine the qnrA6 location. Combinatorial PCRs with primers in qnrA6 and genes usually surrounding qnrA genes were used to determine the genetic environment. The qnrA6 coding region, including or not the promoter region, was cloned into vectors pTOPO and pBR322 and the MICs of six quinolones were measured for transformants of Escherichia coli TOP10 and P. mirabilis ATCC 29906 Rif(R). qnrA6 was shown to be chromosomally encoded in P. mirabilis PS16 and its genetic environment was 81%-87% similar to that of qnrA2 in the Shewanella algae chromosome. The 5138 bp region up- and downstream of qnrA6 contained an IS10 sequence surrounded by two ISCR1. This resulted in qnrA6 being displaced 1.9 kb from its native promoter but supplied a promoter present in ISCR1. qnrA6 cloned into pTOPO and pBR322 conferred a 4-32-fold increase in fluoroquinolone MICs when expressed in E. coli but only 2-3-fold in P. mirabilis. When including the promoter region, a further increase in resistance was observed in both species, reaching MIC values above clinical breakpoints for only P. mirabilis. qnrA6 is the first chromosomally located qnrA gene described in Enterobacteriaceae. The quinolone resistance conferred by qnrA6 depends on the proximity of an efficient promoter and the host strain where it is expressed. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Investigation of intergranular corrosion resistance of Cr16Ni25NMo6 steel

International Nuclear Information System (INIS)

Kamenev, Yu.B.; Nazarov, A.A.; Kuusk, L.V.; Majdeburova, T.F.

1990-01-01

The effect of 08Kh16N25AM6 steel susceptibility to intergranular corrosion on its intergranular cracking resistance in high-temperature water is investigated. In addition, the performed tests point to the susceptibility of sensibilized Kh16N25AM6 steel to intergranular corrosion in media simulating an agressive environment of power generation equipment; the latter requires a strict control over the resistance of weld joints of the above steel to intergranular corrosion. It is shown that Kh16N25AM6 type steel in sensibilized state is susceptible to intercrystalline corrosion cracking in high-temperature water which correlates with its susceptibility to intergranular corrosion established by AM GOST 6032-84 and potentiodynamic reactivation methods

Retrotransposition and mutation events yield Rap1 GTPases with differential signalling capacity

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Penzkofer Tobias

2010-02-01

Full Text Available Abstract Background Retrotransposition of mRNA transcripts gives occasionally rise to functional retrogenes. Through acquiring tempero-spatial expression patterns distinct from their parental genes and/or functional mutations in their coding sequences, such retrogenes may in principle reshape signalling networks. Results Here we present evidence for such a scenario, involving retrogenes of Rap1 belonging to the Ras family of small GTPases. We identified two murine and one human-specific retrogene of Rap1A and Rap1B, which encode proteins that differ by only a few amino acids from their parental Rap1 proteins. Markedly, human hRap1B-retro and mouse mRap1A-retro1 acquired mutations in the 12th and 59th amino acids, respectively, corresponding to residues mutated in constitutively active oncogenic Ras proteins. Statistical and structural analyses support a functional evolution scenario, where Rap1 isoforms of retrogenic origin are functionally distinct from their parental proteins. Indeed, all retrogene-encoded GTPases have an increased GTP/GDP binding ratio in vivo, indicating that their conformations resemble that of active GTP-bound Rap1. We furthermore demonstrate that these three Rap1 isoforms exhibit distinct affinities for the Ras-binding domain of RalGDS. Finally, when tested for their capacity to induce key cellular processes like integrin-mediated cell adhesion or cell spreading, marked differences are seen. Conclusions Together, these data lend strong support for an evolution scenario, where retrotransposition and subsequent mutation events generated species-specific Rap1 isoforms with differential signaling potential. Expression of the constitutively active human Rap1B-retro in cells like those derived from Ramos Burkitt's lymphoma and bone marrow from a patient with myelodysplastic syndrome (MDS warrants further investigation into its role in disease development.

The Arabidopsis Rho of Plants GTPase AtROP6 Functions in Developmental and Pathogen Response Pathways1[C][W][OA

Science.gov (United States)

Poraty-Gavra, Limor; Zimmermann, Philip; Haigis, Sabine; Bednarek, Paweł; Hazak, Ora; Stelmakh, Oksana Rogovoy; Sadot, Einat; Schulze-Lefert, Paul; Gruissem, Wilhelm; Yalovsky, Shaul

2013-01-01

How plants coordinate developmental processes and environmental stress responses is a pressing question. Here, we show that Arabidopsis (Arabidopsis thaliana) Rho of Plants6 (AtROP6) integrates developmental and pathogen response signaling. AtROP6 expression is induced by auxin and detected in the root meristem, lateral root initials, and leaf hydathodes. Plants expressing a dominant negative AtROP6 (rop6DN) under the regulation of its endogenous promoter are small and have multiple inflorescence stems, twisted leaves, deformed leaf epidermis pavement cells, and differentially organized cytoskeleton. Microarray analyses of rop6DN plants revealed that major changes in gene expression are associated with constitutive salicylic acid (SA)-mediated defense responses. In agreement, their free and total SA levels resembled those of wild-type plants inoculated with a virulent powdery mildew pathogen. The constitutive SA-associated response in rop6DN was suppressed in mutant backgrounds defective in SA signaling (nonexpresser of PR genes1 [npr1]) or biosynthesis (salicylic acid induction deficient2 [sid2]). However, the rop6DN npr1 and rop6DN sid2 double mutants retained the aberrant developmental phenotypes, indicating that the constitutive SA response can be uncoupled from ROP function(s) in development. rop6DN plants exhibited enhanced preinvasive defense responses to a host-adapted virulent powdery mildew fungus but were impaired in preinvasive defenses upon inoculation with a nonadapted powdery mildew. The host-adapted powdery mildew had a reduced reproductive fitness on rop6DN plants, which was retained in mutant backgrounds defective in SA biosynthesis or signaling. Our findings indicate that both the morphological aberrations and altered sensitivity to powdery mildews of rop6DN plants result from perturbations that are independent from the SA-associated response. These perturbations uncouple SA-dependent defense signaling from disease resistance execution. PMID

Avaliação da resposta ao fotoperíodo em genótipos de arroz irrigado Evaluation of the photoperiodic response in pad rice genotypes

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Nereu Augusto Streck

2006-01-01

Full Text Available A temperatura e o fotoperíodo são os fatores ambientais principais que influenciam a duração do ciclo de desenvolvimento das plantas de arroz (Oryza sativa L.. A resposta fotoperiódica da maioria dos genótipos modernos de arroz usados no sul do Brasil ainda não foi quantificada. O objetivo do trabalho foi identificar a resposta ao fotoperíodo de algumas cultivares de arroz irrigado cultivadas nessa região. Um experimento de campo foi instalado em Santa Maria (RS, durante os anos agrícolas de 2003/2004 e 2004/2005 com cinco épocas de semeadura por ano. Em 2003/2004, as épocas de semeadura foram 1.º/9/2003, 20/10/2003, 21/11/2003, 5/1/2004 e 29/1/2004 e em 2004/2005, as épocas de semeadura foram 2/9/2004, 7/10/2004, 4/11/2004, 3/12/2004 e 2/3/2005. As cultivares utilizadas foram IRGA 421, IRGA 416, IRGA 417, IRGA 420, BR-IRGA 409, BRS 7 "Taim" e EPAGRI 109, todas do tipo moderno, com diferentes ciclos de desenvolvimento, de muito precoce (IRGA 421 a tardio (EPAGRI 109. O delineamento experimental foi um trifatorial (cultivar, data de semeadura e ano agrícola inteiramente casualizado com quatro repetições. Cada repetição consistiu de um balde de 12 litros de capacidade com 10 plantas, cinco das quais foram marcadas com arames coloridos e avaliadas quanto ao número final de folhas no colmo principal (NFF. Calculou-se o fotoperíodo acumulado na fase em que o arroz é sensível ao fotoperíodo. Foi verificado um aumento linear do NFF com o fotoperíodo acumulado em todas as cultivares, o que indica uma resposta fotoperiódica típica de planta de dia curto nos genótipos de arroz. Houve diferença de sensibilidade ao fotoperíodo entre as cultivares.Temperature and photoperiod are major environmental factors that affect the development of rice (Oryza sativa L.. The photoperiodic response of the majority of modern rice genotypes grown in Southern Brazil has not been quantified yet. The objective of this study was to investigate the

MiR-285 targets P450 (CYP6N23) to regulate pyrethroid resistance in Culex pipiens pallens.

Science.gov (United States)

Tian, Mengmeng; Liu, Bingqian; Hu, Hongxia; Li, Xixi; Guo, Qin; Zou, Feifei; Liu, Xianmiao; Hu, Mengxue; Guo, Juxin; Ma, Lei; Zhou, Dan; Sun, Yan; Shen, Bo; Zhu, Changliang

2016-12-01

MicroRNAs play critical roles in post-transcriptional regulation of gene expression, which participate in the modulation of almost all of the cellular processes. Although emerging evidence indicates that microRNAs are related with antineoplastic drugs resistance, whether microRNAs are responsible for insecticide resistance in mosquitos is poorly understood. In this paper, we found that miR-285 was significantly upregulated in the deltamethrin-resistant strain of Culex pipiens pallens, and overexpression miR-285 through microinjection increased mosquito survival rate against deltamethrin treatement. Using bioinformatic software, quantitative reverse transcription PCR, luciferase reporter assay and microinjection approaches, we conformed that CYP6N23 was the target of miR-285. Lower expression of CYP6N23 was observed in the deltamethrin-resistant strain. While, mosquito mortality rate was decreased after downregulating expression of CYP6N23 by dsRNA against CYP6N23 or miR-285 mimic microinjection. These findings revealed that miR-285 could target CYP6N23 to regulate pyrethroid resistance, providing new insights into mosquito insecticide resistance surveillance and control.

Assessment of insulin resistance in fructose-fed rats with {sup 125}I-6-deoxy-6-iodo-D-glucose, a new tracer of glucose transport

Energy Technology Data Exchange (ETDEWEB)

Perret, Pascale; Slimani, Lotfi; Briat, Arnaud; Villemain, Daniele; Fagret, Daniel; Ghezzi, Catherine [INSERM, E340, 38000 Grenoble, (France); Univ Grenoble, 38000 Grenoble, (France); Halimi, Serge [CHRU Grenoble, Hopital Michallon, Service de Diabetologie, 38000 Grenoble, (France); Demongeot, Jacques [Univ Grenoble, 38000 Grenoble, (France); CNRS, UMR 5525, 38000 Grenoble, (France)

2007-05-15

Insulin resistance, characterised by an insulin-stimulated glucose transport defect, is an important feature of the pre-diabetic state that has been observed in numerous pathological disorders. The purpose of this study was to assess variations in glucose transport in rats using {sup 125}I-6-deoxy-6-iodo-D-glucose (6DIG), a new tracer of glucose transport proposed as an imaging tool to assess insulin resistance in vivo. Two protocols were performed, a hyperinsulinaemic-euglycaemic clamp and a normoinsulinaemic-normoglycaemic protocol, in awake control and insulin-resistant fructose-fed rats. The tracer was injected at steady state, and activity in 11 tissues and the blood was assessed ex vivo at several time points. A multicompartmental mathematical model was developed to obtain fractional transfer coefficients of 6DIG from the blood to the organs. Insulin sensitivity of fructose-fed rats, estimated by the glucose infusion rate, was reduced by 40% compared with control rats. At steady state, 6DIG uptake was significantly stimulated by insulin in insulin-sensitive tissues of control rats (basal versus insulin: diaphragm, p < 0.01; muscle, p < 0.05; heart, p < 0.001), whereas insulin did not stimulate 6DIG uptake in insulin-resistant fructose-fed rats. Moreover, in these tissues, the fractional transfer coefficients of entrance were significantly increased with insulin in control rats (basal vs insulin: diaphragm, p < 0.001; muscle, p < 0.001; heart, p < 0.01) whereas no significant changes were observed in fructose-fed rats. This study sets the stage for the future use of 6DIG as a non-invasive means for the evaluation of insulin resistance by nuclear imaging. (orig.)

Riboflavin-Induced Disease Resistance Requires the Mitogen-Activated Protein Kinases 3 and 6 in Arabidopsis thaliana.

Science.gov (United States)

Nie, Shengjun; Xu, Huilian

2016-01-01

As a resistance elicitor, riboflavin (vitamin B2) protects plants against a wide range of pathogens. At molecular biological levels, it is important to elucidate the signaling pathways underlying the disease resistance induced by riboflavin. Here, riboflavin was tested to induce resistance against virulent Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000) in Arabidopsis. Results showed that riboflavin induced disease resistance based on MAPK-dependent priming for the expression of PR1 gene. Riboflavin induced transient expression of PR1 gene. However, following Pst DC3000 inoculation, riboflavin potentiated stronger PR1 gene transcription. Further was suggested that the transcript levels of mitogen-activated protein kinases, MPK3 and MPK6, were primed under riboflavin. Upon infection by Pst DC3000, these two enzymes were more strongly activated. The elevated activation of both MPK3 and MPK6 was responsible for enhanced defense gene expression and resistance after riboflavin treatment. Moreover, riboflavin significantly reduced the transcript levels of MPK3 and MPK6 by application of AsA and BAPTA, an H2O2 scavenger and a calcium (Ca2+) scavenger, respectively. In conclusion, MPK3 and MPK6 were responsible for riboflavin-induced resistance, and played an important role in H2O2- and Ca2+-related signaling pathways, and this study could provide a new insight into the mechanistic study of riboflavin-induced defense responses.

Distribution and characterization of Podosphaera macularis virulent on hop cultivars possessing R6-based resistance to powdery mildew

Science.gov (United States)

In 2012, an epidemic of powdery mildew occurred in Washington and Idaho on previously resistant cultivars whose resistance was putatively based on the gene designated R6. In 2013, isolates capable of causing severe disease on cultivars with R6-based resistance were confirmed in Oregon and became wid...

Resistance to stem rust Ug99 in six bread wheat cultivars maps to chromosome 6DS.

Science.gov (United States)

Lopez-Vera, Eric E; Nelson, Sarah; Singh, Ravi P; Basnet, Bhoja R; Haley, Scott D; Bhavani, Sridhar; Huerta-Espino, Julio; Xoconostle-Cazares, Beatriz G; Ruiz-Medrano, Roberto; Rouse, Matthew N; Singh, Sukhwinder

2014-01-01

Identified SSR markers ( Xcfd49 and Xbarc183 ) linked with stem rust resistance for efficient use in marker-assisted selection and stacking of resistance genes in wheat breeding programs. More than 80 % of the worldwide wheat (Triticum aestivum L.) area is currently sown with varieties susceptible to the Ug99 race group of stem rust fungus. However, wheat lines Niini, Tinkio, Coni, Pfunye, Blouk, and Ripper have demonstrated Ug99 resistance at the seedling and adult plant stages. We mapped stem rust resistance in populations derived from crosses of a susceptible parent with each of the resistant lines. The segregation of resistance in each population indicated the presence of a single gene. The resistance gene in Niini mapped to short arm of chromosome 6D and was flanked by SSR markers Xcfd49 at distances of 3.9 cM proximal and Xbarc183 8.4 cM distal, respectively. The chromosome location of this resistance was validated in three other populations: PBW343/Coni, PBW343/Tinkio, and Cacuke/Pfunye. Resistance initially postulated to be conferred by the SrTmp gene in Blouk and Ripper was also linked to Xcfd49 and Xbarc183 on 6DS, but it was mapped proximal to Xbarc183 at a similar position to previously mapped genes Sr42 and SrCad. Based on the variation in diagnostic marker alleles, it is possible that Niini and Pfunye may carry different resistance genes/alleles. Further studies are needed to determine the allelic relationships between various genes located on chromosome arm 6DS. Our results provide valuable molecular marker and genetic information for developing Ug99 resistant wheat varieties in diverse germplasm and using these markers to tag the resistance genes in wheat breeding.

Interleukin-6 induces impairment in human subcutaneous adipogenesis in obesity-associated insulin resistance.

Science.gov (United States)

Almuraikhy, Shamma; Kafienah, Wael; Bashah, Moataz; Diboun, Ilhame; Jaganjac, Morana; Al-Khelaifi, Fatima; Abdesselem, Houari; Mazloum, Nayef A; Alsayrafi, Mohammed; Mohamed-Ali, Vidya; Elrayess, Mohamed A

2016-11-01

A subset of obese individuals remains insulin sensitive by mechanisms as yet unclear. The hypothesis that maintenance of normal subcutaneous (SC) adipogenesis accounts, at least partially, for this protective phenotype and whether it can be abrogated by chronic exposure to IL-6 was investigated. Adipose tissue biopsies were collected from insulin-sensitive (IS) and insulin-resistant (IR) individuals undergoing weight-reduction surgery. Adipocyte size, pre-adipocyte proportion of stromal vascular fraction (SVF)-derived cells, adipogenic capacity and gene expression profiles of isolated pre-adipocytes were determined, along with local in vitro IL-6 secretion. Adipogenic capacity was further assessed in response to exogenous IL-6 application. Despite being equally obese, IR individuals had significantly lower plasma leptin and adiponectin levels and higher IL-6 levels compared with age-matched IS counterparts. Elevated systemic IL-6 in IR individuals was associated with hyperplasia of adipose tissue-derived SVF cells, despite higher frequency of hypertrophied adipocytes. SC pre-adipocytes from these tissues exhibited lower adipogenic capacity accompanied by downregulation of PPARγ (also known as PPARG) and CEBPα (also known as CEBPA) and upregulation of GATA3 expression. Impaired adipogenesis in IR individuals was further associated with increased adipose secretion of IL-6. Treatment of IS-derived SC pre-adipocytes with IL-6 reduced their adipogenic capacity to levels of the IR group. Obesity-associated insulin resistance is marked by impaired SC adipogenesis, mediated, at least in a subset of individuals, by elevated local levels of IL-6. Understanding the molecular mechanisms underlying reduced adipogenic capacity in IR individuals could help target appropriate therapeutic strategies aimed at those at greatest risk of insulin resistance and type 2 diabetes mellitus.

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

Rho GTPasas como blancos terapéuticos relevantes en cáncer y otras enfermedades humanas Rho GTPases as therapeutic targets in cancer and other human diseases

Directory of Open Access Journals (Sweden)

Pablo Lorenzano Menna

2010-12-01

Full Text Available Las Rho GTPasas son una familia de proteínas clave en la transmisión de señales provenientes del exterior celular hacia efectores intracelulares tanto citoplasmáticos como nucleares. En los últimos año ha habido un desarrollo vertiginoso de múltiples herramientas genéticas y farmacológicas, lo que ha permitido establecer de manera mucho más precisa las funciones específicas de estas proteínas. El objetivo de la presente revisión es hacer foco en las múltiples funciones celulares reguladas por las Rho GTPasas, describiendo en detalle el mecanismo molecular involucrado. Se discute además la participación de estas proteínas en diversas enfermedades humanas haciendo énfasis en su vinculación con el cáncer. Por último, se hace una actualización detallada sobre las estrategias terapéuticas en experimentación que tienen a las Rho GTPasas como blancos moleculares.Rho GTPases are a key protein family controlling the transduction of external signals to cytoplasmatic and nuclear effectors. In the last few years, the development of genetic and pharmacological tools has allowed a more precise definition of the specific roles of Rho GTPases. The aim of this review is to describe the cellular functions regulated by these proteins with focus on the molecular mechanism involved. We also address the role of Rho GTPases in the development of different human diseases such as cancer. Finally, we describe different experimental therapeutic strategies with Rho GTPases as molecular targets.

Improved ductility and oxidation resistance of cast Ti–6Al–4V alloys by microalloying

International Nuclear Information System (INIS)

Luan, J.H.; Jiao, Z.B.; Chen, G.; Liu, C.T.

2014-01-01

Highlights: • Modified Ti64 alloys with improved ductility and oxidation resistance are developed. • B improves the ductility by refining grain size and enhancing boundary cohesion. • Y enhances the oxidation resistance by possibly slowing down the oxidation kinetics. - Abstract: The effects of B and Y on the mechanical properties and oxidation behavior of cast Ti–6Al–4V alloys were systematically investigated, and the new alloys with improved ductility and oxidation resistance are developed by the microalloying approach. The results indicate that boron is beneficial for improving the ductility by not only grain-size refinement but also grain-boundary enhancement, while yttrium is effective in increasing the oxidation resistance through possibly slowing down the oxidation kinetics. The improved properties, together with their high strength, make the microalloyed cast Ti–6Al–4V alloys competitive for practical engineering applications

CYP6 P450 enzymes and ACE-1 duplication produce extreme and multiple insecticide resistance in the malaria mosquito Anopheles gambiae.

Science.gov (United States)

Edi, Constant V; Djogbénou, Luc; Jenkins, Adam M; Regna, Kimberly; Muskavitch, Marc A T; Poupardin, Rodolphe; Jones, Christopher M; Essandoh, John; Kétoh, Guillaume K; Paine, Mark J I; Koudou, Benjamin G; Donnelly, Martin J; Ranson, Hilary; Weetman, David

2014-03-01

Malaria control relies heavily on pyrethroid insecticides, to which susceptibility is declining in Anopheles mosquitoes. To combat pyrethroid resistance, application of alternative insecticides is advocated for indoor residual spraying (IRS), and carbamates are increasingly important. Emergence of a very strong carbamate resistance phenotype in Anopheles gambiae from Tiassalé, Côte d'Ivoire, West Africa, is therefore a potentially major operational challenge, particularly because these malaria vectors now exhibit resistance to multiple insecticide classes. We investigated the genetic basis of resistance to the most commonly-applied carbamate, bendiocarb, in An. gambiae from Tiassalé. Geographically-replicated whole genome microarray experiments identified elevated P450 enzyme expression as associated with bendiocarb resistance, most notably genes from the CYP6 subfamily. P450s were further implicated in resistance phenotypes by induction of significantly elevated mortality to bendiocarb by the synergist piperonyl butoxide (PBO), which also enhanced the action of pyrethroids and an organophosphate. CYP6P3 and especially CYP6M2 produced bendiocarb resistance via transgenic expression in Drosophila in addition to pyrethroid resistance for both genes, and DDT resistance for CYP6M2 expression. CYP6M2 can thus cause resistance to three distinct classes of insecticide although the biochemical mechanism for carbamates is unclear because, in contrast to CYP6P3, recombinant CYP6M2 did not metabolise bendiocarb in vitro. Strongly bendiocarb resistant mosquitoes also displayed elevated expression of the acetylcholinesterase ACE-1 gene, arising at least in part from gene duplication, which confers a survival advantage to carriers of additional copies of resistant ACE-1 G119S alleles. Our results are alarming for vector-based malaria control. Extreme carbamate resistance in Tiassalé An. gambiae results from coupling of over-expressed target site allelic variants with

Overexpression of cytochrome P450 CYP6BG1 may contribute to chlorantraniliprole resistance in Plutella xylostella (L.).

Science.gov (United States)

Li, Xiuxia; Li, Ran; Zhu, Bin; Gao, Xiwu; Liang, Pei

2018-06-01

The diamondback moth Plutella xylostella (L.) is the most widely distributed pest of cruciferous crops and has developed resistance to most commonly used insecticides, including chlorantraniliprole. Resistance to chlorantraniliprole is likely caused by mutations of the target, the ryanodine receptor, and/or mediated by an increase in detoxification enzyme activities. Although target-site resistance is documented in detail, resistance mediated by increased metabolism has rarely been reported. The activity of cytochrome P450 was significantly higher in two resistant P. xylostella populations than in a susceptible one. Among ten detected cytochrome P450 genes, CYP6BG1 was significantly overexpressed (over 80-fold) in a field-resistant population compared with expression in a susceptible one. Knockdown of CYP6BG1 by RNA interference dramatically reduced the 7-ethoxycoumarin-O-deethylase (7-ECOD) activity of P450 by 45.5% and increased the toxicity of chlorantraniliprole toward P. xylostella by 26.8% at 48 h postinjection of double-stranded RNA. By contrast, overexpression of CYP6BG1 in a transgenic Drosophila melanogaster line significantly decreased the toxicity of the insecticide to the transgenic flies. Overexpression of CYP6BG1 may contribute to chlorantraniliprole resistance in P. xylostella. Our findings will provide new insights into the mechanisms of resistance to diamide insecticides in other insect pests. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

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.

Genetic mapping, marker assisted selection and allelic relationships for the Pu 6 gene conferring rust resistance in sunflower.

Science.gov (United States)

Bulos, Mariano; Vergani, Pablo Nicolas; Altieri, Emiliano

2014-09-01

Rust resistance in the sunflower line P386 is controlled by Pu 6 , a gene which was reported to segregate independently from other rust resistant genes, such as R 4 . The objectives of this work were to map Pu 6 , to provide and validate molecular tools for its identification, and to determine the linkage relationship of Pu 6 and R 4 . Genetic mapping of Pu 6 with six markers covered 24.8 cM of genetic distance on the lower end of linkage Group 13 of the sunflower consensus map. The marker most closely linked to Pu 6 was ORS316 at 2.5 cM in the distal position. ORS316 presented five alleles when was assayed with a representative set of resistant and susceptible lines. Allelism test between Pu 6 and R 4 indicated that both genes are linked at a genetic distance of 6.25 cM. This is the first confirmation based on an allelism test that at least two members of the R adv /R 4 /R 11 / R 13a /R 13b /Pu 6 cluster of genes are at different loci. A fine elucidation of the architecture of this complex locus will allow designing and constructing completely new genomic regions combining genes from different resistant sources and the elimination of the linkage drag around each resistant gene.

The role of inflammation in vascular insulin resistance with focus on IL-6

DEFF Research Database (Denmark)

Andersen, Kirsten; Pedersen, B.K.

2008-01-01

The present review focuses on the possible role of interleukin-(IL)-6 in vascular insulin resistance. The endothelium plays an important role in regulating the tone of the vasculature by releasing nitric oxide (NO) to the smooth muscles of the vessels, thereby regulating the distribution of blood....... It is likely that chronic low-level inflammation plays an important role in developing endothelial dysfunction mainly through proinflammatory actions of tumor necrosis factor alpha (TNF-alpha). TNF-alpha induces production of IL-6 and it has been suggested that a causal relationship exists between endothelial...... dysfunction and these cytokines. With regard to vascular insulin resistance, the available data point to a direct pathogenic role of TNF-alpha in mediating endothelial dysfunction, whereas with regard to IL-6 evidence is sparse and does not allow any firm conclusions Udgivelsesdato: 2008/9...

Functional studies of TcRjl, a novel GTPase of Trypanosoma cruzi, reveals phenotypes related with MAPK activation during parasite differentiation and after heterologous expression in Drosophila model system

International Nuclear Information System (INIS)

Reis Monteiro dos-Santos, Guilherme Rodrigo; Fontenele, Marcio Ribeiro; Dias, Felipe de Almeida; Oliveira, Pedro Lagerblad de; Nepomuceno-Silva, José Luciano

2015-01-01

The life cycle of the protozoan parasite Trypanosoma cruzi comprises rounds of proliferative cycles and differentiation in distinct host environments. Ras GTPases are molecular switches that play pivotal regulatory functions in cell fate. Rjl is a novel GTPase with unknown function. Herein we show that TcRjl blocks in vivo cell differentiation. The forced expression of TcRjl leads to changes in the overall tyrosine protein phosphorylation profile of parasites. TcRjl expressing parasites sustained DNA synthesis regardless the external stimuli for differentiation. Heterologous expression in the Drosophila melanogaster genetic system strongly suggests a role from TcRjl protein in RTK-dependent pathways and MAPK activation.

Functional studies of TcRjl, a novel GTPase of Trypanosoma cruzi, reveals phenotypes related with MAPK activation during parasite differentiation and after heterologous expression in Drosophila model system

Energy Technology Data Exchange (ETDEWEB)

Reis Monteiro dos-Santos, Guilherme Rodrigo [Laboratório de Parasitologia Molecular, Instituto de Biofísica Carlos Chagas Filho, CCS, UFRJ, Rio de Janeiro (Brazil); Fontenele, Marcio Ribeiro [Laboratório de Biologia Molecular do Desenvolvimento Instituto de Ciências Biomédicas, CCS, UFRJ, Rio de Janeiro (Brazil); Dias, Felipe de Almeida [Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica, CCS, UFRJ, Rio de Janeiro (Brazil); Oliveira, Pedro Lagerblad de [Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica, CCS, UFRJ, Rio de Janeiro (Brazil); Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM) (Brazil); Nepomuceno-Silva, José Luciano [Laboratório Integrado de Bioquímica Hatisaburo Masuda, NUPEM/UFRJ, Pólo Barreto, Universidade Federal do Rio de Janeiro, Campus Macaé, Macaé (Brazil); and others

2015-11-06

The life cycle of the protozoan parasite Trypanosoma cruzi comprises rounds of proliferative cycles and differentiation in distinct host environments. Ras GTPases are molecular switches that play pivotal regulatory functions in cell fate. Rjl is a novel GTPase with unknown function. Herein we show that TcRjl blocks in vivo cell differentiation. The forced expression of TcRjl leads to changes in the overall tyrosine protein phosphorylation profile of parasites. TcRjl expressing parasites sustained DNA synthesis regardless the external stimuli for differentiation. Heterologous expression in the Drosophila melanogaster genetic system strongly suggests a role from TcRjl protein in RTK-dependent pathways and MAPK activation.

The effects of 6-mercaptopurine nucleotide derivatives on the growth and survival of 6-mercaptopurine-sensitive and -resistant cell culture lines.

Science.gov (United States)

Johnston, H P; Hawley, P; White, S E; Gibson, I; Tidd, D M

1985-04-01

6-Mercaptopurine (MP)-sensitive and -resistant cell culture lines were used to further characterize the apparent ability of MP nucleotide derivatives to overcome resistance to the parent drug. 6-Mercaptopurine-9-beta-D-ribofuranoside 5'-monophosphate [MPRP], bis(6-mercaptopurine-9-beta-D-ribofuranoside)-5', 5"'-monophosphate [bis(MPR)P], bis(O2',O3'-dibutyryl-6-mercaptopurine-9-beta-D-ribofuranoside)-5', 5"'-monophosphate [bis(dibut.MPR)P], and O2',O3'-dibutyryl-6-mercaptopurine-9-beta-D-ribofuranoside 5'-monophosphate [dibut.MPRP] were tested for cytotoxic and/or growth inhibitory effects against MP-resistant sublines of V79 Chinese hamster lung fibroblasts (CH/TG) and L1210 mouse leukaemia cells (L1210/MPR) in which deficiencies of hypoxanthine-guanine phosphoribosyltransferase, and hence drug nucleotide forming capacity were the basis of resistance. L1210/MPR cells were totally resistant to 1 mM 6-mercaptopurine-9-beta-D-ribofuranoside [MPR] and 2 mM MPRP, but were inhibited by high concentrations (greater than 0.25 mM) of bis(MPR)P. These results suggested that bis(MPR)P was taken up by cells as the intact molecule since MPR and MPRP were its extracellular breakdown products. L1210/MPR cells were much more sensitive to the lipophilic bis(dibut.MPR)P derivative which had a predominantly cytotoxic action as judged by trypan blue staining and the ability of treated cells to produce macroscopic colonies in soft agar medium. However, cells killed by bis(dibut.MPR)P did not disintegrate appreciably over periods of up to 10 days. The effects of bis(dibut.MPR)P were probably the result of cellular uptake of the intact molecule. Dibut.MPRP showed minimal ability to inhibit L1210/MPR cells although this compound was a possible breakdown product of bis(dibut.MPR)P and a source of the same extracellular degradation products. The median cell size decreased in L1210/MPR cultures during exposure to both bis(MPR)P and bis(dibut.MPR)P. This effect was elicited more rapidly and

The effects of 6-mercaptopurine nucleotide derivatives on the growth and survival of 6-mercaptopurine-sensitive and -resistant cell culture lines.

Science.gov (United States)

Johnston, H. P.; Hawley, P.; White, S. E.; Gibson, I.; Tidd, D. M.

1985-01-01

6-Mercaptopurine (MP)-sensitive and -resistant cell culture lines were used to further characterize the apparent ability of MP nucleotide derivatives to overcome resistance to the parent drug. 6-Mercaptopurine-9-beta-D-ribofuranoside 5'-monophosphate [MPRP], bis(6-mercaptopurine-9-beta-D-ribofuranoside)-5', 5"'-monophosphate [bis(MPR)P], bis(O2',O3'-dibutyryl-6-mercaptopurine-9-beta-D-ribofuranoside)-5', 5"'-monophosphate [bis(dibut.MPR)P], and O2',O3'-dibutyryl-6-mercaptopurine-9-beta-D-ribofuranoside 5'-monophosphate [dibut.MPRP] were tested for cytotoxic and/or growth inhibitory effects against MP-resistant sublines of V79 Chinese hamster lung fibroblasts (CH/TG) and L1210 mouse leukaemia cells (L1210/MPR) in which deficiencies of hypoxanthine-guanine phosphoribosyltransferase, and hence drug nucleotide forming capacity were the basis of resistance. L1210/MPR cells were totally resistant to 1 mM 6-mercaptopurine-9-beta-D-ribofuranoside [MPR] and 2 mM MPRP, but were inhibited by high concentrations (greater than 0.25 mM) of bis(MPR)P. These results suggested that bis(MPR)P was taken up by cells as the intact molecule since MPR and MPRP were its extracellular breakdown products. L1210/MPR cells were much more sensitive to the lipophilic bis(dibut.MPR)P derivative which had a predominantly cytotoxic action as judged by trypan blue staining and the ability of treated cells to produce macroscopic colonies in soft agar medium. However, cells killed by bis(dibut.MPR)P did not disintegrate appreciably over periods of up to 10 days. The effects of bis(dibut.MPR)P were probably the result of cellular uptake of the intact molecule. Dibut.MPRP showed minimal ability to inhibit L1210/MPR cells although this compound was a possible breakdown product of bis(dibut.MPR)P and a source of the same extracellular degradation products. The median cell size decreased in L1210/MPR cultures during exposure to both bis(MPR)P and bis(dibut.MPR)P. This effect was elicited more rapidly and

Proteomic analysis of skeletal muscle in insulin-resistant mice: response to 6-week aerobic exercise.

Directory of Open Access Journals (Sweden)

Hairui Yuan

Full Text Available Aerobic exercise has beneficial effects on both weight control and skeletal muscle insulin sensitivity through a number of specific signaling proteins. To investigate the targets by which exercise exerts its effects on insulin resistance, an approach of proteomic screen was applied to detect the potential different protein expressions from skeletal muscle of insulin-resistant mice after prolonged aerobic exercise training and their sedentary controls. Eighteen C57BL/6 mice were divided into two groups: 6 mice were fed normal chow (NC and 12 mice were fed high-fat diet (HFD for 10 weeks to produce an IR model. The model group was then subdivided into HFD sedentary control (HC, n = 6 and HFD exercise groups (HE, n = 6. Mice in HE group underwent 6 weeks of treadmill running. After 6 weeks, mice were sacrificed and skeletal muscle was dissected. Total protein (n = 6, each group was extracted and followed by citrate synthase, 2D proteome profile analysis and immunoblot. Fifteen protein spots were altered between the NC and HC groups and 23 protein spots were changed between the HC and HE groups significantly. The results provided an array of changes in protein abundance in exercise-trained skeletal muscle and also provided the basis for a new hypothesis regarding the mechanism of exercise ameliorating insulin resistance.

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.

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

Science.gov (United States)

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.

Effect of acetic acid on rice seeds coated with rice husk ash

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Lizandro Ciciliano Tavares

2013-06-01

Full Text Available Flooded rice cultivation promotes anaerobic conditions, favoring the formation of short chain organic acids such as acetic acid, which may be toxic to the crop. The objective of this study was to evaluate the effect of acetic acid on rice seeds coated with rice husk ash. The experiment was arranged in a 2 x 5 x 5 factorial randomized design, with two cultivars (IRGA 424 and BRS Querência, five doses of coating material (0, 2, 3,4 e 5 g kg-1 seed and five concentrations of acetic acid (0, 3, 6, 9 and 12 mM, with 4 replications, totaling 50 treatments. The variables first count of germination, germination, shoot and root length, dry weight of shoots and roots were recorded. The results showed that coating rice seeds with rice husk ash up to 5 g kg-1 seed does not influence the performance of rice seeds of cultivars IRGA 424 and BRS Querência when exposed to concentrations of 12 mM acetic acid. The presence of acetic acid in the substrates used for seed germination reduced the vigor and viability of seeds of cultivars IRGA 424 and BRS Querência, as well as seedling development, affecting mainly the roots of BRS Querência.

Fire resistant polyamide based on 1-(diorganooxyphosphonyl)methyl-2,4- and -2,6diamino benzene

Science.gov (United States)

Mikroyannidis, J. A. (Inventor); Kourtides, D. A. (Inventor)

1986-01-01

1-(Diorganooxyphosphonyl)methyl2,4- and-2,6diamino benzenes are reacted with polyacylhalides and optionally comonomers to produce polyamides which have desirable heat and fire resistance properties. These polymers are used to form fibers and fabrics where fire resistance properties are important, e.g., aircraft equipment and structures.

Status of potential PfATP6 molecular markers for artemisinin resistance in Suriname

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Adhin Malti R

2012-09-01

Full Text Available Abstract Background Polymorphisms within the PfATP6 gene have been indicated as potential molecular markers for artemisinin efficacy. Since 2004, the use of artemisinin combination therapy (ACT was introduced as first-line treatment of the uncomplicated malaria cases in Suriname. The aim of this research was to determine changes in Suriname in the status of the polymorphic markers in the PfATP6 gene before and after the adoption of the ACT-regimen, particularly of the S769N mutation, which was reported to be associated with in vitro Artemether resistance in the neighboring country French Guiana. Methods The PfATP6 gene from Plasmodium falciparum parasites in Suriname was investigated in 28 samples using PCR amplification and restriction enzyme analysis, to assess and determine the prevalence of potentially interesting single nucleotide polymorphisms. The polymorphisms [L263E; A623E; S769N], which may be associated with the artemisinin resistant phenotype were characterized in parasites from three endemic regions before and after the adoption of the ACT-regimen. In addition, the status of these molecular markers was compared in paired P. falciparum isolates from patients with recurring malaria after controlled ACT. Results All the investigated samples exhibit the wild-type genotype at all three positions; L263, A623, S769. Conclusion All investigated isolates before and after the adoption of the ACT-regimen and independent of endemic region harbored the wild-type genotype for the three investigated polymorphisms. The study revealed that decreased artemisinin susceptibility could occur independent from PfATP6 mutations, challenging the assumption that artemisinin resistance is associated with these mutations in the PfATP6 gene.

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.

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

Mis-spliced transcripts of nicotinic acetylcholine receptor alpha6 are associated with field evolved spinosad resistance in Plutella xylostella (L..

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Simon W Baxter

2010-01-01

Full Text Available The evolution of insecticide resistance is a global constraint to agricultural production. Spinosad is a new, low-environmental-risk insecticide that primarily targets nicotinic acetylcholine receptors (nAChR and is effective against a wide range of pest species. However, after only a few years of application, field evolved resistance emerged in the diamondback moth, Plutella xylostella, an important pest of brassica crops worldwide. Spinosad resistance in a Hawaiian population results from a single incompletely recessive and autosomal gene, and here we use AFLP linkage mapping to identify the chromosome controlling resistance in a backcross family. Recombinational mapping with more than 700 backcross progeny positioned a putative spinosad target, nAChR alpha 6 (Pxalpha6, at the resistance locus, PxSpinR. A mutation within the ninth intron splice junction of Pxalpha6 results in mis-splicing of transcripts, which produce a predicted protein truncated between the third and fourth transmembrane domains. Additional resistance-associated Pxalpha6 transcripts that excluded the mutation containing exon were detected, and these were also predicted to produce truncated proteins. Identification of the locus of resistance in this important crop pest will facilitate field monitoring of the spread of resistance and offer insights into the genetic basis of spinosad resistance in other species.

The late endocytic Rab39a GTPase regulates the interaction between multivesicular bodies and chlamydial inclusions.

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Gambarte Tudela, Julian; Capmany, Anahi; Romao, Maryse; Quintero, Cristian; Miserey-Lenkei, Stephanie; Raposo, Graca; Goud, Bruno; Damiani, Maria Teresa

2015-08-15

Given their obligate intracellular lifestyle, Chlamydia trachomatis ensure that they have access to multiple host sources of essential lipids by interfering with vesicular transport. These bacteria hijack Rab6-, Rab11- and Rab14-controlled trafficking pathways to acquire sphingomyelin from the Golgi complex. Another important source of sphingolipids, phospholipids and cholesterol are multivesicular bodies (MVBs). Despite their participation in chlamydial inclusion development and bacterial replication, the molecular mechanisms mediating the interaction between MVBs and chlamydial inclusions remain unknown. In the present study, we demonstrate that Rab39a labels a subset of late endocytic vesicles - mainly MVBs - that move along microtubules. Moreover, Rab39a is actively recruited to chlamydial inclusions throughout the pathogen life cycle by a bacterial-driven process that depends on the Rab39a GTP- or GDP-binding state. Interestingly, Rab39a participates in the delivery of MVBs and host sphingolipids to maturing chlamydial inclusions, thereby promoting inclusion growth and bacterial development. Taken together, our findings indicate that Rab39a favours chlamydial replication and infectivity. This is the first report showing that a late endocytic Rab GTPase is involved in chlamydial infection development. © 2015. Published by The Company of Biologists Ltd.

Comparação de parâmetros de crescimento e de desenvolvimento de dois biótipos de arroz vermelho com genótipos de arroz irrigado Growth and developmental of red and pad rice genotypes

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Nereu Augusto Streck

2008-01-01

Full Text Available Caracterizar o crescimento e desenvolvimento de biótipos de arroz vermelho pode ajudar no seu controle e no melhoramento para aumentar a competitividade do arroz cultivado com esta planta daninha. O objetivo deste trabalho foi quantificar parâmetros de crescimento e desenvolvimento de dois biótipos de arroz vermelho e compará-los com genótipos de arroz irrigado. Desenvolveu-se um experimento de campo em Santa Maria (RS, durante o ano agrícola 2004/2005 com cinco épocas de semeadura. O delineamento experimental utilizado foi o inteiramente casualizado com quatro repetições no esquema bifatorial (genótipos e épocas de semeadura. A unidade experimental foi um balde de 12 litros com dez plantas. Os genótipos utilizados foram: IRGA 421, IRGA 416, IRGA 417, IRGA 420, BR-IRGA 409, BRS 7 TAIM e EPAGRI 109 (tipo moderno, EEA 406 (tipo tradicional, um híbrido, arroz vermelho casca amarela com arista (AVCAA e arroz vermelho casca preta com arista (AVCPA. A data dos principais estágios de desenvolvimento da escala de COUNCE, área foliar, comprimento de panícula, estatura de planta, filocrono, número final de folhas no colmo principal e perfilhamento foram avaliados em cinco plantas por balde. Nos biótipos de arroz vermelho geralmente observa-se ciclo de desenvolvimento médio em relação aos genótipos cultivados, sendo o biótipo AVCAA de ciclo mais longo que o biótipo AVCPA. Os biótipos de arroz vermelho tiveram área foliar, estatura, panícula e filocrono maiores que os genótipos modernos de arroz. O perfilhamento dos biótipos de arroz vermelho foi variável e dependente da época de semeadura.The characterization growth and development of red rice biotypes may help controll strategies, the enhancement of competitiveness of cultivated rice with this weed and breeding programs. The objective of this is study was to quantity some growth and development parameters of two red rice biotypes as compared to irrigated genotypes. A field

Characteristics of Resistance Spot Welded Ti6Al4V Titanium Alloy Sheets

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Xinge Zhang

2017-10-01

Full Text Available Ti6Al4V titanium alloy is applied extensively in the aviation, aerospace, jet engine, and marine industries owing to its strength-to-weight ratio, excellent high-temperature properties and corrosion resistance. In order to extend the application range, investigations on welding characteristics of Ti6Al4V alloy using more welding methods are required. In the present study, Ti6Al4V alloy sheets were joined using resistance spot welding, and the weld nugget formation, mechanical properties (including tensile strength and hardness, and microstructure features of the resistance spot-welded joints were analyzed and evaluated. The visible indentations on the weld nugget surfaces caused by the electrode force and the surface expulsion were severe due to the high welding current. The weld nugget width at the sheets’ faying surface was mainly affected by the welding current and welding time, and the welded joint height at weld nugget center was chiefly associated with electrode force. The maximum tensile load of welded joint was up to 14.3 kN in the pullout failure mode. The hardness of the weld nugget was the highest because of the coarse acicular α′ structure, and the hardness of the heat-affected zone increased in comparison to the base metal due to the transformation of the β phase to some fine acicular α′ phase.

Biochemical mechanisms of imidacloprid resistance in Nilaparvata lugens: over-expression of cytochrome P450 CYP6AY1.

Science.gov (United States)

Ding, Zhiping; Wen, Yucong; Yang, Baojun; Zhang, Yixi; Liu, Shuhua; Liu, Zewen; Han, Zhaojun

2013-11-01

Imidacloprid is a key insecticide extensively used for control of Nilaparvata lugens, and its resistance had been reported both in the laboratory selected strains and field populations. A target site mutation Y151S in two nicotinic acetylcholine receptor subunits and enhanced oxidative detoxification have been identified in the laboratory resistant strain, contributing importantly to imidacloprid resistance in N. lugens. To date, however, imidacloprid resistance in field population is primarily attributable to enhanced oxidative detoxification by over-expressed P450 monooxygenases. A resistant strain (Res), originally collected from a field population and continuously selected in laboratory with imidacloprid for more than 40 generations, had 180.8-fold resistance to imidacloprid, compared to a susceptible strain (Sus). Expression of different putative P450 genes at mRNA levels was detected and compared between Res and Sus strains, and six genes were found expressed significantly higher in Res strain than in Sus strain. CYP6AY1 was found to be the most different expressed P450 gene and its mRNA level in Res strain was 17.9 times of that in Sus strain. By expressing in E. coli cells, CYP6AY1 was found to metabolize imidacloprid efficiently with initial velocity calculated of 0.851 ± 0.073 pmol/min/pmol P450. When CYP6AY1 mRNA levels in Res strain was reduced by RNA interference, imidacloprid susceptibility was recovered. In four field populations with different resistance levels, high levels of CYP6AY1 transcript were also found. In vitro and in vivo studies provided evidences that the over-expression of CYP6AY1 was one of the key factors contributing to imidacloprid resistance in the laboratory selected strain Res, which might also be the important mechanism for imidacloprid resistance in field populations, when the target site mutation was not prevalent at present. Copyright © 2013 Elsevier Ltd. All rights reserved.

Inverse Resistance Change Cr2Ge2Te6-Based PCRAM Enabling Ultralow-Energy Amorphization.

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Hatayama, Shogo; Sutou, Yuji; Shindo, Satoshi; Saito, Yuta; Song, Yun-Heub; Ando, Daisuke; Koike, Junichi

2018-01-24

Phase-change random access memory (PCRAM) has attracted much attention for next-generation nonvolatile memory that can replace flash memory and can be used for storage-class memory. Generally, PCRAM relies on the change in the electrical resistance of a phase-change material between high-resistance amorphous (reset) and low-resistance crystalline (set) states. Herein, we present an inverse resistance change PCRAM with Cr 2 Ge 2 Te 6 (CrGT) that shows a high-resistance crystalline reset state and a low-resistance amorphous set state. The inverse resistance change was found to be due to a drastic decrease in the carrier density upon crystallization, which causes a large increase in contact resistivity between CrGT and the electrode. The CrGT memory cell was demonstrated to show fast reversible resistance switching with a much lower operating energy for amorphization than a Ge 2 Sb 2 Te 5 memory cell. This low operating energy in CrGT should be due to a small programmed amorphous volume, which can be realized by a high-resistance crystalline matrix and a dominant contact resistance. Simultaneously, CrGT can break the trade-off relationship between the crystallization temperature and operating speed.

Resistance monitoring and cross-resistance role of CYP6CW1 between buprofezin and pymetrozine in field populations of Laodelphax striatellus (Fallén)

OpenAIRE

Zhang, Yueliang; Han, Yangchun; Liu, Baosheng; Yang, Qiong; Guo, Huifang; Liu, Zewen; Wang, Lihua; Fang, Jichao

2017-01-01

Monitoring resistance and investigating insecticide resistance mechanisms are necessary for controlling the small brown planthopper, Laodelphax striatellus. The susceptibility to four common insecticides of L. striatellus collected from Jiangsu, Anhui, Zhejiang and Jilin provinces of China in 2015 was monitored. The results showed that all field populations remained susceptible to chlorpyrifos and thiamethoxam with resistance ratios (RRs) of 2.3- to 9.5 and 1.6- to 3.3, respectively, while th...

Low temperature resistivity studies of SmB6: Observation of two-dimensional variable-range hopping conductivity

Science.gov (United States)

Batkova, Marianna; Batko, Ivan; Gabáni, Slavomír; Gažo, Emil; Konovalova, Elena; Filippov, Vladimir

2018-05-01

We studied electrical resistance of a single-crystalline SmB6 sample with a focus on the region of the "low-temperature resistivity plateau". Our observations did not show any true saturation of the electrical resistance at temperatures below 3 K down to 70 mK. According to our findings, temperature dependence of the electrical conduction in a certain temperature interval above 70 mK can be decomposed into a temperature-independent term and a temperature-activated term that can be described by variable-range hopping formula for two-dimensional systems, exp [ -(T0 / T) 1 / 3 ]. Thus, our results indicate importance of hopping type of electrical transport in the near-surface region of SmB6.

Sex Differences in the Association between Level of Childhood Interleukin-6 and Insulin Resistance in Adolescence

DEFF Research Database (Denmark)

Bugge, Anna; El-Naaman, Bianca; G McMurray, Robert

2012-01-01

followed for 4 years. Anthropometrics and VO(2peak) were measured. Fasting blood samples were analyzed for IL-6, insulin, and glucose. Homeostasis model assessment (HOMA-IR) was used as a measure of insulin resistance. Results. For girls but not boys, levels of IL-6 at age 9 yrs correlated with HOMA-IR...... at age 13 yrs: r = 0.223, P = 0.008. Girls with IL-6 levels within the highest quartile at age 9 yrs had an odds ratio of 3.68 (CI = 1.58-8.57) being in the highest quartile of HOMA-IR four years later. Conclusion. In this cohort, IL-6 levels in childhood were related to insulin resistance in adolescence...

Development and characterization of japonica rice lines carrying the brown planthopper-resistance genes BPH12 and BPH6.

Science.gov (United States)

Qiu, Yongfu; Guo, Jianping; Jing, Shengli; Zhu, Lili; He, Guangcun

2012-02-01

The brown planthopper (Nilaparvata lugens Stål; BPH) has become a severe constraint on rice production. Identification and pyramiding BPH-resistance genes is an economical and effective solution to increase the resistance level of rice varieties. All the BPH-resistance genes identified to date have been from indica rice or wild species. The BPH12 gene in the indica rice accession B14 is derived from the wild species Oryza latifolia. Using an F(2) population from a cross between the indica cultivar 93-11 and B14, we mapped the BPH12 gene to a 1.9-cM region on chromosome 4, flanked by the markers RM16459 and RM1305. In this population, BPH12 appeared to be partially dominant and explained 73.8% of the phenotypic variance in BPH resistance. A near-isogenic line (NIL) containing the BPH12 locus in the background of the susceptible japonica variety Nipponbare was developed and crossed with a NIL carrying BPH6 to generate a pyramid line (PYL) with both genes. BPH insects showed significant differences in non-preference in comparisons between the lines harboring resistance genes (NILs and PYL) and Nipponbare. BPH growth and development were inhibited and survival rates were lower on the NIL-BPH12 and NIL-BPH6 plants compared to the recurrent parent Nipponbare. PYL-BPH6 + BPH12 exhibited 46.4, 26.8 and 72.1% reductions in population growth rates (PGR) compared to NIL-BPH12, NIL-BPH6 and Nipponbare, respectively. Furthermore, insect survival rates were the lowest on the PYL-BPH6 + BPH12 plants. These results demonstrated that pyramiding different BPH-resistance genes resulted in stronger antixenotic and antibiotic effects on the BPH insects. This gene pyramiding strategy should be of great benefit for the breeding of BPH-resistant japonica rice varieties.

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

Science.gov (United States)

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.

The Cytochrome P450 gene CYP6P12 confers pyrethroid resistance in kdr-free Malaysian populations of the dengue vector Aedes albopictus.

Science.gov (United States)

Ishak, Intan H; Riveron, Jacob M; Ibrahim, Sulaiman S; Stott, Rob; Longbottom, Joshua; Irving, Helen; Wondji, Charles S

2016-04-20

Control of Aedes albopictus, major dengue and chikungunya vector, is threatened by growing cases of insecticide resistance. The mechanisms driving this resistance remain poorly characterised. This study investigated the molecular basis of insecticide resistance in Malaysian populations of Ae. albopictus. Microarray-based transcription profiling revealed that metabolic resistance (cytochrome P450 up-regulation) and possibly a reduced penetration mechanism (consistent over-expression of cuticular protein genes) were associated with pyrethroid resistance. CYP6P12 over-expression was strongly associated with pyrethroid resistance whereas CYP6N3 was rather consistently over-expressed across carbamate and DDT resistant populations. Other detoxification genes also up-regulated in permethrin resistant mosquitoes included a glucuronosyltransferase (AAEL014279-RA) and the glutathione-S transferases GSTS1 and GSTT3. Functional analyses further supported that CYP6P12 contributes to pyrethroid resistance in Ae. albopictus as transgenic expression of CYP6P12 in Drosophila was sufficient to confer pyrethroid resistance in these flies. Furthermore, molecular docking simulations predicted CYP6P12 possessing enzymatic activity towards pyrethroids. Patterns of polymorphism suggested early sign of selection acting on CYP6P12 but not on CYP6N3. The major role played by P450 in the absence of kdr mutations suggests that addition of the synergist PBO to pyrethroids could improve the efficacy of this insecticide class and overcome resistance in field populations of Ae. albopictus.

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.

Identification of QTLs for resistance to sclerotinia stem rot and BnaC.IGMT5.a as a candidate gene of the major resistant QTL SRC6 in Brassica napus.

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Jian Wu

Full Text Available Stem rot caused by Sclerotinia sclerotiorum in many important dicotyledonous crops, including oilseed rape (Brassica napus, is one of the most devastating fungal diseases and imposes huge yield loss each year worldwide. Currently, breeding for Sclerotinia resistance in B. napus, as in other crops, can only rely on germplasms with quantitative resistance genes. Thus, the identification of quantitative trait locus (QTL for S. sclerotiorum resistance/tolerance in this crop holds immediate promise for the genetic improvement of the disease resistance. In this study, ten QTLs for stem resistance (SR at the mature plant stage and three QTLs for leaf resistance (LR at the seedling stage in multiple environments were mapped on nine linkage groups (LGs of a whole genome map for B. napus constructed with SSR markers. Two major QTLs, LRA9 on LG A9 and SRC6 on LG C6, were repeatedly detected across all environments and explained 8.54-15.86% and 29.01%-32.61% of the phenotypic variations, respectively. Genotypes containing resistant SRC6 or LRA9 allele showed a significant reduction in disease lesion after pathogen infection. Comparative mapping with Arabidopsis and data mining from previous gene profiling experiments identified that the Arabidopsis homologous gene of IGMT5 (At1g76790 was related to the SRC6 locus. Four copies of the IGMT5 gene in B. napus were isolated through homologous cloning, among which, only BnaC.IGMT5.a showed a polymorphism between parental lines and can be associated with the SRC6. Furthermore, two parental lines exhibited a differential expression pattern of the BnaC.IGMT5.a gene in responding to pathogen inoculation. Thus, our data suggested that BnaC.IGMT5.a was very likely a candidate gene of this major resistance QTL.

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

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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.

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

Science.gov (United States)

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

Functional analysis of CYP6ER1, a P450 gene associated with imidacloprid resistance in Nilaparvata lugens.

Science.gov (United States)

Pang, Rui; Chen, Meng; Liang, Zhikun; Yue, Xiangzhao; Ge, Hu; Zhang, Wenqing

2016-10-10

The cytochrome P450 CYP6ER1 has been reported to play an important role in imidacloprid resistance of the brown planthopper (BPH), Nilaparvata lugens, and is overexpressed in most resistant populations. In the present study, we confirmed that CYP6ER1 expression can be induced by certain levels of imidacloprid. Developmental expression analysis revealed that CYP6ER1 was expressed highly in the adult stage, and tissue distribution analysis showed that CYP6ER1 was expressed mainly in the fat body and midgut. RNA interference (RNAi) of CYP6ER1 and transgenic expression of CYP6ER1 in Drosophila melanogaster both suggested that the expression of CYP6ER1 is sufficient to confer imidacloprid resistance. Furthermore, we analyzed the interaction of imidacloprid and CYP6ER1 monooxygenase by using dynamic simulations and molecular docking. We found that Nitrogen atoms in the heterocycle of the imidacloprid molecule may bind to iron atoms in the center of the homology model of CYP6ER1 via 4,5-dihedro-1H-imidazole. This finding contributes to a better understanding of how CYP6ER1 takes part in the insecticide metabolism.

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

The interaction properties of the human Rab GTPase family--comparative analysis reveals determinants of molecular binding selectivity.

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Matthias Stein

Full Text Available Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood.Comparative analysis of the amino acid sequences and the three-dimensional electrostatic and hydrophobic molecular interaction fields of 62 human Rab proteins revealed a wide range of binding properties with large differences between some Rab proteins. This analysis assists the functional annotation of Rab proteins 12, 14, 26, 37 and 41 and provided an explanation for the shared function of Rab3 and 27. Rab7a and 7b have very different electrostatic potentials, indicating that they may bind to different effector proteins and thus, exert different functions. The subfamily V Rab GTPases which are associated with endosome differ subtly in the interaction properties of their switch regions, and this may explain exchange factor specificity and exchange kinetics.We have analysed conservation of sequence and of molecular interaction fields to cluster and annotate the human Rab proteins. The analysis of three dimensional molecular interaction fields provides detailed insight that is not available from a sequence-based approach alone. Based on our results, we predict novel functions for some Rab proteins and provide insights into their divergent functions and the determinants of their binding partner selectivity.

Microwave surface resistance of YBa2Cu3O/sub 6.9/ superconducting films

International Nuclear Information System (INIS)

Martens, J.S.; Beyer, J.B.; Ginley, D.S.

1988-01-01

The microwave surface resistance of an YBa 2 Cu 3 O/sub 6.9/ superconducting thick film was measured over the range 7.0--16.7 GHz at 77 K. This was done by placing a sample in a TE 01 /sub n/ wavemeter cavity and observing the change in selectivity of the cavity. The material's surface resistance is of the same order of magnitude as that of silver at 77 K from 8 to 12 GHz and improves about another order at 4.2 K. The power-law behavior of surface resistance with frequency is probably close to quadratic. This is similar to the behavior of low critical temperature superconductors

Improvement of corrosion resistance of carbon steel using chemical vapor deposition from Cr(CO)6 and Mo(CO)6 with an ArF-excimer laser

International Nuclear Information System (INIS)

Okada, Naotada; Katsumura, Yosuke; Ishigure, Kenkichi

1995-01-01

The corrosion resistance of carbon steel has been improved by the deposition from the mixture of Mo(CO) 6 and Cr(CO) 6 as well as from each carbonyl alone with an ArF-excimer (193nm). The corrosion resistance evaluated by multi sweep cyclic voltammetry attained by coating with the films from the mixture is higher than from Mo(CO) 6 alone, while lower than from Cr(CO) 6 alone. While the corrosion resistance increases with beam intensity monotonically over the range 4-25 MWcm -2 for the deposition from Mo(CO) 6 alone, it tends to decrease slightly above 15 MWcm -2 for the deposition from Mo(CO) 6 alone and from the mixture. SEM photographs show that the films from each carbonyl and their mixture consist of small grains that are more densely packed at higher beam intensities. The comparison of the film thickness evaluated from sputtering time to remove the films with that from direct observation with SEM suggests that the density of the film increases with beam intensity. In the films deposited from the mixture, molybdenum is preferentially incorporated from the gas phase. In addition, a model of gas-phase processes including photolysis of Cr(CO) 6 , transportation of photofragments to the substrate surface, and elimination of photofragments through chemical reactions during transportation, is proposed and simulated. Applications of the model will be discussed. (author)

Functional analysis of CYP6ER1, a P450 gene associated with imidacloprid resistance in Nilaparvata lugens

OpenAIRE

Pang, Rui; Chen, Meng; Liang, Zhikun; Yue, Xiangzhao; Ge, Hu; Zhang, Wenqing

2016-01-01

The cytochrome P450 CYP6ER1 has been reported to play an important role in imidacloprid resistance of the brown planthopper (BPH), Nilaparvata lugens, and is overexpressed in most resistant populations. In the present study, we confirmed that CYP6ER1 expression can be induced by certain levels of imidacloprid. Developmental expression analysis revealed that CYP6ER1 was expressed highly in the adult stage, and tissue distribution analysis showed that CYP6ER1 was expressed mainly in the fat bod...

Alantolactone Improves Prolonged Exposure of Interleukin-6-Induced Skeletal Muscle Inflammation Associated Glucose Intolerance and Insulin Resistance

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Minjee Kim

2017-06-01

Full Text Available The pro-inflammatory cytokine, Interleukin-6 (IL-6, has been proposed to be one of the mediators that link chronic inflammation to glucose intolerance and insulin resistance. Many studies have demonstrated the effects of IL-6 on insulin action in the skeletal muscle. However, few studies have investigated the effect of long-term treatment of IL-6, leading to glucose intolerance and insulin resistance. In the present study, we observed protective effects of alantolactone, a sesquiterpene lactone isolated from Inula helenium against glucose intolerance and insulin resistance induced by prolonged exposure of IL-6. Alantolactone has been reported to have anti-inflammatory and anti-cancer effects through IL-6-induced signal transducer and activator of transcription 3 (STAT3 signaling pathway. The relationship between IL-6 exposure and expression of toll-like receptor 4 (TLR4, involved in inflammation in the skeletal muscle, and the underlying mechanisms were investigated. We observed maximum dysregulation of glucose uptake after 40 ng/ml IL-6 induction for 24 h in L6 myotubes. Prolonged IL-6 exposure suppressed glucose uptake regulating alpha serine/threonine-protein kinase (AKT phosphorylation; however, pretreatment with alantolactone activated AKT phosphorylation and improved glucose uptake. Alantolactone also attenuated IL-6-stimulated STAT3 phosphorylation, followed by an increase in expression of negative regulator suppressor of cytokine signaling 3 (SOCS3. Furthermore, IL-6-induced expression of pathogen recognition receptor, TLR4, was also suppressed by alantolactone pretreatment. Post-silencing of STAT3 using siRNA approach, IL-6-stimulated siRNA-STAT3 improved glucose uptake and suppressed TLR4 gene expression. Taken together, we propose that, as a STAT3 inhibitor, alantolactone, improves glucose regulation in the skeletal muscle by inhibiting IL-6-induced STAT3-SOCS3 signaling followed by inhibition of the TLR4 gene expression. Therefore

High-pressure resistivity measurements on the β-pyrochlore oxide KOs2O6

Science.gov (United States)

Ogusu, Hiroki; Takeshita, Nao; Yamaura, Jun-Ichi; Okamoto, Yoshihiko; Hiroi, Zenji

2010-12-01

High-pressure resistivity measurements are performed on a high-quality single crystal of the β-pyrochlore oxide KOs2O6 in the pressure range of 1.0 to 5.0 GPa. The superconducting transition temperature T increases slightly from 9.6 K at ambient pressure to 9.8 K at 1.0 GPa, decreases gradually with increasing pressure, and suddenly drops from 6.5 K to 3.2 K across P=3.6GPa. The drop of T at P is likely to be related to a change in the rattling vibration associated with a symmetry-breaking structural transition.

Soma térmica de algumas fases do ciclo de desenvolvimento da escala de COUNCE para cultivares Sul-Brasileiras de arroz irrigado Thermal time of some developmental phases of the COUNCE scale for irrigated rice cultivars grown in Southern Brazil

Directory of Open Access Journals (Sweden)

Nereu Augusto Streck

2007-01-01

Full Text Available A escala de COUNCE é uma escala de desenvolvimento de arroz recente que vem sendo gradativamente adotada no Brasil. No entanto, a duração das fases em ºC dia dessa escala ainda não está disponível para as cultivares brasileiras de arroz irrigado. Sendo assim, o objetivo deste trabalho foi determinar a soma térmica de algumas fases do ciclo de desenvolvimento da escala de COUNCE para cultivares Sul-Brasileiras de arroz irrigado, utilizando-se diversos métodos de cálculo e diferentes temperaturas cardinais ao longo do ciclo de desenvolvimento. Um experimento a campo foi desenvolvido em Santa Maria, RS, durante dois anos agrícolas (2003/2004 e 2004/2005 e com cinco épocas de semeadura em cada um deles. O delineamento experimental foi o inteiramente casualizado com quatro repetições. Cada repetição consistia de um vaso de 12 litros com 10 plantas. As datas dos estágios de desenvolvimento R2 (aparecimento do colar da folha bandeira e R9 (todos os grãos com casca marrom da escala de COUNCE foram registradas no colmo principal de cinco plantas por vaso. As cultivares utilizadas foram IRGA 421, IRGA 416, IRGA 417, IRGA 420, BR-IRGA 409, BRS 7 TAIM e EPAGRI 109. Considerando-se as temperaturas cardinais de 11 °C, 28 °C e 40 °C, a soma térmica para completar a fase emergência-R2 foi de 700-800 °C dia na cultivar muito precoce, 1000-1400 °C dia nas cultivares precoces e 1300-1700 °C dia nas cultivares médias e tardia, e a soma térmica para completar a fase R2-R9 foi de 400-700 °C dia e sem relação com o ciclo da cultivar.The COUNCE scale is a developmental scale recently proposed for rice and that is being used in Brazil. However, the duration of developmental phases of this scale in degree-days is not available for Brazilian irrigated rice cultivars. Thus, the objective of this study was to determine the thermal time of some developmental phases of the COUNCE scale in irrigated rice cultivars grown in Southern Brazil using

Estimativa do teor de nitrogênio em arroz irrigado com o clorofilômetro e a cartela de cores Nitrogen content estimative in flooded rice by using of chlorophyll meter and color chart

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Elisandra Pocojeski

2012-11-01

Full Text Available O clorofilômetro e a cartela de cores têm sido utilizados para monitorar a adubação nitrogenada em diferentes culturas, com o pressuposto de que há uma relação entre suas leituras e o teor de nitrogênio (N no tecido das plantas. O objetivo deste trabalho foi avaliar a utilização do clorofilômetro e da cartela de cores na estimativa do teor de N em folhas de arroz irrigado por alagamento. Foram utilizados dois experimentos instalados em um Planossolo Háplico, sendo o primeiro conduzido com cinco doses de N (0, 50, 80, 120 e 160kg ha-1 na forma de ureia e a cultivar 'IRGA 417', e o segundo com seis cultivares de arroz irrigado de diferentes ciclos ('BR-IRGA 409', 'BR-IRGA 410', 'IRGA 417', 'IRGA 421', 'EPAGRI 108' e 'HÍBRIDO 2' com uma única dose de N. Foram realizadas avaliações com clorofilômetro e a cartela de cores nas folhas do arroz em diferentes épocas. Simultaneamente às leituras, foram coletadas amostras de folhas e nelas determinado o teor de N. Quando variaram as doses de N, houve correlação significativa entre as leituras do clorofilômetro e da cartela de cores com o teor de N nas folhas, independente da época de avaliação. Já quando variaram as cultivares, as leituras do clorofilômetro se correlacionaram com o teor de N (r=0,78; PThe chlorophyll meter and the color chart have been applied to monitor nitrogen fertilization in different crops, based on the assumption that there is a correlation between their readings and the nitrogen content (N in the plant tissue. This research aims at assessing the use of the chlorophyll meter and the color chart to estimate the N content in leaves of flooded rice plants. Two experiments installed at a Albaqualf (Planossolo Háplico were used, the first using five N doses (0, 50, 80, 120 and 160kg ha-1 as urea and the cultivar 'IRGA 417', and the second with six flooded rice cultivars of different cycles ('BR-IRGA 409', 'BR-IRGA 410', 'IRGA 417', 'IRGA 421', 'EPAGRI 108' and

17β-estradiol exerts anticancer effects in anoikis-resistant hepatocellular carcinoma cell lines by targeting IL-6/STAT3 signaling

Energy Technology Data Exchange (ETDEWEB)

Lee, Seulki, E-mail: sl10f@naver.com [Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799 (Korea, Republic of); Lee, Minjong, E-mail: minjonglee2@naver.com [Division of Gastroenterology, Department of Internal Medicine, Kangwon National University Hospital, 156 Baengnyeong-ro, Chuncheon-si, Gangwon-do (Korea, Republic of); Kim, Jong Bin, E-mail: kkimjp@hanmail.net [Hormel Institute, University of Minnesota, 801 16th Ave NE, Austin, MN, 55912 (United States); Jo, Ara, E-mail: loveara0315@naver.com [Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799 (Korea, Republic of); Cho, Eun Ju, E-mail: creatioex@gmail.com [Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799 (Korea, Republic of); Yu, Su Jong, E-mail: ydoctor2@hanmail.net [Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799 (Korea, Republic of); Lee, Jeong-Hoon, E-mail: pindra@empal.com [Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799 (Korea, Republic of); Yoon, Jung-Hwan, E-mail: yoonjh@snu.ac.kr [Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799 (Korea, Republic of); Kim, Yoon Jun, E-mail: yoonjun@snu.ac.kr [Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799 (Korea, Republic of)

2016-05-13

17β-Estradiol (E2) has been proven to exert protective effects against HCC; however, its mechanism on HCC proliferation and suppression of invasion remains to be further explored. Because HCC up-regulates serum Interleukin-6 (IL-6) levels and Signal Transducer and Activator of Transcription 3 (STAT3), molecular agents that attenuate IL-6/STAT3 signaling can potentially suppress HCC development. In this study, we examined involvement of E2 in anoikis resistance that induces invasion capacities and chemo-resistance. Huh-BAT and HepG2 cells grown under anchorage-independent condition were selected. The anoikis-resistant (AR) cells showed stronger chemo-resistance against sorafenib, doxorubicin, 5-fluorouracil and cisplatin compared to adherent HCC cells. AR HCC cells exhibited decreased expression of E-cadherin and increased expression of the N-cadherin and vimentin compared to adherent HCC cells. We then demonstrated that E2 suppressed cell proliferation in AR HCC cells. IL-6 treatment enhanced invasive characteristics, and E2 reversed it. Regarding mechanism of E2, it decreased in the phosphorylation of STAT3 that overexpressed on AR HCC cells. The inhibitory effect of E2 on cell growth was accompanied with cell cycle arrest at G2/M phase and caspase-3/9/PARP activation through c-Jun N-terminal Kinase (JNK) phosphorylation. Taken together, these findings suggested that E2 inhibited the proliferation of AR HCC cells through down-regulation of IL-6/STAT3 signaling. Thus, E2 can be a potential therapeutic drug for treatment of metastatic or chemo-resistant HCC. -- Highlights: •Anoikis-resistant HCC cells characterized chemo-resistant and metastatic potentials. •17β-Estradiol down-regulated IL-6/STAT3 signaling in anoikis-resistant HCC cells. •17β-Estradiol suppressed cell proliferation by inducing G2/M phase arrest and apoptosis though JNK phosphorylation.

The correlation of adiponectin, IL-6 with insulin resistance and macrovascular lesion in type 2 diabetes

International Nuclear Information System (INIS)

Shi Bimin; Cheng Xingbo

2006-01-01

Objective: To investigate the serum level changes of adiponectin and IL-6 and their relation with macrovascular complications in patients with type 2 diabetes before and after thiazolidinediones intervention. Methods: Serum adiponectin and IL-6 level were examined using ELISA method in 16 patients with obese type 2 diabetes, 24 patients with diabetes with normal body weight, and 14 controls. Their body mass index (BMI) and HOMA-IR were calculated and the internal membrane thickness (IMT) of carotid artery was also observed. These indexes were reexamined after thiazolidinediones (Avandia) intervention and correlation analysis was performed. Results: A decreased serum concentration of adiponectin and an elevated level of IL-6 and HOMA-IR were found in diabetes group especially in those with obesity (P<0.01). Serum adiponectin was negatively correlated with HOMA-IR, BMI, IL-6 and IMT while IL-6 was positively correlated with the above-mentioned indexes. After treatment with thiazolidinediones, the insulin resistance state and adponectin and IL-6 were significantly improved especially in those with obesity (P<0.05, P<0.01). Conclusion: Adiponectin is closed correlated with obesity (P<0.05, P<0.01). Conclusion: Adiponectin is closed correlated with obesity, insulin resistance and macrovascular lesions. IL-6 may be involved in the mechanism of insulin resistance in type 2 diabetes patients especially in the obese diabetes group. In addition to enhance the sensitivity of insulin, thiazolidinediones may play a potential role in anti-inflammation, anti-atherosclerosis and immuno-regulation. (authors)

Contrasting patterns in the evolution of the Rab GTPase family in Archaeplastida

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Romana Petrželková

2014-12-01

Full Text Available Rab GTPases are a vast group of proteins serving a role of master regulators in membrane trafficking in eukaryotes. Previous studies delineated some 23 Rab and Rab-like paralogs ancestral for eukaryotes and mapped their current phylogenetic distribution, but the analyses relied on a limited sampling of the eukaryotic diversity. Taking advantage of the recent growth of genome and transcriptome resources for phylogenetically diverse plants and algae, we reanalyzed the evolution of the Rab family in eukaryotes with the primary plastid, collectively constituting the presumably monophyletic supergroup Archaeplastida. Our most important novel findings are as follows: (i the ancestral set of Rabs in Archaeplastida included not only the paralogs Rab1, Rab2, Rab5, Rab6, Rab7, Rab8, Rab11, Rab18, Rab23, Rab24, Rab28, IFT27, and RTW (=Rabl2, as suggested previously, but also Rab14 and Rab34, because Rab14 exists in glaucophytes and Rab34 is present in glaucophytes and some green algae; (ii except in embryophytes, Rab gene duplications have been rare in Archaeplastida. Most notable is the independent emergence of divergent, possibly functionally novel, in-paralogs of Rab1 and Rab11 in several archaeplastidial lineages; (iii recurrent gene losses have been a significant factor shaping Rab gene complements in archaeplastidial species; for example, the Rab21 paralog was lost at least six times independently within Archaeplastida, once in the lineage leading to the “core” eudicots; (iv while the glaucophyte Cyanophora paradoxa has retained the highest number of ancestral Rab paralogs among all archaeplastidial species studied so far, rhodophytes underwent an extreme reduction of the Rab gene set along their stem lineage, resulting in only six paralogs (Rab1, Rab2, Rab6, Rab7, Rab11, and Rab18 present in modern red algae. Especially notable is the absence of Rab5, a virtually universal paralog essential for the endocytic pathway, suggesting that endocytosis

GTPase ROP2 binds and promotes activation of target of rapamycin, TOR, in response to auxin.

Science.gov (United States)

Schepetilnikov, Mikhail; Makarian, Joelle; Srour, Ola; Geldreich, Angèle; Yang, Zhenbiao; Chicher, Johana; Hammann, Philippe; Ryabova, Lyubov A

2017-04-03

Target of rapamycin (TOR) promotes reinitiation at upstream ORFs (uORFs) in genes that play important roles in stem cell regulation and organogenesis in plants. Here, we report that the small GTPase ROP2, if activated by the phytohormone auxin, promotes activation of TOR, and thus translation reinitiation of uORF-containing mRNAs. Plants with high levels of active ROP2, including those expressing constitutively active ROP2 (CA-ROP2), contain high levels of active TOR ROP2 physically interacts with and, when GTP-bound, activates TOR in vitro TOR activation in response to auxin is abolished in ROP-deficient rop2 rop6 ROP4 RNAi plants. GFP-TOR can associate with endosome-like structures in ROP2-overexpressing plants, indicating that endosomes mediate ROP2 effects on TOR activation. CA-ROP2 is efficient in loading uORF-containing mRNAs onto polysomes and stimulates translation in protoplasts, and both processes are sensitive to TOR inhibitor AZD-8055. TOR inactivation abolishes ROP2 regulation of translation reinitiation, but not its effects on cytoskeleton or intracellular trafficking. These findings imply a mode of translation control whereby, as an upstream effector of TOR, ROP2 coordinates TOR function in translation reinitiation pathways in response to auxin. © 2017 The Authors.

Molecular characterisation of resistance against potato wart races 1, 2, 6 and 18 in a tetraploid population of potato (Solanum tuberosum subsp. tuberosum).

Science.gov (United States)

Groth, Jennifer; Song, Yesu; Kellermann, Adolf; Schwarzfischer, Andrea

2013-05-01

Potato wart is caused by the obligate biotrophic fungus Synchytrium endobioticum, which is subject to quarantine regulations due to the production of long persisting spores in the soil and the lack of effective fungicides. The objective of this study was to identify quantitative trait loci (QTL) for resistance against potato wart races (R) 1, 2, 6 and 18 in a tetraploid potato population developed by crossing cv. Saturna (resistant to R1) with cv. Panda (resistant to R1, R2, R6, R18). A total of 92 progenies were used for phenotyping and genotyping. Resistance tests were performed for races 1 and 18 in 2 years and for races 2 and 6 in 1 year on 10 to 20 eyepieces per genotype. Based on amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers, linkage maps were established for the female and male parent, respectively. Single marker analysis followed by a multiple regression analysis revealed initial marker-trait associations. The interval mapping routine of TetraploidMap was applied for QTL analysis. A major QTL for resistance against race 1 explaining between 46 % and 56 % of the phenotypic variation was identified near Sen1, a known resistance locus for potato wart race 1 on chromosome XI. Other resistance QTL were detected on chromosomes I (to R2), II (to R6, 18), VI (to R1, 2, 6, 18), VII (to R2, 6, 18), VIII (to R1, 2, 6, 18), X (to R2, 6, 18), XI (to R2, 6, 18) and on an unknown linkage group (to R18) explaining minor to moderate effects of the phenotypic variation. Resistance QTL against different potato wart races often overlapped, particularly concerning races 2, 6 and 18. Overall, this study gives a valuable insight into the complex inheritance of resistance against potato wart.

Cisgenic Rvi6 scab-resistant apple lines show no differences in Rvi6 transcription when compared with conventionally bred cultivars

NARCIS (Netherlands)

Chizzali, Cornelia; Gusberti, Michele; Schouten, H.J.; Gessler, Cesare; Broggini, G.A.L.

2016-01-01

Main conclusion: The expression of the apple scab resistance geneRvi6in different apple cultivars and lines is not modulated by biotic or abiotic factors.All commercially important apple cultivars are susceptible to Venturia inaequalis, the causal organism of apple scab. A limited number of apple

Sensory neuropathy with bone destruction due to a mutation in the membrane-shaping atlastin GTPase 3.

Science.gov (United States)

Kornak, Uwe; Mademan, Inès; Schinke, Marte; Voigt, Martin; Krawitz, Peter; Hecht, Jochen; Barvencik, Florian; Schinke, Thorsten; Gießelmann, Sebastian; Beil, F Timo; Pou-Serradell, Adolf; Vílchez, Juan J; Beetz, Christian; Deconinck, Tine; Timmerman, Vincent; Kaether, Christoph; De Jonghe, Peter; Hübner, Christian A; Gal, Andreas; Amling, Michael; Mundlos, Stefan; Baets, Jonathan; Kurth, Ingo

2014-03-01

Many neurodegenerative disorders present with sensory loss. In the group of hereditary sensory and autonomic neuropathies loss of nociception is one of the disease hallmarks. To determine underlying factors of sensory neurodegeneration we performed whole-exome sequencing in affected individuals with the disorder. In a family with sensory neuropathy with loss of pain perception and destruction of the pedal skeleton we report a missense mutation in a highly conserved amino acid residue of atlastin GTPase 3 (ATL3), an endoplasmic reticulum-shaping GTPase. The same mutation (p.Tyr192Cys) was identified in a second family with similar clinical outcome by screening a large cohort of 115 patients with hereditary sensory and autonomic neuropathies. Both families show an autosomal dominant pattern of inheritance and the mutation segregates with complete penetrance. ATL3 is a paralogue of ATL1, a membrane curvature-generating molecule that is involved in spastic paraplegia and hereditary sensory neuropathy. ATL3 proteins are enriched in three-way junctions, branch points of the endoplasmic reticulum that connect membranous tubules to a continuous network. Mutant ATL3 p.Tyr192Cys fails to localize to branch points, but instead disrupts the structure of the tubular endoplasmic reticulum, suggesting that the mutation exerts a dominant-negative effect. Identification of ATL3 as novel disease-associated gene exemplifies that long-term sensory neuronal maintenance critically depends on the structural organisation of the endoplasmic reticulum. It emphasizes that alterations in membrane shaping-proteins are one of the major emerging pathways in axonal degeneration and suggests that this group of molecules should be considered in neuroprotective strategies.

Genome - wide identification, molecular characterization and expression analysis of the rop gtpase family in pepper (capsicum annum)

International Nuclear Information System (INIS)

Huang, D.; Li, M.; He, S.

2015-01-01

ROP/RAC GTPases is a plant-specific subfamily of Rho GTPases that plays a versatile role in the regulation of plant growth, development, in hormone signal transduction and response to the environment. Prior to the present study, only one Rop gene in pepper has been described. However, with the recent release of the draft genome sequence of pepper allowes us to conduct a genome wide search to identify how many Rop family members existed in pepper genome. We carried out bioinformatics analysis to establish the conserved as well as divergent regions on the protein sequences, phylogenetically analysis and the corresponding result shows that, CaROPs could be distributed into four groups as described in the literature for their homologs in Arabidopsis. To understand the function of nine Rop genes in pepper, we accordingly studied the tissue, fruit development and ripening expression patterns of CaRop genes by obtained RNA-seq data from public database. From our analysis, we realized that the expression of CaRop genes shows no total tissue or developmental specific expression. Furthermore, gene expression profiles of CaRop in response to environment stresses and hormone treatment, such as inoculated with Ralstonia solanacearum, by heat stress as well as treated with four phytohormones respectively and evaluated with real time RT-PCR. The potential involvement of specific CaRop genes in growth, fruit development, ripening, environment stresses as well as hormone responses discussed and may lay the foundation for future functional analysis to unravel their biological roles. (author)

Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase

LENUS (Irish Health Repository)

McSherry, Elaine A

2011-03-23

Abstract Introduction The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. Methods MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and β1-integrin, we examined activation of the β1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and β1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. Results JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the β1-integrin substrate fibronectin. This was accompanied by reduced protein expression of β1-integrin and its binding partners αV- and α5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and β1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF-6

Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase.

LENUS (Irish Health Repository)

McSherry, Elaine A

2011-03-23

-6 and the Rap1 activator PDZ-GEF2 in MCF7 cells and in primary cultures from breast cancer patients. CONCLUSIONS: Our findings provide compelling evidence of a novel role for JAM-A in driving breast cancer cell migration via activation of Rap1 GTPase and β1-integrin. We speculate that JAM-A over-expression in some breast cancer patients may represent a novel therapeutic target to reduce the likelihood of metastasis.

Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase.

LENUS (Irish Health Repository)

McSherry, Elaine A

2012-02-01

JAM-A, AF-6 and the Rap1 activator PDZ-GEF2 in MCF7 cells and in primary cultures from breast cancer patients. CONCLUSIONS: Our findings provide compelling evidence of a novel role for JAM-A in driving breast cancer cell migration via activation of Rap1 GTPase and beta1-integrin. We speculate that JAM-A over-expression in some breast cancer patients may represent a novel therapeutic target to reduce the likelihood of metastasis.

Epidermal activation of the small GTPase Rac1 in psoriasis pathogenesis.

Science.gov (United States)

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.

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.

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

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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.

Ameliorative effects of polyunsaturated fatty acids against palmitic acid-induced insulin resistance in L6 skeletal muscle cells

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Sawada Keisuke

2012-03-01

Full Text Available Abstract Background Fatty acid-induced insulin resistance and impaired glucose uptake activity in muscle cells are fundamental events in the development of type 2 diabetes and hyperglycemia. There is an increasing demand for compounds including drugs and functional foods that can prevent myocellular insulin resistance. Methods In this study, we established a high-throughput assay to screen for compounds that can improve myocellular insulin resistance, which was based on a previously reported non-radioisotope 2-deoxyglucose (2DG uptake assay. Insulin-resistant muscle cells were prepared by treating rat L6 skeletal muscle cells with 750 μM palmitic acid for 14 h. Using the established assay, the impacts of several fatty acids on myocellular insulin resistance were determined. Results In normal L6 cells, treatment with saturated palmitic or stearic acid alone decreased 2DG uptake, whereas unsaturated fatty acids did not. Moreover, co-treatment with oleic acid canceled the palmitic acid-induced decrease in 2DG uptake activity. Using the developed assay with palmitic acid-induced insulin-resistant L6 cells, we determined the effects of other unsaturated fatty acids. We found that arachidonic, eicosapentaenoic and docosahexaenoic acids improved palmitic acid-decreased 2DG uptake at lower concentrations than the other unsaturated fatty acids, including oleic acid, as 10 μM arachidonic acid showed similar effects to 750 μM oleic acid. Conclusions We have found that polyunsaturated fatty acids, in particular arachidonic and eicosapentaenoic acids prevent palmitic acid-induced myocellular insulin resistance.

Genetic parameters and selection for resistance to bacterial spot in recombinant F6 lines of Capsicum annuum

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Messias Gonzaga Pereira

2009-01-01

Full Text Available This study aimed to advance generations and select superior sweet pepper genotypes with resistance tobacterial spot, using the breeding method Single Seed Descent (SSD based on the segregating population derived from thecross between Capsicum annuum L. UENF 1421 (susceptible, non-pungent and UENF 1381 (resistant, pungent. Thesegregating F3 generation was grown in pots in a greenhouse until the F5 generation. The F6 generation was grown in fieldconditions. The reaction to bacterial spot was evaluated by inoculation with isolate ENA 4135 of Xanthomonas campestris pv.vesicatoria, based on a score scale and by calculating the area under the disease progress curve (AUDPC. The presence orabsence of capsaicin was also assessed. Eighteen F6 lines were bacterial leaf spot-resistant. Since no capsaicin was detectedin the F6 lines 032, 316, 399, 434, and 517, these will be used in the next steps of the sweet pepper breeding program.

Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology

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Zhao, Guohua; Zhu, Peng-Peng; Renvoisé, Benoît; Maldonado-Báez, Lymarie; Park, Seong Hee; Blackstone, Craig, E-mail: blackstc@ninds.nih.gov

2016-11-15

Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations. - Highlights: • NIH-3T3 cells lacking all three atlastin paralogs were generated using CRISPR/Cas9. • Cells lacking all atlastin GTPases exhibit far fewer 3-way ER tubule junctions. • ER morphology defects in atlastin knockout cells are rescued by distant plant and yeast orthologs. • Atlastin knock out cells also

Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology

International Nuclear Information System (INIS)

Zhao, Guohua; Zhu, Peng-Peng; Renvoisé, Benoît; Maldonado-Báez, Lymarie; Park, Seong Hee; Blackstone, Craig

2016-01-01

Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations. - Highlights: • NIH-3T3 cells lacking all three atlastin paralogs were generated using CRISPR/Cas9. • Cells lacking all atlastin GTPases exhibit far fewer 3-way ER tubule junctions. • ER morphology defects in atlastin knockout cells are rescued by distant plant and yeast orthologs. • Atlastin knock out cells also

Identification of two novel powdery mildew resistance loci, Ren6 and Ren7, from the wild Chinese grape species Vitis piasezkii.

Science.gov (United States)

Pap, Dániel; Riaz, Summaira; Dry, Ian B; Jermakow, Angelica; Tenscher, Alan C; Cantu, Dario; Oláh, Róbert; Walker, M Andrew

2016-07-29

Grapevine powdery mildew Erysiphe necator is a major fungal disease in all grape growing countries worldwide. Breeding for resistance to this disease is crucial to avoid extensive fungicide applications that are costly, labor intensive and may have detrimental effects on the environment. In the past decade, Chinese Vitis species have attracted attention from grape breeders because of their strong resistance to powdery mildew and their lack of negative fruit quality attributes that are often present in resistant North American species. In this study, we investigated powdery mildew resistance in multiple accessions of the Chinese species Vitis piasezkii that were collected during the 1980 Sino-American botanical expedition to the western Hubei province of China. A framework genetic map was developed using simple sequence repeat markers in 277 seedlings of an F1 mapping population arising from a cross of the powdery mildew susceptible Vitis vinifera selection F2-35 and a resistant accession of V. piasezkii DVIT2027. Quantitative trait locus analyses identified two major powdery mildew resistance loci on chromosome 9 (Ren6) and chromosome 19 (Ren7) explaining 74.8 % of the cumulative phenotypic variation. The quantitative trait locus analysis for each locus, in the absence of the other, explained 95.4 % phenotypic variation for Ren6, while Ren7 accounted for 71.9 % of the phenotypic variation. Screening of an additional 259 seedlings of the F1 population and 910 seedlings from four pseudo-backcross populations with SSR markers defined regions of 22 kb and 330 kb for Ren6 and Ren7 in the V. vinifera PN40024 (12X) genome sequence, respectively. Both R loci operate post-penetration through the induction of programmed cell death, but vary significantly in the speed of response and degree of resistance; Ren6 confers complete resistance whereas Ren7 confers partial resistance to the disease with reduced colony size. A comparison of the kinetics of induction of powdery

Glucose-6-Phosphate Dehydrogenase Enhances Antiviral Response through Downregulation of NADPH Sensor HSCARG and Upregulation of NF-κB Signaling

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Yi-Hsuan Wu

2015-12-01

Full Text Available Glucose-6-phosphate dehydrogenase (G6PD-deficient cells are highly susceptible to viral infection. This study examined the mechanism underlying this phenomenon by measuring the expression of antiviral genes—tumor necrosis factor alpha (TNF-α and GTPase myxovirus resistance 1 (MX1—in G6PD-knockdown cells upon human coronavirus 229E (HCoV-229E and enterovirus 71 (EV71 infection. Molecular analysis revealed that the promoter activities of TNF-α and MX1 were downregulated in G6PD-knockdown cells, and that the IκB degradation and DNA binding activity of NF-κB were decreased. The HSCARG protein, a nicotinamide adenine dinucleotide phosphate (NADPH sensor and negative regulator of NF-κB, was upregulated in G6PD-knockdown cells with decreased NADPH/NADP+ ratio. Treatment of G6PD-knockdown cells with siRNA against HSCARG enhanced the DNA binding activity of NF-κB and the expression of TNF-α and MX1, but suppressed the expression of viral genes; however, the overexpression of HSCARG inhibited the antiviral response. Exogenous G6PD or IDH1 expression inhibited the expression of HSCARG, resulting in increased expression of TNF-α and MX1 and reduced viral gene expression upon virus infection. Our findings suggest that the increased susceptibility of the G6PD-knockdown cells to viral infection was due to impaired NF-κB signaling and antiviral response mediated by HSCARG.

Potential Mechanism of Action of 3'-Demethoxy-6-O-demethyl-isoguaiacin on Methicillin Resistant Staphylococcus aureus.

Science.gov (United States)

Favela-Hernández, Juan Manuel J; Clemente-Soto, Aldo F; Balderas-Rentería, Isaías; Garza-González, Elvira; Camacho-Corona, María del Rayo

2015-07-08

Bacterial infections represent one of the main threats to global public health. One of the major causative agents associated with high morbidity and mortality infections in hospitals worldwide is methicillin-resistant Staphylococcus aureus. Therefore, there is a need to develop new antibacterial agents to treat these infections, and natural products are a rich source of them. In previous studies, we reported that lignan 3'-demethoxy-6-O-demethylisoguaiacin, isolated and characterized from Larrea tridentate, showed the best activity towards methicillin-resistant S. aureus. Thus, the aim of this study was to determine the potential molecular mechanism of the antibacterial activity of 3'-demethoxy-6-O-demethylisoguaiacin against methicillin-resistant S. aureus using microarray technology. Results of microarray genome expression were validated by real-time polymerase chain reaction (RT-PCR). The genetic profile expression results showed that lignan 3'-demethoxy-6-O-demethylisoguaiacin had activity on cell membrane affecting proteins of the ATP-binding cassette (ABC) transport system causing bacteria death. This molecular mechanism is not present in any antibacterial commercial drug and could be a new target for the development of novel antibacterial agents.

Radiation-resistant photostructure for Schottky diode based on Cr/In2Hg3Te6

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Ashcheulov A. A.

2016-05-01

Full Text Available Ge, Si, InGaAs, GaInAsP photodiodes are used as optical radiation receivers and function in a spectral range of transparency of quartz fiberglass. For the optical systems operated in the increased radioactivity the photodetectors' application on In2Hg3Te6 crystal base characterized by a photosensitivity in the spectral range of 0,5-1,6 mm and also by increased radiation resistance to alpha, beta and gamma radiation is most acceptable. Schottky photodiode structure was designed on the base of this semiconductor formed by a modified floating zone recrystallization technique where the sedimentation effect was leveled. It consists of n-In2Hg3Te6 substrate and deposited by cathode sputtering Cr barrier layer of thickness within a range 10-11 nm choice of Cr is determined by its optimal optical, electric and adhesive features in high quality radiation-resistant photodiode structures manufacturing. Indium and nichrome are used as ohmic contacts. The barrier structures have the contact area of 1,13 mm2 with photo response of 0,6-1,6 mm at the maximal sensitivity 0,43 A/W on the wavelength l,55 mm. Reverse dark current of these structures do not exceed 4 mA at the bias of 1 V (T=295 K, and the potential barrier height is equal to 0,41 eV. The tests of radiation resistance of these structures demonstrated their ability to function at doses of 2⋅108 rem without evident parameters changes. This allows using them in practical aims in the conditions of high radiation.

Antimicrobial resistance in Pseudomonas sp. causing infections in trauma patients: A 6 year experience from a south asian country

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Nonika Rajkumari

2014-01-01

Full Text Available Drug resistance to Pseudomonas sp. has spread to such a level irrespective of the type of patients, that its pattern of distribution and antibiotic resistance needs to be studied in detail, especially in trauma patients and hence the study. A 6 year study was carried out among trauma patients to see the trend and type of resistance prevalent in the apex hospital for trauma care in India among nonduplicate isolates where multidrug-resistance (MDR, cross-resistance and pan-drug resistance in Pseudomonas sp. were analyzed. Of the total 2,269 isolates obtained, the species, which was maximally isolated was Pseudomonas aeruginosa (2,224, 98%. The highest level of resistance was seen in tetracycline (2,166, 95.5%, P < 0.001 and chloramphenicol (2,160, 95.2%, P < 0.001 and least in meropenem (1,739, 76.7%, P < 0.003. Of the total, 1,692 (74.6% isolates were MDR in which P. aeruginosa (75% were maximum. MDR Pseudomonas is slowing increasing since the beginning of the study period. Of 1,797 imipenem-resistant P. aeruginosa isolated during the study period, 1,763 (98% showed resistance to ciprofloxacin or levofloxacin, suggesting that cross-resistance may have developed for imipenem due to prior use of fluoroquinolones. Antibiotic resistance in Pseudomonas sp. is fast becoming a problem in trauma patients, especially in those who requires prolong hospital stay, which calls for proper antimicrobial stewardship.

Improvements in the corrosion resistance and biocompatibility of biomedical Ti–6Al–7Nb alloy using an electrochemical anodization treatment

Energy Technology Data Exchange (ETDEWEB)

Huang, Her-Hsiung [Department of Dentistry, National Yang-Ming University, Taipei 112, Taiwan (China); Department of Dentistry, Taipei City Hospital, Taipei 115, Taiwan (China); Department of Stomatology, Taipei Veterans General Hospital, Taipei 112, Taiwan (China); Wu, Chia-Ping; Sun, Ying-Sui [Department of Oral Biology, National Yang-Ming University, Taipei 112, Taiwan (China); Lee, Tzu-Hsin, E-mail: biomaterials@hotmail.com [School of Dentistry, Chung Shan Medical University, Taichung 402, Taiwan (China); Department of Dentistry, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China)

2013-01-01

The biocompatibility of an implant material is determined by its surface characteristics. This study investigated the application of an electrochemical anodization surface treatment to improve both the corrosion resistance and biocompatibility of Ti–6Al–7Nb alloy for implant applications. The electrochemical anodization treatment produced an Al-free oxide layer with nanoscale porosity on the Ti–6Al–7Nb alloy surface. The surface topography and microstructure of Ti–6Al–7Nb alloy were analyzed. The corrosion resistance was investigated using potentiodynamic polarization curve measurements in simulated blood plasma (SBP). The adhesion and proliferation of human bone marrow mesenchymal stem cells to test specimens were evaluated using various biological analysis techniques. The results showed that the presence of a nanoporous oxide layer on the anodized Ti–6Al–7Nb alloy increased the corrosion resistance (i.e., increased the corrosion potential and decreased both the corrosion rate and the passive current) in SBP compared with the untreated Ti–6Al–7Nb alloy. Changes in the nanotopography also improved the cell adhesion and proliferation on the anodized Ti–6Al–7Nb alloy. We conclude that a fast and simple electrochemical anodization surface treatment improves the corrosion resistance and biocompatibility of Ti–6Al–7Nb alloy for biomedical implant applications. - Highlights: ► Simple/fast electrochemical anodization was applied to biomedical Ti–6Al–7Nb surface. ► Anodized surface had nano-porous topography and contained Al-free oxide layer. ► Anodized surface raised corrosion resistance in three simulated biological solutions. ► Anodized surface enhanced cell adhesion and cell proliferation. ► Electrochemical anodization has potential as biomedical implant surface treatment.

Improvements in the corrosion resistance and biocompatibility of biomedical Ti–6Al–7Nb alloy using an electrochemical anodization treatment

International Nuclear Information System (INIS)

Huang, Her-Hsiung; Wu, Chia-Ping; Sun, Ying-Sui; Lee, Tzu-Hsin

2013-01-01

The biocompatibility of an implant material is determined by its surface characteristics. This study investigated the application of an electrochemical anodization surface treatment to improve both the corrosion resistance and biocompatibility of Ti–6Al–7Nb alloy for implant applications. The electrochemical anodization treatment produced an Al-free oxide layer with nanoscale porosity on the Ti–6Al–7Nb alloy surface. The surface topography and microstructure of Ti–6Al–7Nb alloy were analyzed. The corrosion resistance was investigated using potentiodynamic polarization curve measurements in simulated blood plasma (SBP). The adhesion and proliferation of human bone marrow mesenchymal stem cells to test specimens were evaluated using various biological analysis techniques. The results showed that the presence of a nanoporous oxide layer on the anodized Ti–6Al–7Nb alloy increased the corrosion resistance (i.e., increased the corrosion potential and decreased both the corrosion rate and the passive current) in SBP compared with the untreated Ti–6Al–7Nb alloy. Changes in the nanotopography also improved the cell adhesion and proliferation on the anodized Ti–6Al–7Nb alloy. We conclude that a fast and simple electrochemical anodization surface treatment improves the corrosion resistance and biocompatibility of Ti–6Al–7Nb alloy for biomedical implant applications. - Highlights: ► Simple/fast electrochemical anodization was applied to biomedical Ti–6Al–7Nb surface. ► Anodized surface had nano-porous topography and contained Al-free oxide layer. ► Anodized surface raised corrosion resistance in three simulated biological solutions. ► Anodized surface enhanced cell adhesion and cell proliferation. ► Electrochemical anodization has potential as biomedical implant surface treatment

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.

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

Science.gov (United States)

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

Biological and Clinical Study of 6-Deoxy-6-Iodo-D-Glucose: a iodinated tracer of glucose transport and of insulin-resistance in human

International Nuclear Information System (INIS)

Barone-Rochette, Gilles

2013-01-01

Insulin resistance (IR), characterized by a depressed cellular sensitivity to insulin in insulin-sensitive organs, is a central feature to obesity, the metabolic syndrome, and diabetes mellitus and leads to increase cardiovascular diseases, particularly heart failure. All these events are today serious public health problems. But actually, there is no simple tool to measure insulin resistance. The gold standard technique remains the hyperinsulinemic euglycemic clamp. However, the complexity and length of this technique render it unsuitable for routine clinical use. Many methods or index have been proposed to assess insulin resistance in human, but none have shown enough relevance to be used in clinical use. The U1039 INSERM unit previously has validated a new tracer of glucose transport, radiolabelled with 123 iodine and has developed a fast and simple imaging protocol with a small animal gamma camera, which allows the obtaining of an IR index for each organ, showing more discriminating for the heart. The project of my thesis was the human transfer of this measurement technique, perfectly validated in animal. The first part of this thesis evaluated to tolerance, in vivo kinetics, distribution and dosimetry of novel tracer of glucose transport, the [ 123 I]-6DIG. The safeties of new tracer and measurement technique were adequate. There were no adverse effects with excellent tolerance of the whole protocol. 6DIG eliminating was fast, primarily in the urine and complete within 72 h. The effective whole-body absorbed dose for a complete scan with injection of 92.5 * 2 MBq was between 3 to 4 mSv. The second part of this thesis evaluated in human feasibility and reproducibility of the measurement technique validated in animal. The third part showed techniques used to allow human transfer of this method. The study protocol was applied on 12 subjects (healthy volunteers (n=6) and type 2 diabetic patients (n=6)). With a method adapted to measure in humans, we determined an

miR Profiling Identifies Cyclin-Dependent Kinase 6 Downregulation as a Potential Mechanism of Acquired Cisplatin Resistance in Non-Small-Cell Lung Carcinoma.

Science.gov (United States)

Bar, Jair; Gorn-Hondermann, Ivan; Moretto, Patricia; Perkins, Theodore J; Niknejad, Nima; Stewart, David J; Goss, Glenwood D; Dimitroulakos, Jim

2015-11-01

To identify the mechanisms of cisplatin resistance, global microRNA (miR) expression was tested. The expression of miR-145 was consistently higher in resistant cells. The expression of cyclin-dependent kinase 6 (CDK6), a potential target of miR-145, was lower in resistant cells, and inhibition of CDK4/6 protected cells from cisplatin. Cell cycle inhibition, currently being tested in clinical trials, might be antagonistic to cisplatin and other cytotoxic drugs. Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death. Platinum-based chemotherapeutic drugs are the most active agents in treating advanced disease. Resistance to these drugs is common and multifactorial; insight into the molecular mechanisms involved will likely enhance efficacy. A set of NSCLC platinum-resistant sublines was created from the Calu6 cell line. Cell viability was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Differentially expressed microRNAs (miRs) in these lines were identified using Affymetrix miR arrays. The potential genes targeted by these miRs were searched using the TargetScan algorithm. The expression levels of miRs and mRNA were tested using real-time polymerase chain reaction. miR-145 was reproducibly elevated in all the resistant sublines tested; however, modulation of miR-145 levels alone in these cells did not affect their response to cisplatin. A potential target of miR-145 is cyclin-dependent kinase 6 (CDK6), an important regulator of cell proliferation. The mRNA and protein levels of CDK6 were both downregulated in the resistant sublines. An inhibitor of CDK4/6 (PD0332991) protected parental NSCLC cells from cisplatin cytotoxicity. In the present study, we identified miRs differentially expressed in cisplatin-resistant cell lines, including miR-145. A predicted target of miR-145 is CDK6, and its expression was found to be downregulated in the resistant sublines, although not directly by miR-145. Inhibition

Imidacloprid Promotes High Fat Diet-Induced Adiposity and Insulin Resistance in Male C57BL/6J Mice.

Science.gov (United States)

Sun, Quancai; Xiao, Xiao; Kim, Yoo; Kim, Daeyoung; Yoon, Kyoon Sup; Clark, John M; Park, Yeonhwa

2016-12-14

Imidacloprid, a neonicotinoid insecticide widely used in agriculture worldwide, has been reported to promote adipogenesis and cause insulin resistance in vitro. The purpose of the current study was to determine the effects of imidacloprid and its interaction with dietary fat in the development of adiposity and insulin resistance using male C57BL/6J mice. Imidacloprid (0.06, 0.6, or 6 mg/kg bw/day) was mixed in a low-fat (4% w/w) or high-fat (20% w/w) diet and given to mice ad libitum for 12 weeks. Imidacloprid significantly promoted high fat diet-induced body weight gain and adiposity. In addition, imidacloprid treatment with the high fat diet resulted in impaired glucose metabolism. Consistently, there were significant effects of imidacloprid on genes regulating lipid and glucose metabolisms, including the AMP-activated protein kinase-α (AMPKα) pathway in white adipose tissue and liver. These results suggest that imidacloprid may potentiate high fat diet-induced adiposity and insulin resistance in male C57BL/6J mice.

S6Ks isoforms contribute to viability, migration, docetaxel resistance and tumor formation of prostate cancer cells

International Nuclear Information System (INIS)

Amaral, Camila L.; Freitas, Lidia B.; Tamura, Rodrigo E.; Tavares, Mariana R.; Pavan, Isadora C. B.; Bajgelman, Marcio C.; Simabuco, Fernando M.

2016-01-01

The S6 Kinase (S6K) proteins are some of the main downstream effectors of the mammalian Target Of Rapamycin (mTOR) and act as key regulators of protein synthesis and cell growth. S6K is overexpressed in a variety of human tumors and is correlated to poor prognosis in prostate cancer. Due to the current urgency to identify factors involved in prostate cancer progression, we aimed to reveal the cellular functions of three S6K isoforms–p70-S6K1, p85-S6K1 and p54-S6K2–in prostate cancer, as well as their potential as therapeutic targets. In this study we performed S6K knockdown and overexpression and investigated its role in prostate cancer cell proliferation, colony formation, viability, migration and resistance to docetaxel treatment. In addition, we measured tumor growth in Nude mice injected with PC3 cells overexpressing S6K isoforms and tested the efficacy of a new available S6K1 inhibitor in vitro. S6Ks overexpression enhanced PC3-luc cell line viability, migration, resistance to docetaxel and tumor formation in Nude mice. Only S6K2 knockdown rendered prostate cancer cells more sensitive to docetaxel. S6K1 inhibitor PF-4708671 was particularly effective for reducing migration and proliferation of PC3 cell line. These findings demonstrate that S6Ks play an important role in prostate cancer progression, enhancing cell viability, migration and chemotherapy resistance, and place both S6K1 and S6K2 as a potential targets in advanced prostate cancer. We also provide evidence that S6K1 inhibitor PF-4708671 may be considered as a potential drug for prostate cancer treatment. The online version of this article (doi:10.1186/s12885-016-2629-y) contains supplementary material, which is available to authorized users

High-fat diet induced insulin resistance in pregnant rats through pancreatic pax6 signaling pathway.

Science.gov (United States)

Wu, Hao; Liu, Yunyun; Wang, Hongkun; Xu, Xianming

2015-01-01

To explore the changes in pancreas islet function of pregnant rats after consumption of high-fat diet and the underlying mechanism. Thirty pregnant Wistar rats were randomly divided into two groups: high-fat diet group and normal control group. Twenty days after gestation, fasting blood glucose concentration (FBG) and fasting serum insulin concentration (FINS) were measured. Then, oral glucose tolerance test (OGTT) and insulin release test (IRT) were performed. Finally, all the rats were sacrificed and pancreas were harvested. Insulin sensitivity index (ISI) and insulin resistance index (HOMA-IR) were calculated according to FBG and FINS. RT-PCR and Real-time PCR were performed to study the expression of paired box 6 transcription factor (Pax6) and its target genes in pancreatic tissues. The body weight was significantly increased in the high-fat diet group compared with that of normal control rats (Pinsulin concentration between the two groups. OGTT and IRT were abnormal in the high-fat diet group. The high-fat diet rats were more prone to impaired glucose tolerance and insulin resistance. The level of the expression of Pax6 transcription factor and its target genes in pancreas, such as pancreatic and duodenal homeobox factor-1 (Pdx1), v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) and glucose transporter 2 (Glut2) were decreased significantly compared with those of normal control group. High-fat diet feeding during pregnancy may induce insulin resistance in maternal rats by inhibiting pancreatic Pax6 and its target genes expression.

Corrosion resistance of welded joints in 08Kh22N6T and 08Kh21N6M2T steels made with new electrodes

International Nuclear Information System (INIS)

Sidorkina, Y.S.; Leibzon, V.M.; Mankevich, T.V.; Sidlin, E.A.; Zheltova, G.A.

1986-01-01

This paper examines the corrosion resistance of 08Kh22N6T and 08Kh21N6M2T steels which would make it possible to ensure equal properties of all the zones of the welded joints. The investigations were carried out on the welded joints in the steels produced with new OZL-40 and OZL-41 electrodes and with standard TsL-11 and EA-400/10u electrodes. The chemical compositions of the steels and deposited metal are presented. The tests of the intercrystalline corrosion resistances of the welded joints in the steels in the as-welded condition and after heat treatment showed that the weld metal is not attacked by intercrystalline corrosion. The tests also showed that the welded joints in 08Kh22N6T steel made with the OZL-40 and TsL-11 electrodes are resistant to corrosion in 55% phosphoric acid at 90 C in 1% sulfuric acid, and in 25% formic acid at 80 C. The investigated steels made with the OZL-40 and OZL-41 electrode respectively show that the welded joints are resistant to intercrystalline and general corrosion, have high mechanical and processing properties and the electrodes, and can be used for welding equipment designed for service in alkaline, neutral, and slightly acid media

Site-directed mutagenesis of Arg58 and Asp86 of elongation factor Tu from Escherichia coli: effects on the GTPase reaction and aminoacyl-tRNA binding

DEFF Research Database (Denmark)

Knudsen, Charlotte Rohde; Clark, Brian F. C.

1996-01-01

Elongation factor Tu from Escherichia coli was mutated separately at positions Asp86 and Arg58, in order to shed light both on the GTPase mechanism of elongation factor Tu and on the binding of aminoacyl-tRNA. In addition, the binding of guanine nucleotides was investigated by determination...

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...

The Rab GTPase Rab8 as a shared regulator of ciliogenesis and immune synapse assembly: From a conserved pathway to diverse cellular structures.

Science.gov (United States)

Patrussi, Laura; Baldari, Cosima T

2016-01-01

Rab GTPases, which form the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Among them, Rab8 is an essential participant in primary cilium formation. In a report recently published in the Journal of Cell Science, Finetti and colleagues identify Rab8 as a novel player in vesicular traffic in the non-ciliated T lymphocytes, which contributes to the assembly of the specialized signaling platform known as the immune synapse. By interacting with the v-SNARE VAMP-3, Rab8 is indeed responsible for the final docking/fusion step in T cell receptor (TCR) recycling to the immune synapse. A second important take-home message which comes to light from this work is that VAMP-3 also interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of Smoothened at the plasma membrane. Hence the data presented in this report, in addition to identifying Rab8 as a novel player in vesicular traffic to the immune synapse, reveal how both ciliated and non-ciliated cells take advantage of a conserved pathway to build highly specific cellular structures.

Extremely low frequency electromagnetic fields promote mesenchymal stem cell migration by increasing intracellular Ca2+ and activating the FAK/Rho GTPases signaling pathways in vitro.

Science.gov (United States)

Zhang, Yingchi; Yan, Jiyuan; Xu, Haoran; Yang, Yong; Li, Wenkai; Wu, Hua; Liu, Chaoxu

2018-05-21

The ability of mesenchymal stem cells (MSCs) to migrate to the desired tissues or lesions is crucial for stem cell-based regenerative medicine and tissue engineering. Optimal therapeutics for promoting MSC migration are expected to become an effective means for tissue regeneration. Electromagnetic fields (EMF), as a noninvasive therapy, can cause a lot of biological changes in MSCs. However, whether EMF can promote MSC migration has not yet been reported. We evaluated the effects of EMF on cell migration in human bone marrow-derived MSCs. With the use of Helmholtz coils and an EMF stimulator, 7.5, 15, 30, 50, and 70 Hz/1 mT EMF was generated. Additionally, we employed the L-type calcium channel blocker verapamil and the focal adhesion kinase (FAK) inhibitor PF-573228 to investigate the role of intracellular calcium content, cell adhesion proteins, and the Rho GTPase protein family (RhoA, Rac1, and Cdc42) in EMF-mediated MSC migration. Cell adhesion proteins (FAK, talin, and vinculin) were detected by Western blot analysis. The Rho GTPase protein family activities were assessed by G-LISA, and F-actin levels, which reflect actin cytoskeletal organization, were detected using immunofluorescence. All the 7.5, 15, 30, 50, and 70 Hz/1 mT EMF promoted MSC migration. EMF increased MSC migration in an intracellular calcium-dependent manner. Notably, EMF-enhanced migration was mediated by FAK activation, which was critical for the formation of focal contacts, as evidenced by increased talin and vinculin expression. Moreover, RhoA, Rac1, and Cdc42 were activated by FAK to increase cytoskeletal organization, thus promoting cell contraction. EMF promoted MSC migration by increasing intracellular calcium and activating the FAK/Rho GTPase signaling pathways. This study provides insights into the mechanisms of MSC migration and will enable the rational design of targeted therapies to improve MSC engraftment.

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

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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.

Evolutionary changes in gene expression, coding sequence and copy-number at the Cyp6g1 locus contribute to resistance to multiple insecticides in Drosophila.

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Thomas W R Harrop

Full Text Available Widespread use of insecticides has led to insecticide resistance in many populations of insects. In some populations, resistance has evolved to multiple pesticides. In Drosophila melanogaster, resistance to multiple classes of insecticide is due to the overexpression of a single cytochrome P450 gene, Cyp6g1. Overexpression of Cyp6g1 appears to have evolved in parallel in Drosophila simulans, a sibling species of D. melanogaster, where it is also associated with insecticide resistance. However, it is not known whether the ability of the CYP6G1 enzyme to provide resistance to multiple insecticides evolved recently in D. melanogaster or if this function is present in all Drosophila species. Here we show that duplication of the Cyp6g1 gene occurred at least four times during the evolution of different Drosophila species, and the ability of CYP6G1 to confer resistance to multiple insecticides exists in D. melanogaster and D. simulans but not in Drosophila willistoni or Drosophila virilis. In D. virilis, which has multiple copies of Cyp6g1, one copy confers resistance to DDT and another to nitenpyram, suggesting that the divergence of protein sequence between copies subsequent to the duplication affected the activity of the enzyme. All orthologs tested conferred resistance to one or more insecticides, suggesting that CYP6G1 had the capacity to provide resistance to anthropogenic chemicals before they existed. Finally, we show that expression of Cyp6g1 in the Malpighian tubules, which contributes to DDT resistance in D. melanogaster, is specific to the D. melanogaster-D. simulans lineage. Our results suggest that a combination of gene duplication, regulatory changes and protein coding changes has taken place at the Cyp6g1 locus during evolution and this locus may play a role in providing resistance to different environmental toxins in different Drosophila species.

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

Genomic and Transcriptomic Associations Identify a New Insecticide Resistance Phenotype for the Selective Sweep at the Cyp6g1 Locus of Drosophila melanogaster.

Science.gov (United States)

Battlay, Paul; Schmidt, Joshua M; Fournier-Level, Alexandre; Robin, Charles

2016-08-09

Scans of the Drosophila melanogaster genome have identified organophosphate resistance loci among those with the most pronounced signature of positive selection. In this study, the molecular basis of resistance to the organophosphate insecticide azinphos-methyl was investigated using the Drosophila Genetic Reference Panel, and genome-wide association. Recently released full transcriptome data were used to extend the utility of the Drosophila Genetic Reference Panel resource beyond traditional genome-wide association studies to allow systems genetics analyses of phenotypes. We found that both genomic and transcriptomic associations independently identified Cyp6g1, a gene involved in resistance to DDT and neonicotinoid insecticides, as the top candidate for azinphos-methyl resistance. This was verified by transgenically overexpressing Cyp6g1 using natural regulatory elements from a resistant allele, resulting in a 6.5-fold increase in resistance. We also identified four novel candidate genes associated with azinphos-methyl resistance, all of which are involved in either regulation of fat storage, or nervous system development. In Cyp6g1, we find a demonstrable resistance locus, a verification that transcriptome data can be used to identify variants associated with insecticide resistance, and an overlap between peaks of a genome-wide association study, and a genome-wide selective sweep analysis. Copyright © 2016 Battlay et al.

GIMAP6 is required for T cell maintenance and efficient autophagy in mice.

Science.gov (United States)

Pascall, John C; Webb, Louise M C; Eskelinen, Eeva-Liisa; Innocentin, Silvia; Attaf-Bouabdallah, Noudjoud; Butcher, Geoffrey W

2018-01-01

The GTPases of the immunity-associated proteins (GIMAP) GTPases are a family of proteins expressed strongly in the adaptive immune system. We have previously reported that in human cells one member of this family, GIMAP6, interacts with the ATG8 family member GABARAPL2, and is recruited to autophagosomes upon starvation, suggesting a role for GIMAP6 in the autophagic process. To study this possibility and the function of GIMAP6 in the immune system, we have established a mouse line in which the Gimap6 gene can be inactivated by Cre-mediated recombination. In mice bred to carry the CD2Cre transgene such that the Gimap6 gene was deleted within the T and B cell lineages there was a 50-70% reduction in peripheral CD4+ and CD8+ T cells. Analysis of splenocyte-derived proteins from these mice indicated increased levels of MAP1LC3B, particularly the lipidated LC3-II form, and S405-phosphorylation of SQSTM1. Electron microscopic measurements of Gimap6-/- CD4+ T cells indicated an increased mitochondrial/cytoplasmic volume ratio and increased numbers of autophagosomes. These results are consistent with autophagic disruption in the cells. However, Gimap6-/- T cells were largely normal in character, could be effectively activated in vitro and supported T cell-dependent antibody production. Treatment in vitro of CD4+ splenocytes from GIMAP6fl/flERT2Cre mice with 4-hydroxytamoxifen resulted in the disappearance of GIMAP6 within five days. In parallel, increased phosphorylation of SQSTM1 and TBK1 was observed. These results indicate a requirement for GIMAP6 in the maintenance of a normal peripheral adaptive immune system and a significant role for the protein in normal autophagic processes. Moreover, as GIMAP6 is expressed in a cell-selective manner, this indicates the potential existence of a cell-restricted mode of autophagic regulation.

Rickettsia parkeri invasion of diverse host cells involves an Arp2/3 complex, WAVE complex and Rho-family GTPase-dependent pathway.

Science.gov (United States)

Reed, Shawna C O; Serio, Alisa W; Welch, Matthew D

2012-04-01

Rickettsiae are obligate intracellular pathogens that are transmitted to humans by arthropod vectors and cause diseases such as spotted fever and typhus. Although rickettsiae require the host cell actin cytoskeleton for invasion, the cytoskeletal proteins that mediate this process have not been completely described. To identify the host factors important during cell invasion by Rickettsia parkeri, a member of the spotted fever group (SFG), we performed an RNAi screen targeting 105 proteins in Drosophila melanogaster S2R+ cells. The screen identified 21 core proteins important for invasion, including the GTPases Rac1 and Rac2, the WAVE nucleation-promoting factor complex and the Arp2/3 complex. In mammalian cells, including endothelial cells, the natural targets of R. parkeri, the Arp2/3 complex was also crucial for invasion, while requirements for WAVE2 as well as Rho GTPases depended on the particular cell type. We propose that R. parkeri invades S2R+ arthropod cells through a primary pathway leading to actin nucleation, whereas invasion of mammalian endothelial cells occurs via redundant pathways that converge on the host Arp2/3 complex. Our results reveal a key role for the WAVE and Arp2/3 complexes, as well as a higher degree of variation than previously appreciated in actin nucleation pathways activated during Rickettsia invasion. © 2011 Blackwell Publishing Ltd.

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

Directory of Open Access Journals (Sweden)

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.

Amylose content in rice (Oryza sativa affects performance, glycemic and lipidic metabolism in rats Teor de amilose do arroz (Oryza sativa afeta o desempenho, metabolismo glicêmico e lipídico em ratos

Directory of Open Access Journals (Sweden)

Cristiane Casagrande Denardin

2012-02-01

Full Text Available This research aimed at evaluating the effect of diets with high, intermediate and low amylose content of rice on performance, glycemic and lipidic metabolism in rats. Male Wistar rats were fed diets with grains of cooked rice of the cultivars 'IRGA 417', 'IRGA 416' and 'MOCHI' with high, intermediate and low amylose content, respectively. Wet and dry fecal production and serum HDL cholesterol were not affected by amylose content. The animals in the treatments with high amylose content ('IRGA 417' presented lower feed intake, body weight gain and apparent digestibility, higher fecal water content and nitrogen excretion, reduced fecal pH, lower postprandial blood glucose response, serum total cholesterol and triglycerides levels and pancreas weight, and higher fasting serum glucose concentration and liver weight. Amylose:amylopectin ratio significantly affects rice starch digestion in the gastrointestinal tract, affecting some biologically relevant parameters.O objetivo deste trabalho foi avaliar o efeito de dietas com alto, intermediário e baixo teor de amilose sobre o desempenho, metabolismo glicêmico e lipídico em ratos. Foram utilizados ratos machos Wistar alimentados com rações experimentais elaboradas com grãos de arroz cozido das cultivares 'IRGA 417', 'IRGA 416' e 'MOCHI' com alto, intermediário e baixo teores de amilose, respectivamente. A produção de fezes úmidas e secas e colesterol HDL não foram afetados pelo teor de amilose dos grãos. Os animais submetidos ao tratamento com alto teor de amilose (IRGA 417 apresentaram menores consumo, ganho de peso e digestibilidade aparente, maiores umidade nas fezes e excreção de nitrogênio, reduzido pH fecal, concentração plasmática posprandial de glicose, colesterol total, triglicerídeos e peso do pâncreas e maior concentração de glicose no jejum e peso do fígado. A proporção amilose e amilopectina nos grãos afeta significativamente a digestão do amido de arroz no trato

Potential Mechanism of Action of 3′-Demethoxy-6-O-demethyl-isoguaiacin on Methicillin Resistant Staphylococcus aureus

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Juan Manuel J. Favela-Hernández

2015-07-01

Full Text Available Bacterial infections represent one of the main threats to global public health. One of the major causative agents associated with high morbidity and mortality infections in hospitals worldwide is methicillin-resistant Staphylococcus aureus. Therefore, there is a need to develop new antibacterial agents to treat these infections, and natural products are a rich source of them. In previous studies, we reported that lignan 3′-demethoxy-6-O-demethylisoguaiacin, isolated and characterized from Larrea tridentate, showed the best activity towards methicillin-resistant S. aureus. Thus, the aim of this study was to determine the potential molecular mechanism of the antibacterial activity of 3′-demethoxy-6-O-demethylisoguaiacin against methicillin-resistant S. aureus using microarray technology. Results of microarray genome expression were validated by real-time polymerase chain reaction (RT-PCR. The genetic profile expression results showed that lignan 3′-demethoxy-6-O-demethylisoguaiacin had activity on cell membrane affecting proteins of the ATP-binding cassette (ABC transport system causing bacteria death. This molecular mechanism is not present in any antibacterial commercial drug and could be a new target for the development of novel antibacterial agents.

Efeito de práticas de manejo sobre o rendimento de grãos e a qualidade industrial dos grãos em arroz irrigado Effects of management practices on rice tield grain and hillquality

Directory of Open Access Journals (Sweden)

Luciano Pasqualoto Canellas

1997-08-01

Full Text Available Foi desenvolvido um experimento a campo num gleissolo em Casemiro de Abreu, RJ, de outubro de 1993 a fevereiro de 1994, com o objetivo de verificar o efeito do uso de calcário em linha de semeadura, de cultivares, densidades de semeadura e épocas de colheita sobre a produção e a qualidade industrial do arroz. Foram utilizadas duas cultivares de arroz (BR-IRGA 412 e BR-IRGA 409, duas doses de calcário aplicadas na linha de semeadura (0 e 200kg ha-1 e três densidades de semeadura (100, 200 e 300kg de sementes ha-1, colhidas aos 140, 147 e 154 dias após a semeadura. O uso do calcário proporcionou aumento de 25% no rendimento de grãos da cultivar BR-IRGA 409. A qualidade industrial do arroz foi influenciada pela época de colheita, sendo a mais indicada quando o teor de umidade do grão se encontra entre 17,5 - 22%.The purpose of the research was to study the use of liming, seeding density of two paddy rice cultivars and harvest time with rice yield and mill quality. The experiment was carried out during 1993/94 growing season in Casemiro de Abreu (RJ, Brazil. The two rice cultivars used were BR-IRGA 409 and BR-IRGA 412, two of limestone doses (0 and 200kg ha-1 and three seeding densities (100, 200 and 300kg ha-1 were distributed in a completely randomized block design. The use of liming and variation of seeding density did not affect rice quality. The moisture content of the rice grains that resulted in highest quality at harvest was between 17,5-22%.

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

International Nuclear Information System (INIS)

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.

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

Identification of a RAPD marker linked to the Co-6 anthracnose resistant gene in common bean cultivar AB 136

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Alzate-Marin Ana Lilia

2000-01-01

Full Text Available The pathogenic variability of the fungus Colletotrichum lindemuthianum represents an obstacle for the creation of resistant common bean (Phaseolus vulgaris L. varieties. Gene pyramiding is an alternative strategy for the development of varieties with durable resistance. RAPD markers have been proposed as a means to facilitate pyramiding of resistance genes without the need for multiple inoculations of the pathogens. The main aims of this work were to define the inheritance pattern of resistance present in common bean cultivar AB 136 in segregating populations derived from crosses with cultivar Rudá (susceptible to most C. lindemuthianum races and to identify RAPD markers linked to anthracnose resistance. The two progenitors, populations F1 and F2, F2:3 families and backcross-derived plants were inoculated with race 89 of C. lindemuthianum under environmentally controlled greenhouse conditions. The results indicate that a single dominant gene, Co-6, controls common bean resistance to this race, giving a segregation ratio between resistant and susceptible plants of 3:1 in the F2, 1:0 in the backcrosses to AB 136 and 1:1 in the backcross to Rudá. The segregation ratio of F2:3 families derived from F2 resistant plants was 1:2 (homozygous to heterozygous resistant. Molecular marker analyses in the F2 population identified a DNA band of approximately 940 base pairs (OPAZ20(940, linked in coupling phase at 7.1 cM of the Co-6 gene. This marker is being used in our backcross breeding program to develop Rudá-derived common bean cultivars resistant to anthracnose and adapted to central Brazil.

The P450 CYP6Z1 confers carbamate/pyrethroid cross-resistance in a major African malaria vector beside a novel carbamate-insensitive N485I acetylcholinesterase-1 mutation.

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Ibrahim, Sulaiman S; Ndula, Miranda; Riveron, Jacob M; Irving, Helen; Wondji, Charles S

2016-07-01

Carbamates are increasingly used for vector control notably in areas with pyrethroid resistance. However, a cross-resistance between these insecticides in major malaria vectors such as Anopheles funestus could severely limit available resistance management options. Unfortunately, the molecular basis of such cross-resistance remains uncharacterized in An. funestus, preventing effective resistance management. Here, using a genomewide transcription profiling, we revealed that metabolic resistance through upregulation of cytochrome P450 genes is driving carbamate resistance. The P450s CYP6P9a, CYP6P9b and CYP6Z1 were the most upregulated detoxification genes in the multiple resistant mosquitoes. However, in silico docking simulations predicted CYP6Z1 to metabolize both pyrethroids and carbamates, whereas CYP6P9a and CYP6P9b were predicted to metabolize only the pyrethroids. Using recombinant enzyme metabolism and inhibition assays, we demonstrated that CYP6Z1 metabolizes bendiocarb and pyrethroids, whereas CYP6P9a and CYP6P9b metabolize only the pyrethroids. Other upregulated gene families in resistant mosquitoes included several cuticular protein genes suggesting a possible reduced penetration resistance mechanism. Investigation of the target-site resistance in acetylcholinesterase 1 (ace-1) gene detected and established the association between the new N485I mutation and bendiocarb resistance (odds ratio 7.3; P resistance and improve the design of effective resistance management strategies to control this malaria vector. © 2016 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

Molecular Cytogenetic Identification of a New Wheat-Rye 6R Chromosome Disomic Addition Line with Powdery Mildew Resistance.

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Diaoguo An

Full Text Available Rye (Secale cereale L. possesses many valuable genes that can be used for improving disease resistance, yield and environment adaptation of wheat (Triticum aestivum L.. However, the documented resistance stocks derived from rye is faced severe challenge due to the variation of virulent isolates in the pathogen populations. Therefore, it is necessary to develop desirable germplasm and search for novel resistance gene sources against constantly accumulated variation of the virulent isolates. In the present study, a new wheat-rye line designated as WR49-1 was produced through distant hybridization and chromosome engineering protocols between common wheat cultivar Xiaoyan 6 and rye cultivar German White. Using sequential GISH (genomic in situ hybridization, mc-FISH (multicolor fluorescence in situ hybridization, mc-GISH (multicolor GISH and EST (expressed sequence tag-based marker analysis, WR49-1 was proved to be a new wheat-rye 6R disomic addition line. As expected, WR49-1 showed high levels of resistance to wheat powdery mildew (Blumeria graminis f. sp. tritici, Bgt pathogens prevalent in China at the adult growth stage and 19 of 23 Bgt isolates tested at the seedling stage. According to its reaction pattern to different Bgt isolates, WR49-1 may possess new resistance gene(s for powdery mildew, which differed from the documented powdery mildew gene, including Pm20 on chromosome arm 6RL of rye. Additionally, WR49-1 was cytologically stable, had improved agronomic characteristics and therefore could serve as an important bridge for wheat breeding and chromosome engineering.

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

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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

Prevalence of quinolone resistance determinant qnrA6 among broad- and extended-spectrum beta-lactam-resistant Proteus mirabilis and Morganella morganii clinical isolates with sul1-type class 1 integron association in a Tunisian Hospital.

Science.gov (United States)

Mahrouki, Sihem; Perilli, Mariagrazia; Bourouis, Amel; Chihi, Hela; Ferjani, Mustapha; Ben Moussa, Mohamed; Amicosante, Gianfranco; Belhadj, Omrane

2013-08-01

The aim of this study was to investigate the prevalence and the emergence of plasmid-mediated quinolone resistance among broad-spectrum beta-lactam-resistant Proteus mirabilis and Morganella morganii clinical isolates recovered in the Military Hospital in Tunisia. Of 200 strains examined, 50 exhibited resistance to quinolones. Quinolone resistance determinants (qnr and aac(6')-Ib-cr) were characterized by multiplex PCR and sequencing. Chromosomal quinolone resistance mutations in the quinolone resistance-determining region (QRDR) and class 1 integron characterization were analysed by PCR and sequencing. The clonal relationship between the isolates was studied by pulsed-field gel electrophoresis (PFGE). Fourteen isolates harboured qnrA6 and among them 8 (57%) were extended-spectrum beta-lactamase (ESBL) producers, whilst 12 (85%) isolates harboured blaDHA-1. Mutations in the QRDR were detected in gyrA (Ser83Ile, Glu87Lys), gyrB (Ser464Phe), and parC (Ser80Ile). qnrA6 and blaDHA-1 genes were found embedded in complex sul1-type class 1 integrons. A gene cassette carrying aac(6')-Ib-cr was found located in the class 1 integron upstream of the qacEΔ1 gene. According to the PFGE analysis, the isolates were clonally unrelated. This is the first description in North Africa of class 1 integrons carrying blaDHA-1, qnrA6 gene, and aac(6')-Ib-cr determinants in clinical strains of Proteus mirabilis and Morganella morganii.

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...

Interaction of LRRK2 with kinase and GTPase signaling cascades

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Joon Y Boon

2014-07-01

Full Text Available LRRK2 is a protein that interacts with a plethora of signaling molecules, but the complexity of LRRK2 function presents a challenge for understanding the role of LRRK2 in the pathophysiology of Parkinson’s disease. Studies of LRRK2 using over-expression in transgenic mice have been disappointing, however studies using invertebrate systems have yielded a much clearer picture, with clear effects of LRRK2 expression, knockdown or deletion in C. elegans and Drosophila on modulation of survival of dopaminergic neurons. Recent studies have begun to focus attention on particular signaling cascades that are a target of LRRK2 function. LRRK2 interacts with members of the MAPK pathway and might regulate the pathway action by acting as a scaffold that directs the location of MAPK pathway activity, without strongly affecting the amount of MAPK pathway activity. Binding to GTPases, GAPs and GEFs are another strong theme in LRRK2 biology, with LRRK2 binding to Rac1, cdc42, rab5, rab7L1, endoA, RGS2, ArfGAP1 and ArhGEF7. All of these molecules appear to feed into a function output for LRRK2 that modulates cytoskeletal outgrowth and vesicular dynamics, including autophagy. These functions likely impact modulation of α-synuclein aggregation and associated toxicity eliciting the disease processes that we term Parkinson’s disease.

Interleukin-6 and Vitamin D Status during High-Intensity Resistance Training in Patients with Chronic Kidney Disease

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Stig Molsted

2014-01-01

Full Text Available Background. The aim of this study was to investigate IL-6 and 25-hydroxyvitamin D (25-OH D associations with muscle size and muscle function in dialysis patients. Methods. Patients were included in a 16-week control period followed by 16 weeks of high-intensity resistance training thrice weekly. IL-6 and 25-OH D were analysed after an over-night fast. Muscle fibre size was analysed in biopsies from m. vastus lateralis. Muscle power was tested using a Leg Extensor Power Rig. Results. Patients (n=36 with IL-6 ≥ 6.49 pg/ml (median were older and had decreased muscle power and a reduced protein intake (P<0.05 compared with patients with IL-6 < 6.49 pg/ml. IL-6 was not associated with muscle fibre size. Vitamin D deficiency (25-OH D < 50 nmol/l was present in 51% of the patients and not associated with muscle power. IL-6 remained unchanged during the training period, whilst muscle power increased by 20–23% (P<0.001. Conclusion. Elevated IL-6 values were associated with decreased muscle power but not with decreased muscle fibre size. Half of the patients were suffering from vitamin D deficiency, which was not associated with muscle power. IL-6 was unchanged by high-intensity resistance training in dialysis patients in this study.

Expression of the cytochrome P450s, CYP6P3 and CYP6M2 are significantly elevated in multiple pyrethroid resistant populations of Anopheles gambiae s.s. from Southern Benin and Nigeria

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Ranson Hilary

2008-11-01

Full Text Available Abstract Background Insecticide resistance in Anopheles mosquitoes is threatening the success of malaria control programmes. This is particularly true in Benin where pyrethroid resistance has been linked to the failure of insecticide treated bed nets. The role of mutations in the insecticide target sites in conferring resistance has been clearly established. In this study, the contribution of other potential resistance mechanisms was investigated in Anopheles gambiae s.s. from a number of localities in Southern Benin and Nigeria. The mosquitoes were sampled from a variety of breeding sites in a preliminary attempt to investigate the role of contamination of mosquito breeding sites in selecting for resistance in adult mosquitoes. Results All mosquitoes sampled belonged to the M form of An. gambiae s.s. There were high levels of permethrin resistance in an agricultural area (Akron and an urban area (Gbedjromede, low levels of resistance in mosquito samples from an oil contaminated site (Ojoo and complete susceptibility in the rural Orogun location. The target site mutation kdrW was detected at high levels in two of the populations (Akron f = 0.86 and Gbedjromede f = 0.84 but was not detected in Ojoo or Orogun. Microarray analysis using the Anopheles gambiae detox chip identified two P450s, CYP6P3 and CYP6M2 up regulated in all three populations, the former was expressed at particularly high levels in the Akron (12.4-fold and Ojoo (7.4-fold populations compared to the susceptible population. Additional detoxification and redox genes were also over expressed in one or more populations including two cuticular pre-cursor genes which were elevated in two of the three resistant populations. Conclusion Multiple resistance mechanisms incurred in the different breeding sites contribute to resistance to permethrin in Benin. The cytochrome P450 genes, CYP6P3 and CYP6M2 are upregulated in all three resistant populations analysed. Several additional potential

Identification and characterization of a lymphocytic Rho-GTPase effector: rhotekin-2

International Nuclear Information System (INIS)

Collier, F.M.; Gregorio-King, C.C.; Gough, T.J.; Talbot, C.D.; Walder, K.; Kirkland, M.A.

2004-01-01

Rhotekin belongs to the group of proteins containing a Rho-binding domain that are target peptides (effectors) for the Rho-GTPases. We previously identified a novel cDNA with homology to human rhotekin and in this study we cloned and characterized the coding region of this novel 12-exon gene. The ORF encodes a 609 amino-acid protein comprising a Class I Rho-binding domain and pleckstrin homology (PH) domain. Cellular cDNA expression of this new protein, designated Rhotekin-2 (RTKN2), was shown in the cytosol and nucleus of CHO cells. Using bioinformatics and RTPCR we identified three major splice variants, which vary in both the Rho-binding and PH domains. Real-time PCR studies showed exclusive RTKN2 expression in pooled lymphocytes and further purification indicated sole expression in CD4 pos T-cells and bone marrow-derived B-cells. Gene expression was increased in quiescent T-cells but negligible in activated proliferating cells. In malignant samples expression was absent in myeloid leukaemias, low in most B-cell malignancies and CD8 pos T-cell malignancies, but very high in CD4 pos /CD8 pos T-lymphoblastic lymphoma. As the Rho family is critical in lymphocyte development and function, RTKN2 may play an important role in lymphopoiesis

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.

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.

Níveis de dano econômico para decisão de controle de capim-arroz (Echinochloa spp. em arroz irrigado (Oryza sativa Economic threshold level for barnyardgrass (Echinochloa spp. control decision in flooded rice (Oryza sativa

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L. Galon

2007-12-01

Full Text Available O nível de dano econômico (NDE para monitoramento das infestações de capim-arroz em lavouras de arroz irrigado permite adotar medidas de manejo na cultura e usar herbicidas de maneira racional. Objetivou-se com o trabalho determinar NDE para capim-arroz, presente em lavouras de arroz irrigado, calculado na base de um único ano, em função de populações de capim-arroz, de cultivares de arroz e de épocas de entrada de água na lavoura. Para isso, foram conduzidos dois experimentos em delineamento experimental completamente casualizado, sem repetição. No primeiro experimento, os tratamentos foram constituídos de seis cultivares de arroz: BRS-Atalanta e IRGA 421 (ciclo muito curto, IRGA 416, IRGA 417 e Avaxi (ciclo curto e BRS-Fronteira (ciclo médio; e dez populações de capim-arroz. No segundo experimento usaram-se como tratamentos épocas de início da irrigação: 1, 10 e 20 dias após aplicação dos tratamentos herbicidas (DAT; e populações de capim-arroz. Os NDEs estimados para o capim-arroz variaram em função das práticas de manejo adotadas na cultura do arroz irrigado. Os NDEs estimados para os cultivares de arroz IRGA 416 e 417 foram superiores aos dos demais cultivares quando em competição com capim-arroz. O atraso da entrada de água diminui os NDEs para o cultivar de arroz BRS-Pelota. Aumentos nas perdas de produtividade por unidade de planta daninha, no potencial de produtividade da cultura, no valor do produto colhido e na eficiência do herbicida e diminuição do custo de controle reduziram os NDEs, tornando econômica a adoção de práticas de manejo sob baixas populações de capim-arroz em competição com arroz irrigado.Economic weed threshold (EWT for monitoring barnyardgrass infestation in flooded rice farms allows the adoption of crop management measurements and a rational use of herbicide. The objective of this study was to determine EWT for barnyardgrass based on a single year in function of

Competitividade relativa entre cultivares de arroz irrigado e biótipo de capim-Arroz (Echinochloa spp. Relative competitivity between flooded rice cultivars and Echinochloa spp.

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D. Agostinetto

2008-01-01

Full Text Available As espécies pertencentes ao gênero Echinochloa se destacam como principais infestantes das lavouras de arroz irrigado do Rio Grande do Sul, causando dano econômico à cultura devido à elevada competitividade pelos recursos do ambiente. O trabalho teve por objetivo comparar as habilidades competitivas relativas entre dois cultivares de arroz e um biótipo de capim-arroz. Para isso, foram realizados experimentos em casa de vegetação na estação de crescimento 2006/07, em delineamento experimental inteiramente casualizado, com quatro repetições. Os tratamentos foram arranjados em série substitutiva, com cinco proporções de plantas de arroz e do competidor (100:0; 75:25; 50:50; 25:75; e 0:100, mantendo a população constante de 24 plantas vaso-1 das espécies associadas. O arroz foi representado pelos cultivares IRGA 417 ou BR-IRGA 410, e o competidor, pelo biótipo de capim-arroz. A análise da competitividade foi efetuada por meio de diagramas aplicados a experimentos substitutivos e através de índices de competitividade relativa. As variáveis estudadas foram área foliar e massa seca aérea das plantas. Os resultados mostram que houve competição entre os cultivares de arroz IRGA 417 ou BR-IRGA 410 com o capim-arroz, independentemente da proporção de plantas na associação, com redução na área foliar e massa seca da parte aérea dos competidores. O capim-arroz apresenta menor perda de produtividade relativa, reduz as variáveis morfológicas do arroz e demonstra possuir superioridade competitiva, comparativamente aos cultivares de arroz.The Echinochloa species are among the most important weeds in flooded rice fields of Rio Grande do Sul, causing economic losses to the culture, due to its high competivity for environmental resources. The objective of this study was to compare the relative competitive abilities between two rice cultivars and an Echinochloa spp. biotype. Greenhouse experiments were conducted in 2006

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

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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.

Early stages of functional diversification in the Rab GTPase gene family revealed by genomic and localization studies in Paramecium species.

Science.gov (United States)

Bright, Lydia J; Gout, Jean-Francois; Lynch, Michael

2017-04-15

New gene functions arise within existing gene families as a result of gene duplication and subsequent diversification. To gain insight into the steps that led to the functional diversification of paralogues, we tracked duplicate retention patterns, expression-level divergence, and subcellular markers of functional diversification in the Rab GTPase gene family in three Paramecium aurelia species. After whole-genome duplication, Rab GTPase duplicates are more highly retained than other genes in the genome but appear to be diverging more rapidly in expression levels, consistent with early steps in functional diversification. However, by localizing specific Rab proteins in Paramecium cells, we found that paralogues from the two most recent whole-genome duplications had virtually identical localization patterns, and that less closely related paralogues showed evidence of both conservation and diversification. The functionally conserved paralogues appear to target to compartments associated with both endocytic and phagocytic recycling functions, confirming evolutionary and functional links between the two pathways in a divergent eukaryotic lineage. Because the functionally diversifying paralogues are still closely related to and derived from a clade of functionally conserved Rab11 genes, we were able to pinpoint three specific amino acid residues that may be driving the change in the localization and thus the function in these proteins. © 2017 Bright et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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.

Bowen ratio/energy balance technique for estimating crop net CO2 assimilation, and comparison with a canopy chamber

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Held, A. A.; Steduto, P.; Orgaz, F.; Matista, A.; Hsiao, T. C.

1990-12-01

This paper describes a Bowen ratio/energy balance (BREB) system which, in conjunction with an infra-red gas analyzer (IRGA), is referred to as BREB+ and is used to estimate evapotranspiration ( ET) and net CO2 flux ( NCF) over crop canopies. The system is composed of a net radiometer, soil heat flux plates, two psychrometers based on platinum resistance thermometers (PRT), bridge circuits to measure resistances, an IRGA, air pumps and switching valves, and a data logger. The psychrometers are triple shielded and aspirated, and with aspiration also between the two inner shields. High resistance (1 000 ohm) PRT's are used for dry and wet bulbs to minimize errors due to wiring and connector resistances. A high (55 K ohm) fixed resistance serves as one arm of the resistance bridge to ensure linearity in output signals. To minimize gaps in data, to allow measurements at short (e.g., 5 min) intervals, and to simplify operation, the psychrometers were fixed at their upper and lower position over the crop and not alternated. Instead, the PRT's, connected to the bridge circuit and the data logger, were carefully calibrated together. Field tests using a common air source showed appartent effects of the local environment around each psychrometer on the temperatures measured. ET rates estimated with the BREB system were compared to those measured with large lysimeters. Daily totals agreed within 5%. There was a tendency, however, for the lysimeter measurements to lag behind the BREB measurements. Daily patterns of NCF estimated with the BREB+ system are consistent with expectations from theories and data in the literature. Side-by-side comparisons with a stirred Mylar canopy chamber showed similar NCF patterns. On the other hand, discrepancies between the results of the two methods were quite marked in the morning or afternoon on certain dates. Part of the discrepancies may be attributed to inaccuracies in the psychrometric temperature measurements. Other possible causes

Human ClC-6 is a late endosomal glycoprotein that associates with detergent-resistant lipid domains.

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Sofie Ignoul

Full Text Available BACKGROUND: The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7. PRINCIPAL FINDINGS: Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432 have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y, endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4 and not with late endosomal/lysosomal markers (LAMP-1, Rab7. Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71-75: KKGRR disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6

Etoxazole resistance in predatory mite Phytoseiulus persimilis A.-H. (Acari: Phytoseiidae): Cross-resistance, inheritance and biochemical resistance mechanisms.

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Yorulmaz Salman, Sibel; Aydınlı, Fatma; Ay, Recep

2015-07-01

Phytoseiulus persimilis of the family Phytoseiidae is an effective predatory mite species that is used to control pest mites. The LC50 and LC60 values of etoxazole were determined on P. persimilis using a leaf-disc method and spraying tower. A laboratory selection population designated ETO6 was found to have a 111.63-fold resistance to etoxazole following 6 selection cycles. This population developed low cross-resistance to spinosad, spiromesifen, acetamiprid, indoxacarb, chlorantraniliprole, milbemectin and moderate cross-resistance to deltamethrin. PBO, IBP and DEM synergised resistance 3.17-, 2.85- and 3.60-fold respectively. Crossing experiments revealed that etoxazole resistance in the ETO6 population was an intermediately dominant and polygenic. In addition, detoxifying enzyme activities were increased 2.71-fold for esterase, 3.09-fold for glutathione S-transferase (GST) and 2.76-fold for cytochrome P450 monooxygenase (P450) in the ETO6 population. Selection for etoxazole under laboratory conditions resulted in the development of etoxazole resistance in the predatory mite P. persimilis that are resistant to pesticides are considered valuable for use in resistance management programmes within integrated pest control strategies. Copyright © 2014 Elsevier Inc. All rights reserved.

Cytochrome P450 CYP4DE1 and CYP6CW3v2 contribute to ethiprole resistance in Laodelphax striatellus (Fallén).

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Elzaki, M E A; Zhang, W; Han, Z

2015-06-01

Laodelphax striatellus Fallén (Hemiptera: Delphacidae), a destructive pest of rice, has developed high resistance to multiple insecticides, threatening the success of pest management programmes. The present study investigated ethiprole resistance mechanisms in a field population that is highly resistant to ethiprole. That population was used to establish a laboratory population that was subjected to further selection to produce a resistant strain. Target genes were cloned and compared between the resistant and the susceptible strains, the role of detoxification enzymes was examined, and the relative expression levels of 71 detoxification enzyme genes were tested using quantitative real time (RT)-PCR. The laboratory selection enhanced the resistance from 107-fold to 180-fold. The Rdl-type target site mutation seldom occurred in the resistant strain and is unlikely to represent the major mechanism underlying the observed resistance. Of the three important detoxification enzymes, only P450 monooxygenase was found to be associated with ethiprole resistance. Moreover, two genes, CYP4DE1 and CYP6CW3v2, were found to be overexpressed in the resistant strain. Furthermore, gene-silencing via a double-stranded RNA feeding test was carried out, and the results showed that the mRNA levels of CYP4DE1 and CYP6CW3v2 were reduced in the resistant strain, whereas ethiprole susceptibility was increased. These results suggest that CYP4DE1 and CYP6CW3v2 play an important role in ethiprole resistance in L. striatellus. © 2015 The Royal Entomological Society.

The correlation of the results of capacitance mapping and of sheet resistance mapping in semi-insulating 6H-SiC

International Nuclear Information System (INIS)

Lin Shenghuang; Chen Zhiming; Liang Peng; Jiang Dong; Xie Huajie; Yang Ying

2010-01-01

A combination of complex surface capacitance mapping and sheet resistance mapping is applied to establish the origin of resistance variations on semi-insulating (SI) 6H-SiC substrates. The direct correlation between the capacitance quadrature and the sheet resistance is found in vanadium-doped SI samples. Regions with low capacitance quadrature show high sheet resistance. This indicates, associated with the nonhomogeneity of sheet resistance on the substrate, that the quality of crystallization is not good enough, which also leads to resistivity nonhomogeneity when comparing with different types of deep defects. According to the capacitance mapping, the region with bad crystallization quality has a high radio absorption coefficient. Another correlation is established between the capacitance in-phase and sheet resistance for the vanadium-doped sample. In this sample, the capacitance in-phase map shows not only the surface topography, but also the same distribution trend as the sheet resistance, namely, regions of high capacitance in-phase reveal high sheet resistance.

α6 Integrin and CD44 enrich for a primary keratinocyte population that displays resistance to UV-induced apoptosis.

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Helen Wray

Full Text Available Epidermal human keratinocytes are exposed to a wide range of environmental genotoxic insults, including the UV component of solar radiation. Epidermal homeostasis in response to cellular or tissue damage is maintained by a population of keratinocyte stem cells (KSC that reside in the basal layer of the epithelium. Using cell sorting based on cell-surface markers, we have identified a novel α6 integrin(high+/CD44(+ sub-population of basal keratinocytes. These α6 integrin(high+/CD44(+ keratinocytes have both high proliferative potential, form colonies in culture that have characteristics of holoclones and have a unique pattern of resistance to apoptosis induced by UVB radiation or by agents that induce single- or double strand DNA breaks. Resistance to UVB induced apoptosis in the α6 integrin(high+/CD44(+ cells involved increased expression of TAp63 and was overcome by PI-3 kinase inhibition. In marked contrast, the α6 integrin(high+/CD44(+ cells were sensitive to apoptosis induced by the cross-linking agent cisplatin, and imatinib inhibition of c-Abl blocked the ability of cisplatin to kill α6 integrin(high+/CD44(+ cells. Our findings reveal a population of basal keratinocytes with long-term proliferative properties that display specific patterns of apoptotic resistance that is dependent upon the genotoxic stimulus, and provide insights into how these cells can be targeted with chemotherapeutic agents.

Cadmium biosorption properties of the metal-resistant ochrobactrum cytisi Azn6.2

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Rodriguez-Llorente, Ignacio D.; Gamane, Djamila; Lafuente, Alejandro; Dary, Mohammed; El Hamdaoui, Abdelaziz; Delgadillo, Julian; Doukkali, Bouchra; Caviedes, Miguel A.; Pajuelo, Eloisa [Departamento de Microbiologia, Facultad de Farmacia, Universidad de Sevilla, Sevilla (Spain)

2010-02-15

The aim of this work was to establish the conditions for using Ochrobactrum cytisi Azn6.2 as a metal biosorbent. Azn6.2 is a novel strain from the legume symbiont O. cytisi that has been isolated from nodules of Medicago polymorpha plants grown on heavy metal-polluted soils. Compared with the strain ESC1, Azn6.2 showed some biochemical differences, as well as antibiotic susceptibility, Azn6.2 was multi-resistant to heavy metals, such as Cu, Cd and Zn, and bacterial pellets were able to biosorb high amounts of Cd and Zn. As shown by scanning electron microscopy coupled to energy dispersive X-ray, most of Cd was attached to the cell surface. Optimal conditions for Cd biosorption were established, being 1 mM Cd ions in solution and 2 h of contact with the biosorbent at room temperature. At these conditions, maximal Cd loading capacity reached 32-34 mg/g. Cd desorption from bacterial pellets was achieved after washing with EDTA or, at higher efficiency, at pH 1.0. These results indicated that biosorption/desorption on O. cytisi Azn6.2 biomass should be a cost-effective method for Cd recovery from contaminated solutions. (Abstract Copyright [2010], Wiley Periodicals, Inc.)

Exploring the correlation between the sequence composition of the nucleotide binding G5 loop of the FeoB GTPase domain (NFeoB) and intrinsic rate of GDP release.

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Guilfoyle, Amy P; Deshpande, Chandrika N; Schenk, Gerhard; Maher, Megan J; Jormakka, Mika

2014-12-12

GDP release from GTPases is usually extremely slow and is in general assisted by external factors, such as association with guanine exchange factors or membrane-embedded GPCRs (G protein-coupled receptors), which accelerate the release of GDP by several orders of magnitude. Intrinsic factors can also play a significant role; a single amino acid substitution in one of the guanine nucleotide recognition motifs, G5, results in a drastically altered GDP release rate, indicating that the sequence composition of this motif plays an important role in spontaneous GDP release. In the present study, we used the GTPase domain from EcNFeoB (Escherichia coli FeoB) as a model and applied biochemical and structural approaches to evaluate the role of all the individual residues in the G5 loop. Our study confirms that several of the residues in the G5 motif have an important role in the intrinsic affinity and release of GDP. In particular, a T151A mutant (third residue of the G5 loop) leads to a reduced nucleotide affinity and provokes a drastically accelerated dissociation of GDP.

HCV Genotype 6a Escape From and Resistance to Velpatasvir, Pibrentasvir, and Sofosbuvir in Robust Infectious Cell Culture Models

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Pham, Long V; Ramirez, Santseharay; Gottwein, Judith M

2018-01-01

V was able to propagate and escape in the presence of pibrentasvir with emergence of NS5A-L28S, conferring a high level of resistance to this inhibitor. CONCLUSIONS: Strains of HCV genotype 6a isolated from patients can be adapted to propagate in cultured cells, permitting studies of the complete...... infectious cell culture models of HCV genotype 6a infection to study the effects of these inhibitors and the development of resistance. METHODS: The consensus sequences of prototype strains HK2 (MG717925) and HK6a (MG717928), originating from serum of patients with chronic HCV infection, were determined...... by Sanger sequencing of genomes amplified by reverse-transcription polymerase chain reaction. In vitro noninfectious full-length clones of these 6a strains were subsequently adapted in Huh7.5 cells, primarily by using substitutions identified in JFH1-based Core-NS5A and Core-NS5B genotype 6a recombinants...

HCV Genotype 6a Escape from and Resistance to Velpatasvir, Pibrentasvir, and Sofosbuvir in Robust Infectious Cell Culture Models

DEFF Research Database (Denmark)

Pham, Long; Ramirez Almeida, Santseharay; Gottwein, Judith Margarete

was able to propagate and escape in the presence of pibrentasvir with emergence of NS5A-L28S, conferring a high level of resistance to this inhibitor. CONCLUSIONS: Strains of HCV genotype 6a isolated from patients can be adapted to propagate in cultured cells, permitting studies of the complete life cycle...... infectious cell culture models of HCV genotype 6a infection to study the effects of these inhibitors and the development of resistance. METHODS: The consensus sequences of prototype strains HK2 (MG717925) and HK6a (MG717928), originating from serum of patients with chronic HCV infection, were determined...... by Sanger sequencing of genomes amplified by reverse transcription-PCR. In vitro non-infectious full-length clones of these 6a strains were subsequently adapted in Huh7.5 cells, primarily by using substitutions identified in JFH1-based core-NS5A and core-NS5B genotype 6a recombinants. We studied...

Genome-Wide Association Study for Identification and Validation of Novel SNP Markers for Sr6 Stem Rust Resistance Gene in Bread Wheat.

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Mourad, Amira M I; Sallam, Ahmed; Belamkar, Vikas; Wegulo, Stephen; Bowden, Robert; Jin, Yue; Mahdy, Ezzat; Bakheit, Bahy; El-Wafaa, Atif A; Poland, Jesse; Baenziger, Peter S

2018-01-01

Stem rust (caused by Puccinia graminis f. sp. tritici Erikss. & E. Henn.), is a major disease in wheat ( Triticum aestivium L.). However, in recent years it occurs rarely in Nebraska due to weather and the effective selection and gene pyramiding of resistance genes. To understand the genetic basis of stem rust resistance in Nebraska winter wheat, we applied genome-wide association study (GWAS) on a set of 270 winter wheat genotypes (A-set). Genotyping was carried out using genotyping-by-sequencing and ∼35,000 high-quality SNPs were identified. The tested genotypes were evaluated for their resistance to the common stem rust race in Nebraska (QFCSC) in two replications. Marker-trait association identified 32 SNP markers, which were significantly (Bonferroni corrected P < 0.05) associated with the resistance on chromosome 2D. The chromosomal location of the significant SNPs (chromosome 2D) matched the location of Sr6 gene which was expected in these genotypes based on pedigree information. A highly significant linkage disequilibrium (LD, r 2 ) was found between the significant SNPs and the specific SSR marker for the Sr6 gene ( Xcfd43 ). This suggests the significant SNP markers are tagging Sr6 gene. Out of the 32 significant SNPs, eight SNPs were in six genes that are annotated as being linked to disease resistance in the IWGSC RefSeq v1.0. The 32 significant SNP markers were located in nine haplotype blocks. All the 32 significant SNPs were validated in a set of 60 different genotypes (V-set) using single marker analysis. SNP markers identified in this study can be used in marker-assisted selection, genomic selection, and to develop KASP (Kompetitive Allele Specific PCR) marker for the Sr6 gene. Novel SNPs for Sr6 gene, an important stem rust resistant gene, were identified and validated in this study. These SNPs can be used to improve stem rust resistance in wheat.

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.

The necrotic signal induced by mycophenolic acid overcomes apoptosis-resistance in tumor cells.

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Gwendaline Guidicelli

Full Text Available BACKGROUND: The amount of inosine monophosphate dehydrogenase (IMPDH, a pivotal enzyme for the biosynthesis of the guanosine tri-phosphate (GTP, is frequently increased in tumor cells. The anti-viral agent ribavirin and the immunosuppressant mycophenolic acid (MPA are potent inhibitors of IMPDH. We recently showed that IMPDH inhibition led to a necrotic signal requiring the activation of Cdc42. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we strengthened the essential role played by this small GTPase in the necrotic signal by silencing Cdc42 and by the ectopic expression of a constitutive active mutant of Cdc42. Since resistance to apoptosis is an essential step for the tumorigenesis process, we next examined the effect of the MPA-mediated necrotic signal on different tumor cells demonstrating various mechanisms of resistance to apoptosis (Bcl2-, HSP70-, Lyn-, BCR-ABL-overexpressing cells. All tested cells remained sensitive to MPA-mediated necrotic signal. Furthermore, inhibition of IMPDH activity in Chronic Lymphocytic Leukemia cells was significantly more efficient at eliminating malignant cells than apoptotic inducers. CONCLUSIONS/SIGNIFICANCE: These findings indicate that necrosis and apoptosis are split signals that share few if any common hub of signaling. In addition, the necrotic signaling pathway induced by depletion of the cellular amount of GTP/GDP would be of great interest to eliminate apoptotic-resistant tumor cells.

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.

SIRT6 knockout cells resist apoptosis initiation but not progression: a computational method to evaluate the progression of apoptosis.

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Domanskyi, Sergii; Nicholatos, Justin W; Schilling, Joshua E; Privman, Vladimir; Libert, Sergiy

2017-11-01

Apoptosis is essential for numerous processes, such as development, resistance to infections, and suppression of tumorigenesis. Here, we investigate the influence of the nutrient sensing and longevity-assuring enzyme SIRT6 on the dynamics of apoptosis triggered by serum starvation. Specifically, we characterize the progression of apoptosis in wild type and SIRT6 deficient mouse embryonic fibroblasts using time-lapse flow cytometry and computational modelling based on rate-equations and cell distribution analysis. We find that SIRT6 deficient cells resist apoptosis by delaying its initiation. Interestingly, once apoptosis is initiated, the rate of its progression is higher in SIRT6 null cells compared to identically cultured wild type cells. However, SIRT6 null cells succumb to apoptosis more slowly, not only in response to nutrient deprivation but also in response to other stresses. Our data suggest that SIRT6 plays a role in several distinct steps of apoptosis. Overall, we demonstrate the utility of our computational model to describe stages of apoptosis progression and the integrity of the cellular membrane. Such measurements will be useful in a broad range of biological applications.

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.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of Pseudomonas aeruginosa MnmE, a GTPase involved in tRNA modification

International Nuclear Information System (INIS)

Lee, Hyung Ho; Suh, Se Won

2010-01-01

MnmE from P. aeruginosa was crystallized and X-ray diffraction data were collected to 2.69 Å resolution. MnmE, an evolutionarily conserved GTPase, is involved in modification of the uridine base (U34) at the wobble position of certain tRNAs. Previous crystal structures of MnmE suggest that it is a dimer with considerable conformational flexibility. To facilitate structural comparisons among MnmE proteins, structural analysis of MnmE from Pseudomonas aeruginosa encoded by the PA5567 gene was initiated. It was overexpressed in Escherichia coli and crystallized at 297 K using a reservoir solution consisting of 100 mM sodium ADA pH 6.5, 12%(w/v) polyethylene glycol 4000, 100 mM lithium sulfate, 2%(v/v) 2-propanol and 2.5 mM dithiothreitol. X-ray diffraction data were collected to 2.69 Å resolution. The crystals belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 96.74, b = 204.66, c = 120.90 Å. Two monomers were present in the asymmetric unit, resulting in a crystal volume per protein mass (V M ) of 2.99 Å 3 Da −1 and a solvent content of 58.8%

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

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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.

Identification of promoter polymorphisms in the cytochrome P450 CYP6AY1 linked with insecticide resistance in the brown planthopper, Nilaparvata lugens.

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Pang, R; Li, Y; Dong, Y; Liang, Z; Zhang, Y; Zhang, W

2014-12-01

Imidacloprid resistance in the brown planthopper, Nilaparvata lugens, is primarily the result of the over-expression of cytochrome P450 monooxygenases. Here, a field-collected strain of N. lugens was shown to be highly resistant to both imidacloprid and buprofezin. Insecticide exposure and quantitative real-time PCR revealed that its resistance was mainly associated with a cytochrome P450 gene, CYP6AY1. CYP6AY1 is known to metabolize imidacloprid but its effect on buprofezin is unclear. In the 5'-untranslated region of CYP6AY1, a novel alternative splicing was detected. After a 1990-bp promoter region was cloned, its basal luciferase activity was assessed. Furthermore, genotyping studies identified 12 variations in the promoter region that discriminated between the field-collected and control strain. Finally, survival bioassays revealed a single nucleotide polymorphism and an insertion-deletion polymorphism linked to buprofezin and imidacloprid resistance. Mutagenesis of these sites enhanced the promoter activity of CYP6AY1. These results suggest that promoter polymorphisms may affect P450-mediated multiple insecticide resistance of pests. © 2014 The Royal Entomological Society.

Proteomics-based identification of midgut proteins correlated with Cry1Ac resistance in Plutella xylostella (L.).

Science.gov (United States)

Xia, Jixing; Guo, Zhaojiang; Yang, Zezhong; Zhu, Xun; Kang, Shi; Yang, Xin; Yang, Fengshan; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Xu, Weijun; Zhang, Youjun

2016-09-01

The diamondback moth, Plutella xylostella (L.), is a worldwide pest of cruciferous crops and can rapidly develop resistance to many chemical insecticides. Although insecticidal crystal proteins (i.e., Cry and Cyt toxins) derived from Bacillus thuringiensis (Bt) have been useful alternatives to chemical insecticides for the control of P. xylostella, resistance to Bt in field populations of P. xylostella has already been reported. A better understanding of the resistance mechanisms to Bt should be valuable in delaying resistance development. In this study, the mechanisms underlying P. xylostella resistance to Bt Cry1Ac toxin were investigated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and ligand blotting for the first time. Comparative analyses of the constitutive expression of midgut proteins in Cry1Ac-susceptible and -resistant P. xylostella larvae revealed 31 differentially expressed proteins, 21 of which were identified by mass spectrometry. Of these identified proteins, the following fell into diverse eukaryotic orthologous group (KOG) subcategories may be involved in Cry1Ac resistance in P. xylostella: ATP-binding cassette (ABC) transporter subfamily G member 4 (ABCG4), trypsin, heat shock protein 70 (HSP70), vacuolar H(+)-ATPase, actin, glycosylphosphatidylinositol anchor attachment 1 protein (GAA1) and solute carrier family 30 member 1 (SLC30A1). Additionally, ligand blotting identified the following midgut proteins as Cry1Ac-binding proteins in Cry1Ac-susceptible P. xylostella larvae: ABC transporter subfamily C member 1 (ABCC1), solute carrier family 36 member 1 (SLC36A1), NADH dehydrogenase iron-sulfur protein 3 (NDUFS3), prohibitin and Rap1 GTPase-activating protein 1. Collectively, these proteomic results increase our understanding of the molecular resistance mechanisms to Bt Cry1Ac toxin in P. xylostella and also demonstrate that resistance to Bt Cry1Ac toxin is complex and multifaceted. Copyright © 2016 Elsevier B.V. All

Effects of SF$_{6}$ on the avalanche mode operation of a real-sized double-gap resistive plate chamber for the Compact Muon Solenoid experiment

CERN Document Server

Ahn Sung Hwan; Hong, B; Hong, S J; Ito, M; Kim, B I; Kim, J H; Kim, Y J; Kim, Y U; Koo, D G; Lee, H W; Lee, K B; Lee, K S; Lee, S J; Lim, J K; Moon, D H; Nam, S K; Park, S; Park, W J; Rhee, J T; Ryu, M S; Shim, H H; Sim, K S; Kang, T I

2005-01-01

We present the design and the test, results for a real-sized prototype resistive plate chamber by using cosmic-ray muons for the forward region of the Compact Muon Solenoid (CMS) experiment at the Large Hadron Collider (LHC). In particular, we investigate the effects of adding SF/sub 6/ to the gas mixture for the avalanche mode operation of a resistive plate chamber. A small fraction of SF/sub 6/ is very effective in suppressing streamer signals in a resistive plate chamber. The shapes of the muon detection efficiency and the muon cluster size remain similar, but are shifted to higher operating voltage by SF/sub 6/. The noise cluster rate and size are not influenced by SF/sub 6/.

Corrosion resistance characteristics of a Ti-6Al-4V alloy scaffold that is fabricated by electron beam melting and selective laser melting for implantation in vivo

Energy Technology Data Exchange (ETDEWEB)

Zhao, Bingjing; Wang, Hong [Department of Stomatology, General Hospital of the PLA, Beijing (China); Department of Stomatology, The Second Affiliated Stomatological Hospital of Liaoning Medical University (China); Qiao, Ning [College of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing (China); Wang, Chao [School of Medicine, Nankai University, Tianjin 300071 (China); Hu, Min, E-mail: humin48@vip.163.com [Department of Stomatology, General Hospital of the PLA, Beijing (China)

2017-01-01

The purpose of this study is to determine the corrosion resistance of Ti-6Al-4V alloy fabricated with electron beam melting and selective laser melting for implantation in vivo. Ti-6Al-4V alloy specimens were fabricated with electron beam melting (EBM) and selective laser melting (SLM). A wrought form of Ti-6Al-4V alloy was used as a control. Surface morphology observation, component analysis, corrosion resistance experimental results, electrochemical impedance spectroscopy, crevice corrosion resistance experimental results, immersion test and metal ions precipitation analysis were processed, respectively. The thermal stability of EBM specimen was the worst, based on the result of open circuit potential (OCP) result. The result of electrochemical impedance spectroscopy indicated that the corrosion resistance of the SLM specimen was the best under the low electric potential. The result of potentiodynamic polarization suggested that the corrosion resistance of the SLM specimen was the best under the low electric potential (< 1.5 V) and EBM specimen was the best under the high electric potential (> 1.5 V).The crevice corrosion resistance of the EBM specimen was the best. The corrosion resistance of SLM specimen was the best, based on the result of immersion test. The content of Ti, Al and V ions of EBM, SLM and wrought specimens was very low. In general, the scaffolds that were fabricated with EBM and SLM had good corrosion resistance, and were suitable for implantation in vivo. - Highlights: • EBM and SLM Ti-6Al-4V alloy have good corrosion resistance, and both of them can be applied in vivo. • SLM Ti-6Al-4V alloy was more suitable for implantation in vivo than that of EBM Ti-6Al-4V alloy. • The crevice corrosion resistance of the EBM specimen is the best. • EBM and SLM specimens can form oxide film.

Magnetic properties and Hall effect of single-crystalline YMn6Sn6

International Nuclear Information System (INIS)

Uhlirova, K.; Sechovsky, V.; Boer, F.R. de; Yoshii, S.; Yamamoto, T.; Hagiwara, M.; Lefevre, C.; Venturini, G.

2007-01-01

Magnetization behavior and Hall resistivity of YMn 6 Sn 6 , which crystallizes in the hexagonal HfFe 6 Ge 6 -type of structure, have been investigated on single crystals at various temperatures in the ordered magnetic state. The field dependence of the Hall resistivity shows anomalies, which are related to the field-induced spin reorientations occurring in YMn 6 Sn 6 . It is also found that the Hall resistivity cannot simply be described by the anomalous contribution proportional to the magnetization, but that an additional field-dependent contribution is present

Acquisition of resistance to 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride in V79 cells through increased removal of O6-alkylguanine.

Science.gov (United States)

Satoh, M S; Huh, N H; Horie, Y; Thomale, J; Rajewsky, M F; Kuroki, T

1987-10-01

The molecular mechanism of acquisition of resistance to 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride (ACNU) was investigated using ACNU-resistant clones (ACNUr-1-4) isolated from the V79 cell line. The binding level of alkyl cyanate, a decomposition product of ACNU, to protein in ACNUr-1 cells was not less than that in the parental V79 cells, indicating that the acquired resistance was not due to a reduced intracellular concentration of ACNU. Because O6-chloroethylguanine, an intermediate in cytotoxic interstrand cross-link formation by ACNU, is known to be repaired by the same mechanism as O6-ethyldeoxyguanosine (O6-EtdGuo), we quantitated O6-EtdGuo by radioimmunoassay at various times after exposure of cells to 100 micrograms/ml N-ethyl-N-nitrosourea for 20 min. In V79 cells, elimination of O6-EtdGuo was negligible, but in all four resistant clones, 30 to 59% of the O6-EtdGuo was removed within 24 hr after exposure. This increased removal of O6-EtdGuo among the resistant clones was associated with the activity of O6-alkylguanine DNA alkyltransferase (O6-AGT) determined using cell extracts. The present results indicate that increased removal of O6-chloroethylguanine in ACNU-resistant clones by O6-AGT is mechanistically linked to the acquisition of resistance to ACNU.

Overexpression of HvIcy6 in Barley Enhances Resistance against Tetranychus urticae and Entails Partial Transcriptomic Reprogramming

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M. Estrella Santamaria

2018-03-01

Full Text Available Cystatins have been largely used for pest control against phytophagous species. However, cystatins have not been commonly overexpressed in its cognate plant species to test their pesticide capacity. Since the inhibitory role of barley HvCPI-6 cystatin against the phytophagous mite Tetranychus urticae has been previously demonstrated, the purpose of our study was to determine if barley transgenic lines overexpressing its own HvIcy6 gene were more resistant against this phytophagous infestation. Besides, a transcriptomic analysis was done to find differential expressed genes among wild-type and transformed barley plants. Barley plants overexpressing HvIcy6 cystatin gene remained less susceptible to T. urticae attack when compared to wild-type plants, with a significant lesser foliar damaged area and a lower presence of the mite. Transcriptomic analysis revealed a certain reprogramming of cellular metabolism and a lower expression of several genes related to photosynthetic activity. Therefore, although caution should be taken to discard potential deleterious pleiotropic effects, cystatins may be used as transgenes with impact on agricultural crops by conferring enhanced levels of resistance to phytophagous pests.

A Bacterial Pathogen Targets a Host Rab-Family GTPase Defense Pathway with a GAP.

Science.gov (United States)

Spanò, Stefania; Gao, Xiang; Hannemann, Sebastian; Lara-Tejero, María; Galán, Jorge E

2016-02-10

Cell-autonomous defense mechanisms are potent strategies that protect individual cells against intracellular pathogens. The Rab-family GTPase Rab32 was previously shown to restrict the intracellular human pathogen Salmonella Typhi, but its potential broader role in antimicrobial defense remains unknown. We show that Rab32 represents a general cell-autonomous, antimicrobial defense that is counteracted by two Salmonella effectors. Mice lacking Rab-32 or its nucleotide exchange factor BLOC-3 are permissive to S. Typhi infection and exhibit increased susceptibility to S. Typhimurium. S. Typhimurium counters this defense pathway by delivering two type III secretion effectors, SopD2, a Rab32 GAP, and GtgE, a specific Rab32 protease. An S. Typhimurium mutant strain lacking these two effectors exhibits markedly reduced virulence, which is fully restored in BLOC-3-deficient mice. These results demonstrate that a cell-autonomous, Rab32-dependent host defense pathway plays a central role in the defense against vacuolar pathogens and describe a mechanism evolved by a bacterial pathogen to counter it. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Improved corrosion resistant and mechanical behavior of distinct composite coatings (silica/titania/zirconia on Ti–6Al–4V deposited by EPD

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M. Chellappa

2017-09-01

Full Text Available Synthesized composite powders (ZrO2/TiO2, SiO2/TiO2, and SiO2/ZrO2 were successfully deposited on Ti–6Al–4V by electrophoretic deposition method (EPD to improve its electrochemical characteristics for better biomedical applications. In the present investigation, the three composite powders were prepared by sol–gel synthesis and its phase purity was analyzed by Powder X-ray diffraction (XRD method. Further, the performance of the deposited coatings was assessed by scanning electron microscopy (SEM coupled with energy dispersive X-ray analysis (EDAX, scratch resistance test. The electrochemical properties of the composite coatings were analyzed by Potentiodynamic (Tafel polarization and electrochemical impedance spectroscopy (EIS studies. From the results, we observed that the corrosion resistance behavior of the different composite coated metallic substrate exhibited divergent corrosion resistance nature than blank Ti–6Al–4V. Of all these coatings on Ti–6Al–4V, the composite made up of, ZrO2/TiO2 has pronounced corrosion resistance behavior in Ringer’s solution when compared to others. This behavior is due to the presence of strong adherent coating owing to the existence of uniform deposition on Ti–6Al–4V.

The effects of RE and Si on the microstructure and corrosion resistance of Zn–6Al–3Mg hot dip coating

International Nuclear Information System (INIS)

Li, Shiwei; Gao, Bo; Yin, Shaohua; Tu, Ganfeng; Zhu, Guanglin; Sun, Shuchen; Zhu, Xiaoping

2015-01-01

Highlights: • ZAM coating has been prepared by using an experimental hot-dip galvanizing simulator. • The corrosion resistance of ZAM coating can be improved by additions of Si and RE. • Zn–6Al–3Mg–Si–RE coating forms a dense and stabilized corrosion product layer. • Zn–6Al–3Mg–Si–RE coating shows uniform corrosion. - Abstract: The effects of Si and RE on the microstructure and corrosion resistance of Zn–6Al–3Mg coating (ZAM) have been investigated. Surface morphology observations of the coating and corrosion products reveal that the additions of Si and rare earth metals (RES) improve the microstructural homogeneity of ZAMSR coating and stability of corrosion products formed on ZAMSR coating. Moreover, only uniform corrosion occurs in ZAMSR coating during the corrosion test, while intergranular corrosion and pitting occur in ZAM. As a result, the corrosion resistance of ZAM coating is improved by the additions of Si and RES.

The effects of RE and Si on the microstructure and corrosion resistance of Zn–6Al–3Mg hot dip coating

Energy Technology Data Exchange (ETDEWEB)

Li, Shiwei [State Key Laboratory of Complex Nonferrous Metal Resources Clean Utilization, Kunming University of Science and Technology, Kunming 650093 (China); School of Materials and Metallurgy, Northeastern University, Shenyang 110819 (China); Faculty of Metallurgical and Energy Engineering, Kunming University of Science and Technology, Kunming 650093 (China); Gao, Bo, E-mail: surfgao@aliyun.com [School of Materials and Metallurgy, Northeastern University, Shenyang 110819 (China); Yin, Shaohua [State Key Laboratory of Complex Nonferrous Metal Resources Clean Utilization, Kunming University of Science and Technology, Kunming 650093 (China); School of Materials and Metallurgy, Northeastern University, Shenyang 110819 (China); Faculty of Metallurgical and Energy Engineering, Kunming University of Science and Technology, Kunming 650093 (China); Tu, Ganfeng; Zhu, Guanglin; Sun, Shuchen; Zhu, Xiaoping [School of Materials and Metallurgy, Northeastern University, Shenyang 110819 (China)

2015-12-01

Highlights: • ZAM coating has been prepared by using an experimental hot-dip galvanizing simulator. • The corrosion resistance of ZAM coating can be improved by additions of Si and RE. • Zn–6Al–3Mg–Si–RE coating forms a dense and stabilized corrosion product layer. • Zn–6Al–3Mg–Si–RE coating shows uniform corrosion. - Abstract: The effects of Si and RE on the microstructure and corrosion resistance of Zn–6Al–3Mg coating (ZAM) have been investigated. Surface morphology observations of the coating and corrosion products reveal that the additions of Si and rare earth metals (RES) improve the microstructural homogeneity of ZAMSR coating and stability of corrosion products formed on ZAMSR coating. Moreover, only uniform corrosion occurs in ZAMSR coating during the corrosion test, while intergranular corrosion and pitting occur in ZAM. As a result, the corrosion resistance of ZAM coating is improved by the additions of Si and RES.

Encephalomyocarditis virus Leader protein hinge domain is responsible for interactions with Ran GTPase

Energy Technology Data Exchange (ETDEWEB)

Bacot-Davis, Valjean R., E-mail: bacotdavis@wisc.edu [Institute for Molecular Virology, University of Wisconsin-Madison, R.M. Bock Laboratories, 1525 Linden Dr. Madison, WI 53706 (United States); Palmenberg, Ann C., E-mail: acpalmen@wisc.edu [Institute for Molecular Virology, University of Wisconsin-Madison, R.M. Bock Laboratories, 1525 Linden Dr. Madison, WI 53706 (United States); Department of Biochemistry, University of Wisconsin-Madison, R.M. Bock Laboratories, 1525 Linden Dr. Madison, WI 53706 (United States)

2013-08-15

Encephalomyocarditis virus (EMCV), a Cardiovirus, initiates its polyprotein with a short 67 amino acid Leader (L) sequence. The protein acts as a unique pathogenicity factor, with anti-host activities which include the triggering of nuclear pore complex hyperphosphorylation and direct binding inhibition of the active cellular transport protein, Ran GTPase. Chemical modifications and protein mutagenesis now map the Ran binding domain to the L hinge-linker region, and in particular, to amino acids 35–40. Large deletions affecting this region were shown previously to diminish Ran binding. New point mutations, especially K35Q, D37A and W40A, preserve the intact L structure, abolish Ran binding and are deficient for nucleoporin (Nup) hyperphosphorylation. Ran itself morphs through multiple configurations, but reacts most effectively with L when in the GDP format, preferably with an empty nucleotide binding pocket. Therefore, L:Ran binding, mediated by the linker-hinge, is a required step in L-induced nuclear transport inhibition. - Highlights: • The hinge domain provides critical residues in Cardiovirus L:Ran complex formation. • Leader prefers to bind Ran in a nucleotide free, GDP-conformation. • L-induced Nup62 phosphorylation is reduced with Ran-deficient binding mutations.

The Effect of Resistance Training on Levels of Interlukine-6 and High-Sensitivity C - reactive protein in Older-Aged Women

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Zahra Mardanpour Shahrekordi

2017-06-01

Full Text Available Introduction: Aging is associated with elevated levels of some proinflammatory factors and exercise is a non-invasive intervention to improve immune function among older adults .The aim of the study was to compare resistance training effects on interlukine-6 (IL-6 and high-sensitivity C-reactive protein (hs-CRP levels in older-aged women. Methods: The study was quasi-experimental and forty healthy females were selected and randomly assigned to one of four groups: strength after endurance training (endurance + strength (E + S, n = 9, strength prior to endurance training (strength + endurance (S + E, n = 10, interval resistance-endurance training (Int, n = 12, and control (n = 9 groups. The training program was performed for eight weeks, three times per week. Human TNF-α and IL-6 sandwich ELISA Kit were used. Within-group differences were analyzed using a paired samples t-test and between-group differences were analyzed using one-way analysis of variance. Results: The intra-session order had not significantly influence on the adaptive response of waist-to-hip ratio (p = 0.55, IL-6 (p = 0.55 and hs-CRP (p = 0.55 throughout the study. However, significant differences were shown following combined training between the S + E, E + S and Int groups for Vo2 max (p = 0.029, body mass (p = 0.016 and BMI (p = 0.023 when comparing pre and posttests. Conclusion: This study confirmed that adaptations to a combination of endurance and resistance training appear to be independent of whether resistance training occurs prior to or following endurance training.

Correlation between Interleukin-6 (IL-6, High Sensitivity C-Reactive Protein (hsCRP, Endothelin-1 (ET-1, Asymmetric Dimethylarginine (ADMA and Insulin Resistance (HOMA-IR in Central Obese Men

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Andri Hidayat

2011-04-01

Full Text Available BACKGROUND: Many studies have shown that obesity was closely related to insulin resistance via several pathways such as inflammation, oxidative stress, lipolysis, and endothelial dysfunction. This study was carried out to observe the correlation between inflammation (IL-6 and hsCRP, lipolysis process (ET-1, and endothelial dysfunction (ADMA and insulin resistance (HOMA-IR in centrally obese men. METHODS: This was a cross sectional study on 62 male subjects aged 30–60 years old with waist circumference (WC >90 cm. IL-6, ET-1 and ADMA levels were measured using ELISA method, while hsCRP and insulin were measured using chemiluminescence method. All blood testings were conducted in Prodia Clinical Laboratory. RESULTS: The results showed that WC was significantly correlated with hsCRP (r=0.294, p=0.022, ET-1 (r=0.257, p=0.047 and ADMA (r=0.338, p=0.009. We also found a significant correlation between hsCRP with HOMA-IR (r=0.324, p=0.021, ADMA with HOMA-IR (r=0.280, p=0.045 and IL-6 with hsCRP (r=0.437, p=0.003. CONCLUSIONS: hsCRP and ADMA have significant correlation with HOMA-IR in centrally obese men. HOMA-IR significantly increases in subjects with ADMA above median and either IL-6 or hsCRP above median, as compared to those in the other groups. Inflammation and endothelial dysfunction are important causal pathways of insulin resistance state in centrally obese men. KEYWORDS: obesity, IL-6, hsCRP, ET-1, ADMA, HOMA-IR.

Overexpression of a cytochrome P450 monooxygenase, CYP6ER1, is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.

Science.gov (United States)

Bass, C; Carvalho, R A; Oliphant, L; Puinean, A M; Field, L M; Nauen, R; Williamson, M S; Moores, G; Gorman, K

2011-12-01

The brown planthopper, Nilaparvata lugens, is an economically significant pest of rice throughout Asia and has evolved resistance to many insecticides including the neonicotinoid imidacloprid. The resistance of field populations of N. lugens to imidacloprid has been attributed to enhanced detoxification by cytochrome P450 monooxygenases (P450s), although, to date, the causative P450(s) has (have) not been identified. In the present study, biochemical assays using the model substrate 7-ethoxycoumarin showed enhanced P450 activity in several resistant N. lugens field strains when compared with a susceptible reference strain. Thirty three cDNA sequences encoding tentative unique P450s were identified from two recent sequencing projects and by degenerate PCR. The mRNA expression level of 32 of these was examined in susceptible, moderately resistant and highly resistant N. lugens strains using quantitative real-time PCR. A single P450 gene (CYP6ER1) was highly overexpressed in all resistant strains (up to 40-fold) and the level of expression observed in the different N. lugens strains was significantly correlated with the resistance phenotype. These results provide strong evidence for a role of CYP6ER1 in the resistance of N. lugens to imidacloprid. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

Bovine α-Lactalbumin Hydrolysates (α-LAH Ameliorate Adipose Insulin Resistance and Inflammation in High-Fat Diet-Fed C57BL/6J Mice

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Jing Gao

2018-02-01

Full Text Available Obesity-induced adipose inflammation has been demonstrated to be a key cause of insulin resistance. Peptides derived from bovine α-lactalbumin have been shown to inhibit the activities of dipeptidyl peptidase IV (DPP-IV and angiotensin converting enzyme (ACE, scavenge 2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonate] (ABTS+ radical and stimulate glucagon-like peptide-2 secretion. In the present study, the effects of bovine α-lactalbumin hydrolysates (α-LAH on adipose insulin resistance and inflammation induced by high-fat diet (HFD were investigated. The insulin resistance model was established by feeding C57BL/6J mice with HFD (60% kcal from fat for eight weeks. Then, the mice were fed with HFD and bovine α-LAH of different doses (100 mg/kg b.w., 200 mg/kg b.w. and 400 mg/kg b.w. for another 12 weeks to evaluate its protective effects against HFD-induced insulin resistance. The oral glucose tolerance test (OGTT and intraperitoneal insulin tolerance test (ipITT were conducted after intervention with α-LAH for 10 weeks and 11 weeks, respectively. Results showed that bovine α-LAH significantly reduced body weight, blood glucose, serum insulin, and HOMA-IR (homeostatic model assessment of insulin resistance levels, lowered the area-under-the-curve (AUC during OGTT and ipITT, and downregulated inflammation-related gene [tumor necrosis factor (TNF-α, interleukin (IL-6, monocyte chemoattractant protein (MCP-1] expression in adipose tissues of HFD-fed C57BL/6J mice. Furthermore, bovine α-LAH also suppressed insulin receptor substrate 1 (IRS-1 serine phosphorylation (Ser307, Ser612, enhanced protein kinase B (known as Akt phosphorylation, and inhibited the activation of inhibitor of kappaB kinase (IKK and mitogen activated protein kinase (MAPK signaling pathways in adipose tissues of HFD-fed C57BL/6J mice. These results suggested that bovine α-LAH could ameliorate adipose insulin resistance and inflammation through IKK and MAPK signaling

NCBI nr-aa BLAST: CBRC-DMEL-03-0035 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-DMEL-03-0035 ref|XP_001650651.1| rab6 gtpase activating protein, gapcena (rabg...ap1 protein) [Aedes aegypti] gb|EAT48197.1| rab6 gtpase activating protein, gapcena (rabgap1 protein) [Aedes aegypti] XP_001650651.1 2e-93 54% ...

NCBI nr-aa BLAST: CBRC-DMEL-03-0035 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-DMEL-03-0035 ref|XP_001650650.1| rab6 gtpase activating protein, gapcena (rabg...ap1 protein) [Aedes aegypti] gb|EAT48196.1| rab6 gtpase activating protein, gapcena (rabgap1 protein) [Aedes aegypti] XP_001650650.1 2e-93 54% ...

NCBI nr-aa BLAST: CBRC-DSIM-03-0031 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-DSIM-03-0031 ref|XP_001650650.1| rab6 gtpase activating protein, gapcena (rabg...ap1 protein) [Aedes aegypti] gb|EAT48196.1| rab6 gtpase activating protein, gapcena (rabgap1 protein) [Aedes aegypti] XP_001650650.1 2e-93 54% ...

NCBI nr-aa BLAST: CBRC-DYAK-03-0017 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-DYAK-03-0017 ref|XP_001650651.1| rab6 gtpase activating protein, gapcena (rabg...ap1 protein) [Aedes aegypti] gb|EAT48197.1| rab6 gtpase activating protein, gapcena (rabgap1 protein) [Aedes aegypti] XP_001650651.1 2e-93 54% ...

NCBI nr-aa BLAST: CBRC-AGAM-01-0094 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-AGAM-01-0094 ref|XP_001650651.1| rab6 gtpase activating protein, gapcena (rabg...ap1 protein) [Aedes aegypti] gb|EAT48197.1| rab6 gtpase activating protein, gapcena (rabgap1 protein) [Aedes aegypti] XP_001650651.1 1e-172 69% ...

NCBI nr-aa BLAST: CBRC-DYAK-03-0017 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-DYAK-03-0017 ref|XP_001650650.1| rab6 gtpase activating protein, gapcena (rabg...ap1 protein) [Aedes aegypti] gb|EAT48196.1| rab6 gtpase activating protein, gapcena (rabgap1 protein) [Aedes aegypti] XP_001650650.1 2e-93 54% ...

NCBI nr-aa BLAST: CBRC-AGAM-01-0094 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-AGAM-01-0094 ref|XP_001650650.1| rab6 gtpase activating protein, gapcena (rabg...ap1 protein) [Aedes aegypti] gb|EAT48196.1| rab6 gtpase activating protein, gapcena (rabgap1 protein) [Aedes aegypti] XP_001650650.1 1e-172 69% ...

Thermally oxidized titania nanotubes enhance the corrosion resistance of Ti6Al4V.

Science.gov (United States)

Grotberg, John; Hamlekhan, Azhang; Butt, Arman; Patel, Sweetu; Royhman, Dmitry; Shokuhfar, Tolou; Sukotjo, Cortino; Takoudis, Christos; Mathew, Mathew T

2016-02-01

The negative impact of in vivo corrosion of metallic biomedical implants remains a complex problem in the medical field. We aimed to determine the effects of electrochemical anodization (60V, 2h) and thermal oxidation (600°C) on the corrosive behavior of Ti-6Al-4V, with serum proteins, at physiological temperature. Anodization produced a mixture of anatase and amorphous TiO2 nanopores and nanotubes, while the annealing process yielded an anatase/rutile mixture of TiO2 nanopores and nanotubes. The surface area was analyzed by the Brunauer-Emmett-Teller method and was estimated to be 3 orders of magnitude higher than that of polished control samples. Corrosion resistance was evaluated on the parameters of open circuit potential, corrosion potential, corrosion current density, passivation current density, polarization resistance and equivalent circuit modeling. Samples both anodized and thermally oxidized exhibited shifts of open circuit potential and corrosion potential in the noble direction, indicating a more stable nanoporous/nanotube layer, as well as lower corrosion current densities and passivation current densities than the smooth control. They also showed increased polarization resistance and diffusion limited charge transfer within the bulk oxide layer. The treatment groups studied can be ordered from greatest corrosion resistance to least as Anodized+Thermally Oxidized > Anodized > Smooth > Thermally Oxidized for the conditions investigated. This study concludes that anodized surface has a potential to prevent long term implant failure due to corrosion in a complex in-vivo environment. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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

Microstructure, microhardness and corrosion resistance of remelted TiG2 and Ti6Al4V by a high power diode laser

International Nuclear Information System (INIS)

Amaya-Vazquez, M.R.; Sánchez-Amaya, J.M.; Boukha, Z.; Botana, F.J.

2012-01-01

Highlights: ► Laser remelting of TiG2 and Ti6Al4V is performed with argon shielded diode laser. ► Microstructure, microhardness and corrosion of remelted samples are deeply analysed. ► Microstructural changes of laser remelted TiG2 lead to microhardness increase. ► Remelted Ti6Al4V presents microhardness increase and corrosion improvement. ► Martensite depth in remelted Ti6Al4V is linearly proportional to laser fluence. - Abstract: The high strength, low density and superior corrosion resistance allow titanium alloys to be widely employed in different industrial applications. The properties of these alloys can be modulated by different heat treatments, including laser processing. In the present paper, laser remelting treatments, performed with a high power diode laser, were applied to samples of two titanium alloys (TiG2 and Ti6Al4V). The influence of the applied laser fluence on microstructure, microhardness and corrosion resistance is investigated. Results show that laser remelting treatments with appropriate fluences provoke microstructural changes leading to microhardness increase and corrosion resistance improvement.

Loss of MutL Disrupts CHK2-Dependent Cell-Cycle Control through CDK4/6 to Promote Intrinsic Endocrine Therapy Resistance in Primary Breast Cancer.

Science.gov (United States)

Haricharan, Svasti; Punturi, Nindo; Singh, Purba; Holloway, Kimberly R; Anurag, Meenakshi; Schmelz, Jacob; Schmidt, Cheryl; Lei, Jonathan T; Suman, Vera; Hunt, Kelly; Olson, John A; Hoog, Jeremy; Li, Shunqiang; Huang, Shixia; Edwards, Dean P; Kavuri, Shyam M; Bainbridge, Matthew N; Ma, Cynthia X; Ellis, Matthew J

2017-10-01

Significant endocrine therapy-resistant tumor proliferation is present in ≥20% of estrogen receptor-positive (ER + ) primary breast cancers and is associated with disease recurrence and death. Here, we uncover a link between intrinsic endocrine therapy resistance and dysregulation of the MutL mismatch repair (MMR) complex ( MLH1/3 , PMS1/2 ), and demonstrate a direct role for MutL complex loss in resistance to all classes of endocrine therapy. We find that MutL deficiency in ER + breast cancer abrogates CHK2-mediated inhibition of CDK4, a prerequisite for endocrine therapy responsiveness. Consequently, CDK4/6 inhibitors (CDK4/6i) remain effective in MutL-defective ER + breast cancer cells. These observations are supported by data from a clinical trial where a CDK4/6i was found to strongly inhibit aromatase inhibitor-resistant proliferation of MutL-defective tumors. These data suggest that diagnostic markers of MutL deficiency could be used to direct adjuvant CDK4/6i to a population of patients with breast cancer who exhibit marked resistance to the current standard of care. Significance: MutL deficiency in a subset of ER + primary tumors explains why CDK4/6 inhibition is effective against some de novo endocrine therapy-resistant tumors. Therefore, markers of MutL dysregulation could guide CDK4/6 inhibitor use in the adjuvant setting, where the risk benefit ratio for untargeted therapeutic intervention is narrow. Cancer Discov; 7(10); 1168-83. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1047 . ©2017 American Association for Cancer Research.

TNP [N2-(m-Trifluorobenzyl, N6-(p-nitrobenzylpurine] ameliorates diet induced obesity and insulin resistance via inhibition of the IP6K1 pathway

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Sarbani Ghoshal

2016-10-01

Full Text Available Objective: Obesity and type 2 diabetes (T2D lead to various life-threatening diseases such as coronary heart disease, stroke, osteoarthritis, asthma, and neurodegeneration. Therefore, extensive research is ongoing to identify novel pathways that can be targeted in obesity/T2D. Deletion of the inositol pyrophosphate (5-IP7 biosynthetic enzyme, inositol hexakisphosphate kinase-1 (IP6K1, protects mice from high fat diet (HFD induced obesity (DIO and insulin resistance. Yet, whether this pathway is a valid pharmacologic target in obesity/T2D is not known. Here, we demonstrate that TNP [N2-(m-Trifluorobenzyl, N6-(p-nitrobenzylpurine], a pan-IP6K inhibitor, has strong anti-obesity and anti-diabetic effects in DIO mice. Methods: Q-NMR, GTT, ITT, food intake, energy expenditure, QRT-PCR, ELISA, histology, and immunoblot studies were conducted in short (2.5-week- and long (10-week-term TNP treated DIO C57/BL6 WT and IP6K1-KO mice, under various diet and temperature conditions. Results: TNP, when injected at the onset of HFD-feeding, decelerates initiation of DIO and insulin resistance. Moreover, TNP facilitates weight loss and restores metabolic parameters, when given to DIO mice. However, TNP does not reduce weight gain in HFD-fed IP6K1-KO mice. TNP specifically enhances insulin sensitivity in DIO mice via Akt activation. TNP decelerates weight gain primarily by enhancing thermogenic energy expenditure in the adipose tissue. Accordingly, TNP's effect on body weight is partly abolished whereas its impact on glucose homeostasis is preserved at thermoneutral temperature. Conclusion: Pharmacologic inhibition of the inositol pyrophosphate pathway has strong therapeutic potential in obesity, T2D, and other metabolic diseases. Author Video: Author Video Watch what authors say about their articles Keywords: IP6K, Inositol pyrophosphate, Obesity, Energy expenditure, Diabetes, Akt

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.

Rho GTPase protein Cdc42 is critical for postnatal cartilage development

Energy Technology Data Exchange (ETDEWEB)

Nagahama, Ryo [Department of Biochemistry, School of Dentistry, Showa University, Tokyo (Japan); Department of Orthodontics, School of Dentistry, Showa University, Tokyo (Japan); Yamada, Atsushi, E-mail: yamadaa@dent.showa-u.ac.jp [Department of Biochemistry, School of Dentistry, Showa University, Tokyo (Japan); Tanaka, Junichi [Department of Oral Diagnostic Sciences, School of Dentistry, Showa University, Tokyo (Japan); Aizawa, Ryo [Department of Periodontology, School of Dentistry, Showa University, Tokyo (Japan); Suzuki, Dai [Department of Biochemistry, School of Dentistry, Showa University, Tokyo (Japan); Kassai, Hidetoshi [Laboratory of Animal Resources, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo (Japan); Yamamoto, Matsuo [Department of Periodontology, School of Dentistry, Showa University, Tokyo (Japan); Mishima, Kenji [Department of Oral Diagnostic Sciences, School of Dentistry, Showa University, Tokyo (Japan); Aiba, Atsu [Laboratory of Animal Resources, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Tokyo (Japan); Maki, Koutaro [Department of Orthodontics, School of Dentistry, Showa University, Tokyo (Japan); Kamijo, Ryutaro [Department of Biochemistry, School of Dentistry, Showa University, Tokyo (Japan)

2016-02-19

Cdc42, a small Rho GTPase family member, has been shown to regulate multiple cellular functions in vitro, including actin cytoskeletal reorganization, cell migration, proliferation, and gene expression. However, its tissue-specific roles in vivo remain largely unknown, especially in postnatal cartilage development, as cartilage-specific Cdc42 inactivated mice die within a few days after birth. In this study, we investigated the physiological functions of Cdc42 during cartilage development after birth using tamoxifen-induced cartilage-specific inactivated Cdc42 conditional knockout (Cdc42 {sup fl/fl}; Col2-CreERT) mice, which were generated by crossing Cdc42 flox mice (Cdc42 {sup fl/fl}) with tamoxifen-induced type II collagen (Col2) Cre transgenic mice using a Cre/loxP system. The gross morphology of the Cdc42 cKO mice was shorter limbs and body, as well as reduced body weight as compared with the controls. In addition, severe defects were found in growth plate chondrocytes of the long bones, characterized by a shorter proliferating zone (PZ), wider hypertrophic zone (HZ), and loss of columnar organization of proliferating chondrocytes, resulting in delayed endochondral bone formation associated with abnormal bone growth. Our findings demonstrate the importance of Cdc42 for cartilage development during both embryonic and postnatal stages. - Highlights: • Tamoxifen-induced cartilage specific inactivated Cdc42 mutant mice were generated. • Cdc42 mutant mice were shorter limbs and body. • Severe defects were found in growth plate chondrocytes.

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.

CARACTERIZAÇÃO FÍSICO-QUÍMICA, FUNCIONAL E NUTRICIONAL DE DUAS CULTIVARES BRASILEIRAS DE ARROZ

OpenAIRE

Polesi, Luis Fernando; Lima, Dâmaris Carvalho; Morais, Pyera Grassi; Romo, Isabela Cristina Freitas; Sarmento, Silene Bruder Silveira; Canniatti-Brazaca, Solange Guidolin

2014-01-01

O objetivo deste trabalho foi realizar a caracterização físico-química, funcional e nutricional de duas cultivares brasileiras de arroz. O arroz da cultivar IAC 202 apresentou maior temperatura de gelatinização, absorção de água, volume de expansão, perda de sólidos, pegajosidade e amido lentamente digerível. O arroz da cultivar IRGA 417 apresentou maior teor de proteínas, lipídeos, amilose aparente, viscosidade em RVA, dureza, amido rapidamente digerível e amido resistente. O arroz IRGA 417 ...

High-temperature resistivity and thermoelectric properties of coupled substituted Ca3Co2O6

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Meenakshisundaram Senthilkumar and Rajagopalan Vijayaraghavan

2009-01-01

Full Text Available Polycrystalline samples of Ca3−xNaxCo2−xMnxO6 (x=0.0–0.5 have been prepared by the sol-gel cum combustion method using sucrose in order to investigate the effects of the coupled substitution of Na and Mn on Ca and Co sites on the transport properties of Ca3Co2O6(Co326. The products were characterized by Fourier transform infrared spectroscopy, powder x-ray diffraction (XRD, thermogravimetry (TGA, differential thermal analysis and scanning electron microscopy. XRD patterns reveal the formation of single-phase products up to x=0.5. Coupled substitution increases the solubility of both Na and Mn on Ca and Co sites, respectively, in contrast to the limited solubility of Na and Mn (x=0.2 when separately substituted. TGA confirms the formation of the Ca3Co2O6 phase at temperatures ~720 °C. The grain size of the parent and substituted products is in the range 150–250 nm. Electrical resistivity and Seebeck coefficient were measured in the temperature range 300–800 K. Resistivity shows semiconducting behavior for all the compositions, particularly in the low-temperature regime. The Seebeck coefficient increases with temperature throughout the measured temperature range for all compositions. The maximum Seebeck coefficient (200 μV K−1 is observed for x=0.5 at 825 K, and this composition may be optimal for high-temperature thermoelectric applications.

Análise microscópica da resistência do arroz à queima das bainhas mediada pelo silício

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Daniel Augusto Schurt

2015-03-01

Full Text Available A queima das bainhas, causada pelo fungo Rhizoctonia solani Kühn, é uma das mais importantes e destrutivas doenças que afetam a produção de arroz no mundo. Embora o silício (Si seja capaz de reduzir a intensidade da queima das bainhas, os mecanismos de resistência mediados por esse elemento permanecem desconhecidos. Assim, este trabalho teve como objetivo investigar a nível microscópico o efeito do Si na resistência do arroz à infecção por R. solani. Plantas de arroz das cultivares BR-Irga-409 e Labelle foram cultivadas em solução nutritiva contendo 0 ou 2 mM de Si e inoculadas no estádio de máximo perfilhamento utilizando-se pedaços de palito de dente colonizados por R. solani. Ambos cultivares supridas com Si apresentaram intensa e homogênea deposição de Si nos tecidos das bainhas colonizados por R. solani. A maior concentração de Si nas bainhas de plantas de arroz de ambas cultivares contribuiu para reduzir os sintomas da queima das bainhas. Com o auxílio da microscopia de luz e da microscopia eletrônica de varredura, observou-se menor crescimento micelial do fungo sobre as bainhas foliares das plantas de ambas cultivares que foram supridas com Si. Secções das bainhas de plantas de ambos cultivares supridas com Si exibiram intensa autofluorescência nos tecidos próximos a regiões necrosadas devido a colonização por R. solani. Em conclusão, o suprimento de Si às plantas de arroz desempenhou um papel importante na redução dos sintomas da queima das bainhas.

Evaluation of the efficacy of ProHeart® 6 (moxidectin) against a resistant isolate of Dirofilaria immitis (JYD-34) in dogs.

Science.gov (United States)

Bowman, Dwight D; McTier, Tom L; Adams, Eric L; Mahabir, Sean P; Login, Joyce A; Bidgood, Tara; Woods, Debra J

2017-11-09

In a previous study, it was demonstrated that ProHeart ® 6 (PH6) (moxidectin, Zoetis) provided only about 20% efficacy in a small six-dog study against a macrocyclic lactone -resistant Dirofilaria immitis isolate (Jd2009-2) when dogs were inoculated with infective third-stage larvae (L3) at the end of the dosing period (ie, 180 days post treatment). The objective of the current study was to determine the prophylactic efficacy of a moxidectin sustained-release formulation (PH6) against a confirmed macrocyclic lactone-resistant isolate of D. immitis (JYD-34) in dogs when administered by subcutaneous injection at the labeled dose of 0.17 mg/kg 2 days before L3 inoculation. This was intended to model the scenario where dogs become infected with resistant heartworms at the end of the PH6 treatment period (ie, 6 months post treatment) when dogs would routinely be given another injection under normal field use. Twelve purpose-bred Beagle dogs (six males and six females) were selected and randomly allocated to two groups, untreated controls and PH6-treated dogs in groups of six each. The dogs were ≥8 months old at the start of the study, and using blood samples collected on Day -7 were shown to be negative for adult heartworm antigen and microfilariae. On Day 0, the dogs in the untreated control group were administered saline subcutaneously by injection, and the dogs in the treated group were administered PH6 according to label instructions. On Day 2, each dog was inoculated in the inguinal area with 50 L3 of D. immitis. The dogs were necropsied on Day 150 (148 days post infection), and the worms were collected and counted. All of the six control dogs were infected and harbored a range of 21 to 37 worms (geometric mean, 25.4; 10.9 males and 13.9 females). Only one of the six PH6 dogs was found to be infected, harboring a single male worm. Efficacy was 99.5% (geometric mean). ProHeart ® 6 was highly effective in preventing the development of heartworms in dogs

Maize ROP2 GTPase provides a competitive advantage to the male gametophyte.

Science.gov (United States)

Arthur, K M; Vejlupkova, Z; Meeley, R B; Fowler, J E

2003-12-01

Rop GTPases have been implicated in the regulation of plant signal transduction and cell morphogenesis. To explore ROP2 function in maize, we isolated five Mutator transposon insertions (rop2::Mu alleles). Transmission frequency through the male gametophyte, but not the female, was lower than expected in three of the rop2::Mu mutants. These three alleles formed an allelic series on the basis of the relative transmission rate of each when crossed as trans-heterozygotes. A dramatic reduction in the level of ROP2-mRNA in pollen was associated with the three alleles causing a transmission defect, whereas a rop2::Mu allele that did not result in a defect had wild-type transcript levels, thus confirming that mutation of rop2 causes the mutant phenotype. These data strongly support a role for rop2 in male gametophyte function, perhaps surprisingly, given the expression in pollen of the nearly identical duplicate gene rop9. However, the transmission defect was apparent only when a rop2::Mu heterozygote was used as the pollen donor or when a mixture of wild-type and homozygous mutant pollen was used. Thus, mutant pollen is at a competitive disadvantage compared to wild-type pollen, although mutant pollen grains lacked an obvious cellular defect. Our data demonstrate the importance in vivo of a specific Rop, rop2, in the male gametophyte.

Longevity and Stress Resistant Property of 6-Gingerol from Zingiber officinale Roscoe in Caenorhabditis elegans.

Science.gov (United States)

Lee, Eun Byeol; Kim, Jun Hyeong; An, Chang Wan; Kim, Yeong Jee; Noh, Yun Jeong; Kim, Su Jin; Kim, Ju-Eun; Shrestha, Abinash Chandra; Ham, Ha-Neul; Leem, Jae-Yoon; Jo, Hyung-Kwon; Kim, Dae-Sung; Moon, Kwang Hyun; Lee, Jeong Ho; Jeong, Kyung Ok; Kim, Dae Keun

2018-03-14

In order to discover lifespan-extending compounds made from natural resources, activity-guided fractionation of Zingiber officinale Roscoe (Zingiberaceae) ethanol extract was performed using the Caenorhabditis elegans ( C. elegans ) model system. The compound 6-gingerol was isolated from the most active ethyl acetate soluble fraction, and showed potent longevity-promoting activity. It also elevated the survival rate of worms against stressful environment including thermal, osmotic, and oxidative conditions. Additionally, 6-gingerol elevated the antioxidant enzyme activities of C. elegans , and showed a dose-depend reduction of intracellular reactive oxygen species (ROS) accumulation in worms. Further studies demonstrated that the increased stress tolerance of 6-gingerol-mediated worms could result from the promotion of stress resistance proteins such as heat shock protein (HSP-16.2) and superoxide dismutase (SOD-3). The lipofuscin levels in 6-gingerol treated intestinal worms were decreased in comparison to the control group. No significant 6-gingerol-related changes, including growth, food intake, reproduction, and movement were noted. These results suggest that 6-gingerol exerted longevity-promoting activities independently of these factors and could extend the human lifespan.

Evaluating the Frequency of aac(6')-IIa, ant(2″)-I, intl1, and intl2 Genes in Aminoglycosides Resistant Klebsiella pneumoniae Isolates Obtained from Hospitalized Patients in Yazd, Iran.

Science.gov (United States)

Mokhtari, Hesam; Eslami, Gilda; Zandi, Hengameh; Dehghan-Banadkouki, Amin; Vakili, Mahmood

2018-01-01

Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen that could be resistant to many antimicrobial agents. Resistance genes can be carried among gram-negative bacteria by integrons. Enzymatic inactivation is the most important mechanism of resistance to aminoglycosides. In this study, the frequencies of two important resistance gene aac(6')-II a and ant(2″)-I, and genes coding integrase I and II, in K. pneumoniae isolates resistant to aminoglycosides were evaluated. In this cross-sectional study, an attempt was made to assess the antibiotic susceptibility of 130 K. pneumoniae isolates obtained from different samples of patients hospitalized in training hospitals of Yazd evaluated by disk diffusion method. The frequencies of aac(6')-II a, ant(2″)-I, intl1 , and intl2 genes were determined by PCR method. Data were analyzed by chi-square method using SPSS software (Ver. 16). our results showed that resistance to gentamicin, tobramycin, kanamycin, and amikacin were 34.6, 33.8, 43.8, and 14.6%, respectively. The frequencies of aac (6')-II a, ant(2″)-I, intl1 , and intl2 genes were 44.6, 27.7, 90, and 0%, respectively. This study showed there are high frequencies of genes coding aminoglycosides resistance in K. pneumoniae isolates. Hence, it is very important to monitor and inhibit the spread of antibiotic resistance genes.

Electrical resistivity tomography for studying liquefaction induced by the May 2012 Emilia-Romagna earthquake (Mw = 6.1, northern Italy)

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Giocoli, A.; Quadrio, B.; Bellanova, J.; Lapenna, V.; Piscitelli, S.

2014-04-01

This work shows the result of an electrical resistivity tomography (ERT) survey carried out for imaging and characterizing the shallow subsurface affected by the coseismic effects of the Mw = 6.1 Emilia-Romagna (northern Italy) earthquake that occurred on 20 May 2012. The most characteristic coseismic effects were ground failure, lateral spreading and liquefaction that occurred extensively along the paleo-Reno River in the urban areas of San Carlo and Mirabello (southwestern portion of Ferrara Province). In total, six electrical resistivity tomographies were performed and calibrated with surface geological surveys, exploratory boreholes and aerial photo interpretations. This was one of first applications of the electrical resistivity tomography method in investigating coseismic liquefaction.

Electrical resistivity tomography for studying liquefaction induced by the May 2012 Emilia-Romagna earthquake (Mw = 6.1, North Italy)

Science.gov (United States)

Giocoli, A.; Quadrio, B.; Bellanova, J.; Lapenna, V.; Piscitelli, S.

2013-10-01

This work shows the result of an Electrical Resistivity Tomography survey carried out for imaging and characterizing the shallow subsurface affected by the coseismic effects of the Mw = 6.1 Emilia-Romagna (North Italy) earthquake occurred on 20 May 2012. The most characteristic coseismic effects were ground failure, lateral spreading and liquefaction that occurred extensively along the paleo-Reno river in the urban areas of San Carlo, a hamlet of Sant'Agostino municipality, and of Mirabello (south-western portion of the Ferrara Province). Totally, six Electrical Resistivity Tomography were performed and calibrated with surface geological surveys, exploratory borehole and aerial photo interpretations. This was one of the first applications of the Electrical Resistivity Tomography method in investigating coseismic liquefaction.

Changing prevalence and resistance patterns in children with drug-resistant tuberculosis in Mumbai.

Science.gov (United States)

Shah, Ira; Shah, Forum

2017-05-01

The prevalence of drug-resistant (DR) tuberculosis (TB) in children is increasing. Although, in India, multi-drug-resistant (MDR) TB rates have been relatively stable, the number of children with pre-extensively drug-resistant and extensively drug-resistant (XDR) TB is increasing. To determine whether the prevalence of DR TB in children in Mumbai is changing and to study the evolving patterns of resistance. A retrospective study was undertaken in 1311 paediatric patients referred between April 2007 and March 2013 to the Paediatric TB clinic at B. J. Wadia Hospital for Children, Mumbai. Children were defined as having DR TB on the basis of drug susceptibility testing (DST) of Mycobacterium tuberculosis grown on culture of body fluids (in the case of extra pulmonary TB) or from gastric lavage/bronchi-alveolar lavage/sputum in patients with pulmonary TB or from DST of the contacts. The prevalence of DR TB was calculated and the type of DR was evaluated yearly and in the pre-2010 and post-2010 eras. The overall prevalence of DR TB was 86 (6.6%) with an increase from 23 (5.6%) patients pre-2010 to 63 (7%) post-2010 (P = 0.40). Nine (10.4%) patients were diagnosed on the basis of contact with a parent with DR TB. Overall fluoroquinolone resistance increased from 9 (39.1%) pre-2010 to 59 (93.7%) post-2010 (P = 0.0001): moxifloxacin resistance increased from 2 (8.7%) to 29 (46%) (P = 0.0018) and ofloxacin resistance increased from 7 (30.4%) to 30 (47.6%) (P = 0.14). Ethionamide resistance also increased from 6 (26.1%) to 31 (49.2%) (P = 0.04), aminoglycoside resistance was one (4.3%) pre-2010 and 12 (19%) post-2010 (P = 0.17) and resistance remained virtually the same for both amikacin [0 pre-2010 and 6 (9.5%) after 2010] and kanamycin [one (4.3%) pre- and 6 (9.5%) post-2010]. Of the first-line drugs, resistance remained the same for isoniazid [23 (100%) to 61 (96.8%)], rifampicin [22 (95.7%) to 51 (80.9%),P = 0.17], pyrazinamide [15 (65.2%) to

Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells

International Nuclear Information System (INIS)

Shigeno, Yuta; Uchiumi, Toshio; Nomura, Takaomi

2016-01-01

Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation. - Highlights: • We constructed an in vivo functional assay system for Escherichia coli ribosomal protein L6. • Growth of an E. coli ΔL6 mutant was biphasic when L6 levels were depleted. • The ΔL6 mutant accumulated 50S ribosomal subunit precursors that sedimented at 45S. • L6 is a key player in the late stage of E. coli 50S subunit assembly.

Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells

Energy Technology Data Exchange (ETDEWEB)

Shigeno, Yuta [Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567 (Japan); Uchiumi, Toshio [Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181 (Japan); Nomura, Takaomi, E-mail: nomurat@shinshu-u.ac.jp [Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567 (Japan)

2016-04-22

Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation. - Highlights: • We constructed an in vivo functional assay system for Escherichia coli ribosomal protein L6. • Growth of an E. coli ΔL6 mutant was biphasic when L6 levels were depleted. • The ΔL6 mutant accumulated 50S ribosomal subunit precursors that sedimented at 45S. • L6 is a key player in the late stage of E. coli 50S subunit assembly.

Microstructure, microhardness and corrosion resistance of remelted TiG2 and Ti6Al4V by a high power diode laser

Energy Technology Data Exchange (ETDEWEB)

Amaya-Vazquez, M.R. [Laboratorio de Corrosion y Proteccion, Universidad de Cadiz, Departamento de Ciencia de los Materiales e Ingenieria Metalurgica y Quimica Inorganica, Avda. Republica Saharaui s/n, 11510 Puerto Real, Cadiz (Spain); Sanchez-Amaya, J.M., E-mail: josemaria.sanchez@uca.es [Titania, Ensayos y Proyectos Industriales S.L., Ctra Sanlucar A-2001 Km 7,5, Parque Tecnologico TecnoBahia-Edif. RETSE Nave 4, 11500 El Puerto de Santa Maria, Cadiz (Spain); Departamento de Fisica Aplicada, CASEM, Avda. Republica Saharaui s/n, 11510-Puerto Real, Cadiz (Spain); Boukha, Z.; Botana, F.J. [Laboratorio de Corrosion y Proteccion, Universidad de Cadiz, Departamento de Ciencia de los Materiales e Ingenieria Metalurgica y Quimica Inorganica, Avda. Republica Saharaui s/n, 11510 Puerto Real, Cadiz (Spain)

2012-03-15

Highlights: Black-Right-Pointing-Pointer Laser remelting of TiG2 and Ti6Al4V is performed with argon shielded diode laser. Black-Right-Pointing-Pointer Microstructure, microhardness and corrosion of remelted samples are deeply analysed. Black-Right-Pointing-Pointer Microstructural changes of laser remelted TiG2 lead to microhardness increase. Black-Right-Pointing-Pointer Remelted Ti6Al4V presents microhardness increase and corrosion improvement. Black-Right-Pointing-Pointer Martensite depth in remelted Ti6Al4V is linearly proportional to laser fluence. - Abstract: The high strength, low density and superior corrosion resistance allow titanium alloys to be widely employed in different industrial applications. The properties of these alloys can be modulated by different heat treatments, including laser processing. In the present paper, laser remelting treatments, performed with a high power diode laser, were applied to samples of two titanium alloys (TiG2 and Ti6Al4V). The influence of the applied laser fluence on microstructure, microhardness and corrosion resistance is investigated. Results show that laser remelting treatments with appropriate fluences provoke microstructural changes leading to microhardness increase and corrosion resistance improvement.

Novel cytochrome P450 (CYP6D1) and voltage sensitive sodium channel (Vssc) alleles of the house fly (Musca domestica) and their roles in pyrethroid resistance.

Science.gov (United States)

Pan, Jing; Yang, Chan; Liu, Yan; Gao, Qi; Li, Mei; Qiu, Xinghui

2018-04-01

The house fly Musca domestica is an important disease vector. Point mutation-mediated target-site insensitivity of the voltage sensitive sodium channel (VSSC) and increased detoxification mediated by cytochrome P450 (CYP6D1) overexpression have been characterized as two major mechanisms of pyrethroid resistance. In this study, genetic mutations in the Vssc and CYP6D1 genes and their contribution to pyrethroid resistance were investigated. Twelve lines of house flies homozygous for four genotypes were established. House flies carrying the VSSC 1014F mutation and overexpressing CYP6D1 had higher resistance to pyrethroids than those carrying 1014F alone. The presence of the 15-bp insert in the promoter region of the CYP6D1 gene did not necessarily result in a significant increase in CYP6D1 mRNA and pyrethroid resistance levels. A novel Vssc allele carrying two mutations (G1924D and G2004S) in combination with the classic 1014F and a novel CYP6D1 allele that is very similar to CYP6D1v1 were identified in Chinese house flies. This work demonstrates the effect of genetic mutations in CYP6D1 and Vssc on the susceptibility of house flies to pyrethroids, and verifies that 15-bp insert-containing CYP6D1 alleles have a single origin. These findings offer insights into the evolution of insecticide resistance and have implications for house fly control. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

In vitro investigation of biodegradable polymeric coating for corrosion resistance of Mg-6Zn-Ca alloy in simulated body fluid

Energy Technology Data Exchange (ETDEWEB)

Gaur, Swati, E-mail: gaurswat@gmail.com [IITB–Monash Research Academy, IIT Bombay, Powai, Mumbai 400076 (India); Singh Raman, R.K. [Department of Mechanical, Monash University, Clayton, VIC-3800 (Australia); Department of Aerospace Engineering, Monash University, Clayton, VIC-3800 (Australia); Department of Chemical Engineering, Monash University, Clayton, VIC-3800 (Australia); Khanna, A.S. [Department of Metallurgical Engineering and Materials Science, IIT Bombay, Powai, Mumbai 400076 (India)

2014-09-01

A silane-based biodegradable coating was developed and investigated to improve corrosion resistance of an Mg-6Zn-Ca magnesium alloy to delay the biodegradation of the alloy in the physiological environment. Conditions were optimized to develop a stable and uniform hydroxide layer on the alloys surface—known to facilitate silane-substrate adhesion. A composite coating of two silanes, namely, diethylphosphatoethyltriethoxysilane (DEPETES) and bis-[3-(triethoxysilyl) propyl] tetrasulfide (BTESPT), was developed, by the sol-gel route. Corrosion resistance of the coated alloy was characterized in a modified-simulated body fluid (m-SBF), using potentiodynamic polarization and electrochemical impedance spectroscopy (EIS). The silane coating provided significant and durable corrosion resistance. During the course of this, hydrogen evolution and pH variation, if any, were monitored for both bare and coated alloys. The coating morphology was characterized using scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDAX) and the cross-linking in the coating was studied using Fourier transform infrared spectroscopy (FTIR). As indicated by X-ray diffraction (XRD) results, an important finding was the presence of hydrated magnesium phosphate on the sample that was subjected to immersion in m-SBF for 216 h. Magnesium phosphate is reported to support osteoblast formation and tissue healing. - Highlights: • A silane-based coating was investigated for improving corrosion resistance. • Coating was developed on Mg-6Zn-Ca alloy to delay its biodegradation in m-SBF. • Corrosion resistance was characterized, using polarization and EIS. • The coating morphology was characterized using SEM, EDAX, XRD and FTIR. • 1:4 volume ratio of DEPETES:BTESPT showed significant corrosion resistance.

In vitro investigation of biodegradable polymeric coating for corrosion resistance of Mg-6Zn-Ca alloy in simulated body fluid

International Nuclear Information System (INIS)

Gaur, Swati; Singh Raman, R.K.; Khanna, A.S.

2014-01-01

A silane-based biodegradable coating was developed and investigated to improve corrosion resistance of an Mg-6Zn-Ca magnesium alloy to delay the biodegradation of the alloy in the physiological environment. Conditions were optimized to develop a stable and uniform hydroxide layer on the alloys surface—known to facilitate silane-substrate adhesion. A composite coating of two silanes, namely, diethylphosphatoethyltriethoxysilane (DEPETES) and bis-[3-(triethoxysilyl) propyl] tetrasulfide (BTESPT), was developed, by the sol-gel route. Corrosion resistance of the coated alloy was characterized in a modified-simulated body fluid (m-SBF), using potentiodynamic polarization and electrochemical impedance spectroscopy (EIS). The silane coating provided significant and durable corrosion resistance. During the course of this, hydrogen evolution and pH variation, if any, were monitored for both bare and coated alloys. The coating morphology was characterized using scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDAX) and the cross-linking in the coating was studied using Fourier transform infrared spectroscopy (FTIR). As indicated by X-ray diffraction (XRD) results, an important finding was the presence of hydrated magnesium phosphate on the sample that was subjected to immersion in m-SBF for 216 h. Magnesium phosphate is reported to support osteoblast formation and tissue healing. - Highlights: • A silane-based coating was investigated for improving corrosion resistance. • Coating was developed on Mg-6Zn-Ca alloy to delay its biodegradation in m-SBF. • Corrosion resistance was characterized, using polarization and EIS. • The coating morphology was characterized using SEM, EDAX, XRD and FTIR. • 1:4 volume ratio of DEPETES:BTESPT showed significant corrosion resistance

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

Science.gov (United States)

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

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

Directory of Open Access Journals (Sweden)

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

Inhibition of p70S6K1 Activation by Pdcd4 Overcomes the Resistance to an IGF-1R/IR Inhibitor in Colon Carcinoma Cells.

Science.gov (United States)

Zhang, Yan; Wang, Qing; Chen, Li; Yang, Hsin-Sheng

2015-03-01

Agents targeting insulin-like growth factor 1 receptor (IGF-1R) are being actively examined in clinical trials. Although there has been some initial success of single-agent targeting IGF-1R, attempts in later studies failed because of resistance. This study aimed to understand the effects of programmed cell death 4 (Pdcd4) on the chemosensitivity of the IGF-1R inhibitor OSI-906 in colorectal cancer cells and the mechanism underlying this impact. Using OSI-906-resistant and -sensitive colorectal cancer cells, we found that the Pdcd4 level directly correlates with cell chemosensitivity to OSI-906. In addition, tumors derived from Pdcd4 knockdown cells resist the growth inhibitory effect of OSI-906 in a colorectal cancer xenograft mouse model. Moreover, Pdcd4 enhances the antiproliferative effect of OSI-906 in resistant cells through suppression of p70S6K1 activation. Knockdown of p70S6K1, but not p70S6K2, significantly increases the chemosensitivity of OSI-906 in cultured colorectal cancer cells. Furthermore, the combination of OSI-906 and PF-4708671, a p70S6K1 inhibitor, efficiently suppresses the growth of OSI-906-resistant colon tumor cells in vitro and in vivo. Taken together, activation of p70S6K1 that is inhibited by Pdcd4 is essential for resistance to the IGF-1R inhibitor in colon tumor cells, and the combinational treatment of OSI-906 and PF-4708671 results in enhanced antiproliferation effects in colorectal cancer cells in vitro and in vivo, providing a novel venue to overcome the resistance to the IGF-1R inhibitor in treating colorectal cancer. ©2015 American Association for Cancer Research.

A chromosome study of 6-thioguanine-resistant mutants in T lymphocytes of Hiroshima atomic bomb survivors

International Nuclear Information System (INIS)

Kodama, Yoshiaki; Hakoda, Masayuki; Shimba, Hachiro; Awa, A.A.; Akiyama, Mitoshi.

1989-07-01

Cytogenetic characterizations were made of lymphocyte colonies established from somatic mutation assays for 6-thioguanine (TG) resistance in Hiroshima atomic bomb survivors. G-banded chromosomes were analyzed in both TG-resistant (TG r ) and wild-type (not TG-selected) colonies. Included were 45 TG r and 19 wild-type colonies derived from proximally exposed A-bomb survivors, as well as colonies from distally exposed control individuals who were not exposed to a significant level of A-bomb radiation (18 TG r and 9 wild-type colonies). Various structural and numerical abnormalities of chromosomes were observed in both TG r and wild-type colonies. Aberrations of the X chromosome, on which the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus is present, were found in six colonies: two resistant colonies from controls [45,X/46,XX; 46,X,ins(X)], three resistant colonies [45,X/46,XX/46,X,+mar; 46,X,t(Xq+;14q-); 46,Y,t(Xq-;5q+)], and one wild-type colony [45,X/47,XXX] from proximally exposed persons. In cases with exchange aberrations, each of the break points on the X chromosome was situated proximally to band q26 where the HPRT locus is known to be assigned. DNA replicating patterns were also studied, and it was found that abnormal X chromosomes showed early replicating patterns, while normal X chromosomes showed late replicating patterns. (author)

Microstructure and Wear Resistance of Composite Coating by Laser Cladding Al/TiN on the Ti-6Al-4V Substrate

Science.gov (United States)

Zhang, H. X.; Yu, H. J.; Chen, C. Z.

2015-05-01

The composite coatings were fabricated by laser cladding Al/TiN pre-placed powders on Ti-6Al-4V substrate for enhancing wear resistance and hardness of the substrate. The composite coatings were analyzed by means of X-ray diffraction (XRD), scanning electron microscope (SEM) and energy dispersive spectrometer (EDS). The sliding wear tests were performed by MM200 wear test machine. The hardness of the coatings was tested by HV-1000 hardness tester. After laser cladding, it was found that there was a good metallurgical bond between the coating and the substrate. The composite coatings were mainly composed of the matrix of β-Ti (Al) and the reinforcements of titanium nitride (TiN), Ti3Al, TiAl and Al3Ti. The hardness and wear resistance of the coatings on four samples were greatly improved, among which sample 4 exhibited the highest hardness and best wear resistance. The hardness of the coating on sample 4 was approximately 2.5 times of the Ti-6Al-4V substrate. And the wear resistance of sample 4 was four times of the substrate.

Mechanisms of Acquired Drug Resistance to the HDAC6 Selective Inhibitor Ricolinostat Reveals Rational Drug-Drug Combination with Ibrutinib.

Science.gov (United States)

Amengual, Jennifer E; Prabhu, Sathyen A; Lombardo, Maximilian; Zullo, Kelly; Johannet, Paul M; Gonzalez, Yulissa; Scotto, Luigi; Serrano, Xavier Jirau; Wei, Ying; Duong, Jimmy; Nandakumar, Renu; Cremers, Serge; Verma, Akanksha; Elemento, Olivier; O'Connor, Owen A

2017-06-15

Purpose: Pan-class I/II histone deacetylase (HDAC) inhibitors are effective treatments for select lymphomas. Isoform-selective HDAC inhibitors are emerging as potentially more targeted agents. ACY-1215 (ricolinostat) is a first-in-class selective HDAC6 inhibitor. To better understand the discrete function of HDAC6 and its role in lymphoma, we developed a lymphoma cell line resistant to ACY-1215. Experimental Design: The diffuse large B-cell lymphoma cell line OCI-Ly10 was exposed to increasing concentrations of ACY-1215 over an extended period of time, leading to the development of a resistant cell line. Gene expression profiling (GEP) was performed to investigate differentially expressed genes. Combination studies of ACY-1215 and ibrutinib were performed in cell lines, primary human lymphoma tissue, and a xenograft mouse model. Results: Systematic incremental increases in drug exposure led to the development of distinct resistant cell lines with IC 50 values 10- to 20-fold greater than that for parental lines. GEP revealed upregulation of MAPK10, HELIOS, HDAC9, and FYN, as well as downregulation of SH3BP5 and LCK. Gene-set enrichment analysis (GSEA) revealed modulation of the BTK pathway. Ibrutinib was found to be synergistic with ACY-1215 in cell lines as well as in 3 primary patient samples of lymphoma. In vivo confirmation of antitumor synergy was demonstrated with a xenograft of DLBCL. Conclusions: The development of this ACY-1215-resistant cell line has provided valuable insights into the mechanistic role of HDAC6 in lymphoma and offered a novel method to identify rational synergistic drug combinations. Translation of these findings to the clinic is underway. Clin Cancer Res; 23(12); 3084-96. ©2016 AACR . ©2016 American Association for Cancer Research.

Rho GTPase protein Cdc42 is critical for postnatal cartilage development

International Nuclear Information System (INIS)

Nagahama, Ryo; Yamada, Atsushi; Tanaka, Junichi; Aizawa, Ryo; Suzuki, Dai; Kassai, Hidetoshi; Yamamoto, Matsuo; Mishima, Kenji; Aiba, Atsu; Maki, Koutaro; Kamijo, Ryutaro

2016-01-01

Cdc42, a small Rho GTPase family member, has been shown to regulate multiple cellular functions in vitro, including actin cytoskeletal reorganization, cell migration, proliferation, and gene expression. However, its tissue-specific roles in vivo remain largely unknown, especially in postnatal cartilage development, as cartilage-specific Cdc42 inactivated mice die within a few days after birth. In this study, we investigated the physiological functions of Cdc42 during cartilage development after birth using tamoxifen-induced cartilage-specific inactivated Cdc42 conditional knockout (Cdc42 "f"l"/"f"l; Col2-CreERT) mice, which were generated by crossing Cdc42 flox mice (Cdc42 "f"l"/"f"l) with tamoxifen-induced type II collagen (Col2) Cre transgenic mice using a Cre/loxP system. The gross morphology of the Cdc42 cKO mice was shorter limbs and body, as well as reduced body weight as compared with the controls. In addition, severe defects were found in growth plate chondrocytes of the long bones, characterized by a shorter proliferating zone (PZ), wider hypertrophic zone (HZ), and loss of columnar organization of proliferating chondrocytes, resulting in delayed endochondral bone formation associated with abnormal bone growth. Our findings demonstrate the importance of Cdc42 for cartilage development during both embryonic and postnatal stages. - Highlights: • Tamoxifen-induced cartilage specific inactivated Cdc42 mutant mice were generated. • Cdc42 mutant mice were shorter limbs and body. • Severe defects were found in growth plate chondrocytes.

A Review of the As-Built SLM Ti-6Al-4V Mechanical Properties towards Achieving Fatigue Resistant Designs

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Dylan Agius

2018-01-01

Full Text Available Ti-6Al-4V has been widely used in both the biomedical and aerospace industry, due to its high strength, corrosion resistance, high fracture toughness and light weight. Additive manufacturing (AM is an attractive method of Ti-6Al-4V parts’ fabrication, as it provides a low waste alternative for complex geometries. With continued progress being made in SLM technology, the influence of build layers, grain boundaries and defects can be combined to improve further the design process and allow the fabrication of components with improved static and fatigue strength in critical loading directions. To initiate this possibility, the mechanical properties, including monotonic, low and high cycle fatigue and fracture mechanical behaviour, of machined as-built SLM Ti-6Al-4V, have been critically reviewed in order to inform the research community. The corresponding crystallographic phases, defects and layer orientations have been analysed to determine the influence of these features on the mechanical behaviour. This review paper intends to enhance our understanding of how these features can be manipulated and utilised to improve the fatigue resistance of components fabricated from Ti-6Al-4V using the SLM technology.

Semeadura direta de genótipos de arroz irrigado em sucessão a espécies de cobertura de inverno

Directory of Open Access Journals (Sweden)

Menezes Valmir Gaedke

2001-01-01

Full Text Available A adoção do sistema de semeadura direta de arroz em sucessão a espécies de cobertura de solo no inverno é uma importante alternativa para possibilitar o cultivo em uma mesma área todos os anos. Com o objetivo de avaliar espécies de cobertura do solo no inverno mais apropriadas para participarem de um sistema de sucessão de culturas com semeadura direta de genótipos de arroz irrigado, conduziu-se um experimento na Estação Experimental do Arroz do Instituto Rio Grandense do Arroz, em Cachoeirinha, RS, nas estações de crescimento de 1996/97, 1997/98 e 1998/99. Os tratamentos constaram de quatro genótipos de arroz irrigado (BRS6, BR-IRGA 409, IRGA 416 e IRGA 417 instalados em semeadura direta, sobre parcelas com as coberturas de aveia-preta (Avena strigosa Scheib, aveia-branca (Avena sativa L., azevém (Lolium multiflorum Lam., serradela nativa [Ornithopus micranthus (Benth. Arechavaleta] e de vegetação espontânea e com solo desnudo (testemunha. Quanto ao rendimento de grãos, houve interação entre sistemas de cobertura de solo e estações de crescimento. Nas três estações de crescimento, somente o arroz cultivado em sucessão à leguminosa serradela nativa apresentou rendimento de grãos similar ao obtido no sistema de cultivo convencional. Por outro lado, o arroz cultivado em sucessão ao azevém foi o único a apresentar rendimento de grãos inferior em relação ao sistema de cultivo convencional nas três estações de crescimento.

High CDK6 protects cells from fulvestrant-mediated apoptosis and is a predictor of resistance to fulvestrant in estrogen receptor-positive metastatic breast cancer

DEFF Research Database (Denmark)

Alves, Carla Maria Lourenco; Elias, Daniel; Lyng, Maria B

2016-01-01

expression impaired fulvestrant-resistant cell growth and induced apoptosis. Treatment with palbociclib re-sensitized fulvestrant-resistant cells to fulvestrant through alteration of retinoblastoma protein phosphorylation. High CDK6 levels in metastatic samples from two independent cohorts of breast cancer...

Progress toward characterization of the group A Streptococcus metagenome: complete genome sequence of a macrolide-resistant serotype M6 strain.

Science.gov (United States)

Banks, David J; Porcella, Stephen F; Barbian, Kent D; Beres, Stephen B; Philips, Lauren E; Voyich, Jovanka M; DeLeo, Frank R; Martin, Judith M; Somerville, Greg A; Musser, James M

2004-08-15

We describe the genome sequence of a macrolide-resistant strain (MGAS10394) of serotype M6 group A Streptococcus (GAS). The genome is 1,900,156 bp in length, and 8 prophage-like elements or remnants compose 12.4% of the chromosome. A 8.3-kb prophage remnant encodes the SpeA4 variant of streptococcal pyrogenic exotoxin A. The genome of strain MGAS10394 contains a chimeric genetic element composed of prophage genes and a transposon encoding the mefA gene conferring macrolide resistance. This chimeric element also has a gene encoding a novel surface-exposed protein (designated "R6 protein"), with an LPKTG cell-anchor motif located at the carboxyterminus. Surface expression of this protein was confirmed by flow cytometry. Humans with GAS pharyngitis caused by serotype M6 strains had antibody against the R6 protein present in convalescent, but not acute, serum samples. Our studies add to the theme that GAS prophage-encoded extracellular proteins contribute to host-pathogen interactions in a strain-specific fashion.

A three amino acid deletion in the transmembrane domain of the nicotinic acetylcholine receptor α6 subunit confers high-level resistance to spinosad in Plutella xylostella.

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Wang, Jing; Wang, Xingliang; Lansdell, Stuart J; Zhang, Jianheng; Millar, Neil S; Wu, Yidong

2016-04-01

Spinosad is a macrocyclic lactone insecticide that acts primarily at the nicotinic acetylcholine receptors (nAChRs) of target insects. Here we describe evidence that high levels of resistance to spinosad in the diamondback moth (Plutella xylostella) are associated with a three amino acid (3-aa) deletion in the fourth transmembrane domain (TM4) of the nAChR α6 subunit (Pxα6). Following laboratory selection with spinosad, the SZ-SpinR strain of P. xylostella exhibited 940-fold resistance to spinosad. In addition, the selected insect population had 1060-fold cross-resistance to spinetoram but, in contrast, no cross-resistance to abamectin was observed. Genetic analysis indicates that spinosad resistance in SZ-SpinR is inherited as a recessive and autosomal trait, and that the 3-aa deletion (IIA) in TM4 of Pxα6 is tightly linked to spinosad resistance. Because of well-established difficulties in functional expression of cloned insect nAChRs, the analogous resistance-associated deletion mutation was introduced into a prototype nAChR (the cloned human α7 subunit). Two-electrode voltage-clamp recording with wild-type and mutated nAChRs expressed in Xenopus laevis oocytes indicated that the mutation causes a complete loss of agonist activation. In addition, radioligand binding studies indicated that the 3-aa deletion resulted in significantly lower-affinity binding of the extracellular neurotransmitter-binding site. These findings are consistent with the 3-amino acid (IIA) deletion within the transmembrane domain of Pxα6 being responsible for target-site resistance to spinosad in the SZ-SpinR strain of P. xylostella. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Evaluation of the efficacy of ProHeart® 6 (moxidectin against a resistant isolate of Dirofilaria immitis (JYD-34 in dogs

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Dwight D. Bowman

2017-11-01

Full Text Available Abstract Background In a previous study, it was demonstrated that ProHeart® 6 (PH6 (moxidectin, Zoetis provided only about 20% efficacy in a small six-dog study against a macrocyclic lactone –resistant Dirofilaria immitis isolate (Jd2009–2 when dogs were inoculated with infective third-stage larvae (L3 at the end of the dosing period (ie, 180 days post treatment. The objective of the current study was to determine the prophylactic efficacy of a moxidectin sustained-release formulation (PH6 against a confirmed macrocyclic lactone–resistant isolate of D. immitis (JYD-34 in dogs when administered by subcutaneous injection at the labeled dose of 0.17 mg/kg 2 days before L3 inoculation. This was intended to model the scenario where dogs become infected with resistant heartworms at the end of the PH6 treatment period (ie, 6 months post treatment when dogs would routinely be given another injection under normal field use. Methods Twelve purpose-bred Beagle dogs (six males and six females were selected and randomly allocated to two groups, untreated controls and PH6-treated dogs in groups of six each. The dogs were ≥8 months old at the start of the study, and using blood samples collected on Day −7 were shown to be negative for adult heartworm antigen and microfilariae. On Day 0, the dogs in the untreated control group were administered saline subcutaneously by injection, and the dogs in the treated group were administered PH6 according to label instructions. On Day 2, each dog was inoculated in the inguinal area with 50 L3 of D. immitis. The dogs were necropsied on Day 150 (148 days post infection, and the worms were collected and counted. Results All of the six control dogs were infected and harbored a range of 21 to 37 worms (geometric mean, 25.4; 10.9 males and 13.9 females. Only one of the six PH6 dogs was found to be infected, harboring a single male worm. Efficacy was 99.5% (geometric mean. Conclusion ProHeart® 6 was highly

Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments

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Juan J. González-Plaza

2018-01-01

Full Text Available Environments polluted by direct discharges of effluents from antibiotic manufacturing are important reservoirs for antibiotic resistance genes (ARGs, which could potentially be transferred to human pathogens. However, our knowledge about the identity and diversity of ARGs in such polluted environments remains limited. We applied functional metagenomics to explore the resistome of two Croatian antibiotic manufacturing effluents and sediments collected upstream of and at the effluent discharge sites. Metagenomic libraries built from an azithromycin-production site were screened for resistance to macrolide antibiotics, whereas the libraries from a site producing veterinary antibiotics were screened for resistance to sulfonamides, tetracyclines, trimethoprim, and beta-lactams. Functional analysis of eight libraries identified a total of 82 unique, often clinically relevant ARGs, which were frequently found in clusters and flanked by mobile genetic elements. The majority of macrolide resistance genes identified from matrices exposed to high levels of macrolides were similar to known genes encoding ribosomal protection proteins, macrolide phosphotransferases, and transporters. Potentially novel macrolide resistance genes included one most similar to a 23S rRNA methyltransferase from Clostridium and another, derived from upstream unpolluted sediment, to a GTPase HflX from Emergencia. In libraries deriving from sediments exposed to lower levels of veterinary antibiotics, we found 8 potentially novel ARGs, including dihydrofolate reductases and beta-lactamases from classes A, B, and D. In addition, we detected 7 potentially novel ARGs in upstream sediment, including thymidylate synthases, dihydrofolate reductases, and class D beta-lactamase. Taken together, in addition to finding known gene types, we report the discovery of novel and diverse ARGs in antibiotic-polluted industrial effluents and sediments, providing a qualitative basis for monitoring the

Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments

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González-Plaza, Juan J.; Šimatović, Ana; Milaković, Milena; Bielen, Ana; Wichmann, Fabienne; Udiković-Kolić, Nikolina

2018-01-01

Environments polluted by direct discharges of effluents from antibiotic manufacturing are important reservoirs for antibiotic resistance genes (ARGs), which could potentially be transferred to human pathogens. However, our knowledge about the identity and diversity of ARGs in such polluted environments remains limited. We applied functional metagenomics to explore the resistome of two Croatian antibiotic manufacturing effluents and sediments collected upstream of and at the effluent discharge sites. Metagenomic libraries built from an azithromycin-production site were screened for resistance to macrolide antibiotics, whereas the libraries from a site producing veterinary antibiotics were screened for resistance to sulfonamides, tetracyclines, trimethoprim, and beta-lactams. Functional analysis of eight libraries identified a total of 82 unique, often clinically relevant ARGs, which were frequently found in clusters and flanked by mobile genetic elements. The majority of macrolide resistance genes identified from matrices exposed to high levels of macrolides were similar to known genes encoding ribosomal protection proteins, macrolide phosphotransferases, and transporters. Potentially novel macrolide resistance genes included one most similar to a 23S rRNA methyltransferase from Clostridium and another, derived from upstream unpolluted sediment, to a GTPase HflX from Emergencia. In libraries deriving from sediments exposed to lower levels of veterinary antibiotics, we found 8 potentially novel ARGs, including dihydrofolate reductases and beta-lactamases from classes A, B, and D. In addition, we detected 7 potentially novel ARGs in upstream sediment, including thymidylate synthases, dihydrofolate reductases, and class D beta-lactamase. Taken together, in addition to finding known gene types, we report the discovery of novel and diverse ARGs in antibiotic-polluted industrial effluents and sediments, providing a qualitative basis for monitoring the dispersal of ARGs

Physicochemical and sensory profile of rice bran roasted in microwave

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Marina Costa Garcia

2012-12-01

Full Text Available The purpose of this study was to evaluate the physical, chemical, and sensory changes in bran from three rice cultivars according to microwave roasting time. This study analyzed three rice cultivars, BRS Sertaneja (S, BRS Primavera (P, and IRGA 417 (I determining the color parameters (L*, a*, and b * at 6, 9, 12, 15, and 18 minutes of roasting time. After applying the difference from control test, the rice brans with different characteristics aroma and flavor were selected: S and P roasted for 9 and 15 minutes and IRGA 417 roasted for 9, 12, and 15 minutes. These samples were characterized by Free-Choice Profile descriptive sensory analysis, and their chemical composition was also determined. The longer the roasting process, the higher the roasted flavor intensity and aroma. The IRG 417 cultivar roasted for 12 minutes showed a sweeter flavor and aroma. After roasting, the brans remained rich in protein and lipid and presented higher fiber content and lower reducing sugar and phytic acid content. Microwave roasting for 12 minutes can be a viable option for improving the sensory functional and nutritional characteristics of the rice bran considering its use in food products.

TNP [N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine] ameliorates diet induced obesity and insulin resistance via inhibition of the IP6K1 pathway.

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Ghoshal, Sarbani; Zhu, Qingzhang; Asteian, Alice; Lin, Hua; Xu, Haifei; Ernst, Glen; Barrow, James C; Xu, Baoji; Cameron, Michael D; Kamenecka, Theodore M; Chakraborty, Anutosh

2016-10-01

Obesity and type 2 diabetes (T2D) lead to various life-threatening diseases such as coronary heart disease, stroke, osteoarthritis, asthma, and neurodegeneration. Therefore, extensive research is ongoing to identify novel pathways that can be targeted in obesity/T2D. Deletion of the inositol pyrophosphate (5-IP7) biosynthetic enzyme, inositol hexakisphosphate kinase-1 (IP6K1), protects mice from high fat diet (HFD) induced obesity (DIO) and insulin resistance. Yet, whether this pathway is a valid pharmacologic target in obesity/T2D is not known. Here, we demonstrate that TNP [N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine], a pan-IP6K inhibitor, has strong anti-obesity and anti-diabetic effects in DIO mice. Q-NMR, GTT, ITT, food intake, energy expenditure, QRT-PCR, ELISA, histology, and immunoblot studies were conducted in short (2.5-week)- and long (10-week)-term TNP treated DIO C57/BL6 WT and IP6K1-KO mice, under various diet and temperature conditions. TNP, when injected at the onset of HFD-feeding, decelerates initiation of DIO and insulin resistance. Moreover, TNP facilitates weight loss and restores metabolic parameters, when given to DIO mice. However, TNP does not reduce weight gain in HFD-fed IP6K1-KO mice. TNP specifically enhances insulin sensitivity in DIO mice via Akt activation. TNP decelerates weight gain primarily by enhancing thermogenic energy expenditure in the adipose tissue. Accordingly, TNP's effect on body weight is partly abolished whereas its impact on glucose homeostasis is preserved at thermoneutral temperature. Pharmacologic inhibition of the inositol pyrophosphate pathway has strong therapeutic potential in obesity, T2D, and other metabolic diseases.

Molecular and functional characterization of CYP6BQ23, a cytochrome P450 conferring resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus.

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Zimmer, Christoph T; Bass, Chris; Williamson, Martin S; Kaussmann, Martin; Wölfel, Katharina; Gutbrod, Oliver; Nauen, Ralf

2014-02-01

The pollen beetle (Meligethes aeneus F.) is widespread throughout much of Europe where it is a major coleopteran pest of oilseed rape (Brassica napus). The reliance on synthetic insecticides for control, particularly the pyrethroid class, has led to the development of populations with high levels of resistance. Resistance to pyrethroids is now widespread throughout Europe and is thought to be mediated by enhanced detoxification by cytochrome P450ś and/or mutation of the pyrethroid target-site, the voltage-gated sodium channel. However, in the case of cytochrome P450 mediated detoxification, the specific enzyme(s) involved has (have) not yet been identified. In this study a degenerate PCR approach was used to identify ten partial P450 gene sequences from pollen beetle. Quantitative PCR was then used to examine the level of expression of these genes in a range of pollen beetle populations that showed differing levels of resistance to pyrethroids in bioassays. The study revealed a single P450 gene, CYP6BQ23, which is significantly and highly overexpressed (up to ∼900-fold) in adults and larvae of pyrethroid resistant strains compared to susceptible strains. CYP6BQ23 overexpression is significantly correlated with both the level of resistance and with the rate of deltamethrin metabolism in microsomal preparations of these populations. Functional recombinant expression of full length CYP6BQ23 along with cytochrome P450 reductase in an insect (Sf9) cell line showed that it is able to efficiently metabolise deltamethrin to 4-hydroxy deltamethrin. Furthermore we demonstrated by detection of 4-hydroxy tau-fluvalinate using ESI-TOF MS/MS that functionally expressed CYP6BQ23 also metabolizes tau-fluvalinate. A protein model was generated and subsequent docking simulations revealed the predicted substrate-binding mode of both deltamethrin and tau-fluvalinate to CYP6BQ23. Taken together these results strongly suggest that the overexpression of CYP6BQ23 is the primary

Resistance to lambda-cyhalothrin in Spanish field populations of Ceratitis capitata and metabolic resistance mediated by P450 in a resistant strain.

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Arouri, Rabeh; Le Goff, Gaelle; Hemden, Hiethem; Navarro-Llopis, Vicente; M'saad, Mariem; Castañera, Pedro; Feyereisen, René; Hernández-Crespo, Pedro; Ortego, Félix

2015-09-01

The withdrawal of malathion in the European Union in 2009 resulted in a large increase in lambda-cyhalothrin applications for the control of the Mediterranean fruit fly, Ceratitis capitata, in Spanish citrus crops. Spanish field populations of C. capitata have developed resistance to lambda-cyhalothrin (6-14-fold), achieving LC50 values (129-287 ppm) higher than the recommended concentration for field treatments (125 ppm). These results contrast with the high susceptibility to lambda-cyhalothrin found in three Tunisian field populations. We have studied the mechanism of resistance in the laboratory-selected resistant strain W-1Kλ (205-fold resistance). Bioassays with synergists showed that resistance was almost completely suppressed by the P450 inhibitor PBO. The study of the expression of 53 P450 genes belonging to the CYP4, CYP6, CYP9 and CYP12 families in C. capitata revealed that CYP6A51 was overexpressed (13-18-fold) in the resistant strain. The W-1Kλ strain also showed high levels of cross-resistance to etofenprox (240-fold) and deltamethrin (150-fold). Field-evolved resistance to lambda-cyhalothrin has been found in C. capitata. Metabolic resistance mediated by P450 appears to be the main resistance mechanism in the resistant strain W-1Kλ. The levels of cross-resistance found may compromise the effectiveness of other pyrethroids for the control of this species. © 2014 Society of Chemical Industry. © 2014 Society of Chemical Industry.

HIV resistance testing and detected drug resistance in Europe

DEFF Research Database (Denmark)

Schultze, Anna; Phillips, Andrew N; Paredes, Roger

2015-01-01

to Southern Europe. CONCLUSIONS: Despite a concurrent decline in virological failure and testing, drug resistance was commonly detected. This suggests a selective approach to resistance testing. The regional differences identified indicate that policy aiming to minimize the emergence of resistance......OBJECTIVES: To describe regional differences and trends in resistance testing among individuals experiencing virological failure and the prevalence of detected resistance among those individuals who had a genotypic resistance test done following virological failure. DESIGN: Multinational cohort...... study. METHODS: Individuals in EuroSIDA with virological failure (>1 RNA measurement >500 on ART after >6 months on ART) after 1997 were included. Adjusted odds ratios (aORs) for resistance testing following virological failure and aORs for the detection of resistance among those who had a test were...

Phosphatidylserine and GTPase activation control Cdc42 nanoclustering to counter dissipative diffusion.

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Sartorel, Elodie; Ünlü, Caner; Jose, Mini; Massoni-Laporte, Aurélie; Meca, Julien; Sibarita, Jean-Baptiste; McCusker, Derek

2018-04-18

The anisotropic organization of plasma membrane constituents is indicative of mechanisms that drive the membrane away from equilibrium. However, defining these mechanisms is challenging due to the short spatio-temporal scales at which diffusion operates. Here, we use high-density single protein tracking combined with photoactivation localization microscopy (sptPALM) to monitor Cdc42 in budding yeast, a system in which Cdc42 exhibits anisotropic organization. Cdc42 exhibited reduced mobility at the cell pole, where it was organized in nanoclusters. The Cdc42 nanoclusters were larger at the cell pole than those observed elsewhere in the cell. These features were exacerbated in cells expressing Cdc42-GTP, and were dependent on the scaffold Bem1, which contributed to the range of mobility and nanocluster size exhibited by Cdc42. The lipid environment, in particular phosphatidylserine levels, also played a role in regulating Cdc42 nanoclustering. These studies reveal how the mobility of a Rho GTPase is controlled to counter the depletive effects of diffusion, thus stabilizing Cdc42 on the plasma membrane and sustaining cell polarity. Movie S1 Movie S1 sptPALM imaging of live yeast expressing Pil1-mEOS expressed at the genomic locus. Pil1-mEOS was simultaneously photo-converted with a 405 nm laser and imaged with a 561 nm laser using HiLo illumination. Images were acquired at 20 ms intervals, of which 300 frames are shown at 7 frames per second.

Interleukin-6 and vitamin D status during high-intensity resistance training in patients with chronic kidney disease

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Molsted, Stig; Eiken, Pia Agnete; Andersen, Jesper L.

2014-01-01

Background. The aim of this study was to investigate IL-6 and 25-hydroxyvitamin D (25-OH D) associations with muscle size and muscle function in dialysis patients. Methods. Patients were included in a 16-week control period followed by 16 weeks of high-intensity resistance training thrice weekly....... IL-6 and 25-OH D were analysed after an over-night fast. Muscle fibre size was analysed in biopsies from m. vastus lateralis. Muscle power was tested using a Leg Extensor Power Rig. Results. Patients (n = 36) with IL-6 ≥ 6.49 pg/ml (median) were older and had decreased muscle power and a reduced...... power increased by 20-23% (P Half of the patients were suffering from vitamin D deficiency, which was not associated with muscle power. IL-6 was unchanged by high...

Genetic Diversity and Selective Pressure in Hepatitis C Virus Genotypes 1-6: Significance for Direct-Acting Antiviral Treatment and Drug Resistance.

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Cuypers, Lize; Li, Guangdi; Libin, Pieter; Piampongsant, Supinya; Vandamme, Anne-Mieke; Theys, Kristof

2015-09-16

Treatment with pan-genotypic direct-acting antivirals, targeting different viral proteins, is the best option for clearing hepatitis C virus (HCV) infection in chronically infected patients. However, the diversity of the HCV genome is a major obstacle for the development of antiviral drugs, vaccines, and genotyping assays. In this large-scale analysis, genome-wide diversity and selective pressure was mapped, focusing on positions important for treatment, drug resistance, and resistance testing. A dataset of 1415 full-genome sequences, including genotypes 1-6 from the Los Alamos database, was analyzed. In 44% of all full-genome positions, the consensus amino acid was different for at least one genotype. Focusing on positions sharing the same consensus amino acid in all genotypes revealed that only 15% was defined as pan-genotypic highly conserved (≥99% amino acid identity) and an additional 24% as pan-genotypic conserved (≥95%). Despite its large genetic diversity, across all genotypes, codon positions were rarely identified to be positively selected (0.23%-0.46%) and predominantly found to be under negative selective pressure, suggesting mainly neutral evolution. For NS3, NS5A, and NS5B, respectively, 40% (6/15), 33% (3/9), and 14% (2/14) of the resistance-related positions harbored as consensus the amino acid variant related to resistance, potentially impeding treatment. For example, the NS3 variant 80K, conferring resistance to simeprevir used for treatment of HCV1 infected patients, was present in 39.3% of the HCV1a strains and 0.25% of HCV1b strains. Both NS5A variants 28M and 30S, known to be associated with resistance to the pan-genotypic drug daclatasvir, were found in a significant proportion of HCV4 strains (10.7%). NS5B variant 556G, known to confer resistance to non-nucleoside inhibitor dasabuvir, was observed in 8.4% of the HCV1b strains. Given the large HCV genetic diversity, sequencing efforts for resistance testing purposes may need to be

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

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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

Key Components of Different Plant Defense Pathways Are Dispensable for Powdery Mildew Resistance of the Arabidopsis mlo2 mlo6 mlo12 Triple Mutant.

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Kuhn, Hannah; Lorek, Justine; Kwaaitaal, Mark; Consonni, Chiara; Becker, Katia; Micali, Cristina; Ver Loren van Themaat, Emiel; Bednarek, Paweł; Raaymakers, Tom M; Appiano, Michela; Bai, Yuling; Meldau, Dorothea; Baum, Stephani; Conrath, Uwe; Feussner, Ivo; Panstruga, Ralph

2017-01-01

Loss of function mutations of particular plant MILDEW RESISTANCE LOCUS O ( MLO ) genes confer durable and broad-spectrum penetration resistance against powdery mildew fungi. Here, we combined genetic, transcriptomic and metabolomic analyses to explore the defense mechanisms in the fully resistant Arabidopsis thaliana mlo2 mlo6 mlo12 triple mutant. We found that this genotype unexpectedly overcomes the requirement for indolic antimicrobials and defense-related secretion, which are critical for incomplete resistance of mlo2 single mutants. Comparative microarray-based transcriptome analysis of mlo2 mlo6 mlo12 mutants and wild type plants upon Golovinomyces orontii inoculation revealed an increased and accelerated accumulation of many defense-related transcripts. Despite the biotrophic nature of the interaction, this included the non-canonical activation of a jasmonic acid/ethylene-dependent transcriptional program. In contrast to a non-adapted powdery mildew pathogen, the adapted powdery mildew fungus is able to defeat the accumulation of defense-relevant indolic metabolites in a MLO protein-dependent manner. We suggest that a broad and fast activation of immune responses in mlo2 mlo6 mlo12 plants can compensate for the lack of single or few defense pathways. In addition, our results point to a role of Arabidopsis MLO2, MLO6, and MLO12 in enabling defense suppression during invasion by adapted powdery mildew fungi.

Phase II trial of carmustine plus O(6)-benzylguanine for patients with nitrosourea-resistant recurrent or progressive malignant glioma.

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Quinn, Jennifer A; Pluda, James; Dolan, M Eileen; Delaney, Shannon; Kaplan, Richard; Rich, Jeremy N; Friedman, Allan H; Reardon, David A; Sampson, John H; Colvin, O Michael; Haglund, Michael M; Pegg, Anthony E; Moschel, Robert C; McLendon, Roger E; Provenzale, James M; Gururangan, Sridharan; Tourt-Uhlig, Sandra; Herndon, James E; Bigner, Darell D; Friedman, Henry S

2002-05-01

We conducted a phase II trial of carmustine (BCNU) plus the O(6)-alkylguanine-DNA alkyltransferase inhibitor O(6)-benzylguanine (O(6)-BG) to define the activity and toxicity of this regimen in the treatment of adults with progressive or recurrent malignant glioma resistant to nitrosoureas. Patients were treated with O(6)-BG at an intravenous dose of 120 mg/m(2) followed 1 hour later by 40 mg/m(2) of BCNU, with cycles repeated at 6-week intervals. Eighteen patients were treated (15 with glioblastoma multiforme, two with anaplastic astrocytoma, and one with malignant glioma). None of the 18 patients demonstrated a partial or complete response. Two patients exhibited stable disease for 12 weeks before their tumors progressed. Three patients demonstrated stable disease for 6, 12, and 18 weeks before discontinuing therapy because of hematopoietic toxicity. Twelve patients experienced reversible > or = grade 3 hematopoietic toxicity. There was no difference in half-lives (0.56 +/- 0.21 hour v 0.54 +/- 0.20 hour) or area under the curve values (4.8 +/- 1.7 microg/mL/h v 5.0 +/- 1.3 microg/mL/h) of O(6)-BG for patients receiving phenytoin and those not treated with this drug. These results indicate that O(6)-BG plus BCNU at the dose schedule used in this trial is unsuccessful in producing tumor regression in patients with nitrosourea-resistant malignant glioma, although stable disease was seen in five patients for 6, 12, 12, 12, and 18 weeks. Future use of this approach will require strategies to minimize dose-limiting toxicity of BCNU such as regional delivery or hematopoietic stem-cell protection.

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

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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

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

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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.

Prevalence of plasmid-mediated quinolone resistance determinants among oxyiminocephalosporin-resistant Enterobacteriaceae in Argentina

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Giovanna Rincon Cruz

2013-11-01

Full Text Available High quinolone resistance rates were observed among oxyiminocephalosporin-resistant enterobacteria. In the present study, we searched for the prevalence of plasmid-mediated quinolone resistance (PMQR genes within the 55 oxyiminocephalosporin-resistant enterobacteria collected in a previous survey. The main PMQR determinants were aac(6'-Ib-cr and qnrB, which had prevalence rates of 42.4% and 33.3%, respectively. The aac(6'-Ib-cr gene was more frequently found in CTX-M-15-producing isolates, while qnrB was homogeneously distributed among all CTX-M producers.

Screening for Neuraminidase Inhibitor Resistance Markers among Avian Influenza Viruses of the N4, N5, N6, and N8 Neuraminidase Subtypes.

Science.gov (United States)

Choi, Won-Suk; Jeong, Ju Hwan; Kwon, Jin Jung; Ahn, Su Jeong; Lloren, Khristine Kaith S; Kwon, Hyeok-Il; Chae, Hee Bok; Hwang, Jungwon; Kim, Myung Hee; Kim, Chul-Joong; Webby, Richard J; Govorkova, Elena A; Choi, Young Ki; Baek, Yun Hee; Song, Min-Suk

2018-01-01

Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs. IMPORTANCE The frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs), as the only available class of drugs for AIVs in humans, have been constantly used for treatment, leading to the inevitable emergence

Corrosion resistance characteristics of a Ti-6Al-4V alloy scaffold that is fabricated by electron beam melting and selective laser melting for implantation in vivo.

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Zhao, Bingjing; Wang, Hong; Qiao, Ning; Wang, Chao; Hu, Min

2017-01-01

The purpose of this study is to determine the corrosion resistance of Ti-6Al-4V alloy fabricated with electron beam melting and selective laser melting for implantation in vivo. Ti-6Al-4V alloy specimens were fabricated with electron beam melting (EBM) and selective laser melting (SLM). A wrought form of Ti-6Al-4V alloy was used as a control. Surface morphology observation, component analysis, corrosion resistance experimental results, electrochemical impedance spectroscopy, crevice corrosion resistance experimental results, immersion test and metal ions precipitation analysis were processed, respectively. The thermal stability of EBM specimen was the worst, based on the result of open circuit potential (OCP) result. The result of electrochemical impedance spectroscopy indicated that the corrosion resistance of the SLM specimen was the best under the low electric potential. The result of potentiodynamic polarization suggested that the corrosion resistance of the SLM specimen was the best under the low electric potential (1.5V).The crevice corrosion resistance of the EBM specimen was the best. The corrosion resistance of SLM specimen was the best, based on the result of immersion test. The content of Ti, Al and V ions of EBM, SLM and wrought specimens was very low. In general, the scaffolds that were fabricated with EBM and SLM had good corrosion resistance, and were suitable for implantation in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of 8 weeks resistance training on plasma levels of nerve growth factor and interlukin-6 in female patients with multiple sclerosis

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Mehrzad Moghadasi

2015-07-01

Full Text Available Background: In recent years, evidence from a number of studies has suggested that exercise is a safe and efficient way to induce improvements in a number of physiological functions in multiple sclerosis (MS patients, but the effects of exercise on nerve growth factor (NGF and interlukin-6 (IL-6 in these patients are not well known. Thus, the purpose of this study was to evaluate the effect of 8 weeks resistance training on plasma NGF and IL-6 in female patients with MS. Materials and methods: Thirty two female MS patients with 32.4±5.5 years of old, and expanded disability status scale (EDSS 0-4.5, participated in this study. The subjects were randomly assigned to control group (n=13 and training group (n=14. Subjects in training group performed selected upper and lower extremities resistance training with weight-training equipment and rubber bands 3 days a week for 8 weeks. Body composition parameters, NGF and IL-6 levels were measured before and after the intervention. Results: The results showed that body weight, body mass index, body fat mass and EDSS were decreased significantly after 8 weeks in training group compared to control group (P<0.05. Also, no significant differences were seen after the intervention in NGF and IL-6 between the training and control groups. Conclusion: The results suggest resistance training with specific intensity and duration utilized in this study improves EDSS in female patients with MS but it has no effecting on NGF and IL-6 in these patients.

Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle.

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Serre, Stéphanie B N; Jensen, Sanne B; Ghanem, Lubna; Humes, Daryl G; Ramirez, Santseharay; Li, Yi-Ping; Krarup, Henrik; Bukh, Jens; Gottwein, Judith M

2016-06-01

Hepatitis C virus (HCV) NS3 protease inhibitors (PIs) are important components of novel HCV therapy regimens. Studies of PI resistance initially focused on genotype 1. Therefore, knowledge about the determinants of PI resistance for the highly prevalent genotypes 2 to 6 remains limited. Using Huh7.5 cell culture-infectious HCV recombinants with genotype 1 to 6 NS3 protease, we identified protease positions 54, 155, and 156 as hot spots for the selection of resistance substitutions under treatment with the first licensed PIs, telaprevir and boceprevir. Treatment of a genotype 2 isolate with the newer PIs vaniprevir, faldaprevir, simeprevir, grazoprevir, paritaprevir, and deldeprevir identified positions 156 and 168 as hot spots for resistance; the Y56H substitution emerged for three newer PIs. Substitution selection also depended on the specific recombinant. The substitutions identified conferred cross-resistance to several PIs; however, most substitutions selected under telaprevir or boceprevir treatment conferred less resistance to certain newer PIs. In a single-cycle production assay, across genotypes, PI treatment primarily decreased viral replication, which was rescued by PI resistance substitutions. The substitutions identified resulted in differential effects on viral fitness, depending on the original recombinant and the substitution. Across genotypes, fitness impairment induced by resistance substitutions was due primarily to decreased replication. Most combinations of substitutions that were identified increased resistance or fitness. Combinations of resistance substitutions with fitness-compensating substitutions either rescued replication or compensated for decreased replication by increasing assembly. This comprehensive study provides insight into the selection patterns and effects of PI resistance substitutions for HCV genotypes 1 to 6 in the context of the infectious viral life cycle, which is of interest for clinical and virological HCV research

GTPase activity, structure, and mechanical properties of filaments assembled from bacterial cytoskeleton protein MreB.

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Esue, Osigwe; Wirtz, Denis; Tseng, Yiider

2006-02-01

MreB, a major component of the recently discovered bacterial cytoskeleton, displays a structure homologous to its eukaryotic counterpart actin. Here, we study the assembly and mechanical properties of Thermotoga maritima MreB in the presence of different nucleotides in vitro. We found that GTP, not ADP or GDP, can mediate MreB assembly into filamentous structures as effectively as ATP. Upon MreB assembly, both GTP and ATP release the gamma phosphate at similar rates. Therefore, MreB is an equally effective ATPase and GTPase. Electron microscopy and quantitative rheology suggest that the morphologies and micromechanical properties of filamentous ATP-MreB and GTP-MreB are similar. In contrast, mammalian actin assembly is favored in the presence of ATP over GTP. These results indicate that, despite high structural homology of their monomers, T. maritima MreB and actin filaments display different assembly, morphology, micromechanics, and nucleotide-binding specificity. Furthermore, the biophysical properties of T. maritima MreB filaments, including high rigidity and propensity to form bundles, suggest a mechanism by which MreB helical structure may be involved in imposing a cylindrical architecture on rod-shaped bacterial cells.

Microfilarial reduction following ProHeart® 6 and ProHeart® SR-12 treatment in dogs experimentally inoculated with a resistant isolate of Dirofilaria immitis.

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McTier, Tom L; Pullins, Aleah; Inskeep, Gregory A; Gagnon, Genevieve; Fan, Huihao; Schoell, Adam; Bidgood, Tara; Login, Joyce; Meeus, Patrick

2017-11-09

Emerging resistance of heartworms (Dirofilaria immitis) to macrocyclic lactone (ML) preventives is an increasing concern for veterinarians, pet owners and animal health companies that supply heartworm preventives, with recent reports of resistant isolates identified from the Mississippi Delta region of the United States. Products that are effective in eliminating microfilariae (MF) in dogs harboring resistant heartworm infections could be important in reducing the spread of heartworm resistance. The current study was conducted to investigate the potential for ProHeart® 6 (PH 6; Zoetis) and ProHeart® SR-12 (PH 12; Zoetis) to reduce MF in dogs experimentally inoculated with an isolate of D. immitis (ZoeMo-2012) confirmed to be resistant to MLs. Twenty-three dogs with preexisting heartworm infections (via surgical transplantation) were randomly allocated to four groups based on pretreatment (Day -14) MF counts. On Day 0, dogs received a subcutaneous injection of either saline (placebo-treated control, 6 dogs), PH 6 (0.17 mg/kg, 6 dogs), PH 12 (0.5 mg/kg, 5 dogs) or a single oral dose of moxidectin powder in a gelatin capsule (0.25 mg/kg, 6 dogs). All dogs were bled for MF counts (modified Knott's test) on Days 0 (pretreatment), 1, 3, 7, 14, 21, 28, 42, 56, and 84. Dogs in control and PH 6 groups were also bled for MF counts on Days 112, 140, and 168. No adverse events associated with treatment were observed for any dog. Average reductions in MF counts compared with controls for PH 6 were 9.7% on Day 1, increasing to 75.0% on Day 7, and further to 86.5% on Day 28. On Day 42, average MF reduction increased to 90.3%. Reductions increased further over the next several months with reductions of 91.3, 96.8, 96.6, and 98.9% on Days 56, 84, 112, and 140, respectively. On Day 168, the reduction was 99.3% (P < 0.0001). Average reductions in MF counts compared with controls for PH 12 were 20.9% on Day 1, increasing to 78.9% on Day 7, and further to 91.2% on Day 28. On Day 84

Metabolism of methoxychlor by the P450-monooxygenase CYP6G1 involved in insecticide resistance of Drosophila melanogaster after expression in cell cultures of Nicotiana tabacum.

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Joussen, Nicole; Schuphan, Ingolf; Schmidt, Burkhard

2010-03-01

Cytochrome P450 monooxygenase CYP6G1 of Drosophila melanogaster was heterologously expressed in a cell suspension culture of Nicotiana tabacum. This in vitro system was used to study the capability of CYP6G1 to metabolize the insecticide methoxychlor (=1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane, 1) against the background of endogenous enzymes of the corresponding non-transgenic culture. The Cyp6g1-transgenic cell culture metabolized 96% of applied methoxychlor (45.8 microg per assay) within 24 h by demethylation and hydroxylation mainly to trishydroxy and catechol methoxychlor (16 and 17%, resp.). About 34% of the metabolism and the distinct formation of trishydroxy and catechol methoxychlor were due to foreign enzyme CYP6G1. Furthermore, methoxychlor metabolism was inhibited by 43% after simultaneous addition of piperonyl butoxide (458 microg), whereas inhibition in the non-transgenic culture amounted to 92%. Additionally, the rate of glycosylation was reduced in both cultures. These results were supported by the inhibition of the metabolism of the insecticide imidacloprid (6; 20 microg, 24 h) in the Cyp6g1-transgenic culture by 82% in the presence of piperonyl butoxide (200 microg). Due to CYP6G1 being responsible for imidacloprid resistance of Drosophila or being involved in DDT resistance, it is likely that CYP6G1 conveys resistance to methoxychlor (1). Furthermore, treating Drosophila with piperonyl butoxide could weaken the observed resistance phenomena.

[Resistance risk, cross-resistance and biochemical resistance mechanism of Laodelphax striatellus to buprofezin].

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Mao, Xu-lian; Liu, Jin; Li, Xu-ke; Chi, Jia-jia; Liu, Yong-jie

2016-01-01

In order to investigate the resistance development law and biochemical resistance mechanism of Laodelphax striatellus to buprofezin, spraying rice seedlings was used to continuously screen resistant strains of L. striatellus and dipping rice seedlings was applied to determine the toxicity and cross-resistance of L. striatellus to insecticides. After 32-generation screening with buprofezin, L. striatellus developed 168.49 folds resistance and its reality heritability (h2) was 0.11. If the killing rate was 80%-90%, L. striatellus was expected to develop 10-fold resistance to buprofezin only after 5 to 6 generations breeding. Because the actual reality heritability of field populations was usually lower than that of the resistant strains, the production of field populations increasing with 10-fold resistance would need much longer time. The results of cross-resistance showed that resistant strain had high level cross-resistance with thiamethoxam and imidacloprid, low level cross-resistance with acetamiprid, and no cross-resistance with pymetrozine and chlorpyrifos. The activity of detoxification enzymes of different strains and the syergism of synergist were measured. The results showed that cytochrome P450 monooxygenase played a major role in the resistance of L. striatellus to buprofezin, the esterase played a minor role and the GSH-S-transferase had no effect. Therefore, L. striatellus would have high risk to develop resistance to buprofezin when used in the field and might be delayed by using pymetrozine and chlorpyrifos.

Comportamento de hidratação e qualidade fisiológica das sementes de arroz Hidration performance and physiological quality of rice seeds

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Rafael Pivotto Bortolotto

2008-12-01

Full Text Available O trabalho teve como objetivo verificar a relação existente entre o comportamento da curva de hidratação de sementes de arroz e sua qualidade fisiológica. Foram utilizados seis lotes de sementes de arroz, sendo três da cultivar IRGA 417 e três da cultivar IRGA 422 CL, inicialmente submetidos a testes para caracterizar o potencial fisiológico dos lotes: germinação, primeira contagem de germinação, envelhecimento acelerado, frio sem terra, condutividade elétrica, comprimento de plântula, massa seca de plântula e emergência em campo. Posteriormente, foi determinada a curva de hidratação das sementes de cada lote. Conclui-se que a qualidade fisiológica afeta o teor de água alcançado pelas sementes de arroz durante a hidratação, e nas sementes menos vigorosas notam-se menor velocidade de hidratação e grau de umidade, comparativamente às sementes de mais vigor.The objective of this study was to verify the relationship between the shape of the hydration curve and the physiological quality of rice seeds. Six lots of rice seeds were used, three of cultivar IRGA 417 and three of cultivar IRGA 422 CL. The rice seeds were submitted to a set of tests in order to measure the physiological potential of the lots: germination, first count germination, accelerated aging, cold test (without soil, electrical conductivity, length and dry mass of seedlings and seedling emergence in the field. After evaluation of the initial quality of the lots, the hydration curve was determined. It was concluded that physiological quality affects the water content reached by rice seeds during hydration, and that seeds with lower vigor present lower hydration rate and water content when compared to seeds with higher vigor.

Influence of supplementation with branched-chain amino acids in combination with resistance exercise on p70S6 kinase phosphorylation in resting and exercising human skeletal muscle.

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Apró, W; Blomstrand, E

2010-11-01

Skeletal muscle growth is thought to be regulated by the mammalian target of rapamycin (mTOR) pathway, which can be activated by resistance exercise and branched-chain amino acids (BCAA). The major aim of the present study was to distinguish between the influence of resistance exercise and BCAA on key enzymes considered to be involved in the regulation of protein synthesis, including p70(S6) kinase (p70(S6k)). Nine healthy subjects (four men and five women) performed unilateral resistance exercise on two occasions separated by 1 month. Subjects were randomly supplied either a mixture of BCAA or flavoured water. Muscle biopsies were taken from both resting and exercising muscle before, after and 1 h after exercise. Phosphorylation of Akt was unaltered by either resistance exercise and/or BCAA supplementation whereas mTOR phosphorylation was enhanced (Pexercising and resting muscle following exercise in the absence (70-90%) and presence of BCAA supplementation (80-130%). Phosphorylation of p70(S6k) was unaffected by resistance exercise alone; however, BCAA intake increased (Pexercise. In resting muscle, a 5- and 16-fold increase in p70(S6k) was observed immediately after and 1 h after exercise, respectively, as compared to 11- and 30-fold increases in the exercising muscle. Phosphorylation of eukaryotic elongation factor 2 was attenuated 1 h after exercise (Pexercising muscle (30-50%) under both conditions. The present findings indicate that resistance exercise and BCAA exert both separate and combined effects on the p70(S6k) phosphorylation in an Akt-independent manner. © 2010 The Authors. Journal compilation © 2010 Scandinavian Physiological Society.

Steroid induction of therapy-resistant cytokeratin-5-positive cells in estrogen receptor-positive breast cancer through a BCL6-dependent mechanism

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Goodman, C R; Sato, T; Peck, A R; Girondo, M A; Yang, N; Liu, C; Yanac, A F; Kovatich, A J; Hooke, J A; Shriver, C D; Mitchell, E P; Hyslop, T; Rui, H

2016-01-01

Therapy resistance remains a major problem in estrogen receptor-α (ERα)-positive breast cancer. A subgroup of ERα-positive breast cancer is characterized by mosaic presence of a minor population of ERα-negative cancer cells expressing the basal cytokeratin-5 (CK5). These CK5-positive cells are therapy resistant and have increased tumor-initiating potential. Although a series of reports document induction of the CK5-positive cells by progestins, it is unknown if other 3-ketosteroids share this ability. We now report that glucocorticoids and mineralocorticoids effectively expand the CK5-positive cell population. CK5-positive cells induced by 3-ketosteroids lacked ERα and progesterone receptors, expressed stem cell marker, CD44, and displayed increased clonogenicity in soft agar and broad drug-resistance in vitro and in vivo. Upregulation of CK5-positive cells by 3-ketosteroids required induction of the transcriptional repressor BCL6 based on suppression of BCL6 by two independent BCL6 small hairpin RNAs or by prolactin. Prolactin also suppressed 3-ketosteroid induction of CK5+ cells in T47D xenografts in vivo. Survival analysis with recursive partitioning in node-negative ERα-positive breast cancer using quantitative CK5 and BCL6 mRNA or protein expression data identified patients at high or low risk for tumor recurrence in two independent patient cohorts. The data provide a mechanism by which common pathophysiological or pharmacologic elevations in glucocorticoids or other 3-ketosteroids may adversely affect patients with mixed ERα+/CK5+ breast cancer. The observations further suggest a cooperative diagnostic utility of CK5 and BCL6 expression levels and justify exploring efficacy of inhibitors of BCL6 and 3-ketosteroid receptors for a subset of ERα-positive breast cancers. PMID:26096934

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.

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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.

Physicochemical and sensory profile of rice bran roasted in microwave Perfil físico-químico e sensorial de farelos de arroz torrados em micro-ondas

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Marina Costa Garcia

2012-12-01

Full Text Available The purpose of this study was to evaluate the physical, chemical, and sensory changes in bran from three rice cultivars according to microwave roasting time. This study analyzed three rice cultivars, BRS Sertaneja (S, BRS Primavera (P, and IRGA 417 (I determining the color parameters (L*, a*, and b * at 6, 9, 12, 15, and 18 minutes of roasting time. After applying the difference from control test, the rice brans with different characteristics aroma and flavor were selected: S and P roasted for 9 and 15 minutes and IRGA 417 roasted for 9, 12, and 15 minutes. These samples were characterized by Free-Choice Profile descriptive sensory analysis, and their chemical composition was also determined. The longer the roasting process, the higher the roasted flavor intensity and aroma. The IRG 417 cultivar roasted for 12 minutes showed a sweeter flavor and aroma. After roasting, the brans remained rich in protein and lipid and presented higher fiber content and lower reducing sugar and phytic acid content. Microwave roasting for 12 minutes can be a viable option for improving the sensory functional and nutritional characteristics of the rice bran considering its use in food products.Este trabalho objetivou avaliar as mudanças físicas, químicas e sensoriais de farelos de três cultivares de arroz em função do tempo de torra em micro-ondas. Foram estudados 3 cultivares de arroz, BRS Sertaneja (S, BRS Primavera (P e Irga 417 (I, determinando-se os parâmetros de cor (L*, a* e b* aos 6, 9, 12, 15 e 18 minutos de torração. Após aplicação do teste de diferença do controle, selecionaram-se farelos de arroz com características de sabor e aroma distintas: S e P torrados por 9 e 15 minutos e I torrado por 9, 12 e 15 minutos. Essas amostras foram caracterizadas pela análise sensorial descritiva de Perfil Livre e quanto composição química. Quanto mais longa a torração, mais intenso o aroma e sabor de torrado dos farelos, apresentando sabor e aroma

Galectins expressed differently in genetically susceptible C57BL/6 and resistant BALB/c mice during acute ocular Toxoplasma gondii infection.

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Chen, S-J; Zhang, Y-X; Huang, S-G; Lu, F-L

2017-07-01

Ocular toxoplasmosis (OT) caused by Toxoplasma gondii is a major cause of infectious uveitis, however little is known about its immunopathological mechanism. Susceptible C57BL/6 (B6) and resistant BALB/c mice were intravitreally infected with 500 tachyzoites of the RH strain of T. gondii. B6 mice showed more severe ocular pathology and higher parasite loads in the eyes. The levels of galectin (Gal)-9 and its receptors (Tim-3 and CD137), interferon (IFN)-γ, IL-6 and IL-10 were significantly higher in the eyes of B6 mice than those of BALB/c mice; however, the levels of IFN-α and -β were significantly decreased in the eyes and CLNs of B6 mice but significantly increased in BALB/c mice after infection. After blockage of galectin-receptor interactions by α-lactose, neither ocular immunopathology nor parasite loads were different from those of infected BALB/c mice without α-lactose treatment. Although the expressions of Gal-9/receptor were significantly increased in B6 mice and Gal-1 and -3 were upregulated in both strains of mice upon ocular T. gondii infection, blockage of galectins did not change the ocular pathogenesis of genetic resistant BALB/c mice. However, IFN-α and -β were differently expressed in B6 and BALB/c mice, suggesting that type I IFNs may play a protective role in experimental OT.

Predicting 6- and 12-Month Risk of Mortality in Patients With Platinum-Resistant Advanced-Stage Ovarian Cancer: Prognostic Model to Guide Palliative Care Referrals.

Science.gov (United States)

Foote, Jonathan; Lopez-Acevedo, Micael; Samsa, Gregory; Lee, Paula S; Kamal, Arif H; Alvarez Secord, Angeles; Havrilesky, Laura J

2018-02-01

Predictive models are increasingly being used in clinical practice. The aim of the study was to develop a predictive model to identify patients with platinum-resistant ovarian cancer with a prognosis of less than 6 to 12 months who may benefit from immediate referral to hospice care. A retrospective chart review identified patients with platinum-resistant epithelial ovarian cancer who were treated at our institution between 2000 and 2011. A predictive model for survival was constructed based on the time from development of platinum resistance to death. Multivariate logistic regression modeling was used to identify significant survival predictors and to develop a predictive model. The following variables were included: time from diagnosis to platinum resistance, initial stage, debulking status, number of relapses, comorbidity score, albumin, hemoglobin, CA-125 levels, liver/lung metastasis, and the presence of a significant clinical event (SCE). An SCE was defined as a malignant bowel obstruction, pleural effusion, or ascites occurring on or before the diagnosis of platinum resistance. One hundred sixty-four patients met inclusion criteria. In the regression analysis, only an SCE and the presence of liver or lung metastasis were associated with poorer short-term survival (P < 0.001). Nine percent of patients with an SCE or liver or lung metastasis survived 6 months or greater and 0% survived 12 months or greater, compared with 85% and 67% of patients without an SCE or liver or lung metastasis, respectively. Patients with platinum-resistant ovarian cancer who have experienced an SCE or liver or lung metastasis have a high risk of death within 6 months and should be considered for immediate referral to hospice care.

The Hv NAC6 transcription factor: a positive regulator of penetration resistance in barley and Arabidopsis

DEFF Research Database (Denmark)

Jensen, Michael Krogh; Rung, Jesper Henrik; Gregersen, Per Langkjaer

2007-01-01

Pathogens induce the expression of many genes encoding plant transcription factors, though specific knowledge of the biological function of individual transcription factors remains scarce. NAC transcription factors are encoded in plants by a gene family with proposed functions in both abiotic...... and biotic stress adaptation, as well as in developmental processes. In this paper, we provide convincing evidence that a barley NAC transcription factor has a direct role in regulating basal defence. The gene transcript was isolated by differential display from barley leaves infected with the biotrophic...... powdery mildew fungus, Blumeria graminis f.sp. hordei (Bgh). The full-length cDNA clone was obtained using 5'-RACE and termed HvNAC6, due to its high similarity to the rice homologue, OsNAC6. Gene silencing of HvNAC6 during Bgh inoculation compromises penetration resistance in barley epidermal cells...

Antibacterial activity of the Antarctic bacterium Janthinobacterium sp. SMN 33.6 against multi-resistant Gram-negative bacteria

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Geraldine Asencio

2014-01-01

Conclusions: The ethanolic extract of Janthinobacterium sp. SMN 33.6 possesses antibacterial activity against a chromosomal AmpC beta-lactamase-producing strain of Serratia marcescens, an extended-spectrum beta-lactamase-producing Escherichia coli and also against carbapenemase-producing strains of Acinetobacter baumannii and Pseudomonas aeruginosa. This becomes a potential and interesting biotechnological tool for the control of bacteria with multi-resistance to commonly used antibiotics.

Evaluation of the effect of Ni-P coating on the corrosion resistance of the aluminium 7075 T6 alloy

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Gil, L.

2008-02-01

Full Text Available The aluminum alloy 7075-T6 is a structural alloy widely used for aeronautical applications due to its high relationship between mechanical resistance and weight. Depending upon the environmental conditions, many types of corrosion mechanisms such as intergranular, exfoliation, have been found to occur in aircraft structural aluminum alloys. A significant advance in order to improve the behavior of this alloy is related to the application of the autocatalytic Ni-P coating which confers an excellent corrosion resistance coupled with both reduced erosive wear and higher hardness. The purpose of this work was to investigate the effect of the application of a Ni-P coating on the corrosion resistance of an aluminum 7075-T6 alloy. The results obtained indicated that the application of the Ni-P coatings diminishes the susceptibility to pitting and makes the aluminum 7075 T6 alloy immune to the exfoliation corrosion attack.

La aleación de aluminio 7075-T6 es una aleación estructural ampliamente utilizada para aplicaciones aeronáuticas, debido a su alta relación entre resistencia mecánica y peso. Dependiendo de las condiciones ambientales, algunos mecanismos de corrosion tales como intergranular, exfoliacion, picadura y crevice se ha encontrado que ocurren en estructuras de aviones de aleaciones de aluminio. Un avance siginificativo para mejorar el comportamiento de esta aleación es la aplicación de recubrimientos autocatalíticos de Ni-P, los cuales confieren una excelente resistencia a la corrosión acoplado con una reducción del desgaste erosivo y un aumento de la dureza. El propósito de este trabajo fue investigar el efecto de la aplicación de un recubrimiento de Ni-P sobre la resistencia a la corrosión de una aleación de aluminio 7075-T6. Los resultados obtenidos indican que la aplicación del recubrimiento de Ni-P disminuye la susceptibilidad a la picadura y hace a la aleación de aluminio 7075 T6, prácticamente inmune al ataque

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.

Ran GTPase protein promotes human pancreatic cancer proliferation by deregulating the expression of Survivin and cell cycle proteins

International Nuclear Information System (INIS)

Deng, Lin; Lu, Yuanyuan; Zhao, Xiaodi; Sun, Yi; Shi, Yongquan; Fan, Hongwei; Liu, Changhao; Zhou, Jinfeng; Nie, Yongzhan; Wu, Kaichun; Fan, Daiming; Guo, Xuegang

2013-01-01

Highlights: •Overexpression of Ran in pancreatic cancer was correlated with histological grade. •Downregulation of Ran could induce cell apoptosis and inhibit cell proliferation. •The effects were mediated by cell cycle proteins, Survivin and cleaved Caspase-3. -- Abstract: Ran, a member of the Ras GTPase family, has important roles in nucleocytoplasmic transport. Herein, we detected Ran expression in pancreatic cancer and explored its potential role on tumour progression. Overexpressed Ran in pancreatic cancer tissues was found highly correlated with the histological grade. Downregulation of Ran led to significant suppression of cell proliferation, cell cycle arrest at the G1/S phase and induction of apoptosis. In vivo studies also validated that result. Further studies revealed that those effects were at least partly mediated by the downregulation of Cyclin A, Cyclin D1, Cyclin E, CDK2, CDK4, phospho-Rb and Survivin proteins and up regulation of cleaved Caspase-3

Patterns of HIV-1 Drug Resistance After First-Line Antiretroviral Therapy (ART) Failure in 6 Sub-Saharan African Countries: Implications for Second-Line ART Strategies

NARCIS (Netherlands)

Hamers, Raph L.; Sigaloff, Kim C. E.; Wensing, Annemarie M.; Wallis, Carole L.; Kityo, Cissy; Siwale, Margaret; Mandaliya, Kishor; Ive, Prudence; Botes, Mariette E.; Wellington, Maureen; Osibogun, Akin; Stevens, Wendy S.; Rinke de Wit, Tobias F.; Schuurman, Rob; Siwale, M.; Njovu, C.; Labib, M.; Menke, J.; Botes, M. E.; Conradie, F.; Ive, P.; Sanne, I.; Wallis, C. L.; Letsoalo, E.; Stevens, W. S.; Hardman, M.; Wellington, M.; Luthy, R.; Mandaliya, K.; Abdallah, S.; Jao, I.; Dolan, M.; Namayanja, G.; Nakatudde, L.; Nankya, I.; Kiconco, M.; Abwola, M.; Mugyenyi, P.; Osibogun, A.; Akanmu, S.; Schuurman, R.; Wensing, A. M.; Straatsma, E.; Wit, F. W.; Dekker, J.; van Vugt, M.; Lange, J. M.

2012-01-01

Background. Human immunodeficiency virus type 1 (HIV-1) drug resistance may limit the benefits of antiretroviral therapy (ART). This cohort study examined patterns of drug-resistance mutations (DRMs) in individuals with virological failure on first-line ART at 13 clinical sites in 6 African

Mutations in circularly permuted GTPase family genes AtNOA1/RIF1/SVR10 and BPG2 suppress var2-mediated leaf variegation in Arabidopsis thaliana.

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Qi, Yafei; Zhao, Jun; An, Rui; Zhang, Juan; Liang, Shuang; Shao, Jingxia; Liu, Xiayan; An, Lijun; Yu, Fei

2016-03-01

Leaf variegation mutants constitute a unique group of chloroplast development mutants and are ideal genetic materials to dissect the regulation of chloroplast development. We have utilized the Arabidopsis yellow variegated (var2) mutant and genetic suppressor analysis to probe the mechanisms of chloroplast development. Here we report the isolation of a new var2 suppressor locus SUPPRESSOR OF VARIEGATION (SVR10). Genetic mapping and molecular complementation indicated that SVR10 encodes a circularly permuted GTPase that has been reported as Arabidopsis thaliana NITRIC OXIDE ASSOCIATED 1 (AtNOA1) and RESISTANT TO INHIBITION BY FOSMIDOMYCIN 1 (RIF1). Biochemical evidence showed that SVR10/AtNOA1/RIF1 likely localizes to the chloroplast stroma. We further demonstrate that the mutant of a close homologue of SVR10/AtNOA1/RIF1, BRASSINAZOLE INSENSITIVE PALE GREEN 2 (BPG2), can also suppress var2 leaf variegation. Mutants of SVR10 and BPG2 are impaired in photosynthesis and the accumulation of chloroplast proteins. Interestingly, two-dimensional blue native gel analysis showed that mutants of SVR10 and BPG2 display defects in the assembly of thylakoid membrane complexes including reduced levels of major photosynthetic complexes and the abnormal accumulation of a chlorophyll-protein supercomplex containing photosystem I. Taken together, our findings suggest that SVR10 and BPG2 are functionally related with VAR2, likely through their potential roles in regulating chloroplast protein homeostasis, and both SVR10 and BPG2 are required for efficient thylakoid protein complex assembly and photosynthesis.

FSL-1, a bacterial-derived toll-like receptor 2/6 agonist, enhances resistance to experimental HSV-2 infection

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Pyles Richard B

2009-11-01

Full Text Available Abstract Background Herpes simplex virus type 2 (HSV-2 is a leading cause of genital ulceration that can predispose individuals to an increased risk of acquiring other sexually transmitted infections. There are no approved HSV-2 vaccines and current suppressive therapies require daily compound administration that does not prevent all recurrences. A promising experimental strategy is the use of toll-like receptor (TLR agonists to induce an innate immune response that provides resistance to HSV-2 infection. Previous studies showed that anti-herpetic activity varied based on origin of the agonists and activation of different TLR indicating that activity likely occurs through elaboration of a specific innate immune response. To test the hypothesis, we evaluated the ability of a bacterial-derived TLR2/6 agonist (FSL-1 to increase resistance to experimental genital HSV-2 infection. Methods Vaginal application of FSL-1 at selected doses and times was evaluated to identify potential increased resistance to genital HSV-2 infection in the mouse model. The FSL-1 induced cytokine profile was quantified using kinetically collected vaginal lavages. Additionally, cytokine elaboration and organ weights were evaluated after single or multiple FSL-1 doses to establish a preliminary safety profile. Human vaginal EC cultures were used to confirm the mouse model outcomes. Results The results showed that vaginally-applied FSL-1 created an environment resistant to a 25-fold higher HSV-2 challenge dose. Mechanistically, vaginal FSL-1 application led to transient elaboration of cytokines linked to anti-herpetic innate immune responses. No gross local or peripheral immunotoxicity was observed even after multiple dosing. FSL-1 also created an anti-herpetic environment in cultures of human vaginal epithelial cells (EC. Conclusion The results showed, for the first time, that the bacterial-derived TLR2/6 agonist FSL-1 induced significant resistance to HSV-2 infection when

Resistance patterns among multidrug-resistant tuberculosis patients in greater metropolitan Mumbai: trends over time.

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Dalal, Alpa; Pawaskar, Akshay; Das, Mrinalini; Desai, Ranjan; Prabhudesai, Pralhad; Chhajed, Prashant; Rajan, Sujeet; Reddy, Deepesh; Babu, Sajit; Jayalakshmi, T K; Saranchuk, Peter; Rodrigues, Camilla; Isaakidis, Petros

2015-01-01

While the high burden of multidrug-resistant tuberculosis (MDR-TB) itself is a matter of great concern, the emergence and rise of advanced forms of drug-resistance such as extensively drug-resistant TB (XDR-TB) and extremely drug-resistant TB (XXDR-TB) is more troubling. The aim of this study was to investigate the trends over time of patterns of drug resistance in a sample of MDR-TB patients in greater metropolitan Mumbai, India. This was a retrospective, observational study of drug susceptibility testing (DST) results among MDR-TB patients from eight health care facilities in greater Mumbai between 2005 and 2013. We classified resistance patterns into four categories: MDR-TB, pre-XDR-TB, XDR-TB and XXDR-TB. A total of 340 MDR-TB patients were included in the study. Pre-XDR-TB was the most common form of drug-resistant TB observed overall in this Mumbai population at 56.8% compared to 29.4% for MDR-TB. The proportion of patients with MDR-TB was 39.4% in the period 2005-2007 and 27.8% in 2011-2013, while the proportion of those with XDR-TB and XXDR-TB was changed from 6.1% and 0% respectively to 10.6% and 5.6% during the same time period. During the same periods, the proportions of patients with ofloxacin, moxifloxacin and ethionamide resistance significantly increased from 57.6% to 75.3%, from 60.0% to 69.5% and from 24.2% to 52.5% respectively (pMumbai highlight the need for individualized drug regimens, designed on the basis of DST results involving first- and second-line anti-TB drugs and treatment history of the patient. A drug-resistant TB case-finding strategy based on molecular techniques that identify only rifampicin resistance will lead to initiation of suboptimal treatment regimens for a significant number of patients, which may in turn contribute to amplification of resistance and transmission of strains with increasingly advanced resistance within the community.

Radiation resistance of Candida parapsilosis

International Nuclear Information System (INIS)

Kristensen, H.

1982-01-01

The radiation resistance of 30 strains classified as Candida parapsilosis was examined. The strains originated partly from environments where ionizing radiation was used for research or routine purposes, partly from environments with no known possibility for selection of strains with unusually high radiation resistance. D-6 values between 1.5 and 2.4 Megarads were found when the cells were irradiated in the dried state, a D-6 value being the dose necessary to reduce the initial number of colony-forming units with a factor of 10 6 . The majority of D-6 values were between 1.9 and 2.1 Megarads. D-6 values for the cells irradiated in liquid media were about 2/3 of tose in the dried state. No difference in resistance was revealed depending on the origin of the strains examined. For radiation sterilization of medical products the demonstrated resistance of Candida parapsilosis might be of importance of routine use of minimum doses below 2.5 Megarads were to be accepted. (author)

Impact of ertapenem on antimicrobial resistance in a sentinel group of Gram-negative bacilli: a 6 year antimicrobial resistance surveillance study.

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Rodriguez-Osorio, Carlos A; Sanchez-Martinez, Cesar O; Araujo-Melendez, Javier; Criollo, Elia; Macias-Hernandez, Alejandro E; Ponce-de-Leon, Alfredo; Ponce-de-Leon, Sergio; Sifuentes-Osornio, Jose

2015-03-01

To determine the association between ertapenem and resistance of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii-calcoaceticus complex to different antimicrobials while adjusting for relevant hospital factors. This was a retrospective time-series study conducted at a tertiary care centre from September 2002 to August 2008. The specific impact of ertapenem on the resistance of these Gram-negative bacilli (GNB) was assessed by multiple linear regression analysis, adjusting for the average length of stay, rate of hospital-acquired infections and use of 10 other antimicrobials, including type 2 carbapenems. Unadjusted analyses revealed significant increases over the duration of the study in the number of GNB resistant to meropenem/imipenem among 1000 isolates each of E. coli (0.46 ± 0.22, P  0.05) with changes in resistance for any pathogen/antimicrobial combination. After controlling for confounders, ertapenem was not associated with changes in resistance in a group of sentinel GNB, although significant variations in resistance to different antimicrobials were observed in the unadjusted analyses. These results emphasize the importance of implementation of local resistance surveillance platforms and stewardship programmes to combat the global emergence and spread of antimicrobial resistance. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Development of a lateral flow test to detect metabolic resistance in Bemisia tabaci mediated by CYP6CM1, a cytochrome P450 with broad spectrum catalytic efficiency.

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Nauen, Ralf; Wölfel, Katharina; Lueke, Bettina; Myridakis, Antonis; Tsakireli, Dimitra; Roditakis, Emmanouil; Tsagkarakou, Anastasia; Stephanou, Euripides; Vontas, John

2015-06-01

Cotton whitefly, Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) is a major sucking pest in many agricultural and horticultural cropping systems globally. The frequent use of insecticides of different mode of action classes resulted in populations resisting treatments used to keep numbers under economic damage thresholds. Recently it was shown that resistance to neonicotinoids such as imidacloprid is linked to the over-expression of CYP6CM1, a cytochrome P450 monooxygenase detoxifying imidacloprid and other neonicotinoid insecticides when recombinantly expressed in insect cells. However over-expression of CYP6CM1 is also known to confer cross-resistance to pymetrozine, an insecticide not belonging to the chemical class of neonicotinoids. In addition we were able to demonstrate by LC-MS/MS analysis the metabolisation of pyriproxyfen by recombinantly expressed CYP6CM1. Based on our results CYP6CM1 is one of the most versatile detoxification enzymes yet identified in a pest of agricultural importance, as it detoxifies a diverse range of chemical classes used to control whiteflies. Therefore we developed a field-diagnostic antibody-based lateral flow assay which detects CYP6CM1 protein at levels providing resistance to neonicotinoids and other insecticides. The ELISA based test kit can be used as a diagnostic tool to support resistance management strategies based on the alternation of different modes of action of insecticides. Copyright © 2014 Elsevier Inc. All rights reserved.

p115 RhoGEF activates the Rac1 GTPase signaling cascade in MCP1 chemokine-induced vascular smooth muscle cell migration and proliferation.

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Singh, Nikhlesh K; Janjanam, Jagadeesh; Rao, Gadiparthi N

2017-08-25

Although the involvement of Rho proteins in the pathogenesis of vascular diseases is well studied, little is known about the role of their upstream regulators, the Rho guanine nucleotide exchange factors (RhoGEFs). Here, we sought to identify the RhoGEFs involved in monocyte chemotactic protein 1 (MCP1)-induced vascular wall remodeling. We found that, among the RhoGEFs tested, MCP1 induced tyrosine phosphorylation of p115 RhoGEF but not of PDZ RhoGEF or leukemia-associated RhoGEF in human aortic smooth muscle cells (HASMCs). Moreover, p115 RhoGEF inhibition suppressed MCP1-induced HASMC migration and proliferation. Consistent with these observations, balloon injury (BI) induced p115 RhoGEF tyrosine phosphorylation in rat common carotid arteries, and siRNA-mediated down-regulation of its levels substantially attenuated BI-induced smooth muscle cell migration and proliferation, resulting in reduced neointima formation. Furthermore, depletion of p115 RhoGEF levels also abrogated MCP1- or BI-induced Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling, which, as we reported previously, is involved in vascular wall remodeling. Our findings also show that protein kinase N1 (PKN1) downstream of Rac1-cyclin D1/CDK6 and upstream of CDK4-PAK1 in the p115 RhoGEF-Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling axis is involved in the modulation of vascular wall remodeling. Of note, we also observed that CCR2-G i/o -Fyn signaling mediates MCP1-induced p115 RhoGEF and Rac1 GTPase activation. These findings suggest that p115 RhoGEF is critical for MCP1-induced HASMC migration and proliferation in vitro and for injury-induced neointima formation in vivo by modulating Rac1-NFATc1-cyclin D1-CDK6-PKN1-CDK4-PAK1 signaling. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Novel trisubstituted benzimidazoles, targeting Mtb FtsZ, as a new class of antitubercular agents.

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Kumar, Kunal; Awasthi, Divya; Lee, Seung-Yub; Zanardi, Ilaria; Ruzsicska, Bela; Knudson, Susan; Tonge, Peter J; Slayden, Richard A; Ojima, Iwao

2011-01-13

Libraries of novel trisubstituted benzimidazoles were created through rational drug design. A good number of these benzimidazoles exhibited promising MIC values in the range of 0.5-6 μg/mL (2-15 μM) for their antibacterial activity against Mtb H37Rv strain. Moreover, five of the lead compounds also exhibited excellent activity against clinical Mtb strains with different drug-resistance profiles. All lead compounds did not show appreciable cytotoxicity (IC(50) > 200 μM) against Vero cells, which inhibited Mtb FtsZ assembly in a dose dependent manner. The two lead compounds unexpectedly showed enhancement of the GTPase activity of Mtb FtsZ. The result strongly suggests that the increased GTPase activity destabilizes FtsZ assembly, leading to efficient inhibition of FtsZ polymerization and filament formation. The TEM and SEM analyses of Mtb FtsZ and Mtb cells, respectively, treated with a lead compound strongly suggest that lead benzimidazoles have a novel mechanism of action on the inhibition of Mtb FtsZ assembly and Z-ring formation.

[Role of immune-related GTPase M1 in cortical neurons autophagy of mice with sepsis-induced brain injury].

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Huang, Qun; Chen, Bin; Li, Yafei; Li, Xihong

2017-12-28

To investigate the role of immune-related GTPase M1 (IRGM1) in cortical neurons autophagy in mice with sepsis induced brain injury (SIBI).
 Methods: Sixty wild-type C57BL/6 mice and sixty IRGM1 gene knockout C57BL/6 mice were randomly divided into 4 groups: a sham-operated wild-type (SWT) group, a cecal ligation and puncture (CLP) model wild-type (MWT) group, a sham-operated knockout (SKO) group, and a CLP model knockout (MKO) group. Models of mice with sepsis were established by CLP. Six hours of after CLP, the neurobehavioral scores for mice were recorded. The mice were diagnosed with SIBI and enrolled for the studies in next step if the neurobehavioral score was less than 6 in the MWT and MKO groups. The sham operation group only opened the abdominal cavity without CLP. Pathological changes in mouse cerebral cortex were observed by HE staining. Electron microscope was used to observe the ultrastructure of autophagy in cortical neurons. The expression of IRGM1 and INF-γ mRNA in the cerebral cortex of mice were detected by Real time quantitative PCR. The protein expression of microtubule-associated protein 1 light chain 3 (LC3)-II, LC3-I, sequestosome-1 (SQSTM1) and IRGM1 were measured by Western blot. Immunofluorescence staining was used to examine the expression of IRGM1 in mouse cortical neurons.
 Results: In the MWT group, the cortical neurons showed dilated endoplasmic reticulum, swelling mitochondria, and increased number of autophagosomes after 6 or 24 h of CLP in contrast to the SWT group. At 6 h after CLP, the expression of LC3-II in the cerebral cortex began to up-regulate, and the up-regulation was maintained till 96 h after CLP; on the contrary, SQSTM1 began to decline after 6 h of CLP. Compared with SWT group, IRGM1 was strongly up-regulated in the cerebral cortex of mice at both mRNA and protein levels in the MWT group after 12 h of CLP, and the mRNA expression of IFN-γ was also increased significantly (PSIBI was 90% (27/30) in the MWT group

Haploinsufficiency of a spliceosomal GTPase encoded by EFTUD2 causes mandibulofacial dysostosis with microcephaly.

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Lines, Matthew A; Huang, Lijia; Schwartzentruber, Jeremy; Douglas, Stuart L; Lynch, Danielle C; Beaulieu, Chandree; Guion-Almeida, Maria Leine; Zechi-Ceide, Roseli Maria; Gener, Blanca; Gillessen-Kaesbach, Gabriele; Nava, Caroline; Baujat, Geneviève; Horn, Denise; Kini, Usha; Caliebe, Almuth; Alanay, Yasemin; Utine, Gulen Eda; Lev, Dorit; Kohlhase, Jürgen; Grix, Arthur W; Lohmann, Dietmar R; Hehr, Ute; Böhm, Detlef; Majewski, Jacek; Bulman, Dennis E; Wieczorek, Dagmar; Boycott, Kym M

2012-02-10

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare sporadic syndrome comprising craniofacial malformations, microcephaly, developmental delay, and a recognizable dysmorphic appearance. Major sequelae, including choanal atresia, sensorineural hearing loss, and cleft palate, each occur in a significant proportion of affected individuals. We present detailed clinical findings in 12 unrelated individuals with MFDM; these 12 individuals compose the largest reported cohort to date. To define the etiology of MFDM, we employed whole-exome sequencing of four unrelated affected individuals and identified heterozygous mutations or deletions of EFTUD2 in all four. Validation studies of eight additional individuals with MFDM demonstrated causative EFTUD2 mutations in all affected individuals tested. A range of EFTUD2-mutation types, including null alleles and frameshifts, is seen in MFDM, consistent with haploinsufficiency; segregation is de novo in all cases assessed to date. U5-116kD, the protein encoded by EFTUD2, is a highly conserved spliceosomal GTPase with a central regulatory role in catalytic splicing and post-splicing-complex disassembly. MFDM is the first multiple-malformation syndrome attributed to a defect of the major spliceosome. Our findings significantly extend the range of reported spliceosomal phenotypes in humans and pave the way for further investigation in related conditions such as Treacher Collins syndrome. Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Rho-GTPase effector ROCK phosphorylates cofilin in actin-meditated cytokinesis during mouse oocyte meiosis.

Science.gov (United States)

Duan, Xing; Liu, Jun; Dai, Xiao-Xin; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Zhen-Bo; Wang, Qiang; Sun, Shao-Chen

2014-02-01

During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.

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

Science.gov (United States)

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

Eficiência no uso de nitrogênio em genótipos de arroz em solos de várzea tropical do Estado do Tocantins = Nitrogen use efficiency in rice genotypes in tropical lowland soils of the State of Tocantins, Brazil

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Nathan Gonçalves dos Passos

2015-03-01

Full Text Available O uso racional de adubação nitrogenada é fundamental para aumentar a produtividade da cultura, assim como reduzir o custo de produção e os riscos de poluição ambiental. Cultivares mais eficientes na absorção e na translocação de nutrientes estão sendo prioridade nos programas de melhoramento, além de que cada vez mais solos pobres são incorporados ao processo de produção agrícola. Assim, objetivou-se, com este trabalho, identificar cultivares melhoradas de arroz, eficientes quanto ao uso de nitrogênio em solos de várzea tropical. As cultivares avaliadas foram BRS-Formoso, Epagri-109, BRSGO-Guará, Metica-1, BRSFronteira, BEST-2000, BRS-Jaburu, BRS-Alvorada, Epagri-116, BRS-Jaçanã, SCSBRS-Piracema, BRSMGOuro Minas, Engopa, IRGA-417 e IRGA-424. Ao simular os ambientes com baixa e alta disponibilidades de nitrogênio, foram aplicadas doses de 20 e 120 kg ha-1 de N, respectivamente. As características analisadas foram altura de plantas, número de panículas por m2, número de dias para maturação, índice de clorofilas a e b e total, massa de 100 grãos e produtividade de grãos. Existe variabilidade genética entre as cultivares avaliadas quanto à eficiência ao uso de nitrogênio em solos de várzea. As cultivares BRSMG-Ouro Minas, SCSBRS-Piracema e IRGA-424 são mais eficientes e responsivas à adubação de nitrogênio = The rational use of nitrogen fertilization is essential to not only increase crop productivity, but also to reduce production costs and risks of environmental pollution. Cultivars more efficient at absorption and translocation of nutrients are taking priority in breeding programs, as well as the poor soils are being increasingly incorporated into the agricultural production process. Thus, the objective of this study was to identify improved cultivars of rice on the efficient use of nitrogen in tropical lowland soils. The evaluated cultivars were BRS-Formoso, Epagri-109, BRSGO-Guará, Metica-1, BRS

Eficiência de diferentes moléculas na redução dos sintomas da queima das bainhas em arroz e no crescimento de Rhizoctonia solani in vitro Efficiency of different molecules on the reduction of sheath blight symptoms in rice and on Rhizoctonia solani growth in vitro

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Daniel Augusto Schurt

2013-04-01

Full Text Available Este estudo verificou a eficiência da aplicação de diferentes moléculas em reduzir o comprimento relativo da lesão (CRL da queima das bainhas em arroz. Plantas dos cultivares BR-Irga 409 e Labelle foram cultivadas em solução nutritiva e inoculadas com Rhizoctonia solani, no estádio de máximo perfilhamento. Às 24 horas antes da inoculação, as bainhas das plantas foram pulverizadas com soluções de silicato de potássio (SP, silicato de potássio + fósforo (SP+F, Acibenzolar-S-Metil (ASM, fungicida Carbendazim, quitosana desacetilada (QD, etileno (ET e fosfito de potássio (FP. Plantas cujas bainhas foram pulverizadas com água destilada serviram como testemunhas. O efeito das moléculas contidas nesses produtos no crescimento micelial de R. solani foi testado in vitro. Para BR-Irga 409, o CRL foi menor com a aplicação do FP, em relação aos demais tratamentos, exceto o Carbendazim. A aplicação do Carbendazim reduziu em 86,1% o CRL, em relação à testemunha. O CRL foi significativamente menor no cultivar BR-Irga 409 do que no 'Labelle', com aplicação do FP. O crescimento micelial de R. solani foi reduzido apenas pelo FP e Carbendazim, em comparação com os demais tratamentos. Não houve diferença significativa entre os tratamentos testemunha, SP e SP+F para a concentração de Si nas bainhas das plantas dos dois cultivares.This study aimed to test the efficiency of different molecules on the reduction of the relative lesion extension (RLE of sheath blight in rice. Plants of cultivars BR-Irga 409 and Labelle were grown in nutrient solution and inoculated with Rhizoctonia solani at the maximum tillering stage. At 24 hours before inoculation, sheaths of plants were sprayed with solutions of potassium silicate (PS, potassium silicate + phosphorus (PS+P, Acibenzolar-S-Methyl (ASM, Carbendazim fungicide, deacetylated chitosan (DC, ethylene (ET and potassium phosphite (PP. Sheaths from plants sprayed with water served as the

Potential Mechanism of Action of 3′-Demethoxy-6-O-demethyl-isoguaiacin on Methicillin Resistant Staphylococcus aureus

OpenAIRE

Favela-Hernández, Juan; Clemente-Soto, Aldo; Balderas-Rentería, Isaías; Garza-González, Elvira; Camacho-Corona, María

2015-01-01

Bacterial infections represent one of the main threats to global public health. One of the major causative agents associated with high morbidity and mortality infections in hospitals worldwide is methicillin-resistant Staphylococcus aureus. Therefore, there is a need to develop new antibacterial agents to treat these infections, and natural products are a rich source of them. In previous studies, we reported that lignan 3′-demethoxy-6-O-demethylisoguaiacin, isolated and characterized from L...

An overexpression screen of Toxoplasma gondii Rab-GTPases reveals distinct transport routes to the micronemes.

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Katrin Kremer

2013-03-01

Full Text Available The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.

Artemether resistance in vitro is linked to mutations in PfATP6 that also interact with mutations in PfMDR1 in travellers returning with Plasmodium falciparum infections.

KAUST Repository

Pillai, Dylan R; Lau, Rachel; Khairnar, Krishna; Lepore, Rosalba; Via, Allegra; Staines, Henry M; Krishna, Sanjeev

2012-01-01

BACKGROUND: Monitoring resistance phenotypes for Plasmodium falciparum, using in vitro growth assays, and relating findings to parasite genotype has proved particularly challenging for the study of resistance to artemisinins. METHODS: Plasmodium falciparum isolates cultured from 28 returning travellers diagnosed with malaria were assessed for sensitivity to artemisinin, artemether, dihydroartemisinin and artesunate and findings related to mutations in pfatp6 and pfmdr1. RESULTS: Resistance to artemether in vitro was significantly associated with a pfatp6 haplotype encoding two amino acid substitutions (pfatp6 A623E and S769N; (mean IC50 (95% CI) values of 8.2 (5.7 - 10.7) for A623/S769 versus 623E/769 N 13.5 (9.8 - 17.3) nM with a mean increase of 65%; p = 0.012). Increased copy number of pfmdr1 was not itself associated with increased IC50 values for artemether, but when interactions between the pfatp6 haplotype and increased copy number of pfmdr1 were examined together, a highly significant association was noted with IC50 values for artemether (mean IC50 (95% CI) values of 8.7 (5.9 - 11.6) versus 16.3 (10.7 - 21.8) nM with a mean increase of 87%; p = 0.0068). Previously described SNPs in pfmdr1 are also associated with differences in sensitivity to some artemisinins. CONCLUSIONS: These findings were further explored in molecular modelling experiments that suggest mutations in pfatp6 are unlikely to affect differential binding of artemisinins at their proposed site, whereas there may be differences in such binding associated with mutations in pfmdr1. Implications for a hypothesis that artemisinin resistance may be exacerbated by interactions between PfATP6 and PfMDR1 and for epidemiological studies to monitor emerging resistance are discussed.

Artemether resistance in vitro is linked to mutations in PfATP6 that also interact with mutations in PfMDR1 in travellers returning with Plasmodium falciparum infections.

KAUST Repository

Pillai, Dylan R

2012-04-27

BACKGROUND: Monitoring resistance phenotypes for Plasmodium falciparum, using in vitro growth assays, and relating findings to parasite genotype has proved particularly challenging for the study of resistance to artemisinins. METHODS: Plasmodium falciparum isolates cultured from 28 returning travellers diagnosed with malaria were assessed for sensitivity to artemisinin, artemether, dihydroartemisinin and artesunate and findings related to mutations in pfatp6 and pfmdr1. RESULTS: Resistance to artemether in vitro was significantly associated with a pfatp6 haplotype encoding two amino acid substitutions (pfatp6 A623E and S769N; (mean IC50 (95% CI) values of 8.2 (5.7 - 10.7) for A623/S769 versus 623E/769 N 13.5 (9.8 - 17.3) nM with a mean increase of 65%; p = 0.012). Increased copy number of pfmdr1 was not itself associated with increased IC50 values for artemether, but when interactions between the pfatp6 haplotype and increased copy number of pfmdr1 were examined together, a highly significant association was noted with IC50 values for artemether (mean IC50 (95% CI) values of 8.7 (5.9 - 11.6) versus 16.3 (10.7 - 21.8) nM with a mean increase of 87%; p = 0.0068). Previously described SNPs in pfmdr1 are also associated with differences in sensitivity to some artemisinins. CONCLUSIONS: These findings were further explored in molecular modelling experiments that suggest mutations in pfatp6 are unlikely to affect differential binding of artemisinins at their proposed site, whereas there may be differences in such binding associated with mutations in pfmdr1. Implications for a hypothesis that artemisinin resistance may be exacerbated by interactions between PfATP6 and PfMDR1 and for epidemiological studies to monitor emerging resistance are discussed.

Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2'')-Ia.

Science.gov (United States)

Smith, Clyde A; Toth, Marta; Bhattacharya, Monolekha; Frase, Hilary; Vakulenko, Sergei B

2014-06-01

The bifunctional acetyltransferase(6')-Ie-phosphotransferase(2'')-Ia [AAC(6')-Ie-APH(2'')-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2'')-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2'')-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2'')-IIa and APH(2'')-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2'')-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2'')-IIIa enzyme. In APH(2'')-Ia this GTP selectivity is governed by the presence of a `gatekeeper' residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2'')-Ia into a dual-specificity enzyme.

Microwave surface resistance of bulk YBa2Cu3O6+x material

Science.gov (United States)

Fathy, A.; Kalokitis, D.; Belohoubek, E.; Sundar, H. G. K.; Safari, A.

1988-10-01

Superconducting Y-Ba-Cu-O samples were prepared by conventional solid-state reaction. The microwave surface resistance of 1:2:3 compound superconductor material was measured in a special disk resonator structure at 10 GHz. At liquid-nitrogen temperatures the microwave surface resistance is comparable to that of Au. At lower temperature (~10 K) the surface resistance is an order of magnitude lower than that of Au at the same temperature.

Two closely related Rho GTPases, Cdc42 and RacA, of the en-dophytic fungus Epichloë festucae have contrasting roles for ROS production and symbiotic infection synchronized with the host plant.

Science.gov (United States)

Kayano, Yuka; Tanaka, Aiko; Takemoto, Daigo

2018-01-01

Epichloë festucae is an endophytic fungus which systemically colonizes temperate grasses to establish symbiotic associations. Maintaining symptomless infection is a key requirement for endophytes, a feature that distinguishes them from pathogenic fungi. While pathogenic fungi extend their hyphae by tip growth, hyphae of E. festucae systemically colonize the intercellular space of expanding host leaves via a unique mechanism of hyphal intercalary growth. This study reports that two homologous Rho GTPases, Cdc42 and RacA, have distinctive roles in the regulation of E. festucae growth in planta. Here we highlight the vital role of Cdc42 for intercalary hyphal growth, as well as involvement of RacA in regulation of hyphal network formation, and demonstrate the consequences of mutations in these genes on plant tissue infection. Functions of Cdc42 and RacA are mediated via interactions with BemA and NoxR respectively, which are expected components of the ROS producing NOX complex. Symbiotic defects found in the racA mutant were rescued by introduction of a Cdc42 with key amino acids substitutions crucial for RacA function, highlighting the significance of the specific interactions of these GTPases with BemA and NoxR for their functional differentiation in symbiotic infection.

The Role of RhoA, RhoB and RhoC GTPases in Cell Morphology, Proliferation and Migration in Human Cytomegalovirus (HCMV Infected Glioblastoma Cells

Directory of Open Access Journals (Sweden)

Melpomeni Tseliou

2016-01-01

Full Text Available Background/Aims: Rho GTPases are crucial regulators of the actin cytoskeleton, membrane trafficking and cell signaling and their importance in cell migration and invasion is well- established. The human cytomegalovirus (HCMV is a widespread pathogen responsible for generally asymptomatic and persistent infections in healthy people. Recent evidence indicates that HCMV gene products are expressed in over 90% of malignant type glioblastomas (GBM. In addition, the HCMV Immediate Early-1 protein (IE1 is expressed in >90% of tumors analyzed. Methods: RhoA, RhoB and RhoC were individually depleted in U373MG glioblastoma cells as well as U373MG cells stably expressing the HCMV IE1 protein (named U373MG-IE1 cells shRNA lentivirus vectors. Cell proliferation assays, migration as well as wound-healing assays were performed in uninfected and HCMV-infected cells. Results: The depletion of RhoA, RhoB and RhoC protein resulted in significant alterations in the morphology of the uninfected cells, which were further enhanced by the cytopathic effect caused by HCMV. Furthermore, in the absence or presence of HCMV, the knockdown of RhoB and RhoC proteins decreased the proliferation rate of the parental and the IE1-expressing glioblastoma cells, whereas the knockdown of RhoA protein in the HCMV infected cell lines restored their proliferation rate. In addition, wound healing assays in U373MG cells revealed that depletion of RhoA, RhoB and RhoC differentially reduced their migration rate, even in the presence or the absence of HCMV. Conclusion: Collectively, these data show for the first time a differential implication of Rho GTPases in morphology, proliferation rate and motility of human glioblastoma cells during HCMV infection, further supporting an oncomodulatory role of HCMV depending on the Rho isoforms' state.

Effect of (NaPO3)6 concentrations on corrosion resistance of plasma electrolytic oxidation coatings formed on AZ91D magnesium alloy

International Nuclear Information System (INIS)

Luo Haihe; Cai Qizhou; Wei Bokang; Yu Bo; Li Dingjun; He Jian; Liu Ze

2008-01-01

Different plasma electrolytic oxidation (PEO) coatings were prepared on AZ91D magnesium alloy in electrolytes containing various concentrations of (NaPO 3 ) 6 . The morphologies, chemical compositions and corrosion resistance of the PEO coatings were characterized by environmental scanning electron microscopy (ESEM), X-ray diffractometer (XRD), energy dispersive analysis of X-rays (EDAX), potentiodynamic polarization and electrochemical impedance spectroscopy (EIS) test. The results showed that the PEO coatings were mainly composed of MgO, Mg 2 SiO 4 , MgAl 2 O 4 and amorphous compounds. As the (NaPO 3 ) 6 concentrations increased from 0 to 10 g/l, the thickness and surface roughness of the coatings approximately linearly increased; the MgO and Mg 2 SiO 4 phase increased within the concentration range of 0-3 and 0-5 g/l, and then decreased within the range of 3-10 and 5-10 g/l, respectively, while the MgAl 2 O 4 phase gradually decreased. Moreover, the corrosion resistance of the coatings increased within the range of 0-5 g/l and then decreased within the range of 5-10 g/l. The best corrosion resistance coating was obtained in electrolyte containing 5 g/l (NaPO 3 ) 6 , it had the most compact microstructure. Besides, a reasonable equivalent circuit was established, and the fitting results were consistent with the results of the EIS test

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

Over-expression of a Rab family GTPase from phreatophyte Prosopis juliflora confers tolerance to salt stress on transgenic tobacco.

Science.gov (United States)

George, Suja; Parida, Ajay

2011-03-01

Plant growth and productivity are adversely affected by various abiotic and biotic stress factors. In our previous study, we used Prosopis juliflora, an abiotic stress tolerant tree species of Fabaceae, as a model plant system for isolating genes functioning in abiotic stress tolerance. Here we report the isolation and characterization of a Rab family GTPase from P. juliflora (Pj Rab7) and the ability of this gene to confer salt stress tolerance in transgenic tobacco. Northern analysis for Pj Rab7 in P. juliflora leaf tissue revealed up-regulation of this gene under salt stress under the concentrations and time points analyzed. Pj Rab7 transgenic tobacco lines survived better under conditions of 150 mM NaCl stress compared to control un-transformed plants. Pj Rab7 transgenic plants were found to accumulate more sodium than control plants during salt stress. The results of our studies could be used as a starting point for generation of crop plants tolerant to abiotic stress.

Identification and Characterization of a Chloroplast-Targeted Obg GTPase in Dendrobium officinale.

Science.gov (United States)

Chen, Ji; Deng, Feng; Deng, Mengsheng; Han, Jincheng; Chen, Jianbin; Wang, Li; Yan, Shen; Tong, Kai; Liu, Fan; Tian, Mengliang

2016-12-01

Bacterial homologous chloroplast-targeted Obg GTPases (ObgCs) belong to the plant-typical Obg group, which is involved in diverse physiological processes during chloroplast development. However, the evolutionarily conserved function of ObgC in plants remains elusive and requires further investigation. In this study, we identified DoObgC from an epiphytic plant Dendrobium officinale and demonstrated the characteristics of DoObgC. Sequence analysis indicated that DoObgC is highly conserved with other plant ObgCs, which contain the chloroplast transit peptide (cTP), Obg fold, G domain, and OCT regions. The C terminus of DoObgC lacking the chloroplast-targeting cTP region, DoObgC Δ1-160 , showed strong similarity to ObgE and other bacterial Obgs. Overexpression of DoObgC Δ1-160 in Escherichia coli caused slow cell growth and an increased number of elongated cells. This phenotype was consistent with the phenotype of cells overexpressing ObgE. Furthermore, the expression of recombinant DoObgC Δ1-160 enhanced the cell persistence of E. coli to streptomycin. Results of transient expression assays revealed that DoObgC was localized to chloroplasts. Moreover, we demonstrated that DoObgC could rescue the embryotic lethal phenotype of the Arabidopsis obgc-t mutant, suggesting that DoObgC is a functional homolog to Arabidopsis AtObgC in D. officinale. Gene expression profiles showed that DoObgC was expressed in leaf-specific and light-dependent patterns and that DoObgC responded to wounding treatments. Our previous and present studies reveal that ObgC has an evolutionarily conserved role in ribosome biogenesis to adapt chloroplast development to the environment.

Interleukin-6 is associated with chronic hyperglycemia and insulin resistance in patients after acute pancreatitis.

Science.gov (United States)

Gillies, Nicola; Pendharkar, Sayali A; Asrani, Varsha M; Mathew, Juby; Windsor, John A; Petrov, Maxim S

2016-01-01

Diabetes is a pervasive disease, with a mounting prevalence and burden on health care systems. Under this collective term of diabetes falls diabetes after diseases of the exocrine pancreas, a condition which was previously under-recognised and often mislabeled as type 2 diabetes mellitus and is now increasingly acknowledged as a stand-alone entity. However, there is a paucity of clinical studies investigating the underlying pathophysiology of diabetes after acute pancreatitis, the most frequent disease of the pancreas. This study aimed to investigate the role of adipocytokines in glucose metabolism after acute pancreatitis. This was a cross-sectional follow-up study of a patient cohort diagnosed with acute pancreatitis. Fasting venous blood samples were collected to analyse markers of glucose metabolism (fasting blood glucose, haemoglobin A1c, homeostasis model assessment (HOMA-IR) as a measure of insulin resistance) and adypocytokines (adiponectin, interleukin-6, leptin, monocyte chemoattractant protein-1, retinol binding protein-4, resistin, and tumor necrosis factor-α). Participants were categorized into two groups: normoglycemia after acute pancreatitis and chronic hyperglycemia after acute pancreatitis (CHAP). Binary logistic regression and linear regression analyses were used to investigate the association between each of the adipocytokines and markers of glucose metabolism. Potential confounders were adjusted for in multivariate analyses. A total of 83 patients with acute pancreatitis were included, of whom 19 developed CHAP. Interleukin-6 was significantly associated with CHAP in both unadjusted and adjusted models (p = 0.030 and p = 0.018, respectively). Further, it was also significantly associated with HOMA-IR in both unadjusted and adjusted models (p = 0.029 and p = 0.037, respectively). Other adipocytokines were not significantly associated with markers of glucose metabolism. Interleukin-6 appears to be implicated in the development of chronic

Are Sewage Treatment Plants Promoting Antibiotic Resistance?

Science.gov (United States)

1. Introduction 1.1. How bacteria exhibit resistance 1.1.1. Resistance to -lactams 1.1.2. Resistance to sulphonamides and trimethoprim 1.1.3. Resistance to macrolides 1.1.4. Resistance to fluoroquinolones 1.1.5. Resistance to tetracyclines 1.1.6. Resistance to nitroimidaz...

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.

Negative differential resistance and switch behavior of T-BxNy (x, y = 5, 6, 11) molecular junctions

Science.gov (United States)

Wang, Shi-Liang; Yang, Chuan-Lu; Wang, Mei-Shan; Ma, Xiao-Guang; Xin, Jian-Guo

2017-05-01

The electronic transport properties of T-BxNy (x, y = 5, 6, 11) molecular junction are investigated based on first-principle density functional theory and non-equilibrium Green's function method. Strong negative differential resistance (NDR) behavior is observed for T-B5N6 molecule under negative and positive bias voltages, with an obvious switch effect for T-B6N5. However, only small NDR is shown for the complex of the two molecules. The projected device density of states, the spatial distribution of molecular orbitals, and the effect of transmission spectra under various bias voltages on the electronic transport properties are analyzed. The obvious effect of bias voltage on the changes in the electronic distribution of frontier molecular orbitals is responsible for the NDR or switch behavior. Therefore, different functional molecular devices can be obtained with different structures of T-BxNy.

Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

Science.gov (United States)

Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

2015-01-01

ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can

Enhanced Macrophage M1 Polarization and Resistance to Apoptosis Enable Resistance to Plague.

Science.gov (United States)

Pachulec, Emilia; Abdelwahed Bagga, Rym Ben; Chevallier, Lucie; O'Donnell, Hope; Guillas, Chloé; Jaubert, Jean; Montagutelli, Xavier; Carniel, Elisabeth; Demeure, Christian E

2017-09-15

Susceptibility to infection is in part genetically driven, and C57BL/6 mice resist various pathogens through the proinflammatory response of their M1 macrophages (MPs). However, they are susceptible to plague. It has been reported elsewhere that Mus spretus SEG mice resist plague and develop an immune response characterized by a strong recruitment of MPs. The responses of C57BL/6 and SEG MPs exposed to Yersinia pestis in vitro were examined. SEG MPs exhibit a stronger bactericidal activity with higher nitric oxide production, a more proinflammatory polarized cytokine response, and a higher resistance to Y. pestis-induced apoptosis. This response was not specific to Y. pestis and involved a reduced sensitivity to M2 polarization/signal transducer and activator of transcription 6 activation and inhibition of caspase 8. The enhanced M1 profile was inducible in C57BL/6 MPs in vitro, and when transferred to susceptible C57BL/6 mice, these MPs significantly increased survival of bubonic plague. MPs can develop an enhanced functional profile beyond the prototypic M1, characterized by an even more potent proinflammatory response coordinated with resistance to killing. This programming plays a key role in the plague-resistance phenotype and may be similarly significant in other highly lethal infections, suggesting that orienting the MP response may represent a new therapeutic approach. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Scanning velocity influence on microstructure, microhardness and wear resistance performance of laser deposited Ti6Al4V/TiC composite

CSIR Research Space (South Africa)

Mahamood, RM

2013-09-01

Full Text Available Ti6Al4V is the most widely used titanium alloy in the aerospace industry because of its excellent properties. However, the wear resistance behaviour of this material is not very impressive and surface damage occurs in applications involving contact...

Preliminary study on the corrosion resistance, antibacterial activity and cytotoxicity of selective-laser-melted Ti6Al4V-xCu alloys.

Science.gov (United States)

Guo, Sai; Lu, Yanjin; Wu, Songquan; Liu, Lingling; He, Mengjiao; Zhao, Chaoqian; Gan, Yiliang; Lin, Junjie; Luo, Jiasi; Xu, Xiongcheng; Lin, Jinxin

2017-03-01

In this study, a series of Cu-bearing Ti6Al4V-xCu (x=0, 2, 4, 6wt%) alloys (shorten by Ti6Al4V, 2C, 4C, and 6C, respectively.) with antibacterial function were successfully fabricated by selective laser melting (SLM) technology with mixed spherical powders of Cu and Ti6Al4V for the first time. In order to systematically investigate the effects of Cu content on the microstructure, phase constitution, corrosion resistance, antibacterial properties and cytotoxicity of SLMed Ti6Al4V-xCu alloys, experiments including XRD, SEM-EDS, electrochemical measurements, antibacterial tests and cytotoxicity tests were conducted with comparison to SLMed Ti6Al4V alloy (Ti6Al4V). Microstructural observations revealed that Cu had completely fused into the Ti6Al4V alloy, and presented in the form of Ti 2 Cu phase at ambient temperature. With Cu content increase, the density of the alloy gradually decreased, and micropores were obviously found in the alloy. Electrochemical measurements showed that corrosion resistance of Cu-bearing alloys were stronger than Cu-free alloy. Antibacterial tests demonstrated that 4C and 6C alloys presented strong and stable antibacterial property against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) compared to the Ti6Al4V and 2C alloy. In addition, similar to the Ti6Al4V alloy, the Cu-bearing alloys also exerted good cytocompatibility to the Bone Marrow Stromal Cells (BMSCs) from Sprague Dawley (SD) rats. Based on those results, the preliminary study verified that it was feasible to fabricated antibacterial Ti6Al4V-xCu alloys direct by SLM processing mixed commercial Ti6Al4V and Cu powder. Copyright © 2016 Elsevier B.V. All rights reserved.

Emergence of fluoroquinolone resistance among drug resistant tuberculosis patients at a tertiary care facility in Karachi, Pakistan.

Science.gov (United States)

Zaidi, Syed Mohammad Asad; Haseeb, Abdul; Habib, Shifa Salman; Malik, Amyn; Khowaja, Saira; SaifUllah, Nausheen; Rizvi, Nadeem

2017-07-25

Pakistan is classified as one of the high multi-drug resistant tuberculosis (MDR-TB) burden countries. A poorly regulated private sector, over-prescription of antibiotics and self-medication has led to augmented rates of drug-resistance in the country. Pakistan's first national anti-tuberculosis drug resistance survey identified high prevalence of fluoroquinolone resistance among MDR-TB patients. Further institutional evidence of fluoroquinolone drug-resistance can support re-evaluation of treatment regimens as well as invigorate efforts to control antibiotic resistance in the country. In this study, data for drug-susceptibility testing (DST) was retrospectively analyzed for a total of 133 patients receiving MDR-TB treatment at the Chest Department of Jinnah Postgraduate Medical Center, Karachi, Pakistan. Frequency analyses for resistance patterns was carried out and association of fluoroquinolone (ofloxacin) resistance with demographics and past TB treatment category were assessed. Within first-line drugs, resistance to isoniazid was detected in 97.7% of cases, followed by rifampicin (96.9%), pyrazinamide (86.4%), ethambutol (69.2%) and streptomycin (64.6%). Within second-line drugs, ofloxacin resistance was detected in 34.6% of cases. Resistance to ethionamide and amikacin was 2.3% and 1.6%, respectively. Combined resistance of oflaxacin and isoniazid was detected in 33.9% of cases. Age, gender and past TB treatment category were not significantly associated with resistance to ofloxacin. Fluoroquinolone resistance was observed in an alarmingly high proportion of MDR-TB cases. Our results suggest caution in their use for empirical management of MDR-TB cases and recommended treatment regimens for MDR-TB may require re-evaluation. Greater engagement of private providers and stringent pharmacy regulations are urgently required.

Experimental investigation on tribological behaviours of PA6, PA6 ...

Indian Academy of Sciences (India)

... wear resistance. Further, the thermal stability of Al2O3 and graphite particles was studied ... no significant improvement in wear resistance, the co-efficient of friction and heat generation. Keywords. ... wear tests for aramid, carbon and glass fibre composites run- ...... PA6/30wt% Al2O3 composites were determined from the.

Suppressing miRNA-15a/-16 expression by interleukin-6 enhances drug-resistance in myeloma cells

Directory of Open Access Journals (Sweden)

Chang Hong

2011-09-01

Full Text Available Abstract The bone marrow microenvironment facilitates the survival, differentiation, and proliferation of myeloma (MM cells. This study identified that microRNA-15a and -16 expressions tightly correlated with proliferation and drug sensitivity of MM cells. miRNA-15a/-16 expression in MM cells was significantly increased after treatment with cytotoxic agents. The interaction of bone marrow stromal cells (BMSC with MM cells resulted in decreased miRNA-15a/-16 expression and promoted the survival of the MM cells. Interleukin-6 (IL-6 produced by BMSCs suppressed the expression of miRNA-15a and 16 in a time- and dose- dependent pattern, with the suppression on miRNA-15a being more significant than on miRNA-16. miRNA-15a-transfected MM cells were found to be arrested in G1/S checkpoint, and the transfected MM cells had decreased growth and survival. In conclusion, our data suggest that via suppressing miRNA-15a and -16 expressions, IL-6 secreted by BMSCs promotes drug-resistance in myeloma cells.