Dissecting the Molecular Mechanism of RhoC GTPase Expression in the Normal and Malignant Breast

DTIC Science & Technology

2011-09-01

and cancer. Nature reviews 2, 133-142. Valastyan, S., Reinhardt, F ., Benaich, N., Calogrias, D., Szasz, A.M., Wang, Z.C., Brock, J.E., Richardson...prostate cancer progression. Cancer cell 17, 443-454.   R ho C F IS H Normal SUM149 SUM190A Figure 1 75 75 75 0 0 0 0 200 R ea ds (R P K M ) R...RhoC A B C D Figure 3 0 5 10 15 20 25 HME MCF7 SUM149 MDA-MB-231 Fo ld E nr ic hm en t o f p 65 B in di ng p65 (-2834) p65 (-2259) p65 (-6) A B

Biochemical magnetic resonance imaging of knee articular cartilage: T1rho and T2 mapping as cartilage degeneration biomarkers.

PubMed

Le, Jenna; Peng, Qi; Sperling, Karen

2016-11-01

Osteoarthritis (OA) is a disease whose hallmark is the degeneration of articular cartilage. There is a worsening epidemic of OA in the United States today, with considerable economic costs. In order to develop more effective treatments for OA, noninvasive biomarkers that permit early diagnosis and treatment monitoring are necessary. T1rho and T2 mapping are two magnetic resonance imaging techniques that have shown great promise as noninvasive biomarkers of cartilage degeneration. Each of the two techniques is endowed with advantages and disadvantages: T1rho can discern earlier biochemical changes of OA than T2 mapping, while T2 mapping is more widely available and can be incorporated into existing imaging protocols in a more time-efficient manner than T1rho. Both techniques have been applied in numerous instances to study how cartilage is affected by OA risk factors, such as age and exercise. Additionally, both techniques have been repeatedly applied to the study of posttraumatic OA in patients with torn anterior cruciate ligaments. © 2016 New York Academy of Sciences.

Small GTPase Tc10 and its homologue RhoT induce N-WASP-mediated long process formation and neurite outgrowth.

PubMed

Abe, Tomoyuki; Kato, Masayoshi; Miki, Hiroaki; Takenawa, Tadaomi; Endo, Takeshi

2003-01-01

Rho family small GTPases regulate multiple cellular functions through reorganization of the actin cytoskeleton. Among them, Cdc42 and Tc10 induce filopodia or peripheral processes in cultured cells. We have identified a member of the family, designated as RhoT, which is closely related to Tc10. Tc10 was highly expressed in muscular tissues and brain and remarkably induced during differentiation of C2 skeletal muscle cells and neuronal differentiation of PC12 and N1E-115 cells. On the other hand, RhoT was predominantly expressed in heart and uterus and induced during neuronal differentiation of N1E-115 cells. Tc10 exogenously expressed in fibroblasts generated actin-filament-containing peripheral processes longer than the Cdc42-formed filopodia, whereas RhoT produced much longer and thicker processes containing actin filaments. Furthermore, both Tc10 and RhoT induced neurite outgrowth in PC12 and N1E-115 cells, but Cdc42 did not do this by itself. Tc10 and RhoT as well as Cdc42 bound to the N-terminal CRIB-motif-containing portion of N-WASP and activated N-WASP to induce Arp2/3-complex-mediated actin polymerization. The formation of peripheral processes and neurites by Tc10 and RhoT was prevented by the coexpression of dominant-negative mutants of N-WASP. Thus, N-WASP is essential for the process formation and neurite outgrowth induced by Tc10 and RhoT. Neuronal differentiation of PC12 and N1E-115 cells induced by dibutyryl cyclic AMP and by serum starvation, respectively, was prevented by dominant-negative Cdc42, Tc10 and RhoT. Taken together, all these Rho family proteins are required for neuronal differentiation, but they exert their functions differentially in process formation and neurite extension. Consequently, N-WASP activated by these small GTPases mediates neuronal differentiation in addition to its recently identified role in glucose uptake.

Inherited pain: sodium channel Nav1.7 A1632T mutation causes erythromelalgia due to a shift of fast inactivation.

PubMed

Eberhardt, Mirjam; Nakajima, Julika; Klinger, Alexandra B; Neacsu, Cristian; Hühne, Kathrin; O'Reilly, Andrias O; Kist, Andreas M; Lampe, Anne K; Fischer, Kerstin; Gibson, Jane; Nau, Carla; Winterpacht, Andreas; Lampert, Angelika

2014-01-24

Inherited erythromelalgia (IEM) causes debilitating episodic neuropathic pain characterized by burning in the extremities. Inherited "paroxysmal extreme pain disorder" (PEPD) differs in its clinical picture and affects proximal body areas like the rectal, ocular, or jaw regions. Both pain syndromes have been linked to mutations in the voltage-gated sodium channel Nav1.7. Electrophysiological characterization shows that IEM-causing mutations generally enhance activation, whereas mutations leading to PEPD alter fast inactivation. Previously, an A1632E mutation of a patient with overlapping symptoms of IEM and PEPD was reported (Estacion, M., Dib-Hajj, S. D., Benke, P. J., Te Morsche, R. H., Eastman, E. M., Macala, L. J., Drenth, J. P., and Waxman, S. G. (2008) NaV1.7 Gain-of-function mutations as a continuum. A1632E displays physiological changes associated with erythromelalgia and paroxysmal extreme pain disorder mutations and produces symptoms of both disorders. J. Neurosci. 28, 11079-11088), displaying a shift of both activation and fast inactivation. Here, we characterize a new mutation of Nav1.7, A1632T, found in a patient suffering from IEM. Although transfection of A1632T in sensory neurons resulted in hyperexcitability and spontaneous firing of dorsal root ganglia (DRG) neurons, whole-cell patch clamp of transfected HEK cells revealed that Nav1.7 activation was unaltered by the A1632T mutation but that steady-state fast inactivation was shifted to more depolarized potentials. This is a characteristic normally attributed to PEPD-causing mutations. In contrast to the IEM/PEPD crossover mutation A1632E, A1632T failed to slow current decay (i.e. open-state inactivation) and did not increase resurgent currents, which have been suggested to contribute to high-frequency firing in physiological and pathological conditions. Reduced fast inactivation without increased resurgent currents induces symptoms of IEM, not PEPD, in the new Nav1.7 mutation, A1632T. Therefore

Topographical Variation of Human Femoral Articular Cartilage Thickness, T1rho and T2 Relaxation Times Is Related to Local Loading during Walking.

PubMed

Van Rossom, Sam; Wesseling, Mariska; Van Assche, Dieter; Jonkers, Ilse

2018-01-01

Objective Early detection of degenerative changes in the cartilage matrix composition is essential for evaluating early interventions that slow down osteoarthritis (OA) initiation. T1rho and T2 relaxation times were found to be effective for detecting early changes in proteoglycan and collagen content. To use these magnetic resonance imaging (MRI) methods, it is important to document the topographical variation in cartilage thickness, T1rho and T2 relaxation times in a healthy population. As OA is partially mechanically driven, the relation between these MRI-based parameters and localized mechanical loading during walking was investigated. Design MR images were acquired in 14 healthy adults and cartilage thickness and T1rho and T2 relaxation times were determined. Experimental gait data was collected and processed using musculoskeletal modeling to identify weight-bearing zones and estimate the contact force impulse during gait. Variation of the cartilage properties (i.e., thickness, T1rho, and T2) over the femoral cartilage was analyzed and compared between the weight-bearing and non-weight-bearing zone of the medial and lateral condyle as well as the trochlea. Results Medial condyle cartilage thickness was correlated to the contact force impulse ( r = 0.78). Lower T1rho, indicating increased proteoglycan content, was found in the medial weight-bearing zone. T2 was higher in all weight-bearing zones compared with the non-weight-bearing zones, indicating lower relative collagen content. Conclusions The current results suggest that medial condyle cartilage is adapted as a long-term protective response to localized loading during a frequently performed task and that the weight-bearing zone of the medial condyle has superior weight bearing capacities compared with the non-weight-bearing zones.

RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA2 Activation.

PubMed

Wu, Xiangbing; Walker, Chandler L; Lu, Qingbo; Wu, Wei; Eddelman, Daniel B; Parish, Jonathan M; Xu, Xiao-Ming

2017-11-01

Activation of RhoA/Rho kinase leads to growth cone collapse and neurite retraction. Although RhoA/Rho kinase inhibition has been shown to improve axon regeneration, remyelination and functional recovery, its role in neuronal cell death remains unclear. To determine whether RhoA/Rho kinase played a role in neuronal death after injury, we investigated the relationship between RhoA/Rho kinase and cytosolic phospholipase A 2 (cPLA 2 ), a lipase that mediates inflammation and cell death, using an in vitro neuronal death model and an in vivo contusive spinal cord injury model performed at the 10th thoracic (T10) vertebral level. We found that co-administration of TNF-α and glutamate induced spinal neuron death, and activation of RhoA, Rho kinase and cPLA 2 . Inhibition of RhoA, Rho kinase and cPLA 2 significantly reduced TNF-α/glutamate-induced cell death by 33, 52 and 43 %, respectively (p < 0.001). Inhibition of RhoA and Rho kinase also significantly downregulated cPLA 2 activation by 66 and 60 %, respectively (p < 0.01). Furthermore, inhibition of RhoA and Rho kinase reduced the release of arachidonic acid, a downstream substrate of cPLA 2 . The immunofluorescence staining showed that ROCK 1 or ROCK 2 , two isoforms of Rho kinase, was co-localized with cPLA 2 in neuronal cytoplasm. Interestingly, co-immunoprecipitation (Co-IP) assay showed that ROCK 1 or ROCK 2 bonded directly with cPLA 2 and phospho-cPLA 2 . When the Rho kinase inhibitor Y27632 was applied in mice with T10 contusion injury, it significantly decreased cPLA 2 activation and expression and reduced injury-induced apoptosis at and close to the lesion site. Taken together, our results reveal a novel mechanism of RhoA/Rho kinase-mediated neuronal death through regulating cPLA 2 activation.

Deregulation of HEF1 Impairs M-Phase Progression by Disrupting the RhoA Activation Cycle

PubMed Central

Dadke, Disha; Jarnik, Michael; Pugacheva, Elena N.; Singh, Mahendra K.; Golemis, Erica A.

2006-01-01

The focal adhesion-associated signaling protein HEF1 undergoes a striking relocalization to the spindle at mitosis, but a function for HEF1 in mitotic signaling has not been demonstrated. We here report that overexpression of HEF1 leads to failure of cells to progress through cytokinesis, whereas depletion of HEF1 by small interfering RNA (siRNA) leads to defects earlier in M phase before cleavage furrow formation. These defects can be explained mechanistically by our determination that HEF1 regulates the activation cycle of RhoA. Inactivation of RhoA has long been known to be required for cytokinesis, whereas it has recently been determined that activation of RhoA at the entry to M phase is required for cellular rounding. We find that increased HEF1 sustains RhoA activation, whereas depleted HEF1 by siRNA reduces RhoA activation. Furthermore, we demonstrate that chemical inhibition of RhoA is sufficient to reverse HEF1-dependent cellular arrest at cytokinesis. Finally, we demonstrate that HEF1 associates with the RhoA-GTP exchange factor ECT2, an orthologue of the Drosophila cytokinetic regulator Pebble, providing a direct means for HEF1 control of RhoA. We conclude that HEF1 is a novel component of the cell division control machinery and that HEF1 activity impacts division as well as cell attachment signaling events. PMID:16394104

17 CFR 240.15b7-3T - Operational capability in a Year 2000 environment.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 17 Commodity and Securities Exchanges 3 2012-04-01 2012-04-01 false Operational capability in a Year 2000 environment. 240.15b7-3T Section 240.15b7-3T Commodity and Securities Exchanges SECURITIES... § 240.15b7-3T Operational capability in a Year 2000 environment. (a) This section applies to every...

17 CFR 240.15b7-3T - Operational capability in a Year 2000 environment.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 17 Commodity and Securities Exchanges 3 2011-04-01 2011-04-01 false Operational capability in a Year 2000 environment. 240.15b7-3T Section 240.15b7-3T Commodity and Securities Exchanges SECURITIES... § 240.15b7-3T Operational capability in a Year 2000 environment. (a) This section applies to every...

17 CFR 240.15b7-3T - Operational capability in a Year 2000 environment.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 17 Commodity and Securities Exchanges 3 2013-04-01 2013-04-01 false Operational capability in a Year 2000 environment. 240.15b7-3T Section 240.15b7-3T Commodity and Securities Exchanges SECURITIES... § 240.15b7-3T Operational capability in a Year 2000 environment. (a) This section applies to every...

17 CFR 240.15b7-3T - Operational capability in a Year 2000 environment.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 17 Commodity and Securities Exchanges 4 2014-04-01 2014-04-01 false Operational capability in a Year 2000 environment. 240.15b7-3T Section 240.15b7-3T Commodity and Securities Exchanges SECURITIES... § 240.15b7-3T Operational capability in a Year 2000 environment. (a) This section applies to every...

17 CFR 240.15b7-3T - Operational capability in a Year 2000 environment.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Operational capability in a Year 2000 environment. 240.15b7-3T Section 240.15b7-3T Commodity and Securities Exchanges SECURITIES... § 240.15b7-3T Operational capability in a Year 2000 environment. (a) This section applies to every...

Insertion of an SVA-E retrotransposon into the CASP8 gene is associated with protection against prostate cancer

PubMed Central

Stacey, Simon N.; Kehr, Birte; Gudmundsson, Julius; Zink, Florian; Jonasdottir, Aslaug; Gudjonsson, Sigurjon A.; Sigurdsson, Asgeir; Halldorsson, Bjarni V.; Agnarsson, Bjarni A.; Benediktsdottir, Kristrun R.; Aben, Katja K.H.; Vermeulen, Sita H.; Cremers, Ruben G.; Panadero, Angeles; Helfand, Brian T.; Cooper, Phillip R.; Donovan, Jenny L.; Hamdy, Freddie C.; Jinga, Viorel; Okamoto, Ichiro; Jonasson, Jon G.; Tryggvadottir, Laufey; Johannsdottir, Hrefna; Kristinsdottir, Anna M.; Masson, Gisli; Magnusson, Olafur T.; Iordache, Paul D.; Helgason, Agnar; Helgason, Hannes; Sulem, Patrick; Gudbjartsson, Daniel F.; Kong, Augustine; Jonsson, Eirikur; Barkardottir, Rosa B.; Einarsson, Gudmundur V.; Rafnar, Thorunn; Thorsteinsdottir, Unnur; Mates, Ioan N.; Neal, David E.; Catalona, William J.; Mayordomo, José I.; Kiemeney, Lambertus A.; Thorleifsson, Gudmar; Stefansson, Kari

2016-01-01

Transcriptional and splicing anomalies have been observed in intron 8 of the CASP8 gene (encoding procaspase-8) in association with cutaneous basal-cell carcinoma (BCC) and linked to a germline SNP rs700635. Here, we show that the rs700635[C] allele, which is associated with increased risk of BCC and breast cancer, is protective against prostate cancer [odds ratio (OR) = 0.91, P = 1.0 × 10−6]. rs700635[C] is also associated with failures to correctly splice out CASP8 intron 8 in breast and prostate tumours and in corresponding normal tissues. Investigation of rs700635[C] carriers revealed that they have a human-specific short interspersed element-variable number of tandem repeat-Alu (SINE-VNTR-Alu), subfamily-E retrotransposon (SVA-E) inserted into CASP8 intron 8. The SVA-E shows evidence of prior activity, because it has transduced some CASP8 sequences during subsequent retrotransposition events. Whole-genome sequence (WGS) data were used to tag the SVA-E with a surrogate SNP rs1035142[T] (r2 = 0.999), which showed associations with both the splicing anomalies (P = 6.5 × 10−32) and with protection against prostate cancer (OR = 0.91, P = 3.8 × 10−7). PMID:26740556

Template based protein structure modeling by global optimization in CASP11.

PubMed

Joo, Keehyoung; Joung, InSuk; Lee, Sun Young; Kim, Jong Yun; Cheng, Qianyi; Manavalan, Balachandran; Joung, Jong Young; Heo, Seungryong; Lee, Juyong; Nam, Mikyung; Lee, In-Ho; Lee, Sung Jong; Lee, Jooyoung

2016-09-01

For the template-based modeling (TBM) of CASP11 targets, we have developed three new protein modeling protocols (nns for server prediction and LEE and LEER for human prediction) by improving upon our previous CASP protocols (CASP7 through CASP10). We applied the powerful global optimization method of conformational space annealing to three stages of optimization, including multiple sequence-structure alignment, three-dimensional (3D) chain building, and side-chain remodeling. For more successful fold recognition, a new alignment method called CRFalign was developed. It can incorporate sensitive positional and environmental dependence in alignment scores as well as strong nonlinear correlations among various features. Modifications and adjustments were made to the form of the energy function and weight parameters pertaining to the chain building procedure. For the side-chain remodeling step, residue-type dependence was introduced to the cutoff value that determines the entry of a rotamer to the side-chain modeling library. The improved performance of the nns server method is attributed to successful fold recognition achieved by combining several methods including CRFalign and to the current modeling formulation that can incorporate native-like structural aspects present in multiple templates. The LEE protocol is identical to the nns one except that CASP11-released server models are used as templates. The success of LEE in utilizing CASP11 server models indicates that proper template screening and template clustering assisted by appropriate cluster ranking promises a new direction to enhance protein 3D modeling. Proteins 2016; 84(Suppl 1):221-232. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

RhoA orchestrates glycolysis for Th2 cell differentiation and allergic airway inflammation

PubMed Central

Yang, Jun-Qi; Kalim, Khalid W.; Li, Yuan; Zhang, Shuangmin; Hinge, Ashwini; Filippi, Marie-Dominique; Zheng, Yi; Guo, Fukun

2015-01-01

Background Mitochondrial metabolism is known to be important for T cell activation. However, its involvement in effector T cell differentiation has just begun to gain attention. Importantly, how metabolic pathways are integrated with T cell activation and effector cell differentiation and function remains largely unknown. Objective We sought to test our hypothesis that RhoA GTPase orchestrates glycolysis for Th2 cell differentiation and Th2-mediated allergic airway inflammation. Methods Conditional RhoA-deficient mice were generated by crossing RhoAflox/flox mice with CD2-Cre transgenic mice. Effects of RhoA on Th2 differentiation were evaluated by in vitro Th2-polarized culture conditions, and in vivo in ovalbumin (OVA)-induced allergic airway inflammation. Cytokines were measured by intracellular staining and ELISA. T cell metabolism was measured by Seahorse XF24 Analyzer and flow cytometry. Results Disruption of RhoA inhibited T cell activation and Th2 differentiation in vitro and prevented the development of allergic airway inflammation in vivo, with no effect on Th1 cells. RhoA deficiency in activated T cells led to multiple defects in metabolic pathways such as glycolysis and oxidative phosphorylation. Importantly, RhoA couples glycolysis to Th2 cell differentiation and allergic airway inflammation via regulating IL-4 receptor mRNA expression and Th2-specific signaling events. Finally, inhibition of Rho-associated protein kinase (ROCK), an immediate downstream effector of RhoA, blocked Th2 differentiation and allergic airway inflammation. Conclusion RhoA is a key component of the signaling cascades leading to Th2-differentiation and allergic airway inflammation, at least in part, through the control of T cell metabolism and via ROCK pathway. PMID:26100081

Challenging the state of the art in protein structure prediction: Highlights of experimental target structures for the 10th Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10.

PubMed

Kryshtafovych, Andriy; Moult, John; Bales, Patrick; Bazan, J Fernando; Biasini, Marco; Burgin, Alex; Chen, Chen; Cochran, Frank V; Craig, Timothy K; Das, Rhiju; Fass, Deborah; Garcia-Doval, Carmela; Herzberg, Osnat; Lorimer, Donald; Luecke, Hartmut; Ma, Xiaolei; Nelson, Daniel C; van Raaij, Mark J; Rohwer, Forest; Segall, Anca; Seguritan, Victor; Zeth, Kornelius; Schwede, Torsten

2014-02-01

For the last two decades, CASP has assessed the state of the art in techniques for protein structure prediction and identified areas which required further development. CASP would not have been possible without the prediction targets provided by the experimental structural biology community. In the latest experiment, CASP10, more than 100 structures were suggested as prediction targets, some of which appeared to be extraordinarily difficult for modeling. In this article, authors of some of the most challenging targets discuss which specific scientific question motivated the experimental structure determination of the target protein, which structural features were especially interesting from a structural or functional perspective, and to what extent these features were correctly reproduced in the predictions submitted to CASP10. Specifically, the following targets will be presented: the acid-gated urea channel, a difficult to predict transmembrane protein from the important human pathogen Helicobacter pylori; the structure of human interleukin (IL)-34, a recently discovered helical cytokine; the structure of a functionally uncharacterized enzyme OrfY from Thermoproteus tenax formed by a gene duplication and a novel fold; an ORFan domain of mimivirus sulfhydryl oxidase R596; the fiber protein gene product 17 from bacteriophage T7; the bacteriophage CBA-120 tailspike protein; a virus coat protein from metagenomic samples of the marine environment; and finally, an unprecedented class of structure prediction targets based on engineered disulfide-rich small proteins. Copyright © 2013 The Authors. Wiley Periodicals, Inc.

Challenging the state-of-the-art in protein structure prediction: Highlights of experimental target structures for the 10th Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10

PubMed Central

Kryshtafovych, Andriy; Moult, John; Bales, Patrick; Bazan, J. Fernando; Biasini, Marco; Burgin, Alex; Chen, Chen; Cochran, Frank V.; Craig, Timothy K.; Das, Rhiju; Fass, Deborah; Garcia-Doval, Carmela; Herzberg, Osnat; Lorimer, Donald; Luecke, Hartmut; Ma, Xiaolei; Nelson, Daniel C.; van Raaij, Mark J.; Rohwer, Forest; Segall, Anca; Seguritan, Victor; Zeth, Kornelius; Schwede, Torsten

2014-01-01

For the last two decades, CASP has assessed the state of the art in techniques for protein structure prediction and identified areas which required further development. CASP would not have been possible without the prediction targets provided by the experimental structural biology community. In the latest experiment, CASP10, over 100 structures were suggested as prediction targets, some of which appeared to be extraordinarily difficult for modeling. In this paper, authors of some of the most challenging targets discuss which specific scientific question motivated the experimental structure determination of the target protein, which structural features were especially interesting from a structural or functional perspective, and to what extent these features were correctly reproduced in the predictions submitted to CASP10. Specifically, the following targets will be presented: the acid-gated urea channel, a difficult to predict trans-membrane protein from the important human pathogen Helicobacter pylori; the structure of human interleukin IL-34, a recently discovered helical cytokine; the structure of a functionally uncharacterized enzyme OrfY from Thermoproteus tenax formed by a gene duplication and a novel fold; an ORFan domain of mimivirus sulfhydryl oxidase R596; the fibre protein gp17 from bacteriophage T7; the Bacteriophage CBA-120 tailspike protein; a virus coat protein from metagenomic samples of the marine environment; and finally an unprecedented class of structure prediction targets based on engineered disulfide-rich small proteins. PMID:24318984

Revisiting the Acanthamoeba species that form star-shaped cysts (genotypes T7, T8, T9, and T17): characterization of seven new Brazilian environmental isolates and phylogenetic inferences.

PubMed

Magliano, Ana C M; Teixeira, Marta M G; Alfieri, Silvia C

2012-01-01

Free-living amoebae of the genus Acanthamoeba are the agents of both opportunistic and non-opportunistic infections and are frequently isolated from the environment. Of the 17 genotypes (T1-T17) identified thus far, 4 (T7, T8, T9, and T17) accommodate the rarely investigated species of morphological group I, those that form large, star-shaped cysts. We report the isolation and characterization of 7 new Brazilian environmental Acanthamoeba isolates, all assigned to group I. Phylogenetic analyses based on partial (~1200 bp) SSU rRNA gene sequences placed the new isolates in the robustly supported clade composed of the species of morphological group I. One of the Brazilian isolates is closely related to A. comandoni (genotype T9), while the other 6, together with 2 isolates recently assigned to genotype T17, form a homogeneous, well-supported group (2·0% sequence divergence) that likely represents a new Acanthamoeba species. Thermotolerance, osmotolerance, and cytophatic effects, features often associated with pathogenic potential, were also examined. The results indicated that all 7 Brazilian isolates grow at temperatures up to 40°C, and resist under hyperosmotic conditions. Additionally, media conditioned by each of the new Acanthamoeba isolates induced the disruption of SIRC and HeLa cell monolayers.

The Jet Stream's Precursor of M7.7 Russia Earthquake on 2017/07/17

NASA Astrophysics Data System (ADS)

Wu, H. C.

2017-12-01

Before M>6.0 earthquakes occurred, jet stream in the epicenter area will interrupt or velocity flow lines cross. That meaning is that before earthquake happen, atmospheric pressure in high altitude suddenly dropped during 6 12 hours (Wu & Tikhonov, 2014; Wu et.al,2015). The 70 knots speed line in jet stream was crossed at the epicenter on 2017/07/13, and then M7.7 Russia earthquake happened on 2017/07/17. Lithosphere-atmosphere-ionosphere (LAI) coupling model may be explained this phenomenon : Ionization of the air produced by an increased emanation of radon at epicenter. The water molecules in the air react with these ions, and then release heat. The heat result in temperature rise and pressure drop in the air(Pulinets, Ouzounov, 2011), and then the speed line of jet stream was changed. ps.Russia earthquake:M7.7 2017-07-17 23:34:13 (UTC) 54.471°N 168.815°E 11.0 kmReference: H.C Wu, I.N. Tikhonov, 2014, "Jet streams anomalies as possible short-term precursors of earthquakes with M>6.0", Research in geophysics. H.C.Wu., Ivan N. Tikhonov, and Ariel R. Ćesped,2015, Multi-parametric analysis of earthquake precursors, Russ. J. Earth. Sci., 15, ES3002, doi:10.2205/2015ES000553 S Pulinets, D Ouzounov, 2011,"Lithosphere-Atmosphere-Ionosphere Coupling (LAIC) model-An unified concept for earthquake precursors validation", Journal of Asian Earth Sciences 41 (4), 371-382.

Ghrelin Inhibits the Differentiation of T Helper 17 Cells through mTOR/STAT3 Signaling Pathway

PubMed Central

Xu, Yanhui; Li, Ziru; Yin, Yue; Lan, He; Wang, Jun; Zhao, Jing; Feng, Juan; Li, Yin; Zhang, Weizhen

2015-01-01

Enhanced activity of interleukin 17 (IL-17) producing T helper 17 (Th17) cells plays an important role in autoimmune and inflammatory diseases. Significant loss of body weight and appetite is associated with chronic inflammation and immune activation, suggesting the cross talk between immune and neuroendocrine systems. Ghrelin has been shown to regulate the organism immune function. However, the effects of ghrelin on the differentiation of Th17 cells remain elusive. In the present study, we observed the enhanced differentiation of Th17 cells in spleens of growth hormone secretagogue receptor 1a (GHSR1a)-/- mice. Treatment of ghrelin repressed Th17 cell differentiation in a time- and concentration-dependent manner. Phosphorylation of mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) was increased in the spleens of GHSR1a-/- mice. Activation of mTOR signaling by injection of Cre-expressiong adenovirus into tuberous sclerosis complex 1 (TSC1) loxp/loxp mice increased the differentiation of Th17 cells in spleen, which was associated with an increment in the phosphorylation of STAT3. Activation of mTOR signaling by leucine or overexpression of p70 ribosome protein subunit 6 kinase 1 (S6K1) activated mTOR signaling in isolated T cells, while reversed the ghrelin-induced inhibition of iTh17 cell differentiation. In conclusion, mTOR mediates the inhibitory effect of ghrelin on the differentiation of Th17 cells by interacting with STAT3. PMID:25658305

The atypical structure and function of newborn arterial endothelium is mediated by Rho/Rho kinase signaling.

PubMed

Flavahan, Sheila; Flavahan, Nicholas A

2014-08-15

Endothelium of fetal or newborn arteries is atypical, displaying actin stress fibers and reduced nitric oxide (NO)-mediated dilatation. This study tested the hypothesis that Rho/Rho kinase signaling, which promotes endothelial stress fibers and inhibits endothelial dilatation, contributed to this phenotype. Carotid arteries were isolated from newborn [postnatal day 1 (P1)], P7, and P21 mice. Endothelial dilatation to acetylcholine (pressure myograph) was minimal at P1, increased at P7, and further increased at P21. Inhibition of Rho (C3 transferase) or Rho kinase (Y27632, fasudil) significantly increased dilatation to acetylcholine in P1 arteries but had no effect in P7 or P21 arteries. After inhibition of NO synthase (N(G)-nitro-l-arginine methyl ester), Rho kinase inhibition no longer increased acetylcholine responses in P1 arteries. Rho kinase inhibition did not affect dilatation to the NO donor DEA-NONOate. The endothelial actin cytoskeleton was labeled with phalloidin and visualized by laser-scanning microscopy. In P1 arteries, the endothelium had prominent transcytoplasmic stress fibers, whereas in P7 and P21 arteries, the actin fibers had a significantly reduced intensity and were restricted to cell borders. Phosphorylation of myosin light chains, a Rho kinase substrate, was highest in P1 endothelium and significantly reduced in P7 and P21 endothelium (laser-scanning microscopy). In P1 arteries, inhibition of Rho (C3 transferase) or Rho kinase (Y27632) significantly reduced the intensity of actin fibers, which were restricted to cell borders. Similarly, in P1 arteries, Rho inhibition significantly reduced endothelial levels of phosphorylated myosin light chains. These results indicate that the atypical function and morphology of newborn endothelium is mediated by Rho/Rho kinase signaling. Copyright © 2014 the American Physiological Society.

Manufacture of a 1.7m prototype of the GMT primary mirror segments

NASA Astrophysics Data System (ADS)

Martin, H. M.; Burge, J. H.; Miller, S. M.; Smith, B. K.; Zehnder, R.; Zhao, C.

2006-06-01

We have nearly completed the manufacture of a 1.7 m off-axis mirror as part of the technology development for the Giant Magellan Telescope. The mirror is an off-axis section of a 5.3 m f/0.73 parent paraboloid, making it roughly a 1:5 model of the outer 8.4 m GMT segment. The 1.7 m mirror will be the primary mirror of the New Solar Telescope at Big Bear Solar Observatory. It has a 2.7 mm peak-to-valley departure from the best-fit sphere, presenting a serious challenge in terms of both polishing and measurement. The mirror was polished with a stressed lap, which bends actively to match the local curvature at each point on the mirror surface, and works for asymmetric mirrors as well as symmetric aspheres. It was measured using a hybrid reflective-diffractive null corrector to compensate for the mirror's asphericity. Both techniques will be applied in scaled-up versions to the GMT segments.

Immunization with M2e-Displaying T7 Bacteriophage Nanoparticles Protects against Influenza A Virus Challenge

PubMed Central

Hashemi, Hamidreza; Pouyanfard, Somayeh; Bandehpour, Mojgan; Noroozbabaei, Zahra; Kazemi, Bahram; Saelens, Xavier; Mokhtari-Azad, Talat

2012-01-01

Considering the emergence of highly pathogenic influenza viruses and threat of worldwide pandemics, there is an urgent need to develop broadly-protective influenza vaccines. In this study, we demonstrate the potential of T7 bacteriophage-based nanoparticles with genetically fused ectodomain of influenza A virus M2 protein (T7-M2e) as a candidate universal flu vaccine. Immunization of mice with non-adjuvanted T7-M2e elicited M2e-specific serum antibody responses that were similar in magnitude to those elicited by M2e peptide administered in Freund’s adjuvant. Comparable IgG responses directed against T7 phage capsomers were induced following vaccination with wild type T7 or T7-M2e. T7-M2e immunization induced balanced amounts of IgG1 and IgG2a antibodies and these antibodies specifically recognized native M2 on the surface of influenza A virus-infected mammalian cells. The frequency of IFN-γ-secreting T cells induced by T7-M2e nanoparticles was comparable to those elicited by M2e peptide emulsified in Freund’s adjuvant. Emulsification of T7-M2e nanoparticles in Freund’s adjuvant, however, induced a significantly stronger T cell response. Furthermore, T7-M2e-immunized mice were protected against lethal challenge with an H1N1 or an H3N2 virus, implying the induction of hetero-subtypic immunity in our mouse model. T7-M2e-immunized mice displayed considerable weight loss and had significantly reduced viral load in their lungs compared to controls. We conclude that display of M2e on the surface of T7 phage nanoparticles offers an efficient and economical opportunity to induce cross-protective M2e-based immunity against influenza A. PMID:23029232

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

PubMed Central

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

2015-01-01

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

Looking at the bright side of the rho Ophiuchi dark cloud. Far infrared spectrophotometric observations of the rho Oph cloud with the ISO

NASA Astrophysics Data System (ADS)

Liseau, R.; White, G. J.; Larsson, B.; Sidher, S.; Olofsson, G.; Kaas, A.; Nordh, L.; Caux, E.; Lorenzetti, D.; Molinari, S.; Nisini, B.; Sibille, F.

1999-04-01

We present far infrared (45-195 mu m) spectrophotometric observations with the Iso-Lws of the active star forming rho Oph main cloud (L 1688). The [C ii] 158 mu m and [O i] 63 mu m lines were detected at each of the 33 positions observed, whereas the [O i] 145 mu m line was clearly seen toward twelve. The principal observational result is that the [C ii] 158 mu m line fluxes exhibit a clear correlation with projected distance from the dominant stellar source in the field (HD 147889). We interpret this in terms of Pdr-type emission from the surface layers of the rho Ophc. The observed [C ii] 158 mu m/[O i] 63 mu m flux ratios are larger than unity everywhere. A comparison of the [C ii] 158 mu m line emission and the Fir dust continuum fluxes yields estimates of the efficiency at which the gas in the cloud converts stellar to [C ii] 158 mu m photons (chi_ {_C II},>_{ ~ },0.5%). We first develop an empirical model, which provides us with a three dimensional view of the far and bright side of the dark rho Ophc, showing that the cloud surface towards the putative energy source is concave. This model also yields quantitative estimates of the incident flux of ultraviolet radiation (G_0 ~ , \\powten{1} - \\powten{2}) and of the degree of clumpiness/texture of the cloud surface (filling of the 80({') '} beam ~ ,0.2). Subsequently, we use theoretical models of Pdr s to derive the particle density, n(H), and the temperature structures, for T_gas and T_dust, in the surface layers of the rho Ophc. T_gas is relatively low, ~ ,60 K, but higher than T_dust ( ~ ,30 K), and densities are generally found within the interval (1-3) \\powten{4} cm(-3) . These Pdr models are moderately successful in explaining the Lws observations. They correctly predict the [O i] 63 mu m and [C ii] 158 mu m line intensities and the observed absence of any molecular line emission. The models do fail, however, to reproduce the observed small [O i] 63 mu m/[O i] 145 mu m ratios. We examine several possible

The Rho regulator Myosin IXb enables nonlymphoid tissue seeding of protective CD8+ T cells.

PubMed

Moalli, Federica; Ficht, Xenia; Germann, Philipp; Vladymyrov, Mykhailo; Stolp, Bettina; de Vries, Ingrid; Lyck, Ruth; Balmer, Jasmin; Fiocchi, Amleto; Kreutzfeldt, Mario; Merkler, Doron; Iannacone, Matteo; Ariga, Akitaka; Stoffel, Michael H; Sharpe, James; Bähler, Martin; Sixt, Michael; Diz-Muñoz, Alba; Stein, Jens V

2018-06-06

T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues (NLTs) with divergent topologies and confinement. How the T cell actomyosin cytoskeleton facilitates this task in distinct environments is incompletely understood. Here, we show that lack of Myosin IXb (Myo9b), a negative regulator of the small GTPase Rho, led to increased Rho-GTP levels and cell surface stiffness in primary T cells. Nonetheless, intravital imaging revealed robust motility of Myo9b -/- CD8 + T cells in lymphoid tissue and similar expansion and differentiation during immune responses. In contrast, accumulation of Myo9b -/- CD8 + T cells in NLTs was strongly impaired. Specifically, Myo9b was required for T cell crossing of basement membranes, such as those which are present between dermis and epidermis. As consequence, Myo9b -/- CD8 + T cells showed impaired control of skin infections. In sum, we show that Myo9b is critical for the CD8 + T cell adaptation from lymphoid to NLT surveillance and the establishment of protective tissue-resident T cell populations. © 2018 Moalli et al.

Association of CASP9, CASP10 gene polymorphisms and tea drinking with colorectal cancer risk in the Han Chinese population.

PubMed

Liu, He; Jiang, Xia; Zhang, Ming-wu; Pan, Yi-feng; Yu, Yun-xian; Zhang, Shan-chun; Ma, Xin-yuan; Li, Qi-long; Chen, Kun

2013-01-01

The initiators caspase-9 (CASP9) and caspase-10 (CASP10) are two key controllers of apoptosis and play important roles in carcinogenesis. This study aims to explore the association between CASPs gene polymorphisms and colorectal cancer (CRC) susceptibility in a population-based study. A two-stage designed population-based case-control study was carried out, including a testing set with 300 cases and 296 controls and a validation set with 206 cases and 845 controls. A total of eight tag selected single nucleotide polymorphisms (SNPs) in CASP9 and CASP10 were chosen based on HapMap and the National Center of Biotechnology Information (NCBI) datasets and genotyped by restriction fragment length polymorphism (RFLP) assay. Multivariate logistic regression models were applied to evaluate the association of SNPs with CRC risk. In the first stage, from eight tag SNPs, three polymorphisms rs4646077 (odds ratio (OR)(AA+AG): 0.654, 95% confidence interval (CI): 0.406-1.055; P=0.082), rs4233532 (OR(CC): 1.667, 95% CI: 0.967-2.876; OR(CT): 1.435, 95% CI: 0.998-2.063; P=0.077), and rs2881930 (OR(CC): 0.263, 95% CI: 0.095-0.728, P=0.036) showed possible association with CRC risk. However, none of the three SNPs, rs4646077 (OR(AA+AG): 1.233, 95% CI: 0.903-1.683), rs4233532 (OR(CC): 0.892, 95% CI: 0.640-1.243; OR(CT): 1.134, 95% CI: 0.897-1.433), and rs2881930 (OR(CC): 1.096, 95% CI: 0.620-1.938; OR(CT): 1.009, 95% CI: 0.801-1.271), remained significant with CRC risk in the validation set, even after stratification for different tumor locations (colon or rectum). In addition, never tea drinking was associated with a significantly increased risk of CRC in testing set together with validation set (OR: 1.755, 95% CI: 1.319-2.334). Our results found that polymorphisms of CASP9 and CASP10 genes may not contribute to CRC risk in Chinese population and thereby the large-scale case-control studies might be in consideration. In addition, tea drinking was a protective factor for CRC.

Association of CASP9, CASP10 gene polymorphisms and tea drinking with colorectal cancer risk in the Han Chinese population*

PubMed Central

Liu, He; Jiang, Xia; Zhang, Ming-wu; Pan, Yi-feng; Yu, Yun-xian; Zhang, Shan-chun; Ma, Xin-yuan; Li, Qi-long; Chen, Kun

2013-01-01

The initiators caspase-9 (CASP9) and caspase-10 (CASP10) are two key controllers of apoptosis and play important roles in carcinogenesis. This study aims to explore the association between CASPs gene polymorphisms and colorectal cancer (CRC) susceptibility in a population-based study. A two-stage designed population-based case-control study was carried out, including a testing set with 300 cases and 296 controls and a validation set with 206 cases and 845 controls. A total of eight tag selected single nucleotide polymorphisms (SNPs) in CASP9 and CASP10 were chosen based on HapMap and the National Center of Biotechnology Information (NCBI) datasets and genotyped by restriction fragment length polymorphism (RFLP) assay. Multivariate logistic regression models were applied to evaluate the association of SNPs with CRC risk. In the first stage, from eight tag SNPs, three polymorphisms rs4646077 (odds ratio (OR)AA+AG: 0.654, 95% confidence interval (CI): 0.406–1.055; P=0.082), rs4233532 (ORCC: 1.667, 95% CI: 0.967–2.876; ORCT: 1.435, 95% CI: 0.998–2.063; P=0.077), and rs2881930 (ORCC: 0.263, 95% CI: 0.095–0.728, P=0.036) showed possible association with CRC risk. However, none of the three SNPs, rs4646077 (ORAA+AG: 1.233, 95% CI: 0.903–1.683), rs4233532 (ORCC: 0.892, 95% CI: 0.640–1.243; ORCT: 1.134, 95% CI: 0.897–1.433), and rs2881930 (ORCC: 1.096, 95% CI: 0.620–1.938; ORCT: 1.009, 95% CI: 0.801–1.271), remained significant with CRC risk in the validation set, even after stratification for different tumor locations (colon or rectum). In addition, never tea drinking was associated with a significantly increased risk of CRC in testing set together with validation set (OR: 1.755, 95% CI: 1.319–2.334). Our results found that polymorphisms of CASP9 and CASP10 genes may not contribute to CRC risk in Chinese population and thereby the large-scale case-control studies might be in consideration. In addition, tea drinking was a protective factor for CRC. PMID

α4+β7hiCD4+ Memory T cells Harbor Most Th-17 cells and are Preferentially Infected During Acute SIV Infection

PubMed Central

Kader, Muhamuda; Wang, Xiaolei; Piatak, Michael; Lifson, Jeffrey; Roederer, Mario; Veazey, Ronald; Mattapallil, Joseph J.

2009-01-01

HIV/SIV are thought to infect minimally activated CD4+ T cells after viral entry. Not much is known about why SIV selectively targets these cells. Here we show that CD4+ T cells that express high levels of the α4β7 heterodimer are preferentially infected very early during the course of SIV infection. At day 2–4 post infection, α4+β7hiCD4+ T cells had ∼ 5x more SIV-gag DNA than β7−CD4+ T cells. α4+β7hiCD4+ T cells displayed a predominantly central memory (CD45RA−CD28+CCR7+) and resting (CD25−CD69−HLA-DR−Ki-67−) phenotype. Though the expression of detectable CCR5 was variable on α4+β7hi and β7−CD4+ T cells, both CCR5+ and CCR5− subsets of α4+β7hi and β7−CD4+ T cells were found to express sufficient levels of CCR5 mRNA suggesting that both these subsets could be efficiently infected by SIV. In line with this, we found similar levels of SIV infection in β7−CD4+CCR5+ and β7−CD4+CCR5− T cells. α4β7hiCD4+ T cells were found to harbor most Th-17 cells that were significantly depleted during acute SIV infection. Taken together, our results show that resting memory α4+β7hiCD4+ T cells in blood are preferentially depleted during acute SIV infection, and the loss of these cells alters the balance between Th-17 and Th-1 responses thereby contributing to disease pathogenesis. PMID:19571800

Evaluation of 3D-Jury on CASP7 models.

PubMed

Kaján, László; Rychlewski, Leszek

2007-08-21

3D-Jury, the structure prediction consensus method publicly available in the Meta Server http://meta.bioinfo.pl/, was evaluated using models gathered in the 7th round of the Critical Assessment of Techniques for Protein Structure Prediction (CASP7). 3D-Jury is an automated expert process that generates protein structure meta-predictions from sets of models obtained from partner servers. The performance of 3D-Jury was analysed for three aspects. First, we examined the correlation between the 3D-Jury score and a model quality measure: the number of correctly predicted residues. The 3D-Jury score was shown to correlate significantly with the number of correctly predicted residues, the correlation is good enough to be used for prediction. 3D-Jury was also found to improve upon the competing servers' choice of the best structure model in most cases. The value of the 3D-Jury score as a generic reliability measure was also examined. We found that the 3D-Jury score separates bad models from good models better than the reliability score of the original server in 27 cases and falls short of it in only 5 cases out of a total of 38. We report the release of a new Meta Server feature: instant 3D-Jury scoring of uploaded user models. The 3D-Jury score continues to be a good indicator of structural model quality. It also provides a generic reliability score, especially important for models that were not assigned such by the original server. Individual structure modellers can also benefit from the 3D-Jury scoring system by testing their models in the new instant scoring feature http://meta.bioinfo.pl/compare_your_model_example.pl available in the Meta Server.

Evaluation of 3D-Jury on CASP7 models

PubMed Central

Kaján, László; Rychlewski, Leszek

2007-01-01

Background 3D-Jury, the structure prediction consensus method publicly available in the Meta Server , was evaluated using models gathered in the 7th round of the Critical Assessment of Techniques for Protein Structure Prediction (CASP7). 3D-Jury is an automated expert process that generates protein structure meta-predictions from sets of models obtained from partner servers. Results The performance of 3D-Jury was analysed for three aspects. First, we examined the correlation between the 3D-Jury score and a model quality measure: the number of correctly predicted residues. The 3D-Jury score was shown to correlate significantly with the number of correctly predicted residues, the correlation is good enough to be used for prediction. 3D-Jury was also found to improve upon the competing servers' choice of the best structure model in most cases. The value of the 3D-Jury score as a generic reliability measure was also examined. We found that the 3D-Jury score separates bad models from good models better than the reliability score of the original server in 27 cases and falls short of it in only 5 cases out of a total of 38. We report the release of a new Meta Server feature: instant 3D-Jury scoring of uploaded user models. Conclusion The 3D-Jury score continues to be a good indicator of structural model quality. It also provides a generic reliability score, especially important for models that were not assigned such by the original server. Individual structure modellers can also benefit from the 3D-Jury scoring system by testing their models in the new instant scoring feature available in the Meta Server. PMID:17711571

RhoA and RhoC are involved in stromal cell-derived factor-1-induced cell migration by regulating F-actin redistribution and assembly.

PubMed

Luo, Jixian; Li, Dingyun; Wei, Dan; Wang, Xiaoguang; Wang, Lan; Zeng, Xianlu

2017-12-01

Stromal cell-derived factor-1 (SDF-1) signaling is important to the maintenance and progression of T-cell acute lymphoblastic leukemia by inducing chemotaxis migration. To identify the mechanism of SDF-1 signaling in the migration of T-ALL, Jurkat acute lymphoblastic leukemia cells were used. Results showed that SDF-1 induces Jurkat cell migration by F-actin redistribution and assembly, which is dependent on Rho activity. SDF-1 induced RhoA and RhoC activation, as well as reactive oxygen species (ROS) production, which was inhibited by Rho inhibitor. The Rho-dependent ROS production led to subsequent cytoskeleton redistribution and assembly in the process of migration. Additionally, RhoA and RhoC were involved in SDF-1-induced Jurkat cell migration. Taken together, we found a SDF-1/CXCR4-RhoA and RhoC-ROS-cytoskeleton pathway that regulates Jurkat cell migration in response to SDF-1. This work will contribute to a clearer insight into the migration mechanism of acute lymphoblastic leukemia.

A T3 and T7 Recombinant Phage Acquires Efficient Adsorption and a Broader Host Range

PubMed Central

Lin, Tiao-Yin; Lo, Yi-Haw; Tseng, Pin-Wei; Chang, Shun-Fu; Lin, Yann-Tsyr; Chen, Ton-Seng

2012-01-01

It is usually thought that bacteriophage T7 is female specific, while phage T3 can propagate on male and female Escherichia coli. We found that the growth patterns of phages T7M and T3 do not match the above characteristics, instead showing strain dependent male exclusion. Furthermore, a T3/7 hybrid phage exhibits a broader host range relative to that of T3, T7, as well as T7M, and is able to overcome the male exclusion. The T7M sequence closely resembles that of T3. T3/7 is essentially T3 based, but a DNA fragment containing part of the tail fiber gene 17 is replaced by the T7 sequence. T3 displays inferior adsorption to strains tested herein compared to T7. The T3 and T7 recombinant phage carries altered tail fibers and acquires better adsorption efficiency than T3. How phages T3 and T7 recombine was previously unclear. This study is the first to show that recombination can occur accurately within only 8 base-pair homology, where four-way junction structures are identified. Genomic recombination models based on endonuclease I cleavages at equivalent and nonequivalent sites followed by strand annealing are proposed. Retention of pseudo-palindromes can increase recombination frequency for reviving under stress. PMID:22347414

A T3 and T7 recombinant phage acquires efficient adsorption and a broader host range.

PubMed

Lin, Tiao-Yin; Lo, Yi-Haw; Tseng, Pin-Wei; Chang, Shun-Fu; Lin, Yann-Tsyr; Chen, Ton-Seng

2012-01-01

It is usually thought that bacteriophage T7 is female specific, while phage T3 can propagate on male and female Escherichia coli. We found that the growth patterns of phages T7M and T3 do not match the above characteristics, instead showing strain dependent male exclusion. Furthermore, a T3/7 hybrid phage exhibits a broader host range relative to that of T3, T7, as well as T7M, and is able to overcome the male exclusion. The T7M sequence closely resembles that of T3. T3/7 is essentially T3 based, but a DNA fragment containing part of the tail fiber gene 17 is replaced by the T7 sequence. T3 displays inferior adsorption to strains tested herein compared to T7. The T3 and T7 recombinant phage carries altered tail fibers and acquires better adsorption efficiency than T3. How phages T3 and T7 recombine was previously unclear. This study is the first to show that recombination can occur accurately within only 8 base-pair homology, where four-way junction structures are identified. Genomic recombination models based on endonuclease I cleavages at equivalent and nonequivalent sites followed by strand annealing are proposed. Retention of pseudo-palindromes can increase recombination frequency for reviving under stress.

Expression of the T Helper 17-Associated Cytokines IL-17A and IL-17F in Asthma and COPD

PubMed Central

Doe, Camille; Bafadhel, Mona; Siddiqui, Salman; Desai, Dhananjay; Mistry, Vijay; Rugman, Paul; McCormick, Margaret; Woods, Joanne; May, Richard; Sleeman, Matthew A.; Anderson, Ian K.

2010-01-01

Background: Asthma and COPD are characterized by airway dysfunction and inflammation. Neutrophilic airway inflammation is a common feature of COPD and is recognized in asthma, particularly in severe disease. The T helper (Th) 17 cytokines IL-17A and IL-17F have been implicated in the development of neutrophilic airway inflammation, but their expression in asthma and COPD is uncertain. Methods: We assessed IL-17A and IL-17F expression in the bronchial submucosa from 30 subjects with asthma, 10 ex-smokers with mild to moderate COPD, and 27 nonsmoking and 14 smoking control subjects. Sputum IL-17 concentration was measured in 165 subjects with asthma and 27 with COPD. Results: The median (interquartile range) IL-17A cells/mm2 submucosa was increased in mild to moderate asthma (2.1 [2.4]) compared with healthy control subjects (0.4 [2.8]) but not in severe asthma (P = .04). In COPD, IL-17A+ cells/mm2 submucosa were increased (0.5 [3.7]) compared with nonsmoking control subjects (0 [0]) but not compared with smoking control subjects (P = .046). IL-17F+ cells/mm2 submucosa were increased in severe asthma (2.7 [3.6]) and mild to moderate asthma (1.6 [1.0]) compared with healthy controls subjects (0.7 [1.4]) (P = .001) but was not increased in subjects with COPD. IL-17A and IL-17F were not associated with increased neutrophilic inflammation, but IL-17F was correlated with the submucosal eosinophil count (rs = 0.5, P = .005). The sputum IL-17 concentration in COPD was increased compared with asthma (2 [0-7] pg/mL vs 0 [0-2] pg/mL, P < .0001) and was correlated with post-bronchodilator FEV1% predicted (r = −0.5, P = .008) and FEV1/FVC (r = −0.4, P = .04). Conclusions: Our findings support a potential role for the Th17 cytokines IL-17A and IL-17F in asthma and COPD, but do not demonstrate a relationship with neutrophilic inflammation. PMID:20538817

UV-induced somatic mutations elicit a functional T cell response in the YUMMER1.7 mouse melanoma model.

PubMed

Wang, Jake; Perry, Curtis J; Meeth, Katrina; Thakral, Durga; Damsky, William; Micevic, Goran; Kaech, Susan; Blenman, Kim; Bosenberg, Marcus

2017-07-01

Human melanomas exhibit relatively high somatic mutation burden compared to other malignancies. These somatic mutations may produce neoantigens that are recognized by the immune system, leading to an antitumor response. By irradiating a parental mouse melanoma cell line carrying three driver mutations with UVB and expanding a single-cell clone, we generated a mutagenized model that exhibits high somatic mutation burden. When inoculated at low cell numbers in immunocompetent C57BL/6J mice, YUMMER1.7 (Yale University Mouse Melanoma Exposed to Radiation) regresses after a brief period of growth. This regression phenotype is dependent on T cells as YUMMER1.7 tumors grow significantly faster in immunodeficient Rag1 -/- mice and C57BL/6J mice depleted of CD4 and CD8 T cells. Interestingly, regression can be overcome by injecting higher cell numbers of YUMMER1.7, which results in tumors that grow without effective rejection. Mice that have previously rejected YUMMER1.7 tumors develop immunity against higher doses of YUMMER1.7 tumor challenge. In addition, escaping YUMMER1.7 tumors are sensitive to anti-CTLA-4 and anti-PD-1 therapy, establishing a new model for the evaluation of immune checkpoint inhibition and antitumor immune responses. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Binding and Translocation of Termination Factor Rho Studied at the Single-Molecule Level

PubMed Central

Koslover, Daniel J.; Fazal, Furqan M.; Mooney, Rachel A.; Landick, Robert; Block, Steven M.

2012-01-01

Rho termination factor is an essential hexameric helicase responsible for terminating 20–50% of all mRNA synthesis in E. coli. We used single- molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho- utilization (rut) site of the ? tR1 terminator. Our results are consistent with Rho complexes adopting two states, one that binds 57 ±2 nucleotides of RNA across all six of the Rho primary binding sites, and another that binds 85 ±2 nucleotides at the six primary sites plus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5′-to-3′ towards RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the rut site and RNAP. These findings lead to a general model for Rho binding and translocation, and establish a novel experimental approach that should facilitate additional single- molecule studies of RNA-binding proteins. PMID:22885804

Pion decay constant and the {rho}-meson mass at finite temperature in hidden local symmetry

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

Harada, M.; Shibata, A.

1997-06-01

We study the temperature dependence of the pion decay constant and {rho}-meson mass in the hidden local symmetry model at one loop. Using the standard imaginary time formalism, we include the thermal effect of the {rho} meson as well as that of the pion. We show that the pion gives a dominant contribution to the pion decay constant and the {rho}-meson contribution slightly decreases the critical temperature. The {rho}-meson pole mass increases as T{sup 4}/m{sub {rho}}{sup 2} at low temperature, dominated by the pion-loop effect. At high temperature, although the pion-loop effect decreases the {rho}-meson mass, the {rho}-loop contribution overcomesmore » the pion-loop contribution and the {rho}-meson mass increases with temperature. We also show that the conventional parameter a is stable as the temperature increases. {copyright} {ital 1997} {ital The American Physical Society}« less

N7-Methylguanine at position 46 (m7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network.

PubMed

Tomikawa, Chie; Yokogawa, Takashi; Kanai, Tamotsu; Hori, Hiroyuki

2010-01-01

N(7)-methylguanine at position 46 (m(7)G46) in tRNA is produced by tRNA (m(7)G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (DeltatrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the DeltatrmB strain and the lack of the m(7)G46 modification in tRNA(Phe) were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the DeltatrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m(7)G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m(1)G37, suggesting that the m(7)G46 positively affects their formations. Although the lack of the m(7)G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNA(Phe), they cause a decrease in melting temperature of class I tRNA and degradation of tRNA(Phe) and tRNA(Ile). (35)S-Met incorporation into proteins revealed that protein synthesis in DeltatrmB cells is depressed above 70 degrees C. At 80 degrees C, the DeltatrmB strain exhibits a severe growth defect. Thus, the m(7)G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m(7)G46 modification supports introduction of other modifications.

Artifacts correction for T1rho imaging with constant amplitude spin-lock

NASA Astrophysics Data System (ADS)

Chen, Weitian

2017-01-01

T1rho imaging with constant amplitude spin-lock is prone to artifacts in the presence of B1 RF and B0 field inhomogeneity. Despite significant technological progress, improvements on the robustness of constant amplitude spin-lock are necessary in order to use it for routine clinical practice. This work proposes methods to simultaneously correct for B1 RF and B0 field inhomogeneity in constant amplitude spin-lock. By setting the maximum B1 amplitude of the excitation adiabatic pulses equal to the expected constant amplitude spin-lock frequency, the spins become aligned along the effective field throughout the spin-lock process. This results in T1rho-weighted images free of artifacts, despite the spatial variation of the effective field caused by B1 RF and B0 field inhomogeneity. When the pulse is long, the relaxation effect during the adiabatic half passage may result in a non-negligible error in the mono-exponential relaxation model. A two-acquisition approach is presented to solve this issue. Simulation, phantom, and in-vivo scans demonstrate the proposed methods achieve superior image quality compared to existing methods, and that the two-acquisition method is effective in resolving the relaxation effect during the adiabatic half passage.

Effects of CASP5 gene overexpression on angiogenesis of HMEC-1 cells.

PubMed

Li, Haiyan; Li, Yuzhen; Cai, Limin; Bai, Bingxue; Wang, Yanhua

2015-01-01

The efficacy of gene overexpression of CASP5, a caspase family member, in angiogenesis in vitro and its mechanisms were clarified. Human full-length CASP5 gene was delivered into human microvascular endothelial HMEC-1 cells by recombinant lentivirus. The infection was estimated by green fluorescent protein. MTT method was used to analyze the efficacy of gene overexpression in cell proliferation ability, and Matrigel was used to estimate its effects in angiogenesis ability of cells. Meanwhile, Western blot was used to analyze the effects of CASP5 gene overexpression on the expression levels of angpt-1, angpt-2, Tie2 and VEGF-1 in the cells, which were signaling pathway factors related to angiogenesis. Recombinant lentivirus containing human full-length CASP5 gene was packed and purified successfully, with virus titer of 1×10(8) TU/ml. The recombinant lentivirus was used to infect HMEC-1 cells with MOI of 1, leading to a cell infection rate of 100%. There were no significant effects of CASP5 gene overexpression on both cell proliferation ability and the expression level of angpt-1. Meanwhile, expressions of angpt-2 and VEGF-1 were both enhanced, while Tie2 expression was inhibited. Results indicated that CASP5 gene overexpression promoted angiogenesis of HMEC-1 cells. CASP5 gene overexpression significantly promoted angiogenesis ability of HMEC-1 cells, which was probably achieved by inhibiting angpt-1/Tie2 and promoting VEGF-1 signal pathway.

Long non-coding RNA CASP5 promotes the malignant phenotypes of human glioblastoma multiforme.

PubMed

Zhou, Yali; Dai, Wei; Wang, Handong; Pan, Hao; Wang, Qiang

2018-06-12

Long non-coding RNAs (lncRNAs) have been demonstrated to be intensively involved in the development of various carcinomas, including glioblastoma multiforme (GBM). However, only a few of them have been well characterized. LncRNA CASP5 have been found to be up-regulated in GBM tissues compared with normal tissues in a microarray-based lncRNA profiling study. In the present study, we further explored the biological role of lncRNA CASP5 in GBM. We examined the expression level of lncRNA CASP5 in GBM tissues as well as GBM cell lines. CCK-8 assay, flow cytometric analysis, western blotting, orthotopic GBM model as well as transwell assay were performed to investigate the biological role of CASP5. We observed that lncRNA CASP5 was highly expressed in GBM tissues and cell lines. Knockdown of CASP5 greatly inhibited GBM proliferation and resulted in G1 cell cycle arrest along with higher apoptosis ratios in vitro and in vivo, while overexpression led to the opposite phenomenon. Furthermore, the migration and invasion ability of GBM cells were significantly decreased after CASP5 down-regulation, while increased migration and invasion can be observed after CASP5 up-regulation. We demonstrate for the first time the potential oncogenic role of lncRNA CASP5 which may be helpful for identifying novel therapeutic targets in GBM. Copyright © 2018 Elsevier Inc. All rights reserved.

Interaction between mDia1 and ROCK in Rho-induced migration and adhesion of human dental pulp cells.

PubMed

Cheng, L; Xu, J; Qian, Y Y; Pan, H Y; Yang, H; Shao, M Y; Cheng, R; Hu, T

2017-01-01

To investigate the effects of mammalian homologue of Drosophila diaphanous-1(mDia1) and Rho-associated coiled-coil-containing protein kinase (ROCK) on the migration and adhesion of dental pulp cells (DPCs). Lysophosphatidic acid (LPA) was used to activate Rho signalling. mDia1 and ROCK were inhibited by short interfering RNA and the specific inhibitor, Y-27632, respectively. The migration of DPCs was assessed using the transwell migration assay and scratch test. Formation of cytoskeleton and focal adhesions(FAs) was observed by confocal laser scanning microscopy. Cell adhesion and spreading assays were performed. Phosphorylation of focal adhesion kinase (FAK) and paxillin was detected by Western blotting, and the bands were analysed using Adobe Photoshop CS5 software. All experiments were performed at least three times, and data were analysed with one-way anova and a post hoc test. LPA-triggered activation of Rho and inhibition of ROCK significantly increased the cell migration rate. Cell migration was inhibited by silencing mDia1. mDia1 silencing and ROCK inhibition suppressed the LPA-induced formation of the cytoskeleton, FA and phosphorylation of FAK and paxillin. Inhibition of ROCK or mDia1 facilitated early cell adhesion and spreading; by contrast, the combined inhibition of ROCK and mDia1 neutralized these effects. mDia1 promoted RhoA-induced migration of DPCs, but ROCK had an opposite effect. Both mDia1 and ROCK participated in cytoskeleton formation and adhesion of DPCs. The interactions between mDia1 and ROCK might influence dental pulp repair by determining the migration and adhesion of DPCs. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

In Vivo Fluorescence Confocal Microscopy to Investigate the Role of RhoC in Inflammatory Breast Cancer

DTIC Science & Technology

2005-04-01

5084. 6. Colpaert CG, Vermeulen PB, Benoy I, Soubry A, van Roy F, van Beest P, Goovaerts G, Dirix LY, van Dam P, Fox SB, Harris AL, van MarckEA...cancer. Cancer Res 1999, 59: 5079-5084. 17 14. Colpaert CG, Vermeulen PB, Benoy I, Soubry A, van Roy F, van Beest P, Goovaerts G, Dirix LY, van Dam P...Ohira S, Feng Y, Nikaido T, Konishi I: Up- regulation of small GTPases, RhoA and RhoC, is associated with tumor progression in ovarian carcinoma. Lab

Quality of life (QOL) among community dwelling older people in Taiwan measured by the CASP-19, an index to capture QOL in old age.

PubMed

Wu, Tai-Yin; Chie, Wei-Chu; Kuo, Kuan-Liang; Wong, Wai-Kuen; Liu, Jen-Pei; Chiu, Shih-Ting; Cheng, Yeung-Hung; Netuveli, Gopal; Blane, David

2013-01-01

There was no existing scale in Mandarin Chinese to specifically measure QOL in old age. We aimed to validate a Chinese Taiwan version of the CASP-19 (control, autonomy, self-realization, pleasure), a QOL questionnaire, in Taiwan. The existing CASP-19 Cantonese version was modified into Chinese Taiwan version and pilot tested. Data were then gathered from 699 older people. Score distribution, exploratory and confirmatory factor structure, reliability and clinical validity of the CASP-19 and its shortened version, the CASP-12, were examined. The mean age of the participants was 75.5 (standard deviation (SD) 6.5), and half (49.5%) were female. The mean CASP-19 score was 38.2 (range 11-56; SD 7.1), lower than that of Western countries. Exploratory factor analysis revealed an additional factor, 'participation' (CASPP-19). There was satisfactory internal consistency (Cronbach's α 0.63-0.85) for the subscales, except for the control domain. For the 19-item scale, the first order five-domain model (CASPP-19) yielded the best fit. For the CASP-12, first and second order original CASP-12 models performed equally well. There was an inverse relationship between the CASP total scores and frailty, chronic diseases, depressive disorders, living alone and fall events in the past 12months, supporting good clinical validity for all versions of the CASP scale (CASP-19, CASPP-19, original and new CASP-12). The original CASP-12 may be presently the best choice for use in China, Taiwan or other Mandarin-speaking populations due to its conciseness and model parsimony. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

RhoA/ROCK pathway activity is essential for the correct localization of the germ plasm mRNAs in zebrafish embryos.

PubMed

Miranda-Rodríguez, Jerónimo Roberto; Salas-Vidal, Enrique; Lomelí, Hilda; Zurita, Mario; Schnabel, Denhi

2017-01-01

Zebrafish germ plasm is composed of mRNAs such as vasa and nanos and of proteins such as Bucky ball, all of which localize symmetrically in four aggregates at the distal region of the first two cleavage furrows. The coordination of actin microfilaments, microtubules and kinesin is essential for the correct localization of the germ plasm. Rho-GTPases, through their effectors, coordinate cytoskeletal dynamics. We address the participation of RhoA and its effector ROCK in germ plasm localization during the transition from two- to eight-cell embryos. We found that active RhoA is enriched along the cleavage furrow during the first two division cycles, whereas ROCK localizes at the distal region of the cleavage furrows in a similar pattern as the germ plasm mRNAs. Specific inhibition of RhoA and ROCK affected microtubules organization at the cleavage furrow; these caused the incorrect localization of the germ plasm mRNAs. The incorrect localization of the germ plasm led to a dramatic change in the number of germ cells during the blastula and 24hpf embryo stages without affecting any other developmental processes. We demonstrate that the Rho/ROCK pathway is intimately related to the determination of germ cells in zebrafish embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

RhoA Regulation of Cardiomyocyte Differentiation

PubMed Central

Kaarbø, Mari; Crane, Denis I.; Murrell, Wayne G.

2013-01-01

Earlier findings from our laboratory implicated RhoA in heart developmental processes. To investigate factors that potentially regulate RhoA expression, RhoA gene organisation and promoter activity were analysed. Comparative analysis indicated strict conservation of both gene organisation and coding sequence of the chick, mouse, and human RhoA genes. Bioinformatics analysis of the derived promoter region of mouse RhoA identified putative consensus sequence binding sites for several transcription factors involved in heart formation and organogenesis generally. Using luciferase reporter assays, RhoA promoter activity was shown to increase in mouse-derived P19CL6 cells that were induced to differentiate into cardiomyocytes. Overexpression of a dominant negative mutant of mouse RhoA (mRhoAN19) blocked this cardiomyocyte differentiation of P19CL6 cells and led to the accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiac α-actin. Taken together, these findings indicate a fundamental role for RhoA in the differentiation of cardiomyocytes. PMID:23935420

Involvement of Rho kinase in the pathogenesis of acute pulmonary embolism-induced polystyrene microspheres in rats.

PubMed

Toba, M; Nagaoka, T; Morio, Y; Sato, K; Uchida, K; Homma, N; Takahashi, K

2010-03-01

Acute pulmonary embolism (PE) is a life-threatening disease, and several vasoconstrictors, including endothelin-1 (ET-1), play a key role in vasoconstriction and hypoxemia during the development of PE. Rho kinase is activated by various vasoconstrictors resulting in vascular contraction and remodeling. Recent evidence has revealed an important role of Rho kinase in the pathogenesis of systemic and pulmonary vascular diseases. However, contribution of Rho kinase in PE remains unclear. We thus investigated the role of Rho kinase in the PE rat model induced by intrajugular administration of polystyrene microspheres (mean diameter, 26 microm). At 6 h following the administration of microspheres (1.5 ml/kg), right ventricular systolic pressure (RVSP) was higher in the PE than in the control rats (15.8 +/- 1.6 vs. 32.9 +/- 7.5 mmHg). Arterial oxygen tension was lower (92.3 +/- 12.5 vs. 66.0 +/- 17.7 Torr), and alveolar-arterial difference in oxygen partial pressure was higher (3.9 +/- 3.8 vs. 36.5 +/- 26.9 Torr) in the PE rats. Western blotting analysis revealed upregulation and downregulation in expression of vascular cell adhesion molecule-1 and endothelial nitric oxide synthase in lungs from the PE rats, respectively, and radioimmunoassay demonstrated an increase in plasma ET-1 levels. Lung Rho kinase alpha expression was greater in the PE rats. At 5 h following administration of microspheres (0.75 ml/kg), intravenous Rho kinase inhibitors HA1077 and Y27632 (3 mg/kg each) attenuated elevation of RVSP (22.0 +/- 3.7, 17.1 +/- 3.2, 14.3 +/- 2.6 mmHg, PE, PE+HA1077, PE+Y27632) and the severity of hypoxemia (66.3 +/- 16.2, 94.9 +/- 23.0, 89.1 +/- 8.5 Torr, PE, PE+HA1077, PE+Y27632) in the PE rats. These results suggest that pulmonary endothelial dysfunction and activation of Rho kinase may contribute to the potentiation of vasoconstriction and hypoxemia in the PE rats.

Evidence of Nematicity in K 0.8Fe 1.7Se 2

DOE PAGES

Duan, Chunruo; Yang, Junjie; Ye, Feng; ...

2015-12-11

We proposed that the superconducting state of K 0.8Fe 1.7Se 2 is phase separated from a non-superconducting magnetic state. These results from a recent neutron diffraction study on a single crystal of K 0.8Fe 1.7Se 2 provide evidence for a continuous transition between the I 4/m m m high temperature phase in which the Fe vacancies are randomly distributed and the I4/m vacancy ordered phase in the temperature range between T (C) and T (S). Upon cooling, the I 4/m phase becomes more populated, increasing the √5 X√5 X 1 superlattice structure, resulting in an enhancement of the (101) superlatticemore » peak. Moreover, the same temperature dependence is observed for the magnetic peak as well. Moreover, due to the Fe site splitting with the transition, its z-coordinate fluctuates, and so must the d xz and d y z orbitals. Finally, the orbital fluctuations couple to the magnetic ordering as seen here and may lead to a realization of nematic order in this system.« less

RhoA/Rho-Kinase in the Cardiovascular System.

PubMed

Shimokawa, Hiroaki; Sunamura, Shinichiro; Satoh, Kimio

2016-01-22

Twenty years ago, Rho-kinase was identified as an important downstream effector of the small GTP-binding protein, RhoA. Thereafter, a series of studies demonstrated the important roles of Rho-kinase in the cardiovascular system. The RhoA/Rho-kinase pathway is now widely known to play important roles in many cellular functions, including contraction, motility, proliferation, and apoptosis, and its excessive activity induces oxidative stress and promotes the development of cardiovascular diseases. Furthermore, the important role of Rho-kinase has been demonstrated in the pathogenesis of vasospasm, arteriosclerosis, ischemia/reperfusion injury, hypertension, pulmonary hypertension, and heart failure. Cyclophilin A is secreted by vascular smooth muscle cells and inflammatory cells and activated platelets in a Rho-kinase-dependent manner, playing important roles in a wide range of cardiovascular diseases. Thus, the RhoA/Rho-kinase pathway plays crucial roles under both physiological and pathological conditions and is an important therapeutic target in cardiovascular medicine. Recently, functional differences between ROCK1 and ROCK2 have been reported in vitro. ROCK1 is specifically cleaved by caspase-3, whereas granzyme B cleaves ROCK2. However, limited information is available on the functional differences and interactions between ROCK1 and ROCK2 in the cardiovascular system in vivo. Herein, we will review the recent advances about the importance of RhoA/Rho-kinase in the cardiovascular system. © 2016 American Heart Association, Inc.

Transcript expression and genetic variability analysis of caspases in breast carcinomas suggests CASP9 as the most interesting target.

PubMed

Brynychova, Veronika; Hlavac, Viktor; Ehrlichova, Marie; Vaclavikova, Radka; Nemcova-Furstova, Vlasta; Pecha, Vaclav; Trnkova, Marketa; Mrhalova, Marcela; Kodet, Roman; Vrana, David; Gatek, Jiri; Bendova, Marie; Vernerova, Zdenka; Kovar, Jan; Soucek, Pavel

2017-01-01

Apoptosis plays a critical role in cancer cell survival and tumor development. We provide a hypothesis-generating screen for further research by exploring the expression profile and genetic variability of caspases (2, 3, 7, 8, 9, and 10) in breast carcinoma patients. This study addressed isoform-specific caspase transcript expression and genetic variability in regulatory sequences of caspases 2 and 9. Gene expression profiling was performed by quantitative real-time PCR in tumor and paired non-malignant tissues of two independent groups of patients. Genetic variability was determined by high resolution melting, allelic discrimination, and sequencing analysis in tumor and peripheral blood lymphocyte DNA of the patients. CASP3 A+B and S isoforms were over-expressed in tumors of both patient groups. The CASP9 transcript was down-regulated in tumors of both groups of patients and significantly associated with expression of hormonal receptors and with the presence of rs4645978-rs2020903-rs4646034 haplotype in the CASP9 gene. Patients with a low intratumoral CASP9A/B isoform expression ratio (predicted to shift equilibrium towards anti-apoptotic isoform) subsequently treated with adjuvant chemotherapy had a significantly shorter disease-free survival than those with the high ratio (p=0.04). Inheritance of CC genotype of rs2020903 in CASP9 was associated with progesterone receptor expression in tumors (p=0.003). Genetic variability in CASP9 and expression of its splicing variants present targets for further study.

Differentially expressed proteins in ER+ MCF7 and ER- MDA- MB-231 human breast cancer cells by RhoGDI-α silencing and overexpression.

PubMed

Hooshmand, Somayeh; Ghaderi, Abbas; Yusoff, Khatijah; Thilakavathy, Karuppiah; Rosli, Rozita; Mojtahedi, Zahra

2014-01-01

The consequence of Rho GDP dissociation inhibitor alpha (RhoGDIα) activity on migration and invasion of estrogen receptor positive (ER+) and negative (ER-) breast cancer cells has not been studied using the proteomic approach. Changes in expression of RhoGDIα and other proteins interacting directly or indirectly with RhoGDIα in MCF7 and MDA-MB-231, with different metastatic potentials is of particular interest. ER+ MCF7 and ER- MDA-MB-231 cell lines were subjected to two-dimensional electrophoresis (2-DE) and spots of interest were identified by matrix-assisted laser desorption/ionization time of- flight/time- of-flight (MALDI-TOF/TOF) mass spectrometry (MS) analysis after downregulation of RhoGDIα using short interfering RNA (siRNA) and upregulated using GFP-tagged ORF clone of RhoGDIα. The results showed a total of 35 proteins that were either up- or down-regulated in these cells. Here we identifed 9 and 15 proteins differentially expressed with silencing of RhoGDIα in MCF-7 and the MDA-MB-231 cells, respectively. In addition, 10 proteins were differentially expressed in the upregulation of RhoGDIα in MCF7, while only one protein was identified in the upregulation of RhoGDIα in MDA-MB-231. Based on the biological functions of these proteins, the results revealed that proteins involved in cell migration are more strongly altered with RhoGDI-α activity. Although several of these proteins have been previously indicated in tumorigenesis and invasiveness of breast cancer cells, some ohave not been previously reported to be involved in breast cancer migration. Hence, these proteins may serve as useful candidate biomarkers for tumorigenesis and invasiveness of breast cancer cells. Future studies are needed to determine the mechanisms by which these proteins regulate cell migration. The combination of RhoGDIα with other potential biomarkers may be a more promising approach in the inhibition of breast cancer cell migration.

17 CFR 249.507 - Form 7-M, consent to service of process by an individual nonresident broker-dealer.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 17 Commodity and Securities Exchanges 3 2011-04-01 2011-04-01 false Form 7-M, consent to service... Forms for Statements Made in Connection With Exempt Tender Offers § 249.507 Form 7-M, consent to service... Federal Register citations affecting Form 7-M, see the List of CFR Sections Affected, which appears in the...

Quantification of M13 and T7 bacteriophages by TaqMan and SYBR green qPCR.

PubMed

Peng, Xiujuan; Nguyen, Alex; Ghosh, Debadyuti

2018-02-01

TaqMan and SYBR Green quantitative PCR (qPCR) methods were developed as DNA-based approaches to reproducibly enumerate M13 and T7 phages from phage display selection experiments individually and simultaneously. The genome copies of M13 and T7 phages were quantified by TaqMan or SYBR Green qPCR referenced against M13 and T7 DNA standard curves of known concentrations. TaqMan qPCR was capable of quantifying M13 and T7 phage DNA simultaneously with a detection range of 2.75*10 1 -2.75*10 8 genome copies(gc)/μL and 2.66*10 1 -2.66*10 8 genome copies(gc)/μL respectively. TaqMan qPCR demonstrated an efficient amplification efficiency (E s ) of 0.97 and 0.90 for M13 and T7 phage DNA, respectively. SYBR Green qPCR was ten-fold more sensitive than TaqMan qPCR, able to quantify 2.75-2.75*10 7 gc/μL and 2.66*10 1 -2.66*10 7 gc/μL of M13 and T7 phage DNA, with an amplification efficiency E s of 1.06 and 0.78, respectively. Due to its superior sensitivity, SYBR Green qPCR was used to enumerate M13 and T7 phage display clones selected against a cell line, and quantified titers demonstrated accuracy comparable to titers from traditional double-layer plaque assay. Compared to enzyme linked immunosorbent assay, both qPCR methods exhibited increased detection sensitivity and reproducibility. These qPCR methods are reproducible, sensitive, and time-saving to determine their titers and to quantify a large number of phage samples individually or simultaneously, thus avoiding the need for time-intensive double-layer plaque assay. These findings highlight the attractiveness of qPCR for phage enumeration for applications ranging from selection to next-generation sequencing (NGS). Copyright © 2017 Elsevier B.V. All rights reserved.

mTORC2 regulates neutrophil chemotaxis in a cAMP- and RhoA-dependent fashion.

PubMed

Liu, Lunhua; Das, Satarupa; Losert, Wolfgang; Parent, Carole A

2010-12-14

We studied the role of the target of rapamycin complex 2 (mTORC2) during neutrophil chemotaxis, a process that is mediated through the polarization of actin and myosin filament networks. We show that inhibition of mTORC2 activity, achieved via knock down (KD) of Rictor, severely inhibits neutrophil polarization and directed migration induced by chemoattractants, independently of Akt. Rictor KD also abolishes the ability of chemoattractants to induce cAMP production, a process mediated through the activation of the adenylyl cyclase 9 (AC9). Cells with either reduced or higher AC9 levels also exhibit specific and severe tail retraction defects that are mediated through RhoA. We further show that cAMP is excluded from extending pseudopods and remains restricted to the cell body of migrating neutrophils. We propose that the mTORC2-dependent regulation of MyoII occurs through a cAMP/RhoA-signaling axis, independently of actin reorganization during neutrophil chemotaxis. Copyright © 2010 Elsevier Inc. All rights reserved.

BECN1-dependent CASP2 incomplete autophagy induction by binding to rabies virus phosphoprotein.

PubMed

Liu, Juan; Wang, Hailong; Gu, Jinyan; Deng, Tingjuan; Yuan, Zhuangchuan; Hu, Boli; Xu, Yunbin; Yan, Yan; Zan, Jie; Liao, Min; DiCaprio, Erin; Li, Jianrong; Su, Shuo; Zhou, Jiyong

2017-04-03

Autophagy is an essential component of host immunity and used by viruses for survival. However, the autophagy signaling pathways involved in virus replication are poorly documented. Here, we observed that rabies virus (RABV) infection triggered intracellular autophagosome accumulation and results in incomplete autophagy by inhibiting autophagy flux. Subsequently, we found that RABV infection induced the reduction of CASP2/caspase 2 and the activation of AMP-activated protein kinase (AMPK)-AKT-MTOR (mechanistic target of rapamycin) and AMPK-MAPK (mitogen-activated protein kinase) pathways. Further investigation revealed that BECN1/Beclin 1 binding to viral phosphoprotein (P) induced an incomplete autophagy via activating the pathways CASP2-AMPK-AKT-MTOR and CASP2-AMPK-MAPK by decreasing CASP2. Taken together, our data first reveals a crosstalk of BECN1 and CASP2-dependent autophagy pathways by RABV infection.

Inhibition of AGEs/RAGE/Rho/ROCK pathway suppresses non-specific neuroinflammation by regulating BV2 microglial M1/M2 polarization through the NF-κB pathway.

PubMed

Chen, Jingkao; Sun, Zhaowei; Jin, Minghua; Tu, Yalin; Wang, Shengnan; Yang, Xiaohong; Chen, Qiuhe; Zhang, Xiao; Han, Yifan; Pi, Rongbiao

2017-04-15

The microglia-mediated neuroinflammation plays an important role in the pathogenesis of Alzheimer's disease (AD). Advanced glycation end products (AGEs)/receptor for advanced glycation end products (RAGE) or Rho/Rho kinase (ROCK) are both involved in the development of non-specific inflammation. However, there are few reports about their effects on neuroinflammation. Here, we explored the mechanism of AGEs/RAGE/Rho/ROCK pathway underlying the non-specific inflammation and microglial polarization in BV2 cells. AGEs could activate ROCK pathway in a concentration-dependent manner. ROCK inhibitor fasudil and RAGE-specific blocker FPS-ZM1 significantly inhibited AGEs-mediated activation of BV2 cells and induction of reactive oxygen species (ROS). FPS-ZM1 and fasudil exerted their anti-inflammatory effects by downregulating inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), NLRP3 and nuclear translocation of nuclear factor kappa B (NF-κB) p65. In addition, AGEs induced both M1 (CD16/32, M1 marker) and M2 (CD206, M2 marker) phenotype in BV2 cells. Fasudil and FPS-ZM1 led to a decreased M1 and increased M2 phenotype. Together, these results indicate that the AGEs/RAGE/Rho/ROCK pathway in BV2 cells could intensify the non-specific inflammation of AD, which will provide novel strategies for the development of anti-AD drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

RhoA/ROCK may involve in cardiac hypertrophy induced by experimental hyperthyroidism.

PubMed

Na, Wang; Peng, Guan; Jianping, Zhang; Yanzhong, Chang; Shengjiang, Guan; Li, Chu

2012-10-01

In this study, the role of the RhoA/Rho-kinase (RhoA/ROCK)-signaling pathway in cardiovascular dysfunction associated with hyperthyroidism was examined with the use of fasudil, a Rho-kinase inhibitor. Male Spraque-Dawley rats were treated with l-thyroxine (T(4)) alone, T(4) + low-dose fasudil (2 mg/kg/day) or T(4) + high-dose fasudil (10 mg/kg/day) and compared with control animals. Rats in the T(4) group showed an increase in the ratio of heart weight to body weight, which was ameliorated by fasudil at both low and high doses. Morphometric and hemodynamic parameters were also evaluated and confirmed that fasudil attenuated the cardiac hypertrophy induced by T(4). The extent of phosphorylation of the myosin phosphatase targeting subunit was quantified by Western blotting to evaluate the activity of Rho-kinase in the heart tissue. Both Western blotting and reverse transcriptase-polymerase chain reaction analyses revealed enhancement of Rho-kinase and activator protein 1 activity and reduction of c-FLIP(L) expression in the T(4) group, and this response was inhibited by fasudil in a dose-dependent manner. Furthermore, fasudil inhibited apoptosis induced by T(4) as evidenced by the detection of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells and the expressions of bax and bcl-2. These results suggested that the RhoA/ROCK pathway is involved in the cardiac hypertrophy induced by experimental hyperthyroidism. The antagonism of this pathway may thus be useful as an alternative target in the treatment of hyperthyroid heart disease.

Center for Advanced Space Propulsion (CASP)

NASA Technical Reports Server (NTRS)

1988-01-01

With a mission to initiate and conduct advanced propulsion research in partnership with industry, and a goal to strengthen U.S. national capability in propulsion technology, the Center for Advanced Space Propulsion (CASP) is the only NASA Center for Commercial Development of Space (CCDS) which focuses on propulsion and associated technologies. Meetings with industrial partners and NASA Headquarters personnel provided an assessment of the constraints placed on, and opportunities afforded commercialization projects. Proprietary information, data rights, and patent rights were some of the areas where well defined information is crucial to project success and follow-on efforts. There were five initial CASP projects. At the end of the first year there are six active, two of which are approaching the ground test phase in their development. Progress in the current six projects has met all milestones and is detailed. Working closely with the industrial counterparts it was found that the endeavors in expert systems development, computational fluid dynamics, fluid management in microgravity, and electric propulsion were well received. One project with the Saturn Corporation which dealt with expert systems application in the assembly process, was placed on hold pending further direction from Saturn. The Contamination Measurment and Analysis project was not implemented since CASP was unable to identify an industrial participant. Additional propulsion and related projects were investigated during the year. A subcontract was let to a small business, MicroCraft, Inc., to study rocket engine certification standards. The study produced valuable results; however, based on a number of factors it was decided not to pursue this project further.

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

PubMed

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.

Rho'ing in and out of cells: viral interactions with Rho GTPase signaling.

PubMed

Van den Broeke, Céline; Jacob, Thary; Favoreel, Herman W

2014-01-01

Rho GTPases are key regulators of actin and microtubule dynamics and organization. Increasing evidence shows that many viruses have evolved diverse interactions with Rho GTPase signaling and manipulate them for their own benefit. In this review, we discuss how Rho GTPase signaling interferes with many steps in the viral replication cycle, especially entry, replication, and spread. Seen the diversity between viruses, it is not surprising that there is considerable variability in viral interactions with Rho GTPase signaling. However, several largely common effects on Rho GTPases and actin architecture and microtubule dynamics have been reported. For some of these processes, the molecular signaling and biological consequences are well documented while for others we just begin to understand them. A better knowledge and identification of common threads in the different viral interactions with Rho GTPase signaling and their ultimate consequences for virus and host may pave the way toward the development of new antiviral drugs that may target different viruses.

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

PubMed

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

2011-08-26

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

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

PubMed Central

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

2011-01-01

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

IL-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4+ T Cells.

PubMed

da Silva, Thiago Aparecido; Mariano, Vania Sammartino; Sardinha-Silva, Aline; de Souza, Maria Aparecida; Mineo, Tiago Wilson Patriarca; Roque-Barreira, Maria Cristina

2016-01-01

ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4+ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4+ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4+ T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4+ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4+ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production

IL-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4+ T Cells

PubMed Central

da Silva, Thiago Aparecido; Mariano, Vania Sammartino; Sardinha-Silva, Aline; de Souza, Maria Aparecida; Mineo, Tiago Wilson Patriarca; Roque-Barreira, Maria Cristina

2016-01-01

ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4+ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4+ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4+ T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4+ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4+ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production

[Construction and immunogenicity of recombinant bacteriophage T7 vaccine expressing M2e peptides of avian influenza virus].

PubMed

Xu, Hai; Wang, Yi-Wei; Tang, Ying-Hua; Zheng, Qi-Sheng; Hou, Ji-Bo

2013-06-01

To construct a recombinant T7 phage expressing matrix protein 2 ectodomain (M2e) peptides of avian influenza A virus and test immunological and protective efficacy in the immunized SPF chickens. M2e gene sequence was obtained from Genbank and two copies of M2e gene were artificially synthesised, the M2e gene was then cloned into the T7 select 415-1b phage in the multiple cloning sites to construct the recombinant phage T7-M2e. The positive recombinant phage was identified by PCR and sequencing, and the expression of surface fusion protein was confirmed by SDS-PAGE and Western-blot. SPF chickens were subcutaneously injected with 1 X 10(10) pfu phage T7-M2e, sera samples were collected pre- and post-vaccination, and were tested for anti-M2e antibody by ELISA. The binding capacity of serum to virus was also examined by indirect immunofluorescence assay in virus- infected CEF. The immunized chickens were challenged with 200 EID50 of H9 type avian influenza virus and viral isolation rate was calculated to evaluate the immune protective efficacy. A recombinant T7 phage was obtained displaying M2e peptides of avian influenza A virus, and the fusion protein had favorable immunoreactivity. All chickens developed a certain amount of anti-M2e antibody which could specially bind to the viral particles. In addition, the protection efficacy of phage T7-M2e vaccine against H9 type avian influenza viruses was 4/5 (80%). These results indicate that the recombinant T7 phage displaying M2e peptides of avian influenza A virus has a great potential to be developed into a novel vaccine for the prevention of avian influenza infection.

Increased pulmonary RhoA expression in the nitrofen-induced congenital diaphragmatic hernia rat model.

PubMed

Takayasu, Hajime; Masumoto, Kouji; Hagiwara, Koki; Sasaki, Takato; Ono, Kentaro; Jimbo, Takahiro; Uesugi, Toru; Gotoh, Chikashi; Urita, Yasuhisa; Shinkai, Toko; Tanaka, Hideaki

2015-09-01

Persistent pulmonary hypertension remains a major cause of mortality and morbidity in cases of congenital diaphragmatic hernia (CDH). Recently, RhoA/Rho-kinase-mediated vasoconstriction has been reported to be important in the pathogenesis of pulmonary hypertension (PH). Several recent reports have described that fasudil, a potent Rho-kinase inhibitor and vasodilator, could represent a potential therapeutic option for PH. We designed this study to investigate the hypothesis that the expression level of RhoA is increased in the nitrofen-induced CDH rat model. The expression level of Wnt11, an activator of RhoA, was also evaluated. Pregnant rats were treated with or without nitrofen on gestational day 9 (D9). Fetuses were sacrificed on D17, D19 and D21 and were divided into control and CDH groups. Quantitative real-time polymerase chain reaction was performed to determine the pulmonary gene expression levels of both Wnt11 and RhoA. An immunofluorescence study was also performed to evaluate the expression and localization of RhoA. The relative mRNA expression levels of pulmonary Wnt11 and RhoA on D21 were significantly increased in the CDH group compared with the control group (p=0.016 and p=0.008, respectively). The immunofluorescence study confirmed the overexpression of RhoA in the pulmonary vessels of CDH rats on D21. Our results provide evidence that the RhoA/Rho-kinase-mediated pathway is involved in the pathogenesis of PH in the nitrofen-induced CDH rat model. Our data also suggest that the fasudil, a Rho-kinase inhibitor, could represent a therapeutic option for the treatment of PH in CDH. Copyright © 2015 Elsevier Inc. All rights reserved.

17 CFR 249.507 - Form 7-M, consent to service of process by an individual nonresident broker-dealer.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Form 7-M, consent to service of process by an individual nonresident broker-dealer. 249.507 Section 249.507 Commodity and... Forms for Statements Made in Connection With Exempt Tender Offers § 249.507 Form 7-M, consent to service...

i3Drefine software for protein 3D structure refinement and its assessment in CASP10.

PubMed

Bhattacharya, Debswapna; Cheng, Jianlin

2013-01-01

Protein structure refinement refers to the process of improving the qualities of protein structures during structure modeling processes to bring them closer to their native states. Structure refinement has been drawing increasing attention in the community-wide Critical Assessment of techniques for Protein Structure prediction (CASP) experiments since its addition in 8(th) CASP experiment. During the 9(th) and recently concluded 10(th) CASP experiments, a consistent growth in number of refinement targets and participating groups has been witnessed. Yet, protein structure refinement still remains a largely unsolved problem with majority of participating groups in CASP refinement category failed to consistently improve the quality of structures issued for refinement. In order to alleviate this need, we developed a completely automated and computationally efficient protein 3D structure refinement method, i3Drefine, based on an iterative and highly convergent energy minimization algorithm with a powerful all-atom composite physics and knowledge-based force fields and hydrogen bonding (HB) network optimization technique. In the recent community-wide blind experiment, CASP10, i3Drefine (as 'MULTICOM-CONSTRUCT') was ranked as the best method in the server section as per the official assessment of CASP10 experiment. Here we provide the community with free access to i3Drefine software and systematically analyse the performance of i3Drefine in strict blind mode on the refinement targets issued in CASP10 refinement category and compare with other state-of-the-art refinement methods participating in CASP10. Our analysis demonstrates that i3Drefine is only fully-automated server participating in CASP10 exhibiting consistent improvement over the initial structures in both global and local structural quality metrics. Executable version of i3Drefine is freely available at http://protein.rnet.missouri.edu/i3drefine/.

i3Drefine Software for Protein 3D Structure Refinement and Its Assessment in CASP10

PubMed Central

Bhattacharya, Debswapna; Cheng, Jianlin

2013-01-01

Protein structure refinement refers to the process of improving the qualities of protein structures during structure modeling processes to bring them closer to their native states. Structure refinement has been drawing increasing attention in the community-wide Critical Assessment of techniques for Protein Structure prediction (CASP) experiments since its addition in 8th CASP experiment. During the 9th and recently concluded 10th CASP experiments, a consistent growth in number of refinement targets and participating groups has been witnessed. Yet, protein structure refinement still remains a largely unsolved problem with majority of participating groups in CASP refinement category failed to consistently improve the quality of structures issued for refinement. In order to alleviate this need, we developed a completely automated and computationally efficient protein 3D structure refinement method, i3Drefine, based on an iterative and highly convergent energy minimization algorithm with a powerful all-atom composite physics and knowledge-based force fields and hydrogen bonding (HB) network optimization technique. In the recent community-wide blind experiment, CASP10, i3Drefine (as ‘MULTICOM-CONSTRUCT’) was ranked as the best method in the server section as per the official assessment of CASP10 experiment. Here we provide the community with free access to i3Drefine software and systematically analyse the performance of i3Drefine in strict blind mode on the refinement targets issued in CASP10 refinement category and compare with other state-of-the-art refinement methods participating in CASP10. Our analysis demonstrates that i3Drefine is only fully-automated server participating in CASP10 exhibiting consistent improvement over the initial structures in both global and local structural quality metrics. Executable version of i3Drefine is freely available at http://protein.rnet.missouri.edu/i3drefine/. PMID:23894517

Dysprosium-doped PbGa2S4 laser generating at 4.3 μm directly pumped by 1.7 μm laser diode.

PubMed

Jelínková, Helena; Doroshenko, Maxim E; Jelínek, Michal; Sulc, Jan; Osiko, Vyacheslav V; Badikov, Valerii V; Badikov, Dmitrii V

2013-08-15

In this Letter, we demonstrate the pulsed and CW operation of the Dy:PbGa(2)S(4) laser directly pumped by the 1.7 μm laser diode. In the pulsed regime (pulse duration 5 ms; repetition rate 20 Hz), the maximum mean output power of 9.5 mW was obtained with the slope efficiency of 9.3% with respect to the absorbed pump power. The generated wavelength was 4.32 μm, and the laser beam cross section was approximately Gaussian on both axes. Stable CW laser generation was also successfully obtained with the maximum output power of 67 mW and the slope efficiency of 8%. Depopulation of the lower laser level by 1.7 μm pump radiation absorption followed by 1.3 μm upconversion fluorescence was demonstrated. These results show the possibility of construction of the compact diode-pumped solid-state pulsed or CW laser generating at 4.3 μm in the power level of tens mW operating at room temperature.

A multipronged strategy of an anti-terminator protein to overcome Rho-dependent transcription termination

PubMed Central

Muteeb, Ghazala; Dey, Debashish; Mishra, Saurabh; Sen, Ranjan

2012-01-01

One of the important role of Rho-dependent transcription termination in bacteria is to prevent gene expressions from the bacteriophage DNA. The transcription anti-termination systems of the lambdoid phages have been designed to overcome this Rho action. The anti-terminator protein N has three interacting regions, which interact with the mRNA, with the NusA and with the RNA polymerase. Here, we show that N uses all these interaction modules to overcome the Rho action. N and Rho co-occupy their overlapping binding sites on the nascent RNA (the nutR/tR1 site), and this configuration slows down the rate of ATP hydrolysis and the rate of RNA release by Rho from the elongation complex. N-RNA polymerase interaction is not too important for this Rho inactivation process near/at the nutR site. This interaction becomes essential when the elongation complex moves away from the nutR site. From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti-termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway. We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals. PMID:23024214

A multipronged strategy of an anti-terminator protein to overcome Rho-dependent transcription termination.

PubMed

Muteeb, Ghazala; Dey, Debashish; Mishra, Saurabh; Sen, Ranjan

2012-12-01

One of the important role of Rho-dependent transcription termination in bacteria is to prevent gene expressions from the bacteriophage DNA. The transcription anti-termination systems of the lambdoid phages have been designed to overcome this Rho action. The anti-terminator protein N has three interacting regions, which interact with the mRNA, with the NusA and with the RNA polymerase. Here, we show that N uses all these interaction modules to overcome the Rho action. N and Rho co-occupy their overlapping binding sites on the nascent RNA (the nutR/tR1 site), and this configuration slows down the rate of ATP hydrolysis and the rate of RNA release by Rho from the elongation complex. N-RNA polymerase interaction is not too important for this Rho inactivation process near/at the nutR site. This interaction becomes essential when the elongation complex moves away from the nutR site. From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti-termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway. We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals.

Inactivation of the small GTP binding protein Rho induces multinucleate cell formation and apoptosis in murine T lymphoma EL4.

PubMed

Moorman, J P; Bobak, D A; Hahn, C S

1996-06-01

The small G-protein Rho regulates the actin microfilament-dependent cytoskeleton. Exoenzyme C3 of Clostridium botulinum ADP-ribosylates Rho at Asn41, a modification that functionally inactivates Rho. Using a Sindbis virus-based transient gene expression system, we studied the role of Rho in murine EL4 T lymphoma cells. We generated a double subgenomic infectious Sindbis virus (dsSIN:C3) recombinant which expressed C3 in >95% of EL4 cells. This intracellular C3 resulted in modification and inactivation of virtually all endogenous Rho. dsSIN:C3 infection led to the formation of multinucleate cells, likely by inhibiting the actin microfilament-dependent step of cytokinesis. Intriguingly, in spite of the inhibition of cytokinesis, karyokinesis continued, with the result that cells containing a nuclear DNA content as high as 16N (eight nuclei) were observed. In addition, dsSIN:C3-mediated inactivation of Rho was a potent activator of apoptosis in EL4 cells. To discern whether the formation of multinucleate cells was responsible for the activation of apoptosis, 5-fluorouracil (5-FUra) was used to induce cell cycle arrest. As expected, EL4 cells treated with 5-FUra were prevented from forming multinucleate cells upon infection with dsSIN:C3. dsSIN:C3 infection, however, still caused marked apoptosis in 5-FUra-treated cells, indicating that this activation of apoptosis was independent of multinucleate cell formation.

Data-assisted protein structure modeling by global optimization in CASP12.

PubMed

Joo, Keehyoung; Heo, Seungryong; Joung, InSuk; Hong, Seung Hwan; Lee, Sung Jong; Lee, Jooyoung

2018-03-01

In CASP12, 2 types of data-assisted protein structure modeling were experimented. Either SAXS experimental data or cross-linking experimental data was provided for a selected number of CASP12 targets that the CASP12 predictor could utilize for better protein structure modeling. We devised 2 separate energy terms for SAXS data and cross-linking data to drive the model structures into more native-like structures that satisfied the given experimental data as much as possible. In CASP11, we successfully performed protein structure modeling using simulated sparse and ambiguously assigned NOE data and/or correct residue-residue contact information, where the only energy term that folded the protein into its native structure was the term which was originated from the given experimental data. However, the 2 types of experimental data provided in CASP12 were far from being sufficient enough to fold the target protein into its native structure because SAXS data provides only the overall shape of the molecule and the cross-linking contact information provides only very low-resolution distance information. For this reason, we combined the SAXS or cross-linking energy term with our regular modeling energy function that includes both the template energy term and the de novo energy terms. By optimizing the newly formulated energy function, we obtained protein models that fit better with provided SAXS data than the X-ray structure of the target. However, the improvement of the model relative to the 1 modeled without the SAXS data, was not significant. Consistent structural improvement was achieved by incorporating cross-linking data into the protein structure modeling. © 2018 Wiley Periodicals, Inc.

Assessment of Template-Based Modeling of Protein Structure in CASP11

PubMed Central

Modi, Vivek; Xu, Qifang; Adhikari, Sam; Dunbrack, Roland L.

2016-01-01

We present the assessment of predictions submitted in the template-based modeling (TBM) category of CASP11 (Critical Assessment of Protein Structure Prediction). Model quality was judged on the basis of global and local measures of accuracy on all atoms including side chains. The top groups on 39 human-server targets based on model 1 predictions were LEER, Zhang, LEE, MULTICOM, and Zhang-Server. The top groups on 81 targets by server groups based on model 1 predictions were Zhang-Server, nns, BAKER-ROSETTASERVER, QUARK, and myprotein-me. In CASP11, the best models for most targets were equal to or better than the best template available in the Protein Data Bank, even for targets with poor templates. The overall performance in CASP11 is similar to the performance of predictors in CASP10 with slightly better performance on the hardest targets. For most targets, assessment measures exhibited bimodal probability density distributions. Multi-dimensional scaling of an RMSD matrix for each target typically revealed a single cluster with models similar to the target structure, with a mode in the GDT-TS density between 40 and 90, and a wide distribution of models highly divergent from each other and from the experimental structure, with density mode at a GDT-TS value of ~20. The models in this peak in the density were either compact models with entirely the wrong fold, or highly non-compact models. The results argue for a density-driven approach in future CASP TBM assessments that accounts for the bimodal nature of these distributions instead of Z-scores, which assume a unimodal, Gaussian distribution. PMID:27081927

Genetic requirements for sensitivity of bacteriophage t7 to dideoxythymidine.

PubMed

Tran, Ngoc Q; Tabor, Stanley; Richardson, Charles C

2014-08-01

We previously reported that the presence of dideoxythymidine (ddT) in the growth medium selectively inhibits the ability of bacteriophage T7 to infect Escherichia coli by inhibiting phage DNA synthese (N. Q. Tran, L. F. Rezende, U. Qimron, C. C. Richardson, and S. Tabor, Proc. Natl. Acad. Sci. U. S. A. 105:9373-9378, 2008, doi:10.1073/pnas.0804164105). In the presence of T7 gene 1.7 protein, ddT is taken up into the E. coli cell and converted to ddTTP. ddTTP is incorporated into DNA as ddTMP by the T7 DNA polymerase, resulting in chain termination. We have identified the pathway by which exogenous ddT is converted to ddTTP. The pathway consists of ddT transport by host nucleoside permeases and phosphorylation to ddTMP by the host thymidine kinase. T7 gene 1.7 protein phosphorylates ddTMP and ddTDP, resulting in ddTTP. A 74-residue peptide of the gene 1.7 protein confers ddT sensitivity to the same extent as the 196-residue wild-type gene 1.7 protein. We also show that cleavage of thymidine to thymine and deoxyribose-1-phosphate by the host thymidine phosphorylase greatly increases the sensitivity of phage T7 to ddT. Finally, a mutation in T7 DNA polymerase that leads to discrimination against the incorporation of ddTMP eliminates ddT sensitivity. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Genetic Requirements for Sensitivity of Bacteriophage T7 to Dideoxythymidine

PubMed Central

Tran, Ngoc Q.; Tabor, Stanley

2014-01-01

We previously reported that the presence of dideoxythymidine (ddT) in the growth medium selectively inhibits the ability of bacteriophage T7 to infect Escherichia coli by inhibiting phage DNA synthese (N. Q. Tran, L. F. Rezende, U. Qimron, C. C. Richardson, and S. Tabor, Proc. Natl. Acad. Sci. U. S. A. 105:9373–9378, 2008, doi:10.1073/pnas.0804164105). In the presence of T7 gene 1.7 protein, ddT is taken up into the E. coli cell and converted to ddTTP. ddTTP is incorporated into DNA as ddTMP by the T7 DNA polymerase, resulting in chain termination. We have identified the pathway by which exogenous ddT is converted to ddTTP. The pathway consists of ddT transport by host nucleoside permeases and phosphorylation to ddTMP by the host thymidine kinase. T7 gene 1.7 protein phosphorylates ddTMP and ddTDP, resulting in ddTTP. A 74-residue peptide of the gene 1.7 protein confers ddT sensitivity to the same extent as the 196-residue wild-type gene 1.7 protein. We also show that cleavage of thymidine to thymine and deoxyribose-1-phosphate by the host thymidine phosphorylase greatly increases the sensitivity of phage T7 to ddT. Finally, a mutation in T7 DNA polymerase that leads to discrimination against the incorporation of ddTMP eliminates ddT sensitivity. PMID:24858186

Some of the most interesting CASP11 targets through the eyes of their authors

PubMed Central

Kryshtafovych, Andriy; Moult, John; Baslé, Arnaud; Burgin, Alex; Craig, Timothy K.; Edwards, Robert A.; Fass, Deborah; Hartmann, Marcus D.; Korycinski, Mateusz; Lewis, Richard J.; Lorimer, Donald; Lupas, Andrei N.; Newman, Janet; Peat, Thomas S.; Piepenbrink, Kurt H.; Prahlad, Janani; van Raaij, Mark J.; Rohwer, Forest; Segall, Anca M.; Seguritan, Victor; Sundberg, Eric J.; Singh, Abhimanyu K.; Wilson, Mark A.

2015-01-01

ABSTRACT The Critical Assessment of protein Structure Prediction (CASP) experiment would not have been possible without the prediction targets provided by the experimental structural biology community. In this article, selected crystallographers providing targets for the CASP11 experiment discuss the functional and biological significance of the target proteins, highlight their most interesting structural features, and assess whether these features were correctly reproduced in the predictions submitted to CASP11. Proteins 2016; 84(Suppl 1):34–50. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:26473983

T7 lytic phage-displayed peptide libraries exhibit less sequence bias than M13 filamentous phage-displayed peptide libraries.

PubMed

Krumpe, Lauren R H; Atkinson, Andrew J; Smythers, Gary W; Kandel, Andrea; Schumacher, Kathryn M; McMahon, James B; Makowski, Lee; Mori, Toshiyuki

2006-08-01

We investigated whether the T7 system of phage display could produce peptide libraries of greater diversity than the M13 system of phage display due to the differing processes of lytic and filamentous phage morphogenesis. Using a bioinformatics-assisted computational approach, collections of random peptide sequences obtained from a T7 12-mer library (X(12)) and a T7 7-mer disulfide-constrained library (CX(7)C) were analyzed and compared with peptide populations obtained from New England BioLabs' M13 Ph.D.-12 and Ph.D.-C7C libraries. Based on this analysis, peptide libraries constructed with the T7 system have fewer amino acid biases, increased peptide diversity, and more normal distributions of peptide net charge and hydropathy than the M13 libraries. The greater diversity of T7-displayed libraries provides a potential resource of novel binding peptides for new as well as previously studied molecular targets. To demonstrate their utility, several of the T7-displayed peptide libraries were screened for streptavidin- and neutravidin-binding phage. Novel binding motifs were identified for each protein.

Quantitative 17O imaging towards oxygen consumption study in tumor bearing mice at 7 T.

PubMed

Narazaki, Michiko; Kanazawa, Yoko; Koike, Sachiko; Ando, Koichi; Ikehira, Hiroo

2013-06-01

(17)O magnetic resonance imaging (MRI) using a conventional pulse sequence was explored as a method of quantitative imaging towards regional oxygen consumption rate measurement for tumor evaluation in mice. At 7 T, fast imaging with steady state (FISP) was the best among gradient echo, fast spin echo and FISP for the purpose. The distribution of natural abundance H2(17)O in mice was visualized under spatial resolution of 2.5 × 2.5mm(2) by FISP in 10 min. The signal intensity by FISP showed a linear relationship with (17)O quantity both in phantom and mice. Following the injection of 5% (17)O enriched saline, (17)O re-distribution was monitored in temporal resolution down to 5 sec with an image quality sufficient to distinguish each organ. The image of labeled water produced from inhaled (17)O2 gas was also obtained. The present method provides quantitative (17)O images under sufficient temporal and spatial resolution for the evaluation of oxygen consumption rate in each organ. Experiments using various model compounds of R-OH type clarified that the signal contribution of body constituents other than water in the present in vivo(17)O FISP image was negligible. Copyright © 2013 Elsevier Inc. All rights reserved.

RhoA/ROCK downregulates FPR2-mediated NADPH oxidase activation in mouse bone marrow granulocytes.

PubMed

Filina, Julia V; Gabdoulkhakova, Aida G; Safronova, Valentina G

2014-10-01

Polymorphonuclear neutrophils (PMNs) express the high and low affinity receptors to formylated peptides (mFPR1 and mFPR2 in mice, accordingly). RhoA/ROCK (Rho activated kinase) pathway is crucial for cell motility and oxidase activity regulated via FPRs. There are contradictory data on RhoA-mediated regulation of NADPH oxidase activity in phagocytes. We have shown divergent Rho GTPases signaling via mFPR1 and mFPR2 to NADPH oxidase in PMNs from inflammatory site. The present study was aimed to find out the role of RhoA/ROCK in the respiratory burst activated via mFPR1 and mFPR2 in the bone marrow PMNs. Different kinetics of RhoA activation were detected with 0.1μM fMLF and 1μM WKYMVM operating via mFPR1 and mFPR2, accordingly. RhoA was translocated in fMLF-activated cells towards the cell center and juxtamembrane space versus uniform allocation in the resting cells. Specific inhibition of RhoA by CT04, Rho inhibitor I, weakly depressed the respiratory burst induced via mFPR1, but significantly increased the one induced via mFPR2. Inhibition of ROCK, the main effector of RhoA, by Y27632 led to the same effect on the respiratory burst. Regulation of mFPR2-induced respiratory response by ROCK was impossible under the cytoskeleton disruption by cytochalasin D, whereas it persisted in the case of mFPR1 activation. Thus we suggest RhoA to be one of the regulatory and signal transduction components in the respiratory burst through FPRs in the mouse bone marrow PMNs. Both mFPR1 and mFPR2 binding with a ligand trigger the activation of RhoA. FPR1 signaling through RhoA/ROCK increases NADPH-oxidase activity. But in FPR2 action RhoA/ROCK together with cytoskeleton-linked systems down-regulates NADPH-oxidase. This mechanism could restrain the reactive oxygen species dependent damage of own tissues during the chemotaxis of PMNs and in the resting cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Some of the most interesting CASP11 targets through the eyes of their authors.

PubMed

Kryshtafovych, Andriy; Moult, John; Baslé, Arnaud; Burgin, Alex; Craig, Timothy K; Edwards, Robert A; Fass, Deborah; Hartmann, Marcus D; Korycinski, Mateusz; Lewis, Richard J; Lorimer, Donald; Lupas, Andrei N; Newman, Janet; Peat, Thomas S; Piepenbrink, Kurt H; Prahlad, Janani; van Raaij, Mark J; Rohwer, Forest; Segall, Anca M; Seguritan, Victor; Sundberg, Eric J; Singh, Abhimanyu K; Wilson, Mark A; Schwede, Torsten

2016-09-01

The Critical Assessment of protein Structure Prediction (CASP) experiment would not have been possible without the prediction targets provided by the experimental structural biology community. In this article, selected crystallographers providing targets for the CASP11 experiment discuss the functional and biological significance of the target proteins, highlight their most interesting structural features, and assess whether these features were correctly reproduced in the predictions submitted to CASP11. Proteins 2016; 84(Suppl 1):34-50. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Target Highlights in CASP9: Experimental Target Structures for the Critical Assessment of Techniques for Protein Structure Prediction

PubMed Central

Kryshtafovych, Andriy; Moult, John; Bartual, Sergio G.; Bazan, J. Fernando; Berman, Helen; Casteel, Darren E.; Christodoulou, Evangelos; Everett, John K.; Hausmann, Jens; Heidebrecht, Tatjana; Hills, Tanya; Hui, Raymond; Hunt, John F.; Jayaraman, Seetharaman; Joachimiak, Andrzej; Kennedy, Michael A.; Kim, Choel; Lingel, Andreas; Michalska, Karolina; Montelione, Gaetano T.; Otero, José M.; Perrakis, Anastassis; Pizarro, Juan C.; van Raaij, Mark J.; Ramelot, Theresa A.; Rousseau, Francois; Tong, Liang; Wernimont, Amy K.; Young, Jasmine; Schwede, Torsten

2011-01-01

One goal of the CASP Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction is to identify the current state of the art in protein structure prediction and modeling. A fundamental principle of CASP is blind prediction on a set of relevant protein targets, i.e. the participating computational methods are tested on a common set of experimental target proteins, for which the experimental structures are not known at the time of modeling. Therefore, the CASP experiment would not have been possible without broad support of the experimental protein structural biology community. In this manuscript, several experimental groups discuss the structures of the proteins which they provided as prediction targets for CASP9, highlighting structural and functional peculiarities of these structures: the long tail fibre protein gp37 from bacteriophage T4, the cyclic GMP-dependent protein kinase Iβ (PKGIβ) dimerization/docking domain, the ectodomain of the JTB (Jumping Translocation Breakpoint) transmembrane receptor, Autotaxin (ATX) in complex with an inhibitor, the DNA-Binding J-Binding Protein 1 (JBP1) domain essential for biosynthesis and maintenance of DNA base-J (β-D-glucosyl-hydroxymethyluracil) in Trypanosoma and Leishmania, an so far uncharacterized 73 residue domain from Ruminococcus gnavus with a fold typical for PDZ-like domains, a domain from the Phycobilisome (PBS) core-membrane linker (LCM) phycobiliprotein ApcE from Synechocystis, the Heat shock protein 90 (Hsp90) activators PFC0360w and PFC0270w from Plasmodium falciparum, and 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae. PMID:22020785

Transport and thermoelectric properties of Sr{sub 3}(Ti{sub 0.95}R{sub 0.05}){sub 2}O{sub 7} (R = Ta, Nb, W) oxides

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

Sun, R. R.; Qin, X. Y.; Li, L. L.

2012-12-15

The Sr{sub 3}(Ti{sub 0.95}R{sub 0.05}){sub 2}O{sub 7} (R = Ta, Nb, W) polycrystalline compounds were fabricated, and their transport and thermoelectric properties were investigated. The results indicate that at T > 300 K electrical resistivity {rho} for all the doped compounds increases monotonically with temperature, and basically can be described by a relation {rho}{proportional_to}T{sup M} at T > {approx}650 K, with M = 1.39, 1.66, and 1.77 for R = Ta, Nb, and W, respectively, implying that at the high temperatures the acoustic phonon scattering dominates the scattering process. Although the resistivity {rho} of Sr{sub 3}(Ti{sub 0.95}Ta{sub 0.05}){sub 2}O{sub 7}more » exhibits a metallic-like behavior at the temperature as low as 5 K, a transition from metallic state (d{rho}/dT > 0) to semiconductor-like state (d{rho}/dT < 0) was observed at a critical low temperature {approx}41 K and {approx}79 K for R = Nb and W, respectively. At T < {approx}22 K, {approx}57 K, and {approx}80 K, a relation of {sigma}{proportional_to}T{sup 1/2} (here conductivity {sigma} = 1/{rho}) holds for the doped compounds with R = Nb, Ta, and W, respectively, suggesting that at the low temperatures the main transport mechanism is electron-electron interaction due to the presence of disorder induced by the dopants. The thermoelectric figure of merit (ZT) for Ta-doped compound increases more steeply with increasing temperature among the three compounds and reaches 0.066 at 1000 K.« less

Sequencing and Analyzing the "t" (1;7) Reciprocal Translocation Breakpoints Associated with a Case of Childhood-Onset Schizophrenia/Autistic Disorder

ERIC Educational Resources Information Center

Idol, Jacquelyn R.; Addington, Anjene M.; Long, Robert T.; Rapoport, Judith L.; Green, Eric D.

2008-01-01

We characterized a "t"(1;7)(p22;q21) reciprocal translocation in a patient with childhood-onset schizophrenia (COS) and autism using genome mapping and sequencing methods. Based on genomic maps of human chromosome 7 and fluorescence in situ hybridization (FISH) studies, we delimited the region of 7q21 harboring the translocation breakpoint to a…

The Aryl Hydrocarbon Receptor: Differential Contribution to T Helper 17 and T Cytotoxic 17 Cell Development

PubMed Central

Hayes, Mark D.; Ovcinnikovs, Vitalijs; Smith, Andrew G.; Kimber, Ian; Dearman, Rebecca J.

2014-01-01

The aryl hydrocarbon receptor (AhR) has been shown to be required for optimal Thelper (Th) 17 cell activation. Th17 cells provide immunity against extracellular pathogens and are implicated in autoimmune diseases. Herein, the role of the AhR in cytokine production by Th17, and by the analogous population of T cytotoxic (Tc)17 cells, has been examined. Lymph node Tc (CD8+) and Th (CD4+) cells were isolated by negative selection from naive AhR+/− and AhR−/− mice and polarised to Tc1/Th1 or Tc17/Th17 phenotypes with appropriate cytokines. Cell differentiation was assessed as a function of mRNA and protein (ELISA and flow cytometry) expression for interferon (IFN)-γ and for key Th17 cytokines. In AhR+/− mice, Th17 cells displayed an exclusive IL-17 profile, which was markedly inhibited by a selective AhR antagonist to levels observed in AhR knockout mice. Addition of the natural AhR agonist 6-formylindolo[3,2-b]carbazole (FICZ) markedly enhanced Th17 cell activity in the heterozygotes. In contrast, Tc17 cells polarised into 3 distinct subsets: producing either IL-17 or IFN-γ alone, or both cytokines. Blocking AhR was also detrimental to Tc17 development, with reduced responses recorded in AhR−/− mice and antagonist-mediated reduction of IL-17 expression in the heterozygotes. However, Tc17 cells were largely refractory to exogenous FICZ, presumably because Tc17 cells express baseline AhR mRNA, but unlike Th17 cells, there is no marked up-regulation during polarisation. Thus, Th17 cell development is more dependent upon AhR activation than is Tc17 cell development, suggesting that endogenous AhR ligands play a much greater role in driving Th17 cell responses. PMID:25203682

Role of RhoA and its effectors ROCK and mDia1 in the modulation of deformation-induced FAK, ERK, p38, and MLC motogenic signals in human Caco-2 intestinal epithelial cells

PubMed Central

Chaturvedi, Lakshmi S.; Marsh, Harold M.

2011-01-01

Repetitive deformation enhances intestinal epithelial migration across tissue fibronectin. We evaluated the contribution of RhoA and its effectors Rho-associated kinase (ROK/ROCK) and mammalian diaphanous formins (mDia1) to deformation-induced intestinal epithelial motility across fibronectin and the responsible focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), p38, and myosin light chain (MLC) signaling. We reduced RhoA, ROCK1, ROCK2, and mDia1 by smart-pool double-stranded short-interfering RNAs (siRNA) and pharmacologically inhibited RhoA, ROCK, and FAK in human Caco-2 intestinal epithelial monolayers on fibronectin-coated membranes subjected to 10% repetitive deformation at 10 cycles/min. Migration was measured by wound closure. Stimulation of migration by deformation was prevented by exoenzyme C3, Y27632, or selective RhoA, ROCK1, and ROCK2 or mDia1 siRNAs. RhoA, ROCK inhibition, or RhoA, ROCK1, ROCK2, mDia1, and FAK reduction by siRNA blocked deformation-induced nuclear ERK phosphorylation without preventing ERK phosphorylation in the cytoplasmic protein fraction. Furthermore, RhoA, ROCK inhibition or RhoA, ROCK1, ROCK2, and mDia1 reduction by siRNA also blocked strain-induced FAK-Tyr925, p38, and MLC phosphorylation. These results suggest that RhoA, ROCK, mDia1, FAK, ERK, p38, and MLC all mediate the stimulation of intestinal epithelial migration by repetitive deformation. This pathway may be an important target for interventions to promote mechanotransduced mucosal healing during inflammation. PMID:21849669

The identification of cellular targets of 17β estradiol using a lytic (T7) cDNA phage display approach.

PubMed

Van Dorst, Bieke; Mehta, Jaytry; Rouah-Martin, Elsa; De Coen, Wim; Blust, Ronny; Robbens, Johan

2011-02-01

To unravel the mechanism of action of chemical compounds, it is crucial to know their cellular targets. A novel in vitro tool that can be used as a fast, simple and cost effective alternative is cDNA phage display. This tool is used in our study to select cellular targets of 17β estradiol (E2). It was possible to select two potential cellular targets of E2 out of the T7 Select™ Human Breast cDNA phage library. The selected cellular targets, autophagy/beclin-1 regulator 1 (beclin 1) and ATP synthase F(0) subunit 6 (ATP6) have so far been unknown as binding proteins of E2. To confirm the E2 binding properties of these selected proteins, surface plasmon resonance (SPR) was used. With SPR the K(d) values were determined to be 0.178±0.031 and 0.401±0.142 nM for the ATP6 phage and beclin 1 phage, respectively. These K(d) values in the low nM range verify that the selected cellular proteins are indeed binding proteins for E2. The selection and identification of these two potential cellular targets of E2, can enhance our current understanding of its mechanism of action. This illustrates the potential of lytic (T7) cDNA phage display in toxicology, to provide important information about cellular targets of chemical compounds. Copyright © 2010 Elsevier Ltd. All rights reserved.

Up-regulation of the RhoA/Rho-kinase signaling pathway in corpus cavernosum from endothelial nitric-oxide synthase (NOS), but not neuronal NOS, null mice.

PubMed

Priviero, Fernanda B M; Jin, Li-Ming; Ying, Zhekang; Teixeira, Cleber E; Webb, R Clinton

2010-04-01

We tested the hypothesis that the basal release of nitric oxide (NO) from endothelial cells modulates contractile activity in the corpus cavernosum (CC) via inhibition of the RhoA/Rho-kinase signaling pathway. Cavernosal strips from wild-type (WT), endothelial nitric-oxide synthase knockout [eNOS(-/-)], and neuronal nitric-oxide synthase knockout [nNOS(-/-)] mice were mounted in myographs, and isometric force was recorded. mRNA and protein expression of key molecules in the RhoA/Rho-kinase pathway were analyzed by real-time polymerase chain reaction and Western blot, respectively. The cGMP levels were determined. The Rho-kinase inhibitors (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl] homopiperazine (H-1152) reduced cavernosal contractions evoked by phenylephrine or electrical field stimulation (EFS) in a concentration-dependent manner, although this inhibition was less effective in tissues from eNOS(-/-) mice. Y-27632 enhanced relaxations induced by sodium nitroprusside, EFS, and NO (administered as acidified NaNO2) without affecting the cGMP content of the cavernosal strips. This enhancement was less prominent in CC from eNOS(-/-). The protein expression of RhoA, Rho-guanine dissociation inhibitor, and Rho-kinase beta did not differ among the strains. However, in eNOS(-/-) CC, the protein expression of Rho-kinase alpha and both mRNA and protein expression of p115-Rho-associated guanine exchange factor (RhoGEF), PDZ-RhoGEF, and leukemia-associated RhoGEF were up-regulated. Phosphorylation of MYPT1 at Thr696 was higher in tissues from eNOS(-/-) mice. A high concentration of Y-27632 significantly enhanced NO release in CC stimulated by EFS. These results suggest a basal release of NO from endothelial cells, which inhibits contractions mediated by the RhoA/Rho-kinase pathway and modulates the expression of proteins related to this pathway in mouse CC. It indicates that

An RNA motif advances transcription by preventing Rho-dependent termination

PubMed Central

Sevostyanova, Anastasia; Groisman, Eduardo A.

2015-01-01

The transcription termination factor Rho associates with most nascent bacterial RNAs as they emerge from RNA polymerase. However, pharmacological inhibition of Rho derepresses only a small fraction of these transcripts. What, then, determines the specificity of Rho-dependent transcription termination? We now report the identification of a Rho-antagonizing RNA element (RARE) that hinders Rho-dependent transcription termination. We establish that RARE traps Rho in an inactive complex but does not prevent Rho binding to its recruitment sites. Although translating ribosomes normally block Rho access to an mRNA, inefficient translation of an open reading frame in the leader region of the Salmonella mgtCBR operon actually enables transcription of its associated coding region by favoring an RNA conformation that sequesters RARE. The discovery of an RNA element that inactivates Rho signifies that the specificity of nucleic-acid binding proteins is defined not only by the sequences that recruit these proteins but also by sequences that antagonize their activity. PMID:26630006

Immunocytochemical evidence for PDBu-induced activation of RhoA/ROCK in human internal anal sphincter smooth muscle cells

PubMed Central

Singh, Jagmohan; Maxwell, Pinckney J.

2011-01-01

Studies were performed to determine the unknown status of PKC and RhoA/ROCK in the phorbol 12,13-dibutyrate (PDBu)-stimulated state in the human internal anal sphincter (IAS) smooth muscle cells (SMCs). We determined the effects of PDBu (10−7 M), the PKC activator, on PKCα and RhoA and ROCK II translocation in the human IAS SMCs. We used immunocytochemistry and fluorescence microcopy in the basal state, following PDBu, and before and after PKC inhibitor calphostin C (10−6 M), cell-permeable RhoA inhibitor C3 exoenzyme (2.5 μg/ml), and ROCK inhibitor Y 27632 (10−6 M). We also determined changes in the SMC lengths via computerized digital micrometry. In the basal state PKCα was distributed almost uniformly throughout the cell, whereas RhoA and ROCK II were located in the higher intensities toward the periphery. PDBu caused significant translocation of PKCα, RhoA, and ROCK II. PDBu-induced translocation of PKCα was attenuated by calphostin C and not by C3 exoenzyme and Y 27632. However, PDBu-induced translocation of RhoA was blocked by C3 exoenzyme, and that of ROCK II was attenuated by both C3 exoenzyme and Y 27632. Contraction of the human IAS SMCs caused by PDBu in parallel with RhoA/ROCK II translocation was attenuated by C3 exoenzyme and Y 27632 but not by calphostin C. In human IAS SMCs RhoA/ROCK compared with PKC are constitutively active, and contractility by PDBu is associated with RhoA/ROCK activation rather than PKC. The relative contribution of RhoA/ROCK vs. PKC in the pathophysiology and potential therapy for the IAS dysfunction remains to be determined. PMID:21566015

Increased T-helper 17 cell differentiation mediated by exosome-mediated microRNA-451 redistribution in gastric cancer infiltrated T cells.

PubMed

Liu, Feng; Bu, Zhouyan; Zhao, Feng; Xiao, Daping

2018-01-01

MicroRNA (miR)-451 is a cell metabolism-related miRNA that can mediate cell energy-consuming models by several targets. As miR-451 can promote mechanistic target of rapamycin (mTOR) activity, and increased mTOR activity is related to increased differentiation of T-helper 17 (Th17) cells, we sought to investigate whether miR-451 can redistribute from cancer cells to infiltrated T cells and enhance the distribution of Th17 cells through mTOR. Real-time PCR was used for detecting expression of miR-451 in gastric cancer, tumor infiltrated T cells and exosomes, and distribution of Th17 was evaluated by both flow cytometry and immunohistochemistry (IHC). Immunofluorescence staining was used in monitoring the exosome-enveloped miR-451 from cancer cells to T cells with different treatments, and signaling pathway change was analyzed by western blot. miR-451 decreased significantly in gastric cancer (GC) tissues but increased in infiltrated T cells and exosomes; tumor miR-451 was negatively related to infiltrated T cells and exosome miR-451. Exosome miR-451 can not only serve as an indicator for poor prognosis of post-operation GC patients but is also related to increased Th17 distribution in gastric cancer. miR-451 can redistribute from cancer cells to T cells with low glucose treatment. Decreased 5' AMP-activated protein kinase (AMPK) and increased mTOR activity was investigated in miR-451 redistributed T cells and the Th17 polarized differentiation of these T cells were also increased. Exosome miR-451 derived from tumor tissues can serve as an indicator for poor prognosis and redistribution of miR-451 from cancer cells to infiltrated T cells in low glucose treatment can enhance Th17 differentiation by enhancing mTOR activity. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

New and Improved T-wave Morphology Parameters to Differentiate Healthy Individuals from those with Cardiomyopathy and Coronary Artery Disease

NASA Technical Reports Server (NTRS)

Greco, E. C.; Schlegel, T. T.; Arenare, B.; DePalma, J. L.; Starc, V.; Rahman, M. A.; Delgado, R.

2007-01-01

We investigated the ability of several known as well as new ECG repolarization parameters to differentiate healthy individuals from patients with obstructive coronary artery disease (CAD) and cardiomyopathy (CM). Advanced high-fidelity 12-lead ECG tests (approx. 5-min supine) were first performed on a "training set" of 99 individuals: 33 with ischemic or dilated CM and low ejection fraction (EF less than 40%); 33 with catheterization-proven obstructive CAD but normal EF; and 33 age-/gender-matched healthy controls. The following multiple parameters of T-wave morphology (TWM) were derived via signal averaging and singular value decomposition (SVD, which yields 8 eigenvalues, rho(sub 1) greater than rho(sub 2)...greater than rho(sub 8) and studied for their retrospective accuracy in detecting underlying disease: 1) Principal component analysis ratio of the T wave (T-PCA) = 100*rho(sub 2)/rho(sub 1); 2) Relative T-wave residuum (rTWR) = 100* SIGMA (rho(sub 4)(sup 2) +...+ rho(sub 8)(sup 2)); 3) Modified complexity ratio of the T wave (T-mCR) = 100*SIGMA(rho(sub 3)(sup 2) +...+rho(sb 8) (sup 2)); and 4) Normalized 3-dimensional volume of the T wave (nTV) = 100*(rho(sub 2)*rho(sub 3)/rho(sub 1)(sup 2). All TWM parameters significantly differentiated CAD from controls (p less than 0.0001), and also CM from controls (p less than 0.0001). Retrospective areas under the ROC curve were 0.77, 0.81, 0.82, and 0.83 (CAD vs. controls) and 0.93, 0.89, 0.95 and 0.96 (CM vs. controls) for T-PCA, rTWR, T-mCR and nTV respectively. The newer TWM parameters (T-mCR and nTV) thus outperformed the more established parameters (T-PCA and rTWR), presumably by putting a greater emphasis on the third T-wave eigenvalue, which in most healthy subjects has little energy compared to the first two eigenvalues. Subsequent prospective analyses have also yielded similar results, such that we conclude that diagnostic differentiation of pathology from non-pathology may be especially aided by detecting

7 CFR 7.17 - Dual office.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 1 2010-01-01 2010-01-01 false Dual office. 7.17 Section 7.17 Agriculture Office of... STATE, COUNTY AND COMMUNITY COMMITTEES § 7.17 Dual office. (a) County committee membership. A member of... any other county office employee. (b) Community committee membership. A member of the community...

PKCε Phosphorylates and Mediates the Cell Membrane Localization of RhoA

PubMed Central

Su, Tizhi; Bao, Liwei; Xie, Xiujie; Lehner, Caryn L.; Cavey, Greg S.; Teknos, Theodoros N.

2013-01-01

Protein kinase Cε (PKCε) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKCε and RhoA was defined. Phosphopeptide mapping revealed that PKCε phosphorylates RhoA at T127 and S188. Recombinant PKCε bound to recombinant RhoA in the absence of ATP indicating that the association between PKCε and RhoA does not require an active ATP-docked PKCε conformation. Activation of PKCε resulted in a dramatic coordinated translocation of PKCε and RhoA from the cytoplasm to the cell membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis revealed that the molecular interaction between PKCε and RhoA is a biphasic event, an initial peak at the cytoplasm and a gradual prolonged increase at the cell membrane for the entire time-course (12.5 minutes). These results suggest that the PKCε-RhoA complex is assembled in the cytoplasm and subsequently recruited to the cell membrane. Kinase inactive (K437R) PKCε is able to recruit RhoA to the cell membrane indicating that the association between PKCε and RhoA is proximal to the active catalytic site and perhaps independent of a PKCε-RhoA phosphorylation event. This work demonstrates, for the first time, that PKCε phosphorylates and modulates the cell membrane translocation of RhoA. PMID:24191200

Analysis of Free Modeling Predictions by RBO Aleph in CASP11

PubMed Central

Mabrouk, Mahmoud; Werner, Tim; Schneider, Michael; Putz, Ines; Brock, Oliver

2015-01-01

The CASP experiment is a biannual benchmark for assessing protein structure prediction methods. In CASP11, RBO Aleph ranked as one of the top-performing automated servers in the free modeling category. This category consists of targets for which structural templates are not easily retrievable. We analyze the performance of RBO Aleph and show that its success in CASP was a result of its ab initio structure prediction protocol. A detailed analysis of this protocol demonstrates that two components unique to our method greatly contributed to prediction quality: residue–residue contact prediction by EPC-map and contact–guided conformational space search by model-based search (MBS). Interestingly, our analysis also points to a possible fundamental problem in evaluating the performance of protein structure prediction methods: Improvements in components of the method do not necessarily lead to improvements of the entire method. This points to the fact that these components interact in ways that are poorly understood. This problem, if indeed true, represents a significant obstacle to community-wide progress. PMID:26492194

ER stress responses in the absence of apoptosome: a comparative study in CASP9 proficient vs deficient mouse embryonic fibroblasts.

PubMed

Deegan, Shane; Saveljeva, Svetlana; Gupta, Sanjeev; MacDonald, David C; Samali, Afshin

2014-08-29

Cells respond to endoplasmic reticulum (ER) stress through the unfolded protein response (UPR), autophagy and cell death. In this study we utilized casp9(+/+) and casp9(-/-) MEFs to determine the effect of inhibition of mitochondrial apoptosis pathway on ER stress-induced-cell death, UPR and autophagy. We observed prolonged activation of UPR and autophagy in casp9(-/-) cells as compared with casp9(+/+) MEFs, which displayed transient activation of both pathways. Furthermore we showed that while casp9(-/-) MEFs were resistant to ER stress, prolonged exposure led to the activation of a non-canonical, caspase-mediated mode of cell death. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed

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

Interleukin 17-Producing γδT Cells Promote Hepatic Regeneration in Mice

PubMed Central

Rao, Raghavendra; Graffeo, Christopher S.; Gulati, Rishabh; Jamal, Mohsin; Narayan, Suchithra; Zambirinis, Constantinos; Barilla, Rocky; Deutsch, Michael; Greco, Stephanie; Ochi, Atsuo; Tomkötter, Lena; Blobstein, Reuven; Avanzi, Antonina; Tippens, Daniel M.; Gelbstein, Yisroel; Heerden, Eliza Van; Miller, George

2014-01-01

Background & Aims Subsets of leukocytes synergize with regenerative growth factors to promote hepatic regeneration. γδT cells are early responders to inflammation-induced injury in a number of contexts. We investigated the role of γδT cells in hepatic regeneration using mice with disruptions in Tcrd (encodes the T cell receptor δ chain) and Clec7a (encodes C-type lectin domain family 7 member a, also known as DECTIN1). Methods We performed partial hepatectomies on wild-type C57BL/6, CD45.1, Tcrd−/−, or Clec7a−/− mice. Cells were isolated from livers of patients and mice via mechanical and enzymatic digestion. γδT cells were purified by fluorescence-activated cell sorting. Results In mice, partial hepatectomy upregulated expression of CCL20 and ligands of Dectin-1, associated with recruitment and activation of γδT cells and their increased production of interleukin (IL)17 family cytokines. Recruited γδT cells induced production of IL6 by antigen-presenting cells and suppressed expression of interferon γ by natural killer T cells, promoting hepatocyte proliferation. Absence of IL17-producing γδT cells or deletion of Dectin-1 prevented development of regenerative phenotypes in subsets of innate immune cells. This slowed liver regeneration and was associated with reduced expression of regenerative growth factors and cell cycle regulators. Conversely, exogenous administration of IL17 family cytokines or Dectin-1 ligands promoted regeneration. More broadly, we found that γδT cells are required for inflammatory responses mediated by IL17 and Dectin-1. Conclusions γδT cells regulate hepatic regeneration by producing IL22 and IL17, which have direct mitogenic effects on hepatocytes and promote a regenerative phenotype in hepatic leukocytes, respectively. Dectin-1 ligation is required for γδT cells to promote hepatic regeneration. PMID:24801349

The effect of the Ras homolog gene family (Rho), member A/Rho associated coiled-coil forming protein kinase pathway in atrial fibrosis of type 2 diabetes in rats.

PubMed

Chen, Jinling; Li, Qingqing; Dong, Ruiqing; Gao, Huikuan; Peng, Hui; Wu, Yongquan

2014-09-01

Diabetes mellitus promotes atrial structural remodeling, thereby producing atrial arrhythmogenicity. Atrial arrhythmia can substantially increase the risk of premature death. The aim of this study was to investigate the role of Ras homolog gene family, member A (RhoA)/Rho associated coiled-coil forming protein kinase (ROCK) in atrial fibrosis in diabetic hearts, and the effects of fasudil hydrochloride hydrate on atrial fibrosis. An eight-week-old male Sprague-Dawley rat model of type 2 diabetes was established using a high-fat diet combined with streptozotocin [30 mg/kg, once, intraperitoneal (i.p.)]. Animals were randomly divided into three groups: Control rats, untreated diabetic rats that received vehicle, and treated diabetic rats that received Rho kinase inhibitor fasudil hydrochloride hydrate (10 mg/kg/day, i.p., for 14 weeks). The morphological features of atrial fibrosis were observed using Masson staining. The mRNA expression levels of RhoA, ROCK1, ROCK2, type-I and type-III procollagen were assessed with quantitative polymerase chain reaction. The protein levels of RhoA, ROCK1 and ROCK2 were evaluated using western blot analysis. The atria of untreated diabetic rats showed evident atrial fibrosis as compared to the control rats; the mRNA expression levels of RhoA, ROCK1, ROCK2, type-I and type-III procollagen were upregulated; and the protein levels of RhoA, ROCK1 and ROCK2 were increased. The treatment with fasudil hydrochloride hydrate significantly reduced atrial fibrosis, mRNA levels of RhoA, ROCK1, ROCK2, type-I and type-III procollagen, and the protein levels of RhoA, ROCK1 and ROCK2. The results suggested that RhoA/ROCK was involved in atrial fibrosis, and that fasudil hydrochloride hydrate ameliorates atrial fibrosis through the RhoA/ROCK pathway in rats with type 2 diabetes.

RhoA/ROCK pathway is the major molecular determinant of basal tone in intact human internal anal sphincter.

PubMed

Rattan, Satish; Singh, Jagmohan

2012-04-01

The knowledge of molecular control mechanisms underlying the basal tone in the intact human internal anal sphincter (IAS) is critical for the pathophysiology and rational therapy for a number of debilitating rectoanal motility disorders. We determined the role of RhoA/ROCK and PKC pathways by comparing the effects of ROCK- and PKC-selective inhibitors Y 27632 and Gö 6850 (10(-8) to 10(-4) M), respectively, on the basal tone in the IAS vs. the rectal smooth muscle (RSM). Western blot studies were performed to determine the levels of RhoA/ROCK II, PKC-α, MYPT1, CPI-17, and MLC(20) in the unphosphorylated and phosphorylated forms, in the IAS vs. RSM. Confocal microscopic studies validated the membrane distribution of ROCK II. Finally, to confirm a direct relationship, we examined the enzymatic activities and changes in the basal IAS tone and p-MYPT1, p-CPI-17, and p-MLC(20), before and after Y 27632 and Gö 6850. Data show higher levels of RhoA/ROCK II and related downstream signal transduction proteins in the IAS vs. RSM. In addition, data show a significant correlation between the active RhoA/ROCK levels, ROCK enzymatic activity, downstream proteins, and basal IAS tone, before and after ROCK inhibitor. From these data we conclude 1) RhoA/ROCK and downstream signaling are constitutively active in the IAS, and this pathway (in contrast with PKC) is the critical determinant of the basal tone in intact human IAS; and 2) RhoA and ROCK are potential therapeutic targets for a number of rectoanal motility disorders for which currently there is no satisfactory treatment.

RhoA/ROCK pathway is the major molecular determinant of basal tone in intact human internal anal sphincter

PubMed Central

Singh, Jagmohan

2012-01-01

The knowledge of molecular control mechanisms underlying the basal tone in the intact human internal anal sphincter (IAS) is critical for the pathophysiology and rational therapy for a number of debilitating rectoanal motility disorders. We determined the role of RhoA/ROCK and PKC pathways by comparing the effects of ROCK- and PKC-selective inhibitors Y 27632 and Gö 6850 (10−8 to 10−4 M), respectively, on the basal tone in the IAS vs. the rectal smooth muscle (RSM). Western blot studies were performed to determine the levels of RhoA/ROCK II, PKC-α, MYPT1, CPI-17, and MLC20 in the unphosphorylated and phosphorylated forms, in the IAS vs. RSM. Confocal microscopic studies validated the membrane distribution of ROCK II. Finally, to confirm a direct relationship, we examined the enzymatic activities and changes in the basal IAS tone and p-MYPT1, p-CPI-17, and p-MLC20, before and after Y 27632 and Gö 6850. Data show higher levels of RhoA/ROCK II and related downstream signal transduction proteins in the IAS vs. RSM. In addition, data show a significant correlation between the active RhoA/ROCK levels, ROCK enzymatic activity, downstream proteins, and basal IAS tone, before and after ROCK inhibitor. From these data we conclude 1) RhoA/ROCK and downstream signaling are constitutively active in the IAS, and this pathway (in contrast with PKC) is the critical determinant of the basal tone in intact human IAS; and 2) RhoA and ROCK are potential therapeutic targets for a number of rectoanal motility disorders for which currently there is no satisfactory treatment. PMID:22241857

Two-level inhibition of galK expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the galK gene

PubMed Central

Wang, Xun; Ji, Sang Chun; Jeon, Heung Jin; Lee, Yonho; Lim, Heon M.

2015-01-01

The Escherichia coli gal operon has the structure Pgal-galE-galT-galK-galM. During early log growth, a gradient in gene expression, named type 2 polarity, is established, as follows: galE > galT > galK > galM. However, during late-log growth, type 1 polarity is established in which galK is greater than galT, as follows: galE > galK > galT > galM. We found that type 2 polarity occurs as a result of the down-regulation of galK, which is caused by two different molecular mechanisms: Spot 42-mediated degradation of the galK-specific mRNA, mK2, and Spot 42-mediated Rho-dependent transcription termination at the end of galT. Because the concentration of Spot 42 drops during the transition period of the polarity type switch, these results demonstrate that type 1 polarity is the result of alleviation of Spot 42-mediated galK down-regulation. Because the Spot 42-binding site overlaps with a putative Rho-binding site, a molecular mechanism is proposed to explain how Spot 42, possibly with Hfq, enhances Rho-mediated transcription termination at the end of galT. PMID:26045496

Membrane depolarization-induced RhoA/Rho-associated kinase activation and sustained contraction of rat caudal arterial smooth muscle involves genistein-sensitive tyrosine phosphorylation

PubMed Central

Mita, Mitsuo; Tanaka, Hitoshi; Yanagihara, Hayato; Nakagawa, Jun-ichi; Hishinuma, Shigeru; Sutherland, Cindy; Walsh, Michael P.; Shoji, Masaru

2013-01-01

Rho-associated kinase (ROK) activation plays an important role in K+-induced contraction of rat caudal arterial smooth muscle (Mita et al., Biochem J. 2002; 364: 431–40). The present study investigated a potential role for tyrosine kinase activity in K+-induced RhoA activation and contraction. The non-selective tyrosine kinase inhibitor genistein, but not the src family tyrosine kinase inhibitor PP2, inhibited K+-induced sustained contraction (IC50 = 11.3 ± 2.4 µM). Genistein (10 µM) inhibited the K+-induced increase in myosin light chain (LC20) phosphorylation without affecting the Ca2+ transient. The tyrosine phosphatase inhibitor vanadate induced contraction that was reversed by genistein (IC50 = 6.5 ± 2.3 µM) and the ROK inhibitor Y-27632 (IC50 = 0.27 ± 0.04 µM). Vanadate also increased LC20 phosphorylation in a genistein- and Y-27632-dependent manner. K+ stimulation induced translocation of RhoA to the membrane, which was inhibited by genistein. Phosphorylation of MYPT1 (myosin-targeting subunit of myosin light chain phosphatase) was significantly increased at Thr855 and Thr697 by K+ stimulation in a genistein- and Y-27632-sensitive manner. Finally, K+ stimulation induced genistein-sensitive tyrosine phosphorylation of proteins of ∼55, 70 and 113 kDa. We conclude that a genistein-sensitive tyrosine kinase, activated by the membrane depolarization-induced increase in [Ca2+]i, is involved in the RhoA/ROK activation and sustained contraction induced by K+. Ca2+ sensitization, myosin light chain phosphatase, RhoA, Rho-associated kinase, tyrosine kinase PMID:24133693

REVISITING {rho}{sup 1} CANCRI e: A NEW MASS DETERMINATION OF THE TRANSITING SUPER-EARTH

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

Endl, Michael; Cochran, William D.; MacQueen, Phillip J.

2012-11-01

We present a mass determination for the transiting super-Earth {rho}{sup 1} Cancri e based on nearly 700 precise radial velocity (RV) measurements. This extensive RV data set consists of data collected by the McDonald Observatory planet search and published data from Lick and Keck observatories. We obtained 212 RV measurements with the Tull Coude Spectrograph at the Harlan J. Smith 2.7 m Telescope and combined them with a new Doppler reduction of the 131 spectra that we have taken in 2003-2004 with the High-Resolution Spectrograph (HRS) at the Hobby-Eberly Telescope for the original discovery of {rho}{sup 1} Cancri e. Usingmore » this large data set we obtain a five-planet Keplerian orbital solution for the system and measure an RV semi-amplitude of K = 6.29 {+-} 0.21 m s{sup -1} for {rho}{sup 1} Cnc e and determine a mass of 8.37 {+-} 0.38 M {sub Circled-Plus }. The uncertainty in mass is thus less than 5%. This planet was previously found to transit its parent star, which allowed them to estimate its radius. Combined with the latest radius estimate from Gillon et al., we obtain a mean density of {rho} = 4.50 {+-} 0.20 g cm{sup -3}. The location of {rho}{sup 1} Cnc e in the mass-radius diagram suggests that the planet contains a significant amount of volatiles, possibly a water-rich envelope surrounding a rocky core.« less

Suppressed translation as a mechanism of initiation of CASP8 (caspase 8)-dependent apoptosis in autophagy-deficient NSCLC cells under nutrient limitation.

PubMed

Allavena, Giulia; Cuomo, Francesca; Baumgartner, Georg; Bele, Tadeja; Sellgren, Alexander Yarar; Oo, Kyaw Soe; Johnson, Kaylee; Gogvadze, Vladimir; Zhivotovsky, Boris; Kaminskyy, Vitaliy O

2018-01-01

Macroautophagy/autophagy inhibition under stress conditions is often associated with increased cell death. We found that under nutrient limitation, activation of CASP8/caspase-8 was significantly increased in autophagy-deficient lung cancer cells, which precedes mitochondria outer membrane permeabilization (MOMP), CYCS/cytochrome c release, and activation of CASP9/caspase-9, indicating that under such conditions the activation of CASP8 is a primary event in the initiation of apoptosis as well as essential to reduce clonogenic survival of autophagy-deficient cells. Starvation leads to suppression of CFLAR proteosynthesis and accumulation of CASP8 in SQSTM1 puncta. Overexpression of CFLARs reduces CASP8 activation and apoptosis during starvation, while its silencing promotes efficient activation of CASP8 and apoptosis in autophagy-deficient U1810 lung cancer cells even under nutrient-rich conditions. Similar to starvation, inhibition of protein translation leads to efficient activation of CASP8 and cell death in autophagy-deficient lung cancer cells. Thus, here for the first time we report that suppressed translation leads to activation of CASP8-dependent apoptosis in autophagy-deficient NSCLC cells under conditions of nutrient limitation. Our data suggest that targeting translational machinery can be beneficial for elimination of autophagy-deficient cells via the CASP8-dependent apoptotic pathway.

The Oncogenic Role of RhoGAPs in Basal-Like Breast Cancer

DTIC Science & Technology

2016-04-01

somatic mutations of RhoA in peripheral T cell lymphomas (PTCLs) (16-18) and in diffuse-type gastric carcinomas (19-21). Surprisingly, unlike Rac1...Diffuse-type gastric cancers exhibited mutations in the effector binding domain of RhoA, most commonly Y42C (19-21), which prevents binding to the...Impiombato A, Perez-Garcia A, et al. Recurrent mutations in epigenetic regulators, RHOA and FYN kinase in peripheral T cell lymphomas . Nat Genet 2014;46

Liquid chromatographic determination of chloramine-T and its primary degradation product, p-toluenesulfonamide, in water

USGS Publications Warehouse

Dawson, Verdel K.; Davis, Ruth A.

1997-01-01

N-sodium-N-chloro-rho-toluenesulfonamide (chloramine-T) effectively controls bacterial gill disease (BGD) in cultured fishes, BGD, a common disease of hatchery-reared salmonids, causes more fish losses than any other disease among these species. This study describes a liquid chromatographic (LC) method that is capable of direct, simultaneous analysis of chloramine-T and its primary degradation product, rho-toluenesulfonamide (rho-TSA), in water. The procedure involves reversed-phase (C-18) LC analysis with ion suppression, using 0.01 M phosphate buffer at pH 3. The mobile phase is phosphate buffer-acetonitrile (60 + 40) at 1 mL/min. Both chemicals can be detected with a UV spectrophotometer at 229 nm; the method is linear up to 40 mg, chloramine-T or rho-TSA/L. Mean recoveries were 96.4 +/- 6.1% for water samples fortified with 0.03 mg chloramine-T/L and 95.3 +/- 4.6% for water samples fortified with 0.005 mg rho-TSA/L. Limits of detection without sample enrichment for chloramine-T and rho-TSA are 0.01 mg/L and 0.001 mg/L, respectively.

Liquid chromatographic determination of chloramine-T and its primary degradation product, p-toluenesulfonamide, in water

USGS Publications Warehouse

Dawson, V.K.; Davis, R.A.

1997-01-01

N-sodium-N-chloro-rho-toluenesulfonamide (chloramine-T) effectively controls bacterial gill disease (BGD) in cultured fishes, BGD, a common disease of hatchery-reared salmonids, causes more fish losses than any other disease among these species, This study describes a liquid chromatographic (LC) method that is capable of direct, simultaneous analysis of chloramine-T and its primary degradation product, rho-toluenesulfonamide (rho-TSA), in water. The procedure involves reversed-phase (C-18) LC analysis with ion suppression, using 0.01 M phosphate buffer at pH 3. The mobile phase is phosphate buffer-acetonitrile (60 + 40) at 1 mL/min. Both chemicals can be detected with a UV spectrophotometer at 229 nm; the method is linear up to 40 mg, chloramine-T or rho-TSA/L. Mean recoveries were 96.4 +/- 6.1% for water samples fortified with 0.03 mg chloramine-T/L and 95.3 +/- 4.6% for water samples fortified with 0.005 mg rho-TSA/L. Limits of detection without sample enrichment for chloramine-T and rho-TSA are 0.01 mg/L and 0.001 mg/L, respectively.

17{alpha}-Estradiol arrests cell cycle progression at G{sub 2}/M and induces apoptotic cell death in human acute leukemia Jurkat T cells

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

Jun, Do Youn; Park, Hae Sun; Kim, Jun Seok

2008-09-15

A pharmacological dose (2.5-10 {mu}M) of 17{alpha}-estradiol (17{alpha}-E{sub 2}) exerted a cytotoxic effect on human leukemias Jurkat T and U937 cells, which was not suppressed by the estrogen receptor (ER) antagonist ICI 182,780. Along with cytotoxicity in Jurkat T cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, PARP degradation, and DNA fragmentation were induced. The cytotoxicity of 17{alpha}-E{sub 2} was not blocked by the anti-Fas neutralizing antibody ZB-4. While undergoing apoptosis, there was a remarkable accumulation of G{sub 2}/M cells with the upregulatoin of cdc2 kinase activity, which was reflected in the Thr56more » phosphorylation of Bcl-2. Dephosphorylation at Tyr15 and phosphorylation at Thr161 of cdc2, and significant increase in the cyclin B1 level were underlying factors for the cdc2 kinase activation. Whereas the 17{alpha}-E{sub 2}-induced apoptosis was completely abrogated by overexpression of Bcl-2 or by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G{sub 2}/M cells significantly increased. The caspase-8 inhibitor z-IETD-fmk failed to influence 17{alpha}-E{sub 2}-mediated caspase-9 activation, but it markedly reduced caspase-3 activation and PARP degradation with the suppression of apoptosis, indicating the contribution of caspase-8; not as an upstream event of the mitochondrial cytochrome c release, but to caspase-3 activation. In the presence of hydroxyurea, which blocked the cell cycle progression at the G{sub 1}/S boundary, 17{alpha}-E{sub 2} failed to induce the G{sub 2}/M arrest as well as apoptosis. These results demonstrate that the cytotoxicity of 17{alpha}-E{sub 2} toward Jurkat T cells is attributable to apoptosis mainly induced in G{sub 2}/M-arrested cells, in an ER-independent manner, via a mitochondria-dependent caspase pathway regulated by Bcl-2.« less

Overexpression of caspase 7 is ERα dependent to affect proliferation and cell growth in breast cancer cells by targeting p21(Cip).

PubMed

Chaudhary, S; Madhukrishna, B; Adhya, A K; Keshari, S; Mishra, S K

2016-04-18

Caspase 7 (CASP7) expression has important function during cell cycle progression and cell growth in certain cancer cells and is also involved in the development and differentiation of dental tissues. However, the function of CASP7 in breast cancer cells is unclear. The aim of this study was to analyze the expression of CASP7 in breast carcinoma patients and determine the role of CASP7 in regulating tumorigenicity in breast cancer cells. In this study, we show that the CASP7 expression is high in breast carcinoma tissues compared with normal counterpart. The ectopic expression of CASP7 is significantly associated with ERα expression status and persistently elevated in different stages of the breast tumor grades. High level of CASP7 expression showed better prognosis in breast cancer patients with systemic endocrine therapy as observed from Kaplan-Meier analysis. S3 and S4, estrogen responsive element (ERE) in the CASP7 promoter, is important for estrogen-ERα-mediated CASP7 overexpression. Increased recruitment of p300, acetylated H3 and pol II in the ERE region of CASP7 promoter is observed after hormone stimulation. Ectopic expression of CASP7 in breast cancer cells results in cell growth and proliferation inhibition via p21(Cip) reduction, whereas small interfering RNA (siRNA) mediated reduction of CASP7 rescued p21(Cip) levels. We also show that pro- and active forms of CASP7 is located in the nucleus apart from cytoplasmic region of breast cancer cells. The proliferation and growth of breast cancer cells is significantly reduced by broad-spectrum peptide inhibitors and siRNA of CASP7. Taken together, our findings show that CASP7 is aberrantly expressed in breast cancer and contributes to cell growth and proliferation by downregulating p21(Cip) protein, suggesting that targeting CASP7-positive breast cancer could be one of the potential therapeutic strategies.

Rare NaV1.7 variants associated with painful diabetic peripheral neuropathy

PubMed Central

Blesneac, Iulia; Themistocleous, Andreas C.; Fratter, Carl; Conrad, Linus J.; Ramirez, Juan D.; Cox, James J.; Tesfaye, Solomon; Shillo, Pallai R.; Rice, Andrew S.C.; Tucker, Stephen J.

2018-01-01

Abstract Diabetic peripheral neuropathy (DPN) is a common disabling complication of diabetes. Almost half of the patients with DPN develop neuropathic pain (NeuP) for which current analgesic treatments are inadequate. Understanding the role of genetic variability in the development of painful DPN is needed for improved understanding of pain pathogenesis for better patient stratification in clinical trials and to target therapy more appropriately. Here, we examined the relationship between variants in the voltage-gated sodium channel NaV1.7 and NeuP in a deeply phenotyped cohort of patients with DPN. Although no rare variants were found in 78 participants with painless DPN, we identified 12 rare NaV1.7 variants in 10 (out of 111) study participants with painful DPN. Five of these variants had previously been described in the context of other NeuP disorders and 7 have not previously been linked to NeuP. Those patients with rare variants reported more severe pain and greater sensitivity to pressure stimuli on quantitative sensory testing. Electrophysiological characterization of 2 of the novel variants (M1852T and T1596I) demonstrated that gain of function changes as a consequence of markedly impaired channel fast inactivation. Using a structural model of NaV1.7, we were also able to provide further insight into the structural mechanisms underlying fast inactivation and the role of the C-terminal domain in this process. Our observations suggest that rare NaV1.7 variants contribute to the development NeuP in patients with DPN. Their identification should aid understanding of sensory phenotype, patient stratification, and help target treatments effectively. PMID:29176367

Analysis of free modeling predictions by RBO aleph in CASP11.

PubMed

Mabrouk, Mahmoud; Werner, Tim; Schneider, Michael; Putz, Ines; Brock, Oliver

2016-09-01

The CASP experiment is a biannual benchmark for assessing protein structure prediction methods. In CASP11, RBO Aleph ranked as one of the top-performing automated servers in the free modeling category. This category consists of targets for which structural templates are not easily retrievable. We analyze the performance of RBO Aleph and show that its success in CASP was a result of its ab initio structure prediction protocol. A detailed analysis of this protocol demonstrates that two components unique to our method greatly contributed to prediction quality: residue-residue contact prediction by EPC-map and contact-guided conformational space search by model-based search (MBS). Interestingly, our analysis also points to a possible fundamental problem in evaluating the performance of protein structure prediction methods: Improvements in components of the method do not necessarily lead to improvements of the entire method. This points to the fact that these components interact in ways that are poorly understood. This problem, if indeed true, represents a significant obstacle to community-wide progress. Proteins 2016; 84(Suppl 1):87-104. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Differential binding of RhoA, RhoB, and RhoC to protein kinase C-related kinase (PRK) isoforms PRK1, PRK2, and PRK3: PRKs have the highest affinity for RhoB.

PubMed

Hutchinson, Catherine L; Lowe, Peter N; McLaughlin, Stephen H; Mott, Helen R; Owen, Darerca

2013-11-12

Protein kinase C-related kinases (PRKs) are members of the protein kinase C superfamily of serine-threonine kinases and can be activated by binding to members of the Rho family of GTPases via a Rho-binding motif known as an HR1 domain. Three tandem HR1 domains reside at the N-terminus of the PRKs. We have assessed the ability of the HR1a and HR1b domains from the three PRK isoforms (PRK1, PRK2, and PRK3) to interact with the three Rho isoforms (RhoA, RhoB, and RhoC). The affinities of RhoA and RhoC for a construct encompassing both PRK1 HR1 domains were similar to those for the HR1a domain alone, suggesting that these interactions are mediated solely by the HR1a domain. The affinities of RhoB for both the PRK1 HR1a domain and the HR1ab didomain were higher than those of RhoA or RhoC. RhoB also bound more tightly to the didomain than to the HR1a domain alone, implicating the HR1b domain in the interaction. As compared with PRK1 HR1 domains, PRK2 and PRK3 domains bind less well to all Rho isoforms. Uniquely, however, the PRK3 domains display a specificity for RhoB that requires both the C-terminus of RhoB and the PRK3 HR1b domain. The thermal stability of the HR1a and HR1b domains was also investigated. The PRK2 HR1a domain was found to be the most thermally stable, while PRK2 HR1b, PRK3 HR1a, and PRK3 HR1b domains all exhibited lower melting temperatures, similar to that of the PRK1 HR1a domain. The lower thermal stability of the PRK2 and PRK3 HR1b domains may impart greater flexibility, driving their ability to interact with Rho isoforms.

Pathophysiological effects of RhoA and Rho-associated kinase on cardiovascular system.

PubMed

Cai, Anping; Li, Liwen; Zhou, Yingling

2016-01-01

In past decades, growing evidence from basic and clinical researches reveal that small guanosine triphosphate binding protein ras homolog gene family, member A (RhoA) and its main effector Rho-associated kinase (ROCK) play central and complex roles in cardiovascular systems, and increasing RhoA and ROCK activity is associated with a broad range of cardiovascular diseases such as congestive heart failure, atherosclerosis, and hypertension. Favorable outcomes have been observed with ROCK inhibitors treatment. In this review, we briefly summarize the pathophysiological roles of RhoA/ROCK signaling pathway on cardiovascular system, displaying the potential benefits in the cardiovascular system with controlling RhoA/ROCK signaling pathway.

Tetramethylpyrazine Protects Against Oxygen-Glucose Deprivation-Induced Brain Microvascular Endothelial Cells Injury via Rho/Rho-kinase Signaling Pathway.

PubMed

Yang, Guang; Qian, Chen; Wang, Ning; Lin, Chenyu; Wang, Yan; Wang, Guangyun; Piao, Xinxin

2017-05-01

Tetramethylpyrazine (TMP, also known as Ligustrazine), which is isolated from Chinese Herb Medicine Ligustium wollichii Franchat (Chuan Xiong), has been widely used in China for the treatment of ischemic stroke by Chinese herbalists. Brain microvascular endothelial cells (BMECs) are the integral parts of the blood-brain barrier (BBB), protecting BMECs against oxygen-glucose deprivation (OGD) which is important for the treatment of ischemic stroke. Here, we investigated the protective mechanisms of TMP, focusing on OGD-injured BMECs and the Rho/Rho-kinase (Rho-associated kinases, ROCK) signaling pathway. The model of OGD-injured BMECs was established in this study. BMECs were identified by von Willebrand factor III staining and exposed to fasudil, or TMP at different concentrations (14.3, 28.6, 57.3 µM) for 2 h before 24 h of OGD injury. The effect of each treatment was examined by cell viability assays, measurement of intracellular reactive oxygen species (ROS), and transendothelial electric resistance and western blot analysis (caspase-3, endothelial nitric oxide synthase (eNOS), RhoA, Rac1). Our results show that TMP significantly attenuated apoptosis and the permeability of BMECs induced by OGD. In addition, TMP could notably down-regulate the characteristic proteins in Rho/ROCK signaling pathway such as RhoA and Rac1, which triggered abnormal changes of eNOS and ROS, respectively. Altogether, our results show that TMP has a strong protective effect against OGD-induced BMECs injury and suggest that the mechanism might be related to the inhibition of the Rho/ROCK signaling pathway.

Pathway-focused gene expression profiles and immunohistochemistry detection identify contrasting association of caspase 3 (CASP3) expression with prognosis in pediatric classical Hodgkin lymphoma.

PubMed

Vera-Lozada, Gabriela; Segges, Priscilla; Stefanoff, Claudio Gustavo; Barros, Mário Henrique M; Niedobitek, Gerald; Hassan, Rocio

2018-06-14

The search for clinically relevant molecular markers in classical Hodgkin lymphoma (cHL) is hampered by the histopathological complexity of the disease, resulting from the admixture of a small number of neoplastic Hodgkin and Reed-Sternberg (H-RS) cells with an abundant and heterogeneous microenvironment. In this study, we evaluated gene expression profiles of 11 selected genes previously proposed as a molecular score for adult cHL, aiming to validate its application in the pediatric setting. Assays were performed by RT-qPCR from formalin-fixed paraffin-embedded (FFPE) lymph nodes in 80 patients with cHL. Selected genes were associated with cell cycle (CENPF, CDK1, CCNA2, CCNE2, and HMMR), apoptosis (BCL2, BCL2L1, and CASP3), and monocytes/macrophages (LYZ and STAT1). Despite using controlled preanalytical and analytical strategies, we were not able to validate the 11-gene score to be applied in pediatric cHL. Principal component analysis (PCA) disclosed 3 components that accounted for 65.7% of the total variability. The second PC included microenvironment and apoptosis genes, from which CASP3 expression was associated with a short time of progression-free survival, which impact was maintained in the unfavorable risk group, Epstein-Barr virus-negative cases, and multivariate analysis (P < .05). Because this is a counterintuitive association, CASP3 active expression was assessed at the protein level in H-RS cells by double immunohistochemistry. In contrast to the association of mRNA levels with a poor therapeutic response, a high number of cleaved CASP3+ cells were associated with longer progression-free survival (P = .03) and overall survival (P = .002). Our results demonstrate the feasibility of using FFPE samples as RNA source for molecular prognostication, but argue against the concept of direct and wide applicability of molecular scores in cHL. We reinforce the potential of CASP3 as an interesting target to be explored in adult and pediatric cHL, and

The Andes Virus Nucleocapsid Protein Directs Basal Endothelial Cell Permeability by Activating RhoA

PubMed Central

Gorbunova, Elena E.; Simons, Matthew J.; Gavrilovskaya, Irina N.

2016-01-01

ABSTRACT Andes virus (ANDV) predominantly infects microvascular endothelial cells (MECs) and nonlytically causes an acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). In HPS patients, virtually every pulmonary MEC is infected, MECs are enlarged, and infection results in vascular leakage and highly lethal pulmonary edema. We observed that MECs infected with the ANDV hantavirus or expressing the ANDV nucleocapsid (N) protein showed increased size and permeability by activating the Rheb and RhoA GTPases. Expression of ANDV N in MECs increased cell size by preventing tuberous sclerosis complex (TSC) repression of Rheb-mTOR-pS6K. N selectively bound the TSC2 N terminus (1 to 1403) within a complex containing TSC2/TSC1/TBC1D7, and endogenous TSC2 reciprocally coprecipitated N protein from ANDV-infected MECs. TSCs normally restrict RhoA-induced MEC permeability, and we found that ANDV infection or N protein expression constitutively activated RhoA. This suggests that the ANDV N protein alone is sufficient to activate signaling pathways that control MEC size and permeability. Further, RhoA small interfering RNA, dominant-negative RhoA(N19), and the RhoA/Rho kinase inhibitors fasudil and Y27632 dramatically reduced the permeability of ANDV-infected MECs by 80 to 90%. Fasudil also reduced the bradykinin-directed permeability of ANDV and Hantaan virus-infected MECs to control levels. These findings demonstrate that ANDV activation of RhoA causes MEC permeability and reveal a potential edemagenic mechanism for ANDV to constitutively inhibit the basal barrier integrity of infected MECs. The central importance of RhoA activation in MEC permeability further suggests therapeutically targeting RhoA, TSCs, and Rac1 as potential means of resolving capillary leakage during hantavirus infections. PMID:27795403

Interleukin 17-producing γδT cells promote hepatic regeneration in mice.

PubMed

Rao, Raghavendra; Graffeo, Christopher S; Gulati, Rishabh; Jamal, Mohsin; Narayan, Suchithra; Zambirinis, Constantinos P; Barilla, Rocky; Deutsch, Michael; Greco, Stephanie H; Ochi, Atsuo; Tomkötter, Lena; Blobstein, Reuven; Avanzi, Antonina; Tippens, Daniel M; Gelbstein, Yisroel; Van Heerden, Eliza; Miller, George

2014-08-01

Subsets of leukocytes synergize with regenerative growth factors to promote hepatic regeneration. γδT cells are early responders to inflammation-induced injury in a number of contexts. We investigated the role of γδT cells in hepatic regeneration using mice with disruptions in Tcrd (encodes the T-cell receptor δ chain) and Clec7a (encodes C-type lectin domain family 7 member a, also known as DECTIN1). We performed partial hepatectomies on wild-type C57BL/6, CD45.1, Tcrd(-/-), or Clec7a(-/-) mice. Cells were isolated from livers of patients and mice via mechanical and enzymatic digestion. γδT cells were purified by fluorescence-activated cell sorting. In mice, partial hepatectomy up-regulated expression of CCL20 and ligands of Dectin-1, which was associated with recruitment and activation of γδT cells and their increased production of interleukin (IL)-17 family cytokines. Recruited γδT cells induced production of IL-6 by antigen-presenting cells and suppressed expression of interferon gamma by natural killer T cells, promoting hepatocyte proliferation. Absence of IL-17-producing γδT cells or deletion of Dectin-1 prevented development of regenerative phenotypes in subsets of innate immune cells. This slowed liver regeneration and was associated with reduced expression of regenerative growth factors and cell cycle regulators. Conversely, exogenous administration of IL-17 family cytokines or Dectin-1 ligands promoted regeneration. More broadly, we found that γδT cells are required for inflammatory responses mediated by IL-17 and Dectin-1. γδT cells regulate hepatic regeneration by producing IL-22 and IL-17, which have direct mitogenic effects on hepatocytes and promote a regenerative phenotype in hepatic leukocytes, respectively. Dectin-1 ligation is required for γδT cells to promote hepatic regeneration. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Processing of the Escherichia coli leuX tRNA transcript, encoding tRNA(Leu5), requires either the 3'-->5' exoribonuclease polynucleotide phosphorylase or RNase P to remove the Rho-independent transcription terminator.

PubMed

Mohanty, Bijoy K; Kushner, Sidney R

2010-01-01

Here we report a unique processing pathway in Escherichia coli for tRNA(Leu5) in which the exoribonuclease polynucleotide phosphorylase (PNPase) removes the Rho-independent transcription terminator from the leuX transcript without requiring the RhlB RNA helicase. Our data demonstrate for the first time that PNPase can efficiently degrade an RNA substrate containing secondary structures in vivo. Furthermore, RNase P, an endoribonuclease that normally generates the mature 5'-ends of tRNAs, removes the leuX terminator inefficiently independent of PNPase activity. RNase P cleaves 4-7 nt downstream of the CCA determinant generating a substrate for RNase II, which removes an additional 3-4 nt. Subsequently, RNase T completes the 3' maturation process by removing the remaining 1-3 nt downstream of the CCA determinant. RNase E, G and Z are not involved in terminator removal. These results provide further evidence that the E. coli tRNA processing machinery is far more diverse than previously envisioned.

Functionalized Fullerene Targeting Human Voltage-Gated Sodium Channel, hNav1.7.

PubMed

Hilder, Tamsyn A; Robinson, Anna; Chung, Shin-Ho

2017-08-16

Mutations of hNa v 1.7 that cause its activities to be enhanced contribute to severe neuropathic pain. Only a small number of hNa v 1.7 specific inhibitors have been identified, most of which interact with the voltage-sensing domain of the voltage-activated sodium ion channel. In our previous computational study, we demonstrated that a [Lys 6 ]-C 84 fullerene binds tightly (affinity of 46 nM) to Na v Ab, the voltage-gated sodium channel from the bacterium Arcobacter butzleri. Here, we extend this work and, using molecular dynamics simulations, demonstrate that the same [Lys 6 ]-C 84 fullerene binds strongly (2.7 nM) to the pore of a modeled human sodium ion channel hNa v 1.7. In contrast, the fullerene binds only weakly to a mutated model of hNa v 1.7 (I1399D) (14.5 mM) and a model of the skeletal muscle hNa v 1.4 (3.7 mM). Comparison of one representative sequence from each of the nine human sodium channel isoforms shows that only hNa v 1.7 possesses residues that are critical for binding the fullerene derivative and blocking the channel pore.

Role of RhoA, mDia, and ROCK in cell shape-dependent control of the Skp2-p27kip1 pathway and the G1/S transition.

PubMed

Mammoto, Akiko; Huang, Sui; Moore, Kimberly; Oh, Philmo; Ingber, Donald E

2004-06-18

Cell shape-dependent control of cell-cycle progression underlies the spatial differentials of growth that drive tissue morphogenesis, yet little is known about how cell distortion impacts the biochemical signaling machinery that is responsible for growth control. Here we show that the Rho family GTPase, RhoA, conveys the "cell shape signal" to the cell-cycle machinery in human capillary endothelial cells. Cells accumulating p27(kip1) and arrested in mid G(1) phase when spreading were inhibited by restricted extracellular matrix adhesion, whereas constitutively active RhoA increased expression of the F-box protein Skp2 required for ubiquitination-dependent degradation of p27(kip1) and restored G(1) progression in these cells. Studies with dominant-negative and constitutively active forms of mDia1, a downstream effector of RhoA, and with a pharmacological inhibitor of ROCK, another RhoA target, revealed that RhoA promoted G(1) progression by altering the balance of activities between these two downstream effectors. These data indicate that signaling proteins such as mDia1 and ROCK, which are thought to be involved primarily in cytoskeletal remodeling, also mediate cell growth regulation by coupling cell shape to the cell-cycle machinery at the level of signal transduction.

Targeting RhoA/Rho kinase and p21-activated kinase signaling to prevent cancer development and progression.

PubMed

Chang, Yu-Wen E; Bean, Ronald R; Jakobi, Rolf

2009-06-01

Elevated RhoA/Rho kinase and p21-activated kinase signaling have been shown to promote cancer development and metastasis and have drawn much attention as potential targets of anti-cancer therapy. Elevated RhoA and Rho kinase activity promote cancer cell invasion and eventually lead to metastasis by disrupting E-cadherin-mediated adherens junctions and degradation of the extracellular matrix. Elevated p21-activated kinase activity promotes invasion by stimulating cell motility but also promotes cancer cell survival and growth. In this review we describe normal functions of RhoA/Rho kinase and p21-activated kinase signaling, mechanisms that lead to constitutive activation of RhoA/Rho kinase and p21-activated kinase pathways, and processes by which constitutive RhoA/Rho kinase and p21-activated kinase activity promote cancer development and progression to more aggressive and metastatic phenotypes. In addition, we summarize relevant patents on RhoA/Rho kinase and p21-activated kinase as targets of anti-cancer therapy and discuss the clinical potential of different approaches to modulate RhoA/Rho kinase and p21-activated kinase signaling.

HIV infection impairs Th1 and Th17 Mycobacterium tuberculosis-specific T cell responses

PubMed Central

Murray, Lyle W; Satti, Iman; Meyerowitz, Jodi; Jones, Matthew; Willberg, Christian B; Ussher, James E; Goedhals, Dominique; Hurst, Jacob; Phillips, Rodney E; McShane, Helen

2018-01-01

Background HIV-infected individuals have a higher risk of developing active tuberculosis than HIV-uninfected individuals, but the mechanisms underpinning this are unclear. We hypothesized that depletion of specific components of Mycobacterium tuberculosis (M.tb)-specific CD4+ and CD8+ T cell responses contributed to this increased risk. Methods M.tb-specific T cell responses in 147 HIV-infected and 44 HIV-uninfected control subjects in a TB-endemic setting in Bloemfontein, South Africa were evaluated. Using a whole-blood flow cytometry assay, we measured expression of IFNγ, TNFα, IL-2 and IL-17 in CD4+ and CD8+ T cells in response to M.tb antigens (PPD, ESAT-6/CFP-10 (EC) and DosR regulon-encoded α-crystallin (Rv2031c)). Results Fewer HIV-infected individuals had detectable CD4+ and CD8+ T cell responses to PPD and Rv2031c than HIV-uninfected subjects. M.tb-specific T cells showed distinct patterns of cytokine expression comprising both Th1 (CD4 and CD8) and Th17 (CD4) cytokines, the latter at highest frequency for Rv2031c. Th17 antigen-specific responses to all antigens tested were specifically impaired in HIV-infected individuals. Conclusions HIV-associated impairment of CD4+ and CD8+ M.tb-specific T cell responses is antigen-specific, particularly impacting responses to PPD and Rv2031c. Preferential depletion of Th17 cytokine-expressing CD4+ T cells suggests this T cell subset may be key to TB susceptibility in HIV-infected individuals. PMID:29546381

Low-level laser therapy (LLLT) attenuates RhoA mRNA expression in the rat bronchi smooth muscle exposed to tumor necrosis factor-alpha.

PubMed

de Lima, Flávia Mafra; Bjordal, Jan M; Albertini, Regiane; Santos, Fábio V; Aimbire, Flavio

2010-09-01

Low-level laser therapy (LLLT) has been found to produce anti-inflammatory effects in a variety of disorders. Bronchial smooth muscle (BSM) hyperreactivity is associated with increased Ca+2 sensitivity and increased RhoA mRNA expression. In the current study, we investigated if LLLT could reduce BSM contraction force and RhoA mRNA expression in tumor necrosis factor-alpha (TNF-alpha)-induced BSM hyperreactivity. In the study, 112 male Wistar rats were divided randomly into 16 groups, and BSM was harvested and suspended in TNF-alpha baths for 6 and 24 h, respectively. Irradiation with LLLT was performed with a wavelength of 660 nm for 42 s with a dose of 1.3 J/cm2. This LLLT dose was administered once in the 6-h group and twice in the 24-h group. LLLT significantly decreased contraction force in BSM at 6 h (TNF-alpha + LLLT: 11.65+/-1.10 g/100 mg of tissue) (F=3115) and at 24 h (TNF-alpha+ LLLT: 14.15+/-1.1 g/100 mg of tissue) (F=3245, p<0.05) after TNF-alpha, respectively, when compared to vehicle-bathed groups (control). LLLT also significantly decreased the expression of RhoA mRNA in BSM segments at 6 h (1.22+/-0.20) (F=2820, p<0.05) and 24 h (2.13+/-0.20) (F=3324, p<0.05) when compared to BSM segments incubated with TNF-alpha without LLLT irradiation. We conclude that LLLT administered with this protocol, reduces RhoA mRNA expression and BSM contraction force in TNF-alpha-induced BSM hyperreactivity.

[Changes of CD(4)(+)Foxp3(+) regulatory T cells and CD(4)(+)IL-17(+)T cells in cigarette smoke-exposed rats].

PubMed

Meng, Jing-jing; Zhong, Xiao-ning; Bai, Jing; He, Zhi-yi; Zhang, Jian-quan; Huang, Qiu-pin

2012-01-01

To evaluate the changes of CD(4)(+)IL-17(+) T (Th17) and CD(4)(+)Foxp3(+) regulatory T (Treg) cells in peripheral blood and bronchoalveolar lavage fluid (BALF), and therefore to explore the role of Th17 and Treg in cigarette smoke-induced airway inflammation/COPD in rats. Forty male Wistar rats were randomly divided into 4 groups: a 12 wk smoke-exposure group, a 24 wk smoke-exposure group, a 12 wk control group and a 24 wk control group (n = 10 each). Cells in BALF were collected and analyzed by absolute and differential cell counts. IL-17 and IL-6 levels in serum and BALF were tested by enzyme linked immunosorbent assay (ELISA). The proportion of CD(4)(+)IL-17(+) T and CD(4)(+)Foxp3(+) Treg in peripheral blood and BALF were determined by flow cytometry. The mRNA expressions of IL-17 and Foxp3 were measured by real-time PCR. Comparisons of the data between different groups were performed using one-way ANOVA, and SNK and Games-Howell test were used for comparison between 2 groups. Levels of IL-17 were remarkable increased in the 12 wk smoke-exposure group and the 24 wk smoke-exposure group in serum [(52.6 ± 1.8) ng/L, (75.4 ± 6.0) ng/L] and BALF [(78.1 ± 5.8) ng/L, (95.0 ± 6.8) ng/L] compared with the 12 wk control group [(40.0 ± 3.2)ng/L, (54.5 ± 4.6) ng/L] and the 24 wk control group [(36.7 ± 3.2) ng/L, (53.9 ± 3.7) ng/L], all P < 0.05. IL-6 in serum was significantly increased in the 24 wk smoke-exposure group [(31.4 ± 2.1) ng/L] compared with the 24 wk control group [(11.5 ± 0.5) ng/L], and it was increased in the 12 wk and the 24 wk smoke-exposure group [(33.3 ± 2.3) ng/L, (44.6 ± 3.0) ng/L] compared with the 12 wk and the 24 wk control group [(15.6 ± 1.8) ng/L, (18.0 ± 1.9) ng/L] in BALF. Ratio of Th17 was higher in the 12 wk and the 24 wk smoke-exposure groups in peripheral blood [(1.81 ± 0.19)%, (3.74 ± 0.55)%] and BALF [(7.84 ± 0.28)%, (8.01 ± 0.39)%] compared with the12 wk [(0.97 ± 0.08)%, (5.64 ± 0.54)%] and the 24 wk control group [(1

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

PubMed

Yu, Xuan; Zhang, Qiao; Zhao, Yan; Schwarz, Benjamin J; Stallone, John N; Heaps, Cristine L; Han, Guichun

2017-01-01

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

CASP10-BCL::Fold efficiently samples topologies of large proteins.

PubMed

Heinze, Sten; Putnam, Daniel K; Fischer, Axel W; Kohlmann, Tim; Weiner, Brian E; Meiler, Jens

2015-03-01

During CASP10 in summer 2012, we tested BCL::Fold for prediction of free modeling (FM) and template-based modeling (TBM) targets. BCL::Fold assembles the tertiary structure of a protein from predicted secondary structure elements (SSEs) omitting more flexible loop regions early on. This approach enables the sampling of conformational space for larger proteins with more complex topologies. In preparation of CASP11, we analyzed the quality of CASP10 models throughout the prediction pipeline to understand BCL::Fold's ability to sample the native topology, identify native-like models by scoring and/or clustering approaches, and our ability to add loop regions and side chains to initial SSE-only models. The standout observation is that BCL::Fold sampled topologies with a GDT_TS score > 33% for 12 of 18 and with a topology score > 0.8 for 11 of 18 test cases de novo. Despite the sampling success of BCL::Fold, significant challenges still exist in clustering and loop generation stages of the pipeline. The clustering approach employed for model selection often failed to identify the most native-like assembly of SSEs for further refinement and submission. It was also observed that for some β-strand proteins model refinement failed as β-strands were not properly aligned to form hydrogen bonds removing otherwise accurate models from the pool. Further, BCL::Fold samples frequently non-natural topologies that require loop regions to pass through the center of the protein. © 2015 Wiley Periodicals, Inc.

Thermoelectric Properties of Pulsed Electric Current Sintered Samples of AgPb m SbSe17 ( m = 16 or 17)

NASA Astrophysics Data System (ADS)

Wu, Chun-I.; Todorov, Ilyia; Kanatzidis, Mercouri G.; Timm, Edward; Case, Eldon D.; Schock, Harold; Hogan, Timothy P.

2012-06-01

Lead chalcogenide materials have drawn attention in recent years because of their outstanding thermoelectric properties. Bulk n-type materials of AgPb m SbTe2+ m have been reported to exhibit high figure of merit, ZT, as high as 1.7 at 700 K. Recent reports have shown p-type lead selenide-based compounds with comparable ZT. The analogous material AgPb m SbSe17 shares a similar cubic rock-salt structure with PbTe-based compounds; however, it exhibits a higher melting point, and selenium is more abundant than tellurium. Using solid solution chemistry, we have fabricated cast AgPb15SbSe17 samples that show a peak power factor of approximately 17 μW/cm K2 at 450 K. Increasing the strength of such materials is commonly achieved through powder processing, which also helps to homogenize the source materials. Pulsed electric current sintering (PECS) is a hot-pressing technique that utilizes electric current through the die and sample for direct Joule heating during pressing. The mechanisms present during PECS processing have captured significant research interest and have led to some notable improvements in sample properties compared with other densification techniques. We report the thermoelectric properties of PECS samples of AgPb m SbSe17 along with sample fabrication and processing details.

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

DTIC Science & Technology

2008-08-01

structures, termed the terminal ductal–lobular units (TDLUs), together with interlobular fat and fibrous tissue [16,17]. Most breast cancers arise in the...that benign stages (such as typical and atypical hyperplasia ), noninvasive cancers (such as carcinoma in situ – ductal or lobular), and invasive cancers... inflammatory breast cancer and overexpression of RhoC in immortalized hMECs induces their transformation (35). Impor- tantly, given the linkage of Rho

Princeton_TIGRESS 2.0: High refinement consistency and net gains through support vector machines and molecular dynamics in double-blind predictions during the CASP11 experiment.

PubMed

Khoury, George A; Smadbeck, James; Kieslich, Chris A; Koskosidis, Alexandra J; Guzman, Yannis A; Tamamis, Phanourios; Floudas, Christodoulos A

2017-06-01

Protein structure refinement is the challenging problem of operating on any protein structure prediction to improve its accuracy with respect to the native structure in a blind fashion. Although many approaches have been developed and tested during the last four CASP experiments, a majority of the methods continue to degrade models rather than improve them. Princeton_TIGRESS (Khoury et al., Proteins 2014;82:794-814) was developed previously and utilizes separate sampling and selection stages involving Monte Carlo and molecular dynamics simulations and classification using an SVM predictor. The initial implementation was shown to consistently refine protein structures 76% of the time in our own internal benchmarking on CASP 7-10 targets. In this work, we improved the sampling and selection stages and tested the method in blind predictions during CASP11. We added a decomposition of physics-based and hybrid energy functions, as well as a coordinate-free representation of the protein structure through distance-binning Cα-Cα distances to capture fine-grained movements. We performed parameter estimation to optimize the adjustable SVM parameters to maximize precision while balancing sensitivity and specificity across all cross-validated data sets, finding enrichment in our ability to select models from the populations of similar decoys generated for targets in CASPs 7-10. The MD stage was enhanced such that larger structures could be further refined. Among refinement methods that are currently implemented as web-servers, Princeton_TIGRESS 2.0 demonstrated the most consistent and most substantial net refinement in blind predictions during CASP11. The enhanced refinement protocol Princeton_TIGRESS 2.0 is freely available as a web server at http://atlas.engr.tamu.edu/refinement/. Proteins 2017; 85:1078-1098. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Heat and moisture fluxes within a nighttime maritime stratus cloud during CASP II

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

Gultepe, I.; Issac, G.

Stratus clouds in the lower part of the atmosphere over the ocean or land can play an important role in boundary layer processes and in climate change. Physical, dynamical, and radiative processes within marine stratus clouds on both cloud and regional scale are studied for the first time during the First ISCCP (International Satellite Cloud Climatology Project) Regional Experiment (FIRE) (Albrecht et al., 1988). These clouds can effect the nowcasting, pollution transfer, and radiative processes (Nicholls and Leighton, 1986). Similar to the FIRE stratus project, the Canadian Atlantic Storms Program (CASP) II field project was planned to obtain a bettermore » understanding of cloud physical, dynamical, radiative characteristics, and mesoscale structure of Canadian east coast storms. Here the dynamical and microphysical data, and a radiative transfer model are used to better understand a developing nighttime stratus cloud over the ocean during CASP II which took place over Atlantic Canada. Observations collected by the Convair aircraft of the National Research Council (NRC) of Canada during the CASP II field project on February 6, 1991 are presented.« less

Angiotensin 1-7 Is a Negative Modulator of Aldosterone Secretion In Vitro and In Vivo.

PubMed

Shefer, Gabi; Marcus, Yonit; Knoll, Esther; Dolkart, Oleg; Foichtwanger, Shulamit; Nevo, Nava; Limor, Rona; Stern, Naftali

2016-08-01

Angiotensin (1-7) [Ang 1-7] is a 7 amino acid peptide generated predominantly from Ang II by the action of Ang-converting enzyme 2. We previously showed that Ang 1-7 reduced plasma aldosterone and plasma renin activity in high fructose-fed rats, and that the reduction in circulating aldosterone seemed to accord a parallel reduction in plasma renin activity. Here, we tested the possibility that Ang 1-7 affects aldosterone secretion acting directly in glomerulosa cells. First, as detected by immunofluorescence, the receptor for Ang 1-7, Mas1 is localized predominantly at the rat adrenal subcapsular region. Second, in isolated rat glomerulosa cells incubates, Ang 1-7 attenuated the aldosterone response to Ang II, with the strongest effect seen on Ang II (10(-9) M) (control 22±2.5 pg/10(5) cells; Ang II [10(-9) M] 189±11 pg/10(5) cells; Ang II [10(-9) M]+Ang 1-7 [10(-6) M] 33±3.6 pg/10(5) cells; P<0.001) and the largest effect on adrenocorticotropic hormone (10(-8) M) (control 30±3.4 pg/10(5) cells; ACTH [10(-8) M] 409±32.5 pg/10(5) cells; ACTH [10(-8) M]+Ang 1-7 [10(-6) M] 280±12.5 pg/10(5) cells; P<0.001). In contrast, Ang 1-7 did not affect the aldosterone response to potassium (K(+)). In rats subjected to a low-salt diet for 7 days, continuous infusion of Ang 1-7 (576 μg/kg per day) resulted in a lesser rise in aldosterone (salt deplete+Ang 1-7, 16.4±4.8 ng/dL) compared with rats receiving vehicle (salt deplete+vehicle, 27.6±5.3 ng/dL; P<0.01) but did not modify plasma renin activity. Taken together, these results indicate that Ang 1-7 can act as a negative modulator of aldosterone secretion in vitro and in vivo. © 2016 American Heart Association, Inc.

Rho-kinase signaling controls nucleocytoplasmic shuttling of class IIa Histone Deacetylase (HDAC7) and transcriptional activation of orphan nuclear receptor NR4A1

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

Compagnucci, Claudia; Barresi, Sabina; Petrini, Stefania

2015-04-03

Rho-kinase (ROCK) has been well documented to play a key role in RhoA-induced actin remodeling. ROCK activation results in myosin light chain (MLC) phosphorylation either by direct action on MLC kinase (MLCK) or by inhibition of MLC phosphatase (MLCP), modulating actin–myosin contraction. We found that inhibition of the ROCK pathway in induced pluripotent stem cells, leads to nuclear export of HDAC7 and transcriptional activation of the orphan nuclear receptor NR4A1 while in cells with constitutive ROCK hyperactivity due to loss of function of the RhoGTPase activating protein Oligophrenin-1 (OPHN1), the orphan nuclear receptor NR4A1 is downregulated. Our study identify amore » new target of ROCK signaling via myosin phosphatase subunit (MYPT1) and Histone Deacetylase (HDAC7) at the nuclear level and provide new insights in the cellular functions of ROCK. - Highlights: • ROCK regulates nucleocytoplasmic shuttling of HDAC7 via phosphorylation of MYPT1. • Nuclear export of HDAC7 and upregulation of NR4A1 occurs with low ROCK activity. • High levels of ROCK activity due to OPHN1 loss of function downregulate NR4A1.« less

Enhanced activity of an angiotensin-(1-7) neuropeptidase in glucocorticoid-induced fetal programming.

PubMed

Marshall, Allyson C; Shaltout, Hossam A; Pirro, Nancy T; Rose, James C; Diz, Debra I; Chappell, Mark C

2014-02-01

We previously identified angiotensin converting enzyme (ACE) and an endopeptidase activity that degraded angiotensin-(1-7) [Ang-(1-7)] to Ang-(1-5) and Ang-(1-4), respectively, in the cerebrospinal fluid (CSF) of 6-month old male sheep. The present study undertook a more comprehensive analysis of the CSF peptidase that converts Ang-(1-7) to Ang-(1-4) in control and in utero betamethasone-exposed sheep (BMX). Characterization of the Ang-(1-7) peptidase revealed that the thiol agents 4-aminophenylmercuric acetate (APMA) and p-chloromercuribenzoic acid (PCMB), as well as the metallo-chelators o-phenanthroline and EDTA essentially abolished the enzyme activity. Additional inhibitors for serine, aspartyl, and cysteine proteases, as well as selective inhibitors against the endopeptidases neprilysin, neurolysin, prolyl and thimet oligopeptidases did not attenuate enzymatic activity. Competition studies against the peptidase revealed similar IC50s for Ang-(1-7) (5μM) and Ang II (3μM), but lower values for Ala(1)-Ang-(1-7) and Ang-(2-7) of 1.8 and 2.0μM, respectively. In contrast, bradykinin exhibited a 6-fold higher IC50 (32μM) than Ang-(1-7) while neurotensin was a poor competitor. Mean arterial pressure (78±1 vs. 94±2mmHg, N=4-5, P<0.01) and Ang-(1-7) peptidase activity (14.2±1 vs 32±1.5fmol/min/ml CSF, N=5, P<0.01) were higher in the BMX group, and enzyme activity inversely correlated with Ang-(1-7) content in CSF. Lower Ang-(1-7) expression in brain is linked to baroreflex impairment in hypertension and aging, thus, increased activity of an Ang-(1-7) peptidase may contribute to lower CSF Ang-(1-7) levels, elevated blood pressure and impaired reflex function in this model of fetal programming. Copyright © 2013 Elsevier Inc. All rights reserved.

17β-Estradiol Induces Overproliferation in Adenomyotic Human Uterine Smooth Muscle Cells of the Junctional Zone Through Hyperactivation of the Estrogen Receptor-Enhanced RhoA/ROCK Signaling Pathway.

PubMed

Sun, Fu-Qing; Duan, Hua; Wang, Sha; Li, Jin-Jiao

2015-11-01

Adenomyosis (ADS) is a common estrogen-dependent gynecological disease with unknown etiology. Recent models favor abnormal thickening of the junctional zone (JZ) may be the causative factor in the development of ADS. RhoA, a small guanosine triphosphatase which controls multiple cellular processes, is involved in the control of cell proliferation. Here we demonstrate that treatment of human uterine smooth muscle cells (SMCs) of the JZ with 17β-estradiol (E2) increased expression of RhoA and its downstream effectors (-associated coiled coil containing protein kinase [ROCK] 1 and ROCK2). Compared with non-ADS cells, RhoA, ROCK1, and ROCK2 were overexpressed and hyperactivated in ADS cells. These effects were suppressed in the presence of ICI 182,780, supporting an estrogen receptor (ER)-dependent mechanism. Hyperactivation of ER-enhanced RhoA/ROCK signaling was associated with overproliferation in ADS human uterine SMCs of the JZ. Moreover, E2-induced overproliferation was accompanied by downregulation of cyclin-dependent kinases inhibitors (CKIs; p21(Waf1/Cip1) and p27(Kip1)) and upregulation of cyclin-dependent kinases (CDKs) and cyclins (cyclin D1, cyclin E1, CDK2, CDK4, and CDK6). © The Author(s) 2015.

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

PubMed Central

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

2012-01-01

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

17 CFR 230.702(T)-230.703(T) - [Reserved

Code of Federal Regulations, 2011 CFR

2011-04-01

... 17 Commodity and Securities Exchanges 2 2011-04-01 2011-04-01 false [Reserved] 230.702(T)-230.703(T) Section 230.702(T)-230.703(T) Commodity and Securities Exchanges SECURITIES AND EXCHANGE... Small Business Investment Companies §§ 230.702(T)-230.703(T) [Reserved] Exemptions for Cross-Border...

Measurement of branching fractions and charge asymmetries in B+/--->rho+/-pi0 and B+/--->rho0pi+/- decays, and search for B0-->rho0pi0.

PubMed

Aubert, B; Barate, R; Boutigny, D; Couderc, F; Gaillard, J-M; Hicheur, A; Karyotakis, Y; Lees, J P; Robbe, P; Tisserand, V; Zghiche, A; Palano, A; Pompili, A; Chen, J C; Qi, N D; Rong, G; Wang, P; Zhu, Y S; Eigen, G; Ofte, I; Stugu, B; Abrams, G S; Borgland, A W; Breon, A B; Brown, D N; Button-Shafer, J; Cahn, R N; Charles, E; Day, C T; Gill, M S; Gritsan, A V; Groysman, Y; Jacobsen, R G; Kadel, R W; Kadyk, J; Kerth, L T; Kolomensky, Yu G; Kukartsev, G; LeClerc, C; Levi, M E; Lynch, G; Mir, L M; Oddone, P J; Orimoto, T J; Pripstein, M; Roe, N A; Romosan, A; Ronan, M T; Shelkov, V G; Telnov, A V; Wenzel, W A; Ford, K; Harrison, T J; Hawkes, C M; Knowles, D J; Morgan, S E; Penny, R C; Watson, A T; Watson, N K; Goetzen, K; Held, T; Koch, H; Lewandowski, B; Pelizaeus, M; Peters, K; Schmuecker, H; Steinke, M; Boyd, J T; Chevalier, N; Cottingham, W N; Kelly, M P; Latham, T E; Mackay, C; Wilson, F F; Abe, K; Cuhadar-Donszelmann, T; Hearty, C; Mattison, T S; McKenna, J A; Thiessen, D; Kyberd, P; McKemey, A K; Teodorescu, L; Blinov, V E; Bukin, A D; Golubev, V B; Ivanchenko, V N; Kravchenko, E A; Onuchin, A P; Serednyakov, S I; Skovpen, Yu I; Solodov, E P; Yushkov, A N; Best, D; Bruinsma, M; Chao, M; Kirkby, D; Lankford, A J; Mandelkern, M; Mommsen, R K; Roethel, W; Stoker, D P; Buchanan, C; Hartfiel, B L; Gary, J W; Layter, J; Shen, B C; Wang, K; del Re, D; Hadavand, H K; Hill, E J; MacFarlane, D B; Paar, H P; Rahatlou, Sh; Sharma, V; Berryhill, J W; Campagnari, C; Dahmes, B; Levy, S L; Long, O; Lu, A; Mazur, M A; Richman, J D; Verkerke, W; Beck, T W; Beringer, J; Eisner, A M; Heusch, C A; Lockman, W S; Schalk, T; Schmitz, R E; Schumm, B A; Seiden, A; Spradlin, P; Turri, M; Walkowiak, W; Williams, D C; Wilson, M G; Albert, J; Chen, E; Dubois-Felsmann, G P; Dvoretskii, A; Erwin, R J; Hitlin, D G; Narsky, I; Piatenko, T; Porter, F C; Ryd, A; Samuel, A; Yang, S; Jayatilleke, S; Mancinelli, G; Meadows, B T; Sokoloff, M D; Abe, T; Blanc, F; Bloom, P; Chen, S; Clark, P J; Ford, W T; Nauenberg, U; Olivas, A; Rankin, P; Roy, J; Smith, J G; van Hoek, W C; Zhang, L; Harton, J L; Hu, T; Soffer, A; Toki, W H; Wilson, R J; Zhang, J; Altenburg, D; Brandt, T; Brose, J; Colberg, T; Dickopp, M; Dubitzky, R S; Hauke, A; Lacker, H M; Maly, E; Müller-Pfefferkorn, R; Nogowski, R; Otto, S; Schubert, J; Schubert, K R; Schwierz, R; Spaan, B; Wilden, L; Bernard, D; Bonneaud, G R; Brochard, F; Cohen-Tanugi, J; Grenier, P; Thiebaux, Ch; Vasileiadis, G; Verderi, M; Khan, A; Lavin, D; Muheim, F; Playfer, S; Swain, J E; Andreotti, M; Azzolini, V; Bettoni, D; Bozzi, C; Calabrese, R; Cibinetto, G; Luppi, E; Negrini, M; Piemontese, L; Sarti, A; Treadwell, E; Baldini-Ferroli, R; Calcaterra, A; de Sangro, R; Falciai, D; Finocchiaro, G; Patteri, P; Piccolo, M; Zallo, A; Buzzo, A; Capra, R; Contri, R; Crosetti, G; Lo Vetere, M; Macri, M; Monge, M R; Passaggio, S; Patrignani, C; Robutti, E; Santroni, A; Tosi, S; Bailey, S; Morii, M; Won, E; Bhimji, W; Bowerman, D A; Dauncey, P D; Egede, U; Eschrich, I; Gaillard, J R; Morton, G W; Nash, J A; Taylor, G P; Grenier, G J; Lee, S-J; Mallik, U; Cochran, J; Crawley, H B; Lamsa, J; Meyer, W T; Prell, S; Rosenberg, E I; Yi, J; Davier, M; Grosdidier, G; Höcker, A; Laplace, S; Le Diberder, F; Lepeltier, V; Lutz, A M; Petersen, T C; Plaszczynski, S; Schune, M H; Tantot, L; Wormser, G; Brigljević, V; Cheng, C H; Lange, D J; Simani, M C; Wright, D M; Bevan, A J; Coleman, J P; Fry, J R; Gabathuler, E; Gamet, R; Kay, M; Parry, R J; Payne, D J; Sloane, R J; Touramanis, C; Back, J J; Harrison, P F; Shorthouse, H W; Vidal, P B; Brown, C L; Cowan, G; Flack, R L; Flaecher, H U; George, S; Green, M G; Kurup, A; Marker, C E; McMahon, T R; Ricciardi, S; Salvatore, F; Vaitsas, G; Winter, M A; Brown, D; Davis, C L; Allison, J; Barlow, N R; Barlow, R J; Hart, P A; Hodgkinson, M C; Jackson, F; Lafferty, G D; Lyon, A J; Weatherall, J H; Williams, J C; Farbin, A; Jawahery, A; Kovalskyi, D; Lae, C K; Lillard, V; Roberts, D A; Blaylock, G; Dallapiccola, C; Flood, K T; Hertzbach, S S; Kofler, R; Koptchev, V B; Moore, T B; Saremi, S; Staengle, H; Willocq, S; Cowan, R; Sciolla, G; Taylor, F; Yamamoto, R K; Mangeol, D J J; Patel, P M; Robertson, S H; Lazzaro, A; Palombo, F; Bauer, J M; Cremaldi, L; Eschenburg, V; Godang, R; Kroeger, R; Reidy, J; Sanders, D A; Summers, D J; Zhao, H W; Brunet, S; Cote-Ahern, D; Taras, P; Nicholson, H; Cartaro, C; Cavallo, N; De Nardo, G; Fabozzi, F; Gatto, C; Lista, L; Paolucci, P; Piccolo, D; Sciacca, C; Baak, M A; Raven, G; LoSecco, J M; Gabriel, T A; Brau, B; Gan, K K; Honscheid, K; Hufnagel, D; Kagan, H; Kass, R; Pulliam, T; Wong, Q K; Brau, J; Frey, R; Igonkina, O; Potter, C T; Sinev, N B; Strom, D; Torrence, E; Colecchia, F; Dorigo, A; Galeazzi, F; Margoni, M; Morandin, M; Posocco, M; Rotondo, M; Simonetto, F; Stroili, R; Tiozzo, G; Voci, C; Benayoun, M; Briand, H; Chauveau, J; David, P; de la Vaissière, Ch; Del Buono, L; Hamon, O; John, M J J; Leruste, Ph; Ocariz, J; Pivk, M; Roos, L; Stark, J; T'Jampens, S; Therin, G; Manfredi, P F; Re, V; Behera, P K; Gladney, L; Guo, Q H; Panetta, J; Anulli, F; Biasini, M; Peruzzi, I M; Pioppi, M; Angelini, C; Batignani, G; Bettarini, S; Bondioli, M; Bucci, F; Calderini, G; Carpinelli, M; Del Gamba, V; Forti, F; Giorgi, M A; Lusiani, A; Marchiori, G; Martinez-Vidal, F; Morganti, M; Neri, N; Paoloni, E; Rama, M; Rizzo, G; Sandrelli, F; Walsh, J; Haire, M; Judd, D; Paick, K; Wagoner, D E; Danielson, N; Elmer, P; Lu, C; Miftakov, V; Olsen, J; Smith, A J S; Tanaka, H A; Varnes, E W; Bellini, F; Cavoto, G; Faccini, R; Ferrarotto, F; Ferroni, F; Gaspero, M; Mazzoni, M A; Morganti, S; Pierini, M; Piredda, G; Safai Tehrani, F; Voena, C; Christ, S; Wagner, G; Waldi, R; Adye, T; De Groot, N; Franek, B; Geddes, N I; Gopal, G P; Olaiya, E O; Xella, S M; Aleksan, R; Emery, S; Gaidot, A; Ganzhur, S F; Giraud, P-F; Hamel de Monchenault, G; Kozanecki, W; Langer, M; Legendre, M; London, G W; Mayer, B; Schott, G; Vasseur, G; Yeche, Ch; Zito, M; Purohit, M V; Weidemann, A W; Yumiceva, F X; Aston, D; Bartoldus, R; Berger, N; Boyarski, A M; Buchmueller, O L; Convery, M R; Cristinziani, M; Dong, D; Dorfan, J; Dujmic, D; Dunwoodie, W; Elsen, E E; Field, R C; Glanzman, T; Gowdy, S J; Grauges-Pous, E; Hadig, T; Halyo, V; Hast, C; Hryn'ova, T; Innes, W R; Jessop, C P; Kelsey, M H; Kim, P; Kocian, M L; Langenegger, U; Leith, D W G S; Libby, J; Luitz, S; Luth, V; Lynch, H L; Marsiske, H; Messner, R; Muller, D R; O'Grady, C P; Ozcan, V E; Perazzo, A; Perl, M; Petrak, S; Ratcliff, B N; Roodman, A; Salnikov, A A; Schindler, R H; Schwiening, J; Simi, G; Snyder, A; Soha, A; Stelzer, J; Su, D; Sullivan, M K; Va'vra, J; Wagner, S R; Weaver, M; Weinstein, A J R; Wisniewski, W J; Wright, D H; Young, C C; Burchat, P R; Edwards, A J; Meyer, T I; Petersen, B A; Roat, C; Ahmed, M; Ahmed, S; Alam, M S; Ernst, J A; Saeed, M A; Saleem, M; Wappler, F R; Bugg, W; Krishnamurthy, M; Spanier, S M; Eckmann, R; Kim, H; Ritchie, J L; Schwitters, R F; Izen, J M; Kitayama, I; Lou, X C; Ye, S; Bianchi, F; Bona, M; Gallo, F; Gamba, D; Borean, C; Bosisio, L; Della Ricca, G; Dittongo, S; Grancagnolo, S; Lanceri, L; Poropat, P; Vitale, L; Vuagnin, G; Panvini, R S; Banerjee, Sw; Brown, C M; Fortin, D; Jackson, P D; Kowalewski, R; Roney, J M; Band, H R; Dasu, S; Datta, M; Eichenbaum, A M; Johnson, J R; Kutter, P E; Li, H; Liu, R; Di Lodovico, F; Mihalyi, A; Mohapatra, A K; Pan, Y; Prepost, R; Sekula, S J; von Wimmersperg-Toeller, J H; Wu, J; Wu, S L; Yu, Z; Neal, H

2004-07-30

We present measurements of branching fractions and charge asymmetries in B-meson decays to rho(+)pi(0), rho(0)pi(+), and rho(0)pi(0). The data sample comprises 89x10(6) Upsilon(4S)-->BBmacr; decays collected with the BABAR detector at the PEP-II asymmetric-energy B Factory at SLAC. We find the charge-averaged branching fractions B(B+-->rho(+)pi(0))=[10.9+/-1.9(stat)+/-1.9(syst)]x10(-6) and B(B+-->rho(0)pi(+))=(9.5+/-1.1+/-0.9)x10(-6), and we set a 90% confidence-level upper limit B(B0-->rho(0)pi(0))<2.9x10(-6). We measure the charge asymmetries ACP(pi(0))(rho(+))=0.24+/-0.16+/-0.06 and ACP(pi(+))(rho(0))=-0.19+/-0.11+/-0.02.

Rho/Rho kinase and phosphoinositide 3-kinase are parallel pathways in the development of spontaneous arterial tone in deoxycorticosterone acetate-salt hypertension.

PubMed

Wehrwein, Erica A; Northcott, Carrie A; Loberg, Robert D; Watts, Stephanie W

2004-06-01

Hypertension is characterized by abnormal vascular contractility and function. Arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive rats develop spontaneous tone that is not observed in arteries from normotensive rats. Inhibition of phosphoinositide 3-kinase (PI3-kinase) by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) reduces spontaneous tone development. The Rho/Rho-kinase pathway has been suggested to play a role in hypertension and may be dependent on PI3-kinase activity. We hypothesized that Rhokinase is involved in spontaneous tone development and that Rho/Rho-kinase is a downstream effector of PI3-kinase. Using endothelium-denuded aortic strips in isolated tissue bath, we demonstrated that (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) (Y27632) (1 microM), a Rho-kinase inhibitor, significantly reduced spontaneous tone in the DOCA aorta but that it did not affect sham aorta basal tone (DOCA 63.5 +/- 15.9 versus sham 1.2 +/- 0.4 total change in percentage of phenylephrine contraction). We examined the interaction between the PI3-kinase and Rho pathways by observing the effects of LY294002 on a Rhokinase effector, myosin phosphatase (MYPT), and Y27632 on a PI3-kinase effector, Akt, using Western blot analysis. Inhibition of PI3-kinase reduced spontaneous tone, but it had no effect on the phosphorylation status of MYPT, indicating that PI3-kinase is not a downstream effector of Rho/Rho-kinase. These data indicate that there is little interaction between the Rho/Rhokinase and PI3-kinase pathways in the DOCA-salt aorta, and the two pathways seem to operate in parallel in supporting spontaneous arterial tone. These data reflect spontaneous tone only and do not rule out the possibility of interaction between these pathways in agonist-stimulated tone.

Quality of life amongst older Brazilians: a cross-cultural validation of the CASP-19 into Brazilian-Portuguese.

PubMed

Lima, Fábia M; Hyde, Martin; Chungkham, Holendro Singh; Correia, Clarice; Siqueira Campos, Alexsandra; Campos, Marília; Novaes, Moacir; Laks, Jerson; Petribu, Kátia

2014-01-01

As population ageing becomes a global phenomenon the need to understand the quality of life of older people around the world has become increasingly salient. The CASP-19 is a well established measure of quality of later life. The scale is composed of 19 items which map onto the four domains of control (C), Autonomy (A), Self-Realisation (S) and Pleasure (P). It has already been translated to 12 languages and has been used in a number of national and international studies. However use of the scale outside of Europe has been very limited. The objective of this study was to translate and evaluate the use of the CASP-19 amongst older Brazilians. The CASP-19 was translated from English to Portuguese, back-translated and submitted to an analysis of equivalence by a committee of judges. The scale was then administered to a sample of community dwelling older people in Recife, Brazil (n = 87), and tested for psychometric properties. The Control and Pleasure domains exhibited good internal consistency. By removing one item from each of the Autonomy and Self Realisation domains their internal consistency was improved. The mean age of the sample was 75.6±0.7 years, subjects were mainly female (52.9%), white (52.9%), who lived without a partner (54%), and had a monthly income varying from USD 340.00 to USD 850.00. Translation and cross-cultural adaptation permitted good understanding and applicability of final version. Psychometric analyses revealed that the removal of two items improved the internal consistency of the Autonomy and Pleasure domains. Confirmatory factor analyses suggest that a 16 item, four factor, model best fits the data. In this small exploratory study the CASP-19 Brazil demonstrated good psychometric properties. It was easy to use for both participants and researchers. Hopefully future studies in Brazil will employ the scale so that more direct cross national comparisons can be made with older people in Europe and the US.

Redundancy of primary RNA-binding functions of the bacterial transcription terminator Rho

PubMed Central

Shashni, Rajesh; Qayyum, M. Zuhaib; Vishalini, V.; Dey, Debashish; Sen, Ranjan

2014-01-01

The bacterial transcription terminator, Rho, terminates transcription at half of the operons. According to the classical model derived from in vitro assays on a few terminators, Rho is recruited to the transcription elongation complex (EC) by recognizing specific sites (rut) on the nascent RNA. Here, we explored the mode of in vivo recruitment process of Rho. We show that sequence specific recognition of the rut site, in majority of the Rho-dependent terminators, can be compromised to a great extent without seriously affecting the genome-wide termination function as well as the viability of Escherichia coli. These terminators function optimally only through a NusG-assisted recruitment and activation of Rho. Our data also indicate that at these terminators, Rho-EC-bound NusG interaction facilitates the isomerization of Rho into a translocase-competent form by stabilizing the interactions of mRNA with the secondary RNA binding site, thereby overcoming the defects of the primary RNA binding functions. PMID:25081210

The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration

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

Blom, Magdalena; Reis, Katarina; Heldin, Johan

RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as corticalmore » actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration. - Highlights: • Increased RhoD expression leads to loss of actin structures, e.g. stress fibers and gives rise to decreased actin dynamics. • RhoD knockdown induces various actin-containing structures such as edge ruffles, stress fibers and cortical actin, in a cell-type specific manner. • RhoD induces specific actin rearrangements depending on its subcellular localization. • RhoD knockdown has effects on cellular processes, such as directed cell migration and proliferation.« less

Antenatal/early postnatal hypothyroidism increases the contribution of Rho-kinase to contractile responses of mesenteric and skeletal muscle arteries in adult rats.

PubMed

Gaynullina, Dina K; Sofronova, Svetlana I; Shvetsova, Anastasia A; Selivanova, Ekaterina K; Sharova, Anna P; Martyanov, Andrey A; Tarasova, Olga S

2018-05-23

Maternal thyroid deficiency can increase Rho-kinase procontractile influence in arteries of 2-week-old progeny. Here we hypothesized that augmented role of Rho-kinase persists in arteries from adult progeny of hypothyroid rats. Dams were treated with 6-propyl-2-thiouracil (PTU) in drinking water (0.0007%) during pregnancy and 2 weeks postpartum; control (CON) females received PTU-free water. At the age of 10-12-weeks, serum T 3 /T 4 levels did not differ between PTU and CON male offspring. Cutaneous (saphenous), mesenteric, and skeletal muscle (sural) arteries were studied by wire myography, qPCR, and Western blotting. Saphenous arteries of PTU and CON groups showed similar responses to α 1 -adrenoceptor agonist methoxamine and were equally suppressed by Rho-kinase inhibitor Y27632. Responses of mesenteric arteries also did not differ between PTU and CON, but the effects of Y27632 were more prominent in the PTU group. Sural arteries of PTU rats compared to CON demonstrated augmented responses to methoxamine, increased RhoA mRNA contents and higher levels of MYPT1 phosphorylation at Thr 855 . Intergroup differences in contractile responses and phospho-MYPT1-Thr 855 were eliminated by Y27632. Rho-kinase contribution to contractile responses of mesenteric and especially sural arteries is augmented in adult PTU rats. Therefore, maternal thyroid deficiency may have long-term detrimental consequences for vasculature in adult offspring.

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

PubMed Central

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

2013-01-01

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

Protein structure modeling and refinement by global optimization in CASP12.

PubMed

Hong, Seung Hwan; Joung, InSuk; Flores-Canales, Jose C; Manavalan, Balachandran; Cheng, Qianyi; Heo, Seungryong; Kim, Jong Yun; Lee, Sun Young; Nam, Mikyung; Joo, Keehyoung; Lee, In-Ho; Lee, Sung Jong; Lee, Jooyoung

2018-03-01

For protein structure modeling in the CASP12 experiment, we have developed a new protocol based on our previous CASP11 approach. The global optimization method of conformational space annealing (CSA) was applied to 3 stages of modeling: multiple sequence-structure alignment, three-dimensional (3D) chain building, and side-chain re-modeling. For better template selection and model selection, we updated our model quality assessment (QA) method with the newly developed SVMQA (support vector machine for quality assessment). For 3D chain building, we updated our energy function by including restraints generated from predicted residue-residue contacts. New energy terms for the predicted secondary structure and predicted solvent accessible surface area were also introduced. For difficult targets, we proposed a new method, LEEab, where the template term played a less significant role than it did in LEE, complemented by increased contributions from other terms such as the predicted contact term. For TBM (template-based modeling) targets, LEE performed better than LEEab, but for FM targets, LEEab was better. For model refinement, we modified our CASP11 molecular dynamics (MD) based protocol by using explicit solvents and tuning down restraint weights. Refinement results from MD simulations that used a new augmented statistical energy term in the force field were quite promising. Finally, when using inaccurate information (such as the predicted contacts), it was important to use the Lorentzian function for which the maximal penalty arising from wrong information is always bounded. © 2017 Wiley Periodicals, Inc.

17 CFR 240.17Ad-21T - Operational capability in a Year 2000 environment.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Operational capability in a Year 2000 environment. 240.17Ad-21T Section 240.17Ad-21T Commodity and Securities Exchanges SECURITIES... Company Rules § 240.17Ad-21T Operational capability in a Year 2000 environment. (a) This section applies...

17 CFR 230.702(T)-230.703(T) - [Reserved

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 2 2010-04-01 2010-04-01 false [Reserved] 230.702(T)-230.703(T) Section 230.702(T)-230.703(T) Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION GENERAL RULES AND REGULATIONS, SECURITIES ACT OF 1933 Regulation E-Exemption for Securities of...

IL-23 Activated γδ T Cells Affect Th17 Cells and Regulatory T Cells by Secreting IL-21 in Children with Primary Nephrotic Syndrome.

PubMed

Zhang, L; Yan, J; Yang, B; Zhang, G; Wang, M; Dong, S; Liu, W; Yang, H; Li, Q

2018-01-01

This study (1) analysed the percentage of γδ T cells, γδ T cell subsets, Th17 cells and regulatory T cells (Treg cells) and (2) determined the role of IL-23 in primary nephrotic syndrome (PNS) patients with active disease and in remission. Eighty-four patients with PNS and 51 healthy age-matched controls were included in this study. The percentage of γδ T cells, γδ T cell subsets, Th17 cells and Treg cells in peripheral blood mononuclear cells (PBMCs) were analysed by fluorescence-activated cell sorting. PMBCs from PNS patients with active disease were cultured in the presence of IL-23, IL-23 and an IL-23 antagonist, or IL23 and an anti-IL-21 monoclonal antibody (mAb). The percentage of γδ T cells, IL-23R + γδ T cells and IL-17 + γδ T cells were significantly increased in PNS patients with active disease. There was a positive correlation between the percentage of γδ T cells, IL-23R + γδ T cells, IL-17 + γδ T cells and the Th17/Treg ratio. IL-23 increased the percentage of γδ T cells and Th17 cells and decreased the percentage of Treg cells in PBMCs isolated from PNS patients with active disease. Anti-IL-21 mAb reduced the percentage of γδ T cells and Th17 cells, but increased the percentage of Treg cells. γδ T cells, IL-17 + γδ T cells and IL-23R + γδ T cells may be involved in the pathogenesis of paediatric PNS by modulating the balance of Th17/Treg cells. γδ T cells may cause an imbalance in Th17/Treg cells by secreting IL-21 in the presence of IL-23. © 2017 The Foundation for the Scandinavian Journal of Immunology.

The RhoU/Wrch1 Rho GTPase gene is a common transcriptional target of both the gp130/STAT3 and Wnt-1 pathways

PubMed Central

SCHIAVONE, Davide; DEWILDE, Sarah; VALLANIA, Francesco; TURKSON, James; CUNTO, Ferdinando DI; POLI, Valeria

2010-01-01

STAT3 (signal transducer and activator of transcription 3) is a transcription factor activated by cytokines, growth factors and oncogenes, whose activity is required for cell survival/proliferation of a wide variety of primary tumours and tumour cell lines. Prominent among its multiple effects on tumour cells is the stimulation of cell migration and metastasis, whose functional mechanisms are however not completely characterized. RhoU/Wrch1 (Wnt-responsive Cdc42 homologue) is an atypical Rho GTPase thought to be constitutively bound to GTP. RhoU was first identified as a Wnt-1-inducible mRNA and subsequently shown to act on the actin cytoskeleton by stimulating filopodia formation and stress fibre dissolution. It was in addition recently shown to localize to focal adhesions and to Src-induced podosomes and enhance cell migration. RhoU overexpression in mammary epithelial cells stimulates quiescent cells to re-enter the cell cycle and morphologically phenocopies Wnt-1-dependent transformation. In the present study we show that Wnt-1-mediated RhoU induction occurs at the transcriptional level. Moreover, we demonstrate that RhoU can also be induced by gp130 cytokines via STAT3, and we identify two functional STAT3-binding sites on the mouse RhoU promoter. RhoU induction by Wnt-1 is independent of β-catenin, but does not involve STAT3. Rather, it is mediated by the Wnt/planar cell polarity pathway through the activation of JNK (c-Jun N-terminal kinase). Both the so-called non-canonical Wnt pathway and STAT3 are therefore able to induce RhoU, which in turn may be involved in mediating their effects on cell migration. PMID:19397496

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

PubMed Central

Zhang, Donglei; Glotzer, Michael

2015-01-01

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

RORγt antagonist suppresses M3 muscarinic acetylcholine receptor-induced Sjögren's syndrome-like sialadenitis.

PubMed

Tahara, M; Tsuboi, H; Segawa, S; Asashima, H; Iizuka-Koga, M; Hirota, T; Takahashi, H; Kondo, Y; Matsui, M; Matsumoto, I; Sumida, T

2017-02-01

We showed recently that M3 muscarinic acetylcholine receptor (M3R)-reactive CD3 + T cells play a pathogenic role in the development of murine autoimmune sialadenitis (MIS), which mimics Sjögren's syndrome (SS). The aim of this study was to determine the effectiveness and mechanism of action of retinoic acid-related orphan receptor-gamma t (RORγt) antagonist (A213) in MIS. Splenocytes from M3R knockout (M3R -/- ) mice immunized with murine M3R peptide mixture were inoculated into recombination-activating gene 1 knockout (Rag-1 -/- ) mice (M3R -/- →Rag-1 -/- ) with MIS. Immunized M3R -/- mice (pretransfer treatment) and M3R -/- →Rag-1 -/- mice (post-transfer treatment) were treated with A213 every 3 days. Salivary volume, severity of sialadenitis and cytokine production from M3R peptide-stimulated splenocytes and lymph node cells were examined. Effects of A213 on cytokine production were analysed by enzyme-linked immunosorbent assay (ELISA) and on T helper type 1 (Th1), Th17 and Th2 differentiation from CD4 + T cells by flow cytometry. Pretransfer A213 treatment maintained salivary volume, improved MIS and reduced interferon (IFN)-γ and interleukin (IL)-17 production significantly compared with phosphate-buffered saline (PBS) (P < 0·05). These suppressive effects involved CD4 + T cells rather than CD11c + cells. Post-transfer treatment with A213 increased salivary volume (P < 0·05), suppressed MIS (P < 0·005) and reduced IFN-γ and IL-17 production (P < 0·05). In vitro, A213 suppressed IFN-γ and IL-17 production from M3R-stimulated splenocytes and CD4 + T cells of immunized M3R -/- mice (P < 0·05). In contrast with M3R specific responses, A213 suppressed only IL-17 production from Th17 differentiated CD4 + T cells without any effect on Th1 and Th2 differentiation in vitro. Our findings suggested that RORγt antagonism is potentially suitable treatment strategy for SS-like sialadenitis through suppression of IL-17 and IFN-γ production

[Changes of CD(4)(+) Foxp3+ regulatory T cells and CD(4)(+)IL-17+T cells in acrolein exposure rats].

PubMed

Wei, Ming; Tu, Ling; Liang, Yinghong; Li, Jia; Gong, Yanjie; Zhang, Yihua; Yang, Lu

2015-09-01

To evaluate the changes of CD(4)(+) IL-17+T (Th17) and CD(4)(+)Foxp3+regulatory T (Treg) cells in peripheral blood and bronchoalveolar lavage fluid (BALF) , and therefore to explore the role of Th17 and Treg in acrolein exposure airway inflammation in rats. Forty male Wistar rats were randomly divided into 4 groups: a 2 wk acrolein exposure group, a 4 wk acrolein exposure group, a 2 wk control group and a 4 wk control group (n=10 each). Cells in BALF were collected and analyzed by absolute and differential cell counts.IL-17 and IL-6 levels in serum and BALF were tested by enzyme linked immunosorbent assay (ELISA). The proportion of CD(4)(+)IL-17+T and CD(4)(+) Foxp3+Treg in peripheral blood and BALF were determined by flow cytometry.The mRNA expressions of IL-17 and Foxp3 were measured by real-time PCR. Comparisons of the data between different groups were performed using one-way ANOVA, and SNK and Games-Howell test were used for comparison between 2 groups. Levels of IL-17 were remarkable increased in the 2 wk acrolein exposure group and the 4 wk acrolein exposure group in serum [(52.64 ± 1.89) ng/L, (76.73 ± 5.57) ng/L], and BALF [(79.07 ± 5.67) ng/L, (96.61 ± 6.44) ng/L] compared with the 2 wk control group [(40.05 ± 3.12) ng/L, (56.75 ± 4.37) ng/L] and the 4 wk control group [(38.75 ± 3.23) ng/L, (53.27 ± 4.48) ng/L], all P<0.01. IL-6 was increased in the 2 wk and the 4 wk acrolein exposure group [ (33.28 ± 2.27) ng/L, (46.24 ± 3.16) ng/L] compared with the 2 wk and the 4 wk control group [ (16.37 ± 1.49) ng/L, (17.02 ± 1.43) ng/L] in BALF.Ratio of Th17 was higher in the 2 wk and the 4 wk acrolein exposure groups in peripheral blood (1.82 ± 0.18) %, (3.75 ± 0.48) % and BALF [(7.23 ± 0.27) %, (8.12 ± 0.38) %] compared with the 2 wk [(0.96 ± 0.07) %, (5.64 ± 0.63) %] and the 4 wk control group [(1.01 ± 0.08) %, (5.86 ± 0.57) %]. Ratio of Treg in BALF was higher in the acrolein exposure groups [ (8.83 ± 0.52) %, (12.05 ± 0.74) %] compared

Expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in control of GnRH secretion.

PubMed

Yang, Ying; Zhou, Li-bin; Liu, Shang-quan; Tang, Jing-feng; Li, Feng-yin; Li, Rong-ying; Song, Huai-dong; Chen, Ming-dao

2005-08-01

To investigate the expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in the control of GnRH secretion. Receptors of bombesin3, cholecystokinin (CCK)-A, CCK-B, glucagon-like peptide (GLP)1, melanin-concentrating hormone (MCH)1, orexin1, orexin2, neuromedin-B, neuropeptide Y (NPY)1 and NPY5, neurotensin (NT)1, NT2, NT3, and leptin receptor long form mRNA in GT1-7 cells were detected by reversed transcriptase-polymerase chain reaction. GT1-7 cells were treated with leptin, orexin A and orexin B at a cohort of concentrations for different lengths of time, and GnRH in medium was determined by radioimmunoassay (RIA). Receptors of bombesin 3, CCK-B, GLP1, MCH1, orexin1, neuromedin-B, NPY1, NPY5, NT1, NT3, and leptin receptor long form mRNA were expressed in GT1-7 cells, of which, receptors of GLP1, neuromedin-B, NPY1, and NT3 were highly expressed. No amplified fragments of orexin2, NT2, and CCK-A receptor cDNA were generated with GT1-7 RNA, indicating that the GT1-7 cells did not express mRNA of them. Leptin induced a significant stimulation of GnRH release, the results being most significant at 0.1 nmol/L for 15 min. In contrast to other studies in hypothalamic explants, neither orexin A nor orexin B affected basal GnRH secretion over a wide range of concentrations ranging from 1 nmol/L to 500 nmol/Lat 15, 30, and 60 min. Feeding and reproductive function are closely linked. Many orexigenic and anorexigenic signals may control feeding behavior as well as alter GnRH secretion through their receptors on GnRH neurons.

Rho/Rho-dependent kinase affects locomotion and actin-myosin II activity of Amoeba proteus.

PubMed

Kłopocka, W; Redowicz, M J

2004-10-01

The highly motile free-living unicellular organism Amoeba proteus has been widely used as a model to study cell motility. However, the molecular mechanisms underlying its unique locomotion are still scarcely known. Recently, we have shown that blocking the amoebae's endogenous Rac- and Rho-like proteins led to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. In order to elucidate the mechanism of the Rho pathway, we tested the effects of blocking the endogenous Rho-dependent kinase (ROCK) by anti-ROCK antibodies and Y-27632, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, a specific inhibitor of ROCK, on migrating amoebae and the effect of the Rho and ROCK inhibition on the actin-activated Mg-ATPase of the cytosolic fraction of the amoebae. Amoebae microinjected with anti-ROCK inhibitors remained contracted and strongly attached to the glass surface and exhibited an atypical locomotion. Despite protruding many pseudopodia that were advancing in various directions, the amoebae could not effectively move. Immunofluorescence studies showed that ROCK-like protein was dispersed throughout the cytoplasm and was also found in the regions of actin-myosin II interaction during both isotonic and isometric contraction. The Mg-ATPase activity was about two- to threefold enhanced, indicating that blocking the Rho/Rho-dependent kinase activated myosin. It is possible then that in contrast to the vertebrate cells, the inactivation of Rho/Rho-dependent kinase in amoebae leads to the activation of myosin II and to the observed hypercontracted cells which cannot exert effective locomotion.

Redundancy of primary RNA-binding functions of the bacterial transcription terminator Rho.

PubMed

Shashni, Rajesh; Qayyum, M Zuhaib; Vishalini, V; Dey, Debashish; Sen, Ranjan

2014-09-01

The bacterial transcription terminator, Rho, terminates transcription at half of the operons. According to the classical model derived from in vitro assays on a few terminators, Rho is recruited to the transcription elongation complex (EC) by recognizing specific sites (rut) on the nascent RNA. Here, we explored the mode of in vivo recruitment process of Rho. We show that sequence specific recognition of the rut site, in majority of the Rho-dependent terminators, can be compromised to a great extent without seriously affecting the genome-wide termination function as well as the viability of Escherichia coli. These terminators function optimally only through a NusG-assisted recruitment and activation of Rho. Our data also indicate that at these terminators, Rho-EC-bound NusG interaction facilitates the isomerization of Rho into a translocase-competent form by stabilizing the interactions of mRNA with the secondary RNA binding site, thereby overcoming the defects of the primary RNA binding functions. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

CpG island methylation of TMS1/ASC and CASP8 genes in cervical cancer

PubMed Central

2009-01-01

Background Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration that serves as an alternative to genetic defects in the inactivation of tumor suppressor and other genes in human cancers. Aims This study describes the methylation status of TMS1/ASC and CASP8 genes in cervical cancer. We also examined the prevalence of TMS1/ASC and CASP8 genes methylation in cervical cancer tissue and none - neo plastic samples in an effort to correlate with smoking habit and clinicopathological features. Method Target DNA was modified by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequently amplified by Methylation Specific (MS) PCR with primers specific for methylated versus unmethylated DNA. The PCR product was detected by gel electrophoresis and combined with the clinical records of patients. Results The methylation pattern of the TMS1/ASC and CASP8 genes in specimens of cervical cancer and adjacent normal tissues were detected [5/80 (6.2%), 3/80 (3.75%)-2/80 (2.5%), 1/80 (1.2%) respectively]. No statistical differences were seen in the extent of differentiation, invasion, pathological type and smoking habit between the methylated and unmethylated tissues (P > 0.05). Conclusion The present study conclude that the frequency of TMS1/ASC and CASP8 genes methylation in cervical cancer are rare (< 6%), and have no any critical role in development of cervical cancer. PMID:19258216

Advances in the T7 phage display system (Review).

PubMed

Deng, Xiangying; Wang, Li; You, Xiaolong; Dai, Pei; Zeng, Yanhua

2018-01-01

The present review describes the advantages and updated applications of the T7 phage display system in bioscience and medical science. Current phage display systems are based on various bacteriophage vectors, including M13, T7, T4 and f1. Of these, the M13 phage display is the most frequently used, however, the present review highlights the advantages of the T7 system. As a phage display platform, M13 contains single‑stranded DNA, while the T7 phage consists of double‑stranded DNA, which exhibits increased stability and is less prone to mutation during replication. Additional characteristics of the T7 phage include the following: The T7 phage does not depend on a protein secretion pathway in the lytic cycle; expressed peptides and proteins are usually located on the C‑terminal region of capsid protein gp10B, which avoids problems associated with steric hindrance; and T7 phage particles exhibit high stability under various extreme conditions, including high temperature and low pH, which facilitates effective high‑throughput affinity elutriation. Recent applications of the T7 phage display system have been instrumental in uncovering mechanisms of molecular interaction, particularly in the fields of antigen discovery, vaccine development, protein interaction, and cancer diagnosis and treatment.

[Tryptase inhibits cell apoptosis through upregulating PAR-2 and Rho kinase in rheumatoid arthritis synovial fibroblasts].

PubMed

Zheng, Qianqian; Li, Shigang; Jia, Yunli; Liu, Wei; Yu, Lingling; Chen, Xianyong; Wang, Jinling

2016-12-01

Objective To investigate the regulatory effects of tryptase on protease-activated receptors 2 (PAR-2), Rho signal pathway and apoptosis of MH7A rheumatoid arthritis synovial fibroblasts. Methods In MH7A cells, the expression of PAR-2 was measured by flow cytometry; cell apoptosis was examined by annexin V- FITC/PI staining combined with flow cytometry; the expression of Rho kinase was detected by Pull-down assay and Western botting. Results Tryptase upregulated the expression of PAR-2 in MH7A cells, and suppressed Fas-mediated apoptosis of MH7A cells in a dose-dependent manner. Meanwhile, PAR-2 inhibitor, FSLLRY-NH2 significantly reduced anti-apoptotic effects of tryptase in MH7A cells, which was related with the increase of activated Rho kinase expression. Conclusion Tryptase plays a role in resistance to the apoptosis of MH7A cells through raising PAR-2 and activating Rho kinase.

Role of interleukin (IL)-17 and T-helper (Th)17 cells in cancer.

PubMed

Song, Yang; Yang, Jian Ming

2017-11-04

Interleukin-17 (IL-17), a pleiotropic proinflammatory cytokine, is reported to be significantly generated by a distinct subset of CD4 + T-cells, upgrading cancer-elicited inflammation and preventing cancer cells from immune surveillance. T-helper (Th)17 cells produced from naive CD4 + T cells have recently been renowned and generally accepted, gaining eminence in cancer studies and playing the effective role in context of cancer. Th17 cells are the main source of IL-17-secreting cells, It was found that other cell types produced this cytokine as well, including Group 3 innate lymphoid cells (ILC3), δγT cells, invariant natural killer T (iNKT) cells, lymphoid-tissue inducer (LTi)-like cells and Natural killer (NK) cells. Th17-associated cytokines give impetus to tumor progression, or inducing angiogenesis and metastasis. This review demonstrates an understanding on how the pro- or antitumor function of Th17 cells and IL-17 may change cancer progression, leading to the appearance of complex and pivotal biologic activities in tumor. Copyright © 2017 Elsevier Inc. All rights reserved.

Brain distribution and molecular cloning of the bovine GABA rho1 receptor.

PubMed

Rosas-Arellano, Abraham; Ochoa-de la Paz, Lenin David; Miledi, Ricardo; Martínez-Torres, Ataúlfo

2007-03-01

GABA(C) receptors were originally found in the mammalian retina and recent evidence shows that they are also expressed in several areas of the brain, including caudate nucleus, brain stem, pons and corpus callosum. In this study, plasma membranes from the caudate nucleus were microinjected into X. laevis oocytes. This led the oocyte plasma membrane to incorporate functional bicuculline-resistant, Cl(-) conducting bovine GABA receptors, similar to those of the retina. Immunolocalization of the GABA rho1 subunit revealed its expression in bovine neurons in the head of the caudate as well as in the olive, cuneiform and reticular nuclei of the brain stem. The same antibodies failed to show expression in the callosum and pons, where the GABA rho1 mRNA was previously detected. The cloned GABA rho1 sequence predicts a protein with 473 amino acids and 74-93% similarity to other GABA rho1 subunits. Oocytes injected with the cDNA express a non-desensitizing, homomeric receptor with a GABA EC(50)=6.0 microM and a Hill coefficient of 1.8. The results confirm the presence of GABA(C) receptor mRNAs in several areas of the mammalian brain and show that some of these areas express functional GABA rho1 receptors that have the classic GABA(C) receptor characteristics.

MTORC1 EXPANDS TH17 AND IL-4+ DN T CELLS AND CONTRACTS TREGS IN SLE

PubMed Central

Kato, Hiroshi; Perl, Andras

2014-01-01

The mechanistic target of rapamycin (mTOR) is activated in CD4−CD8− double-negative (DN) T cells and its blockade is therapeutic in systemic lupus erythematosus (SLE) patients. Murine studies showed the involvement of mTOR complex 1 (mTORC1) and 2 (mTORC2) in the differentiation of Th1/Th17 cells and Th2 cells, respectively. Here, we investigated the roles of mTORC1 and mTORC2 in T-cell lineage development in SLE and matched healthy control (HC) subjects. mTORC1 activity was increased while mTORC2 was reduced as assessed by phosphorylation of their substrates pS6K or pS6RP and pAkt, respectively. Rapamycin inhibited mTORC1 and enhanced mTORC2. IL-4 expression was increased in freshly isolated CD8+ lupus T cells (SLE: 8.09±1.93%, HC: 3.61±0.49%; p=0.01). DN T cells had greater IL-4 expression than CD4+ or CD8+ T cells of SLE patients after 3 day in vitro stimulation, which was suppressed by rapamycin (control: 9.26±1.48%, rapamycin: 5.03±0.66%; p<0.001). GATA-3 expression was increased in CD8+ lupus T cells (p<0.01) and insensitive to rapamycin treatment. IFN-γ expression was reduced in all lupus T cell subsets (p=1.0×10−5) and also resisted rapamycin. IL-17 expression was increased in CD4+ lupus T cells (SLE: 3.62±0.66%, HC: 2.29±0.27%; p=0.019), which was suppressed by rapamycin (control: 3.91±0.79%, rapamycin: 2.22±0.60%; p<0.001). Frequency of Tregs was reduced in SLE (SLE: 1.83±0.25%, HC: 2.97±0.27%; p=0.0012). Rapamycin inhibited mTORC1 in Tregs and promoted their expansion. Neutralization of IL-17 but not IL-4 also expanded Tregs in SLE and HC subjects. These results indicate that mTORC1 expands IL-4+ DN T and Th17 cells and contracts Tregs in SLE. PMID:24683191

Characterization of αβ and γδ T cell subsets expressing IL-17A in ruminants and swine.

PubMed

Elnaggar, Mahmoud M; Abdellrazeq, Gaber S; Dassanayake, Rohana P; Fry, Lindsay M; Hulubei, Victoria; Davis, William C

2018-08-01

As part of our ongoing program to expand immunological reagents available for research in cattle, we developed a monoclonal antibody (mAb) to bovine interleukin-17A (IL-17A), a multifunctional cytokine centrally involved in regulating innate and adaptive immune responses. Initial comparative studies demonstrated the mAb recognizes a conserved epitope expressed on orthologues of IL-17A in sheep, goats and pigs. Comparative flow cytometric analyses of lymphocyte subsets stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin revealed differences in expression of IL-17A by CD4, CD8, and γδ T cells across ruminants and swine species. Results in cattle showed the largest proportion of IL-17A + cells were CD4 + followed by γδ and CD8 + T cells. Further analysis revealed the IL-17A + γδ T cell subset was comprised of WC1.1 + , WC1.2 + , and WC1 - subsets. Analysis of the IL-17A + CD8 + T cell subset revealed it was comprised of αβ and γδ T cell subsets. Results in sheep and goats revealed IL-17A is expressed mainly by CD4 + and CD8 + T cells, with little expression by γδ T cells. Analysis of IL-17A + CD8 + T cells showed the majority were CD8 + αβ in sheep, whereas they were CD8 + γδ in goats. The majority of the sheep and goat IL-17A + γδ T cells were WC1 + . Results obtained in swine showed expression of IL-17A by CD4, CD8, and γδ T cell subsets were similar to results reported in other studies. Comparison of expression of IL-17A with IFN-γ revealed subsets co-expressed IL-17A and IFN-γ in cattle, sheep, and goats. The new mAb expands opportunities for immunology research in ruminants and swine. Copyright © 2018 Elsevier Ltd. All rights reserved.

Prime, Shock, and Kill: Priming CD4 T Cells from HIV Patients with a BCL-2 Antagonist before HIV Reactivation Reduces HIV Reservoir Size

PubMed Central

Cummins, Nathan W.; Sainski, Amy M.; Dai, Haiming; Natesampillai, Sekar; Pang, Yuan-Ping; Bren, Gary D.; de Araujo Correia, Maria Cristina Miranda; Sampath, Rahul; Rizza, Stacey A.; O'Brien, Daniel; Yao, Joseph D.

2016-01-01

ABSTRACT Understanding how some HIV-infected cells resist the cytotoxicity of HIV replication is crucial to enabling HIV cure efforts. HIV killing of CD4 T cells that replicate HIV can involve HIV protease-mediated cleavage of procaspase 8 to generate a fragment (Casp8p41) that directly binds and activates the mitochondrial proapoptotic protein BAK. Here, we demonstrate that Casp8p41 also binds with nanomolar affinity to the antiapoptotic protein Bcl-2, which sequesters Casp8p41 and prevents apoptosis. Further, we show that central memory CD4 T cells (TCM) from HIV-infected individuals have heightened expression of BCL-2 relative to procaspase 8, possibly explaining the persistence of HIV-infected TCM despite generation of Casp8p41. Consistent with this hypothesis, the selective BCL-2 antagonist venetoclax induced minimal killing of uninfected CD4 T cells but markedly increased the death of CD4 T cells and diminished cell-associated HIV DNA when CD4 T cells from antiretroviral therapy (ART)-suppressed HIV patients were induced with αCD3/αCD28 to reactivate HIV ex vivo. Thus, priming CD4 T cells from ART suppressed HIV patients with a BCL-2 antagonist, followed by HIV reactivation, achieves reductions in cell-associated HIV DNA, whereas HIV reactivation alone does not. IMPORTANCE HIV infection is incurable due to a long-lived reservoir of HIV+ memory CD4 T cells, and no clinically relevant interventions have been identified that reduce the number of these HIV DNA-containing cells. Since postintegration HIV replication can result in HIV protease generation of Casp8p41, which activates BAK, causing infected CD4 T cell death, we sought to determine whether this occurs in memory CD4 T cells. Here, we demonstrate that memory CD4 T cells can generate Casp8p41 and yet are intrinsically resistant to death induced by diverse stimuli, including Casp8p41. Furthermore, BCL-2 expression is relatively increased in these cells and directly binds and inhibits Casp8p41's

Template-free modeling by LEE and LEER in CASP11.

PubMed

Joung, InSuk; Lee, Sun Young; Cheng, Qianyi; Kim, Jong Yun; Joo, Keehyoung; Lee, Sung Jong; Lee, Jooyoung

2016-09-01

For the template-free modeling of human targets of CASP11, we utilized two of our modeling protocols, LEE and LEER. The LEE protocol took CASP11-released server models as the input and used some of them as templates for 3D (three-dimensional) modeling. The template selection procedure was based on the clustering of the server models aided by a community detection method of a server-model network. Restraining energy terms generated from the selected templates together with physical and statistical energy terms were used to build 3D models. Side-chains of the 3D models were rebuilt using target-specific consensus side-chain library along with the SCWRL4 rotamer library, which completed the LEE protocol. The first success factor of the LEE protocol was due to efficient server model screening. The average backbone accuracy of selected server models was similar to that of top 30% server models. The second factor was that a proper energy function along with our optimization method guided us, so that we successfully generated better quality models than the input template models. In 10 out of 24 cases, better backbone structures than the best of input template structures were generated. LEE models were further refined by performing restrained molecular dynamics simulations to generate LEER models. CASP11 results indicate that LEE models were better than the average template models in terms of both backbone structures and side-chain orientations. LEER models were of improved physical realism and stereo-chemistry compared to LEE models, and they were comparable to LEE models in the backbone accuracy. Proteins 2016; 84(Suppl 1):118-130. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Mesophilic-hydrothermal-thermophilic (M-H-T) digestion of green corn straw.

PubMed

Li, Dong; Wang, Qingjing; Li, Jiang; Li, Zhidong; Yuan, Yuexiang; Yan, Zhiying; Mei, Zili; Liu, Xiaofeng

2016-02-01

Mesophilic-hydrothermal (80-160 °C, 30 min)-thermophilic (M-H-T) digestion and control tests of mesophilic (M), thermophilic (T), hydrothermal-mesophilic (H-M), and mesophilic-thermophilic digestion (M-T) of green corn straw were conducted for a 20-day fermentation period. The results indicate that M-H-T is an efficient method to improve methane production. A maximum methane yield of 371.74 mL/g volatile solid was obtained by the M (3 days)-H (140 °C)-T (17 days) process, which was 20.44%, 16.55%, 31.44%, and 14.31% higher than the yields of the M, T, 140-M, and M-T processes. The enhanced methane production was attributed to (1) the improved hemicellulose degradation and lignin disorganization; (2) prevention of the degradation of soluble sugar, easily hydrolyzed hemicellulose and cellulose into furfural and methylfurfural; and (3) lack of formation of Maillard reaction products during initial hydrothermal treatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transglutaminase-dependent RhoA activation and depletion by serotonin in vascular smooth muscle cells.

PubMed

Guilluy, Christophe; Rolli-Derkinderen, Malvyne; Tharaux, Pierre-Louis; Melino, Gerry; Pacaud, Pierre; Loirand, Gervaise

2007-02-02

The small G protein RhoA plays a major role in several vascular processes and cardiovascular disorders. Here we analyze the mechanisms of RhoA regulation by serotonin (5-HT) in arterial smooth muscle. 5-HT (0.1-10 microM) induced activation of RhoA followed by RhoA depletion at 24-72 h. Inhibition of 5-HT1 receptors reduced the early phase of RhoA activation but had no effect on 5-HT-induced delayed RhoA activation and depletion, which were suppressed by the 5-HT transporter inhibitor fluoxetine and the transglutaminase inhibitor monodansylcadaverin and in type 2 transglutaminase-deficient smooth muscle cells. Coimmunoprecipitations demonstrated that 5-HT associated with RhoA both in vitro and in vivo. This association was calcium-dependent and inhibited by fluoxetine and monodansylcadaverin. 5-HT promotes the association of RhoA with the E3 ubiquitin ligase Smurf1, and 5-HT-induced RhoA depletion was inhibited by the proteasome inhibitor MG132 and the RhoA inhibitor Tat-C3. Simvastatin, the Rho kinase inhibitor Y-27632, small interfering RNA-mediated RhoA gene silencing, and long-term 5-HT stimulation induced Akt activation. In contrast, inhibition of 5-HT-mediated RhoA degradation by MG132 prevented 5-HT-induced Akt activation. Long-term 5-HT stimulation also led to the inhibition of the RhoA/Rho kinase component of arterial contraction. Our data provide evidence that 5-HT, internalized through the 5-HT transporter, is transamidated to RhoA by transglutaminase. Transamidation of RhoA leads to RhoA activation and enhanced proteasomal degradation, which in turn is responsible for Akt activation and contraction inhibition. The observation of transamidation of 5-HT to RhoA in pulmonary artery of hypoxic rats suggests that this process could participate in pulmonary artery remodeling and hypertension.

Vibrio parahaemolyticus Inhibition of Rho Family GTPase Activation Requires a Functional Chromosome I Type III Secretion System▿

PubMed Central

Casselli, Timothy; Lynch, Tarah; Southward, Carolyn M.; Jones, Bryan W.; DeVinney, Rebekah

2008-01-01

Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis; however, its virulence mechanisms are not well understood. The identification of type III secreted proteins has provided candidate virulence factors whose functions are still being elucidated. Genotypic strain variability contributes a level of complexity to understanding the role of different virulence factors. The ability of V. parahaemolyticus to inhibit Rho family GTPases and cause cytoskeletal disruption was examined with HeLa cells. After HeLa cells were infected, intracellular Rho activation was inhibited in response to external stimuli. In vitro activation of Rho, Rac, and Cdc42 isolated from infected HeLa cell lysates was also inhibited, indicating that the bacteria were specifically targeting GTPase activation. The inhibition of Rho family GTPase activation was retained for clinical and environmental isolates of V. parahaemolyticus and was dependent on a functional chromosome I type III secretion system (CI-T3SS). GTPase inhibition was independent of hemolytic toxin genotype and the chromasome II (CII)-T3SS. Rho inhibition was accompanied by a shift in the total actin pool to its monomeric form. These phenotypes were abrogated in a mutant strain lacking the CI-T3S effector Vp1686, suggesting that the inhibiting actin polymerization may be a downstream effect of Vp1686-dependent GTPase inhibition. Although Vp1686 has been previously characterized as a potential virulence factor in macrophages, our findings reveal an effect on cultured HeLa cells. The ability to inhibit Rho family GTPases independently of the CII-T3SS and the hemolytic toxins may provide insight into the mechanisms of virulence used by strains lacking these virulence factors. PMID:18347050

Psychometric properties and confirmatory factor analysis of the CASP-19, a measure of quality of life in early old age: the HAPIEE study

PubMed Central

Kim, Gyu Ri; Netuveli, Gopalakrishnan; Blane, David; Peasey, Anne; Malyutina, Sofia; Simonova, Galina; Kubinova, Ruzena; Pajak, Andrzej; Croezen, Simone; Bobak, Martin; Pikhart, Hynek

2015-01-01

Objectives: The aim was to assess the reliability and validity of the quality of life (QoL) instrument CASP-19, and three shorter versions of CASP-12 in large population sample of older adults from the HAPIEE (Health, Alcohol, and Psychosocial factors In Eastern Europe) study. Methods: From the Czech Republic, Russia, and Poland, 13,210 HAPIEE participants aged 50 or older completed the retirement questionnaire including CASP-19 at baseline. Three shorter 12-item versions were also derived from original 19-item instrument. Psychometric validation used confirmatory factor analysis, Cronbach's alpha, Pearson's correlation, and construct validity. Results: The second-order four-factor model of CASP-19 did not provide a good fit to the data. Two-factor CASP-12v.3 including residual covariances for negative items to account for the method effect of negative items had the best fit to the data in all countries (CFI = 0.98, TLI = 0.97, RMSEA = 0.05, and WRMR = 1.65 in the Czech Republic; 0.96, 0.94, 0.07, and 2.70 in Poland; and 0.93, 0.90, 0.08, and 3.04 in Russia). Goodness-of-fit indices for the two-factor structure were substantially better than second-order models. Conclusions: This large population-based study is the first validation study of CASP scale in Central and Eastern Europe (CEE), which includes a general population sample in Russia, Poland, and the Czech Republic. The results of this study have demonstrated that the CASP-12v.3 is a valid and reliable tool for assessing QoL among adults aged 50 years or older. This version of CASP is recommended for use in future studies investigating QoL in the CEE populations. PMID:25059754

Inhibition of RhoA/Rho kinase pathway and smooth muscle contraction by hydrogen sulfide.

PubMed

Nalli, Ancy D; Wang, Hongxia; Bhattacharya, Sayak; Blakeney, Bryan A; Murthy, Karnam S

2017-10-01

Hydrogen sulfide (H 2 S) plays an important role in smooth muscle relaxation. Here, we investigated the expression of enzymes in H 2 S synthesis and the mechanism regulating colonic smooth muscle function by H 2 S. Expression of cystathionine-γ-lyase (CSE), but not cystathionine-β-synthase (CBS), was identified in the colonic smooth muscle of rabbit, mouse, and human. Carbachol (CCh)-induced contraction in rabbit muscle strips and isolated muscle cells was inhibited by l-cysteine (substrate of CSE) and NaHS (an exogenous H 2 S donor) in a concentration-dependent fashion. H 2 S induced S-sulfhydration of RhoA that was associated with inhibition of RhoA activity. CCh-induced Rho kinase activity also was inhibited by l-cysteine and NaHS in a concentration-dependent fashion. Inhibition of CCh-induced contraction by l-cysteine was blocked by the CSE inhibitor, dl-propargylglycine (DL-PPG) in dispersed muscle cells. Inhibition of CCh-induced Rho kinase activity by l-cysteine was blocked by CSE siRNA in cultured cells and DL-PPG in dispersed muscle cells. Stimulation of Rho kinase activity and muscle contraction in response to CCh was also inhibited by l-cysteine or NaHS in colonic muscle cells from mouse and human. Collectively, our studies identified the expression of CSE in colonic smooth muscle and determined that sulfhydration of RhoA by H 2 S leads to inhibition of RhoA and Rho kinase activities and muscle contraction. The mechanism identified may provide novel therapeutic approaches to mitigate gastrointestinal motility disorders. © 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

Neural activation-based sexual orientation and its correlation with free testosterone level in postoperative female-to-male transsexuals: preliminary study with 3.0-T fMRI.

PubMed

Kim, Gwang-Won; Kim, Seok-Kwun; Jeong, Gwang-Woo

2016-03-01

The purpose of this study was to evaluate the brain activation pattern associated with sexual orientation and its correlation with the level of the free testosterone (free T) in postoperative female-to-male (FtM) transsexuals using a 3.0-Tesla functional magnetic resonance imaging (fMRI). Eleven postoperative FtM transsexuals with sex reassignment surgery underwent fMRI on a 3.0-T MR scanner. Brain activity was measured while viewing erotic male and female nude pictures. The average level of free T in the FtM transsexuals was in the normal range of heterosexual men. The brain areas with predominant activities during viewing female nude pictures in contrast to male pictures included the hippocampus, parahippocampal gyrus, anterior cingulate gyrus, putamen, amygdala, hypothalamus, and insula (p < 0.005). The free T levels were positively correlated with the BOLD signal changes in the parahippocampal gyrus (Spearman's rho = 0.91, p < 0.001), hippocampus (rho = 0.90, p < 0.001), insula (rho = 0.68, p < 0.05), putamen (rho = 0.66, p < 0.05), and amygdala (rho = 0.64, p < 0.05). Compared to FtM transsexuals with deficient level of free T, the FtM transsexuals with normal range of free T showed significantly higher activities in the parahippocampal gyrus, hippocampus, insula, putamen, and amygdala during viewing female nude pictures (p < 0.005). This study revealed the specific brain activation pattern associated with sexual orientation and its correlation with free T in the postoperative FtM transsexuals. These findings are applicable in understanding the neural mechanism on sexual arousal in FtM transsexuals and their sexual orientation in connection with the free T levels.

Off-resonance R1rho relaxation outside of the fast exchange limit: an experimental study of a cavity mutant of T4 lysozyme.

PubMed

Korzhnev, Dmitry M; Orekhov, Vladislav Yu; Dahlquist, Frederick W; Kay, Lewis E

2003-05-01

An (15)N off-resonance R(1rho) spin relaxation study of an L99A point mutant of T4 lysozyme is presented. Previous CPMG-based relaxation dispersion studies of exchange in this protein have established that the molecule interconverts between a populated ground state and an excited state (3.4%) with an exchange rate constant of 1450 s(-1) at 25 degrees C. It is shown that for the majority of residues in this protein the offset dependence of the R(1rho) relaxation rates cannot be well fit using models which are only valid in the fast exchange regime. In contrast, a recently derived expression by Trott and Palmer (J. Magn. Reson., 154, 157-160, 2002) which is valid over a wider window of exchange than other relations, is shown to fit the data well. Values of (signed) chemical shift differences between exchanging sites have been extracted and are in reasonable agreement with shift differences measured using CPMG methods. A set of simulations is presented which help establish the exchange regimes that are best suited to analysis by off-resonance R(1rho) techniques.

Site preference, magnetism and lattice vibrations of intermetallics Lu₂Fe 17–xT x (T=Cr, Mn, Ru)

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

Li, Jin-Chun; Qian, Ping, E-mail: qianpinghu@sohu.com; Zhang, Zhen-Feng

We present an atomistic study on the phase stability, site preference and lattice constants of the rare earth intermetallics Lu₂Fe 17–xT x (T=Cr, Mn, Ru). The calculated preferential occupation site of ternary element T is found to be the 4f site. The order of site preference is given as 4f, 12k, 12j and 6g for Lu₂Fe 17–xT x. The calculated lattice parameters are corresponding to the experimental results. We have calculated the magnetic moments of Lu₂Fe 17–xT x compounds. Results show that the calculated total magnetic moment of Lu₂Fe₁₇ compound is M=37.34 μ B/f.u. In addition, the total and partialmore » phonon densities of states are evaluated first for these complicated structures. - Graphical abstract: The vibrational modes are mostly excited by Fe atoms, Lu contributes to the lower frequencies modes, and the contribution of Ru atoms is the same as Fe atoms. Highlights: • There are no reports on lattice vibrations of Lu₂(Fe, T) 17–x (T=Cr, Mn, Ru) compounds. • The phase stability and site preference are evaluated first for the complex structures of Lu₂(Fe, T) 17–x (T=Cr, Mn, Ru) compounds. • The lattice inversion method to obtain the interatomic pair potential is the unique one.« less

Unique Structural and Nucleotide Exchange Features of the Rho1 GTPase of Entamoeba histolytica

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

Bosch, Dustin E.; Wittchen, Erika S.; Qiu, Connie

The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engagesmore » a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.« less

Fibroblast growth factor 2 restrains Ras-driven proliferation of malignant cells by triggering RhoA-mediated senescence.

PubMed

Costa, Erico T; Forti, Fábio L; Matos, Tatiana G F; Dermargos, Alexandre; Nakano, Fábio; Salotti, Jacqueline; Rocha, Kátia M; Asprino, Paula F; Yoshihara, Celina K; Koga, Marianna M; Armelin, Hugo A

2008-08-01

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant Y1 adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated beta-galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Y1 adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either Y1 or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Ras-dependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RhoA-GTP. Surprisingly, attempts to select FGF2-resistant cells from the Y1 and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Ras-dependent malignant cells could rarely overcome.

Myxoma virus M-T7, a secreted homolog of the interferon-gamma receptor, is a critical virulence factor for the development of myxomatosis in European rabbits.

PubMed

Mossman, K; Nation, P; Macen, J; Garbutt, M; Lucas, A; McFadden, G

1996-01-01

Myxoma virus is a leporipoxvirus of New World rabbits (Sylvilagus sp.) that induces a rapidly lethal infection known as myxomatosis in the European rabbit (Oryctolagus cuniculus). Like all poxviruses, myxoma virus encodes a plethora of proteins to circumvent or inhibit a variety of host antiviral immune mechanisms. M-T7, the most abundantly secreted protein of myxoma virus-infected cells, was originally identified as an interferon-gamma receptor homolog (Upton, Mossman, and McFadden, Science 258, 1369-1372, 1992). Here, we demonstrate that M-T7 is dispensable for virus replication in cultured cells but is a critical virulence factor for virus pathogenesis in European rabbits. Disruption of both copies of the M-T7 gene in myxoma virus was achieved by the deletion of 372 bp of M-T7 coding sequences, replacement with a selectable marker, p7.5Ecogpt, and selection of a recombinant virus (vMyxlac-T7gpt) resistant to mycophenolic acid. vMyxlac-T7gpt expressed no detectable M-T7 protein and infected cells supernatants were devoid of any detectable interferon-gamma binding activities. Immunohistochemical staining with anti-beta-galactosidase and anti-CD43 antibodies demonstrated that in vMyxlac-T7gpt-infected rabbits the loss of M-T7 not only caused a dramatic reduction in disease symptoms and viral dissemination to secondary sites, but also dramatically influenced host leukocyte behavior. Notably, primary lesions in wild-type virus infections were generally underlayed by large masses of inflammatory cells that did not effectively migrate into the dermal sites of viral replication, whereas in vMyxlac-T7gpt infections this apparent block to leukocyte influx was relieved. A second major phenotypic distinction noted for the M-T7 knockout virus was the extensive activation of lymphocytes in secondary immune organs, particularly the spleen and lymph nodes, by Day 4 of the infection. This is in stark contrast to infection by wild-type myxoma virus, which results in relatively

RhoGAP18B Isoforms Act on Distinct Rho-Family GTPases and Regulate Behavioral Responses to Alcohol via Cofilin

PubMed Central

Kalahasti, Geetha; Rodan, Aylin R.; Rothenfluh, Adrian

2015-01-01

Responses to the effects of ethanol are highly conserved across organisms, with reduced responses to the sedating effects of ethanol being predictive of increased risk for human alcohol dependence. Previously, we described that regulators of actin dynamics, such as the Rho-family GTPases Rac1, Rho1, and Cdc42, alter Drosophila’s sensitivity to ethanol-induced sedation. The GTPase activating protein RhoGAP18B also affects sensitivity to ethanol. To better understand how different RhoGAP18B isoforms affect ethanol sedation, we examined them for their effects on cell shape, GTP-loading of Rho-family GTPase, activation of the actin-severing cofilin, and actin filamentation. Our results suggest that the RhoGAP18B-PA isoform acts on Cdc42, while PC and PD act via Rac1 and Rho1 to activate cofilin. In vivo, a loss-of-function mutation in the cofilin-encoding gene twinstar leads to reduced ethanol-sensitivity and acts in concert with RhoGAP18B. Different RhoGAP18B isoforms, therefore, act on distinct subsets of Rho-family GTPases to modulate cofilin activity, actin dynamics, and ethanol-induced behaviors. PMID:26366560

Progression of Human Renal Cell Carcinoma via Inhibition of RhoA-ROCK Axis by PARG1.

PubMed

Miyazaki, Junichiro; Ito, Keiichi; Fujita, Tomonobu; Matsuzaki, Yuriko; Asano, Takako; Hayakawa, Masamichi; Asano, Tomohiko; Kawakami, Yutaka

2017-04-01

Renal cell carcinoma (RCC) is the most lethal urological malignancy with high risk of recurrence; thus, new prognostic biomarkers are needed. In this study, a new RCC antigen, PTPL1 associated RhoGAP1 (PARG1), was identified by using serological identification of recombinant cDNA expression cloning with sera from RCC patients. PARG1 protein was found to be differentially expressed in RCC cells among patients. High PARG1 expression is significantly correlated with various clinicopathological factors relating to cancer cell proliferation and invasion, including G3 percentage (P = .0046), Ki-67 score (p expression is also correlated with high recurrence of N0M0 patients (P = .0084) and poor prognosis in RCC patients (P = .0345). Multivariate analysis has revealed that high PARG1 expression is an independent factor for recurrence (P = .0149) of N0M0 RCC patients. In in vitro studies, depletion of PARG1by siRNA in human RCC cell lines inhibited their proliferation through inducing G1 cell cycle arrest via upregulation of p53 and subsequent p21 Cip1/Waf1 , which are mediated by increased RhoA-ROCK activities. Similarly, PARG1 depletion cells inhibited invasion ability via increasing RhoA-ROCK activities in the RCC cell lines. Conversely, overexpression of PARG1 on human embryonic kidney cell line HEK293T promotes its cell proliferation and invasion. These results indicate that PARG1 plays crucial roles in progression of human RCC in increasing cell proliferation and invasion ability via inhibition of the RhoA-ROCK axis, and PARG1 is a poor prognostic marker, particularly for high recurrence of N0M0 RCC patients. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Favorable Vascular Actions of Angiotensin-(1-7) in Human Obesity.

PubMed

Schinzari, Francesca; Tesauro, Manfredi; Veneziani, Augusto; Mores, Nadia; Di Daniele, Nicola; Cardillo, Carmine

2018-01-01

Obese patients have vascular dysfunction related to impaired insulin-stimulated vasodilation and increased endothelin-1-mediated vasoconstriction. In contrast to the harmful vascular actions of angiotensin (Ang) II, the angiotensin-converting enzyme 2 product Ang-(1-7) has shown to exert cardiovascular and metabolic benefits in experimental models through stimulation of the Mas receptor. We, therefore, examined the effects of exogenous Ang-(1-7) on vasodilator tone and endothelin-1-dependent vasoconstriction in obese patients. Intra-arterial infusion of Ang-(1-7) (10 nmol/min) resulted in significant increase in unstimulated forearm flow ( P =0.03), an effect that was not affected by the Mas receptor antagonist A779 (10 nmol/min; P >0.05). In the absence of hyperinsulinemia, however, forearm flow responses to graded doses of acetylcholine and sodium nitroprusside were not different during Ang-(1-7) administration compared with saline (both P >0.05). During infusion of regular insulin (0.15 mU/kg per minute), by contrast, endothelium-dependent vasodilator response to acetylcholine was significantly enhanced by Ang-(1-7) ( P =0.04 versus saline), whereas endothelium-independent response to sodium nitroprusside was not modified ( P =0.91). Finally, Ang-(1-7) decreased the vasodilator response to endothelin A receptor blockade (BQ-123; 10 nmol/min) compared with saline (6±1% versus 93±17%; P <0.001); nitric oxide inhibition by l- N -monomethylarginine (4 µmol/min) during concurrent endothelin A antagonism resulted in similar vasoconstriction in the absence or presence of Ang-(1-7 Ang-(1-7) ( P =0.69). Our findings indicate that in obese patients Ang-(1-7) has favorable effects not only to improve insulin-stimulated endothelium-dependent vasodilation but also to blunt endothelin-1-dependent vasoconstrictor tone. These findings provide support for targeting Ang-(1-7) to counteract the hemodynamic abnormalities of human obesity. © 2017 American Heart Association, Inc.

[Construction of autocatalytic caspase-3 driven by amplified human telomerase reverse transcriptase promoter and its enhanced efficacy of inducing apoptosis in human ovarian carcinoma].

PubMed

Song, Yue; Shen, Keng; He, Chun-Xia

2007-09-01

To construct recombinant adenoviral vector expressing autocatalysis caspase-3 driven by human telomerase reverse transcriptase promoter amplified by two-step transcription amplification (hTERTp-TSTA), and investigate its antitumor effect in ovarian cancer in vitro and in vivo. Recombinant adenoviruses expressing autocatalytic caspase-3 (rev-caspase-3) driven by hTERTp-TSTA were prepared, which were named as AdHTVP2G5-rev-casp3. AdHT-rev-casp3, Ad-rev-casp3 and AdHTVP2G5-EGEP, which express rev-caspase-3 driven by hTERTp, cytomegalovirus promoter (CMVp) and enhanced green fluorescent protein (EGFP), respectively, were used as controls. Western blot, cell counting kit (CCK-8), flow cytometry (FCM) and TdT-mediated dUTP-biotin nick end labeling (TUNEL) were used to detect the expression of p17, active subunit of caspase-3, and p85, and to measure cell survival rates, apoptotic rates and cell cycle distribution in ovarian cell line AO and normal human umbilical vein endothelial cell line HUVEC, following treatments of AdHTVP2G5-rev-casp3. subcutaneous tumor models and abdominally spread tumor models of human ovarian carcinoma using AO cells in BALB/c nude mice were established. Following treatments of AdHTVP2G5-rev-casp3, western blot was used to detect the expression of active caspase-3 in abdominally spread tumors and liver tissues, respectively, and the mouse survival rates and the volume of tumor nodules were measured, and the serum level of alanine transaminase (ALT) and aspartate transaminase (AST) were analyzed to monitor liver damages and HE staining was used to detect the histopathological changes of various organs. The levels of p17 expression in AdHTVP2G5-rev-casp3-treated AO cells were significantly higher than that in Ad-rev-casp3 or AdHT-rev-casp3 treated AO cells, while no expression was observed in AdHTVP2G5-rev-casp3-treated HUVEC. There was strong cell killing of AdHTVP2G5-rev-casp3 of hTERT positive AO cells, but not of the hTERT-negative HUVEC cells

Bioequivalence of clavulanate potassium and amoxicillin (1:7) dispersible tablets in healthy volunteers.

PubMed

Hu, Guoxin; Dai, Zongshun; Long, Lihong; Han, Ying; Hou, Shuxian; Wu, Li

2002-01-01

To study the bioequivalence of Clavulanate Potassium and Amoxicillin (1:7) dispersible tablets, a randomized cross-over study was conducted in 18 healthy volunteers. A single oral dose of 1,000 mg Clavulanate Potassium and Amoxicillin (1:7) dispersible tablets (Tested formulation, T) or Augmentin syrup (Reference formulation, R). Concentrations in plasma were determined with high-performance liquid chromatography. The main parameters of T were: for Clavulanate Potassium and Amoxicillin, Cmax: 2.46 +/- 1.11 micrograms/ml and 18.81 +/- 7.26 micrograms/ml, Tmax: 1.12 +/- 0.23 h and 1.30 +/- 0.34 h, AUC(0-6 h): 5.18 +/- 2.24 micrograms.h/ml and 45.09 +/- 14.53 micrograms.h/ml, t1/2: 1.43 +/- 0.44 h and 1.09 +/- 0.22 h., respectively. The relative bioavailability of T to R were 96.5 +/- 19.2% and 98.4 +/- 26.1%, respectively. Statistical analysis showed that the two formulations were bioequivalent.

Regulated Localization Is Sufficient for Hormonal Control of Regulator of G Protein Signaling Homology Rho Guanine Nucleotide Exchange Factors (RH-RhoGEFs)*

PubMed Central

Carter, Angela M.; Gutowski, Stephen; Sternweis, Paul C.

2014-01-01

The regulator of G protein signaling homology (RH) Rho guanine nucleotide exchange factors (RhoGEFs) (p115RhoGEF, leukemia-associated RhoGEF, and PDZ-RhoGEF) contain an RH domain and are specific GEFs for the monomeric GTPase RhoA. The RH domains interact specifically with the α subunits of G12 heterotrimeric GTPases. Activated Gα13 modestly stimulates the exchange activity of both p115RhoGEF and leukemia-associated RhoGEF but not PDZ-RhoGEF. Because all three RH-RhoGEFs can localize to the plasma membrane upon expression of activated Gα13, cellular localization of these RhoGEFs has been proposed as a mechanism for controlling their activity. We use a small molecule-regulated heterodimerization system to rapidly control the localization of RH-RhoGEFs. Acute localization of the proteins to the plasma membrane activates RhoA within minutes and to levels that are comparable with activation of RhoA by hormonal stimulation of G protein-coupled receptors. The catalytic activity of membrane-localized RhoGEFs is not dependent on activated Gα13. We further show that the conserved RH domains can rewire two different RacGEFs to activate Rac1 in response to a traditional activator of RhoA. Thus, RH domains act as independent detectors for activated Gα13 and are sufficient to modulate the activity of RhoGEFs by hormones via mediating their localization to substrate, membrane-associated RhoA. PMID:24855647

Contact-assisted protein structure modeling by global optimization in CASP11.

PubMed

Joo, Keehyoung; Joung, InSuk; Cheng, Qianyi; Lee, Sung Jong; Lee, Jooyoung

2016-09-01

We have applied the conformational space annealing method to the contact-assisted protein structure modeling in CASP11. For Tp targets, where predicted residue-residue contact information was provided, the contact energy term in the form of the Lorentzian function was implemented together with the physical energy terms used in our template-free modeling of proteins. Although we observed some structural improvement of Tp models over the models predicted without the Tp information, the improvement was not substantial on average. This is partly due to the inaccuracy of the provided contact information, where only about 18% of it was correct. For Ts targets, where the information of ambiguous NOE (Nuclear Overhauser Effect) restraints was provided, we formulated the modeling in terms of the two-tier optimization problem, which covers: (1) the assignment of NOE peaks and (2) the three-dimensional (3D) model generation based on the assigned NOEs. Although solving the problem in a direct manner appears to be intractable at first glance, we demonstrate through CASP11 that remarkably accurate protein 3D modeling is possible by brute force optimization of a relevant energy function. For 19 Ts targets of the average size of 224 residues, generated protein models were of about 3.6 Å Cα atom accuracy. Even greater structural improvement was observed when additional Tc contact information was provided. For 20 out of the total 24 Tc targets, we were able to generate protein structures which were better than the best model from the rest of the CASP11 groups in terms of GDT-TS. Proteins 2016; 84(Suppl 1):189-199. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Anatomic & metabolic brain markers of the m.3243A>G mutation: A multi-parametric 7T MRI study.

PubMed

Haast, Roy A M; Ivanov, Dimo; IJsselstein, Rutger J T; Sallevelt, Suzanne C E H; Jansen, Jacobus F A; Smeets, Hubert J M; de Coo, Irenaeus F M; Formisano, Elia; Uludağ, Kâmil

2018-01-01

One of the most common mitochondrial DNA (mtDNA) mutations, the A to G transition at base pair 3243, has been linked to changes in the brain, in addition to commonly observed hearing problems, diabetes and myopathy. However, a detailed quantitative description of m.3243A>G patients' brains has not been provided so far. In this study, ultra-high field MRI at 7T and volume- and surface-based data analyses approaches were used to highlight morphology (i.e. atrophy)-, microstructure (i.e. myelin and iron concentration)- and metabolism (i.e. cerebral blood flow)-related differences between patients (N = 22) and healthy controls (N = 15). The use of quantitative MRI at 7T allowed us to detect subtle changes of biophysical processes in the brain with high accuracy and sensitivity, in addition to typically assessed lesions and atrophy. Furthermore, the effect of m.3243A>G mutation load in blood and urine epithelial cells on these MRI measures was assessed within the patient population and revealed that blood levels were most indicative of the brain's state and disease severity, based on MRI as well as on neuropsychological data. Morphometry MRI data showed a wide-spread reduction of cortical, subcortical and cerebellar gray matter volume, in addition to significantly enlarged ventricles. Moreover, surface-based analyses revealed brain area-specific changes in cortical thickness (e.g. of the auditory cortex), and in T 1 , T 2 * and cerebral blood flow as a function of mutation load, which can be linked to typically m.3243A>G-related clinical symptoms (e.g. hearing impairment). In addition, several regions linked to attentional control (e.g. middle frontal gyrus), the sensorimotor network (e.g. banks of central sulcus) and the default mode network (e.g. precuneus) were characterized by alterations in cortical thickness, T 1 , T 2 * and/or cerebral blood flow, which has not been described in previous MRI studies. Finally, several hypotheses, based either on vascular

A self-initiating eukaryotic transient gene expression system based on contransfection of bacteriophage T7 RNA polymerase and DNA vectors containing a T7 autogene.

PubMed Central

Chen, X; Li, Y; Xiong, K; Wagner, T E

1994-01-01

A novel cytoplasmic gene expression system has been developed. This system differs from other expression systems in that it relies on the co-delivery of plasmid DNA and T7 RNA polymerase (RNAP) during transfection. The plasmid contains a T7 RNAP gene driven by the T7 promoter (T7 autogene) and a functional/reporter gene driven by another T7 promoter (T7T7/T7-gene construct). Once this DNA-enzyme complex is introduced into eukaryotic cells, the transcription of the T7 RNAP and the functional/reporter genes is initiated by the co-delivered T7 RNAP. The T7 RNAP, which is responsible for the initiation and maintenance of expression of both T7 and functional/reporter genes, is replenished by translation of newly synthesized T7 mRNA. This T7 system was designed in such a manner that the expression of the functional/reporter genes can occur in the cytoplasm and does not require any nuclear involvement. When transfected by either a pT7T7/T7Luc or a pT7T7/T7hGH plasmids with the cointroduced T7 RNAP, mouse L cells were found to express high levels of luciferase immediately after transfection, apparently due to the cytoplasmic gene expression; the expression of human growth hormone (hGH) could be sustained for at least 6 days. Both T7 and hGH mRNA were expressed by the cells transfected with pT7T7/T7hGH. These results suggest that this cytoplasmic expression system may be used for certain targets of somatic gene therapy. Images PMID:8029020

Enhanced p122RhoGAP/DLC-1 Expression Can Be a Cause of Coronary Spasm

PubMed Central

Kinjo, Takahiko; Tanaka, Makoto; Osanai, Tomohiro; Shibutani, Shuji; Narita, Ikuyo; Tanno, Tomohiro; Nishizaki, Kimitaka; Ichikawa, Hiroaki; Kimura, Yoshihiro; Ishida, Yuji; Yokota, Takashi; Shimada, Michiko; Homma, Yoshimi; Tomita, Hirofumi; Okumura, Ken

2015-01-01

Background We previously showed that phospholipase C (PLC)-δ1 activity was enhanced by 3-fold in patients with coronary spastic angina (CSA). We also reported that p122Rho GTPase-activating protein/deleted in liver cancer-1 (p122RhoGAP/DLC-1) protein, which was discovered as a PLC-δ1 stimulator, was upregulated in CSA patients. We tested the hypothesis that p122RhoGAP/DLC-1 overexpression causes coronary spasm. Methods and Results We generated transgenic (TG) mice with vascular smooth muscle (VSM)-specific overexpression of p122RhoGAP/DLC-1. The gene and protein expressions of p122RhoGAP/DLC-1 were markedly increased in the aorta of homozygous TG mice. Stronger staining with anti-p122RhoGAP/DLC-1 in the coronary artery was found in TG than in WT mice. PLC activities in the plasma membrane fraction and the whole cell were enhanced by 1.43 and 2.38 times, respectively, in cultured aortic vascular smooth muscle cells from homozygous TG compared with those from WT mice. Immediately after ergometrine injection, ST-segment elevation was observed in 1 of 7 WT (14%), 6 of 7 heterozygous TG (84%), and 7 of 7 homozygous TG mice (100%) (p<0.05, WT versus TGs). In the isolated Langendorff hearts, coronary perfusion pressure was increased after ergometrine in TG, but not in WT mice, despite of the similar response to prostaglandin F2α between TG and WT mice (n = 5). Focal narrowing of the coronary artery after ergometrine was documented only in TG mice. Conclusions VSM-specific overexpression of p122RhoGAP/DLC-1 enhanced coronary vasomotility after ergometrine injection in mice, which is relevant to human CSA. PMID:26624289

46 CFR 34.17-1 - Application-T/ALL.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Application-T/ALL. 34.17-1 Section 34.17-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-1 Application—T/ALL. (a) Where a fixed foam extinguishing system is...

46 CFR 34.17-1 - Application-T/ALL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Application-T/ALL. 34.17-1 Section 34.17-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-1 Application—T/ALL. (a) Where a fixed foam extinguishing system is...

50 CFR 17.7 - Raptor exemption.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 50 Wildlife and Fisheries 2 2010-10-01 2010-10-01 false Raptor exemption. 17.7 Section 17.7....7 Raptor exemption. (a) The prohibitions found in §§ 17.21 and 17.31 do not apply to any raptor [a... permittee's possession on November 10, 1978, or as the progeny of such a raptor. (b) This section does not...

Monitoring Progress in Vocal Development in Young Cochlear Implant Recipients: Relationships between Speech Samples and Scores from the Conditioned Assessment of Speech Production (CASP)

PubMed Central

Ertmer, David J.; Jung, Jongmin

2012-01-01

Background Evidence of auditory-guided speech development can be heard as the prelinguistic vocalizations of young cochlear implant recipients become increasingly complex, phonetically diverse, and speech-like. In research settings, these changes are most often documented by collecting and analyzing speech samples. Sampling, however, may be too time-consuming and impractical for widespread use in clinical settings. The Conditioned Assessment of Speech Production (CASP; Ertmer & Stoel-Gammon, 2008) is an easily administered and time-efficient alternative to speech sample analysis. The current investigation examined the concurrent validity of the CASP and data obtained from speech samples recorded at the same intervals. Methods Nineteen deaf children who received CIs before their third birthdays participated in the study. Speech samples and CASP scores were gathered at 6, 12, 18, and 24 months post-activation. Correlation analyses were conducted to assess the concurrent validity of CASP scores and data from samples. Results CASP scores showed strong concurrent validity with scores from speech samples gathered across all recording sessions (6 – 24 months). Conclusions The CASP was found to be a valid, reliable, and time-efficient tool for assessing progress in vocal development during young CI recipient’s first 2 years of device experience. PMID:22628109

46 CFR 34.17-15 - Piping-T/ALL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Piping-T/ALL. 34.17-15 Section 34.17-15 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-15 Piping—T/ALL. (a) All piping, valves, and fittings shall meet the applicable...

Gadolinium heteropoly complex K 17[Gd(P 2W 17O 61) 2] as a potential MRI contrast agent

NASA Astrophysics Data System (ADS)

Sun, Guoying; Feng, Jianghua; Wu, Huifeng; Pei, Fengkui; Fang, Ke; Lei, Hao

2004-10-01

Gadolinium heteropoly complex K17[Gd(P2W17O61)2] has been evaluated by in vitro and in vivo experiments as a potential contrast agent for magnetic resonance imaging (MRI). The thermal analysis and conductivity study indicate that this complex has good thermal stability and wide pH stability range. The T1 relaxivity is 7.59 mM-1 s-1 in aqueous solution and 7.97 mM-1 s-1 in 0.725 mmol l-1 bovine serum albumin (BSA) solution at 25 °C and 9.39 T, respectively. MR imaging of three male Sprague-Dawley rats showed remarkable enhancement in rat liver after intravenous injection, which persisted longer than with Gd-DTPA. The signal intensity increased by 57.1±16.9% during the whole imaging period at 0.082 mmol kg-1dose. Our preliminary in vitro and in vivo studies indicate that K17[Gd(P2W17O61)2] is a potential liver-specific MRI contrast agent.

Expression of gamma-aminobutyric acid rho 1 and rho 1 Delta 450 as gene fusions with the green fluorescent protein.

PubMed

Martinez-Torres, A; Miledi, R

2001-02-13

The functional characteristics and cellular localization of the gamma aminobutyric acid (GABA) rho 1 receptor and its nonfunctional isoform rho 1 Delta 450 were investigated by expressing them as gene fusions with the enhanced version of the green fluorescent protein (GFP). Oocytes injected with rho 1-GFP had receptors that gated chloride channels when activated by GABA. The functional characteristics of these receptors were the same as for those of wild-type rho 1 receptors. Fluorescence, because of the chimeric receptors expressed, was over the whole oocyte but was more intense near the cell surface and more abundant in the animal hemisphere. Similar to the wild type, rho 1 Delta 450-GFP did not lead to the expression of functional GABA receptors, and injected oocytes failed to generate currents even after exposure to high concentrations of GABA. Nonetheless, the fluorescence displayed by oocytes expressing rho 1 Delta 450-GFP was distributed similarly to that of rho 1-GFP. Mammalian cells transfected with the rho 1-GFP or rho 1 Delta 450-GFP constructs showed mostly intracellularly distributed fluorescence in confocal microscope images. A sparse localization of fluorescence was observed in the plasma membrane regardless of the cell line used. We conclude that rho 1 Delta 450 is expressed and transported close to, and perhaps incorporated into, the plasma membrane. Thus, rho 1- and rho 1 Delta 450-GFP fusions provide a powerful tool to visualize the traffic of GABA type C receptors.

The sequence of the CA-SP1 junction accounts for the differential sensitivity of HIV-1 and SIV to the small molecule maturation inhibitor 3-O-{3',3'-dimethylsuccinyl}-betulinic acid.

PubMed

Zhou, Jing; Chen, Chin Ho; Aiken, Christopher

2004-06-29

Despite the effectiveness of currently available antiretroviral therapies in the treatment of HIV-1 infection, a continuing need exists for novel compounds that can be used in combination with existing drugs to slow the emergence of drug-resistant viruses. We previously reported that the small molecule 3-O-{3',3'-dimethylsuccinyl}-betulinic acid (DSB) specifically inhibits HIV-1 replication by delaying the processing of the CA-SP1 junction in Pr55Gag. By contrast, SIVmac239 replicates efficiently in the presence of high concentrations of DSB. To determine whether sequence differences in the CA-SP1 junction can fully account for the differential sensitivity of HIV-1 and SIV to DSB, we engineered mutations in this region of two viruses and tested their sensitivity to DSB in replication assays using activated human primary CD4+ T cells. Substitution of the P2 and P1 residues of HIV-1 by the corresponding amino acids of SIV resulted in strong resistance to DSB, but the mutant virus replicated with reduced efficiency. Conversely, replication of an SIV mutant containing three amino acid substitutions in the CA-SP1 cleavage site was highly sensitive to DSB, and the mutations resulted in delayed cleavage of the CA-SP1 junction in the presence of the drug. These results demonstrate that the CA-SP1 junction in Pr55Gag represents the primary viral target of DSB. They further suggest that the therapeutic application of DSB will be accompanied by emergence of mutant viruses that are highly resistant to the drug but which exhibit reduced fitness relative to wild type HIV-1.

Molecular characterization of a novel RhoGAP, RRC-1 of the nematode Caenorhabditis elegans

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

Delawary, Mina; Nakazawa, Takanobu; Tezuka, Tohru

2007-06-01

The GTPase-activating proteins for Rho family GTPases (RhoGAP) transduce diverse intracellular signals by negatively regulating Rho family GTPase-mediated pathways. In this study, we have cloned and characterized a novel RhoGAP for Rac1 and Cdc42, termed RRC-1, from Caenorhabditis elegans. RRC-1 was highly homologous to mammalian p250GAP and promoted GTP hydrolysis of Rac1 and Cdc42 in cells. The rrc-1 mRNA was expressed in all life stages. Using an RRC-1::GFP fusion protein, we found that RRC-1 was localized to the coelomocytes, excretory cell, GLR cells, and uterine-seam cell in adult worms. These data contribute toward understanding the roles of Rho family GTPasesmore » in C. elegans.« less

EspO1-2 Regulates EspM2-Mediated RhoA Activity to Stabilize Formation of Focal Adhesions in Enterohemorrhagic Escherichia coli-Infected Host Cells

PubMed Central

Iyoda, Sunao; Izumiya, Hidemasa; Watanabe, Haruo; Ohnishi, Makoto; Terajima, Jun

2013-01-01

Enterohemorrhagic Escherichia coli (EHEC) Sakai strain encodes two homologous type III effectors, EspO1-1 and EspO1-2. These EspO1s have amino acid sequence homology with Shigella OspE, which targets integrin-linked kinase to stabilize formation of focal adhesions (FAs). Like OspE, EspO1-1 was localized to FAs in EHEC-infected cells, but EspO1-2 was localized in the cytoplasm. An EHEC ΔespO1-1ΔespO1-2 double mutant induced cell rounding and FA loss in most of infected cells, but neither the ΔespO1-1 nor ΔespO1-2 single mutant did. These results suggested that EspO1-2 functioned in the cytoplasm by a different mechanism from EspO1-1 and OspE. Since several type III effectors modulate Rho GTPase, which contributes to FA formation, we investigated whether EspO1-2 modulates the function of these type III effectors. We identified a direct interaction between EspO1-2 and EspM2, which acts as a RhoA guanine nucleotide exchange factor. Upon ectopic co-expression, EspO1-2 co-localized with EspM2 in the cytoplasm and suppressed EspM2-mediated stress fiber formation. Consistent with these findings, an ΔespO1-1ΔespO1-2ΔespM2 triple mutant did not induce cell rounding in epithelial cells. These results indicated that EspO1-2 interacted with EspM2 to regulate EspM2-mediated RhoA activity and stabilize FA formation during EHEC infection. PMID:23409096

IκB Kinase γ/Nuclear Factor-κB-Essential Modulator (IKKγ/NEMO) Facilitates RhoA GTPase Activation, which, in Turn, Activates Rho-associated Kinase (ROCK) to Phosphorylate IKKβ in Response to Transforming Growth Factor (TGF)-β1*

PubMed Central

Kim, Hee-Jun; Kim, Jae-Gyu; Moon, Mi-Young; Park, Seol-Hye; Park, Jae-Bong

2014-01-01

Transforming growth factor (TGF)-β1 plays several roles in a variety of cellular functions. TGF-β1 transmits its signal through Smad transcription factor-dependent and -independent pathways. It was reported that TGF-β1 activates NF-κB and RhoA, and RhoA activates NF-κB in several kinds of cells in a Smad-independent pathway. However, the activation molecular mechanism of NF-κB by RhoA upon TGF-β1 has not been clearly elucidated. We observed that RhoA-GTP level was increased by TGF-β1 in RAW264.7 cells. RhoA-GDP and RhoGDI were bound to N- and C-terminal domains of IKKγ, respectively. Purified IKKγ facilitated GTP binding to RhoA complexed with RhoGDI. Furthermore, Dbs, a guanine nucletotide exchange factor of RhoA much more enhanced GTP binding to RhoA complexed with RhoGDI in the presence of IKKγ. Indeed, si-IKKγ abolished RhoA activation in response to TGF-β1 in cells. However, TGF-β1 stimulated the release of RhoA-GTP from IKKγ and Rho-associated kinase (ROCK), an active RhoA effector protein, directly phosphorylated IKKβ in vitro, whereas TGF-β1-activated kinase 1 activated RhoA upon TGF-β1 stimulation. Taken together, our data indicate that IKKγ facilitates RhoA activation via a guanine nucletotide exchange factor, which in turn activates ROCK to phosphorylate IKKβ, leading to NF-κB activation that induced the chemokine expression and cell migration upon TGF-β1. PMID:24240172

Rho-associated coiled-coil containing kinases (ROCK)

PubMed Central

Julian, Linda; Olson, Michael F

2014-01-01

Rho-associated coiled-coil containing kinases (ROCK) were originally identified as effectors of the RhoA small GTPase.1–5 They belong to the AGC family of serine/threonine kinases6 and play vital roles in facilitating actomyosin cytoskeleton contractility downstream of RhoA and RhoC activation. Since their discovery, ROCK kinases have been extensively studied, unveiling their manifold functions in processes including cell contraction, migration, apoptosis, survival, and proliferation. Two mammalian ROCK homologs have been identified, ROCK1 (also called ROCK I, ROKβ, Rho-kinase β, or p160ROCK) and ROCK2 (also known as ROCK II, ROKα, or Rho kinase), hereafter collectively referred to as ROCK. In this review, we will focus on the structure, regulation, and functions of ROCK. PMID:25010901

Differential Responsiveness of Innate-like IL-17- and IFN-γ-Producing γδ T Cells to Homeostatic Cytokines.

PubMed

Corpuz, Theresa M; Stolp, Jessica; Kim, Hee-Ok; Pinget, Gabriela V; Gray, Daniel H D; Cho, Jae-Ho; Sprent, Jonathan; Webster, Kylie E

2016-01-15

γδ T cells respond to molecules upregulated following infection or cellular stress using both TCR and non-TCR molecules. The importance of innate signals versus TCR ligation varies greatly. Both innate-like IL-17-producing γδ T (γδT-17) and IFN-γ-producing γδ T (γδT-IFNγ) subsets tune the sensitivity of their TCR following thymic development, allowing robust responses to inflammatory cytokines in the periphery. The remaining conventional γδ T cells retain high TCR responsiveness. We determined homeostatic mechanisms that govern these various subsets in the peripheral lymphoid tissues. We found that, although innate-like γδT-17 and γδT-IFNγ cells share elements of thymic development, they diverge when it comes to homeostasis. Both exhibit acute sensitivity to cytokines compared with conventional γδ T cells, but they do not monopolize the same cytokine. γδT-17 cells rely exclusively on IL-7 for turnover and survival, aligning them with NKT17 cells; IL-7 ligation triggers proliferation, as well as promotes survival, upregulating Bcl-2 and Bcl-xL. γδT-IFNγ cells instead depend heavily on IL-15. They display traits analogous to memory CD8(+) T cells and upregulate Bcl-xL and Mcl-1 upon cytokine stimulation. The conventional γδ T cells display low sensitivity to cytokine-alone stimulation and favor IL-7 for their turnover, characteristics reminiscent of naive αβ T cells, suggesting that they may also require tonic TCR signaling for population maintenance. These survival constraints suggest that γδ T cell subsets do not directly compete with each other for cytokines, but instead fall into resource niches with other functionally similar lymphocytes. Copyright © 2016 by The American Association of Immunologists, Inc.

Expression loss and revivification of RhoB gene in ovary carcinoma carcinogenesis and development.

PubMed

Liu, Yingwei; Song, Na; Ren, Kexing; Meng, Shenglan; Xie, Yao; Long, Qida; Chen, Xiancheng; Zhao, Xia

2013-01-01

RhoB, a member of small GTPases belonging to the Ras protein superfamily, might have a suppressive activity in cancer progression. Here, expression of RhoB gene was evaluated in human benign, borderline and malignant ovary tumors by immunostaining, with normal ovary tissue as control. Malignant tumors were assessed according to Federation Internationale de Gynecologie Obstetrique (FIGO) guidelines and classified in stage I-IV. Revivification of RhoB gene was investigated by analyzing the effect of histone deacetylase (HDAC) inhibitor trichostatin (TSA) and methyltransferase inhibitor 5-azacytidine (5-Aza) on ovarian cancer cells via RT-PCR and western blot. Apoptosis of ovary cancer cells was detected using flowcytometry and fluorescence microscopy. Subsequently, RhoB expression is detected in normal ovary epithelium, borderline tumors, and decreases significantly or lost in the majority of ovarian cancer specimen (P<0.05). RhoB expression decreases significantly from stage II (71.4%) to stage III (43.5%) to stage IV (18.2%, P<0.05). TSA can both significantly revive the RhoB gene and mediate apoptosis of ovarian cancer cells, but 5-Aza couldn't. Interference into Revivification of RhoB gene results in reduction of ovary carcinoma cell apoptosis. It is proposed that loss of RhoB expression occurs frequently in ovary carcinogenesis and progression and its expression could be regulated by histone deacetylation but not by promoter hypermethylation, which may serve as a prospective gene treatment target for the patients with ovarian malignancy not responding to standard therapies.

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

PubMed Central

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

1997-01-01

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

7 CFR 1.7 - Agency response to requests for records.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 1 2013-01-01 2013-01-01 false Agency response to requests for records. 1.7 Section 1.7 Agriculture Office of the Secretary of Agriculture ADMINISTRATIVE REGULATIONS Official Records § 1.7 Agency response to requests for records. (a) 5 U.S.C. 552(a)(6)(A)(i) provides that each agency of...

IL-17+ γδ T cells as kick-starters of inflammation.

PubMed

Papotto, Pedro H; Ribot, Julie C; Silva-Santos, Bruno

2017-05-18

Shortly after the discovery of interleukin 17 (IL-17)-producing CD4 + helper T cells (T H 17 cells), it was found that γδ T cells can also secrete large amounts of this pro-inflammatory cytokine. A decade later, it is now known that IL-17 + γδ T cells (γδ17 T cells) are often the main providers of IL-17A in various models of inflammatory diseases, while they also contribute to protective immune responses to infectious organisms. Due to an intricate thymic program of differentiation, γδ17 T cells are able to respond faster than T H 17 cells do and thus predominate in the early stages of inflammatory responses. Here we review the current knowledge of the development, activation and pathophysiological functions of γδ17 T cells, aiming to increase the awareness in the community of the therapeutic potential of this 'other side' of IL-17-mediated immune responses.

17 CFR 7.200-7.201 - [Reserved

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 1 2010-04-01 2010-04-01 false [Reserved] 7.200-7.201 Section 7.200-7.201 Commodity and Securities Exchanges COMMODITY FUTURES TRADING COMMISSION CONTRACT MARKET RULES ALTERED OR SUPPLEMENTED BY THE COMMISSION Board of Trade of the City of Chicago Rules § 7.200-7...

17 CFR 7.200-7.201 - [Reserved

Code of Federal Regulations, 2011 CFR

2011-04-01

... 17 Commodity and Securities Exchanges 1 2011-04-01 2011-04-01 false [Reserved] 7.200-7.201 Section 7.200-7.201 Commodity and Securities Exchanges COMMODITY FUTURES TRADING COMMISSION CONTRACT MARKET RULES ALTERED OR SUPPLEMENTED BY THE COMMISSION Board of Trade of the City of Chicago Rules § 7.200-7...

Small-molecule RORγt antagonists inhibit T helper 17 cell transcriptional network by divergent mechanisms.

PubMed

Xiao, Sheng; Yosef, Nir; Yang, Jianfei; Wang, Yonghui; Zhou, Ling; Zhu, Chen; Wu, Chuan; Baloglu, Erkan; Schmidt, Darby; Ramesh, Radha; Lobera, Mercedes; Sundrud, Mark S; Tsai, Pei-Yun; Xiang, Zhijun; Wang, Jinsong; Xu, Yan; Lin, Xichen; Kretschmer, Karsten; Rahl, Peter B; Young, Richard A; Zhong, Zhong; Hafler, David A; Regev, Aviv; Ghosh, Shomir; Marson, Alexander; Kuchroo, Vijay K

2014-04-17

We identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity. Copyright © 2014 Elsevier Inc. All rights reserved.

Human exposure to power frequency magnetic fields up to 7.6 mT: An integrated EEG/fMRI study.

PubMed

Modolo, Julien; Thomas, Alex W; Legros, Alexandre

2017-09-01

We assessed the effects of power-line frequency (60 Hz in North America) magnetic fields (MF) in humans using simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI). Twenty-five participants were enrolled in a pseudo-double-blind experiment involving "real" or "sham" exposure to sinusoidal 60 Hz MF exposures delivered using the gradient coil of an MRI scanner following two conditions: (i) 10 s exposures at 3 mT (10 repetitions); (ii) 2 s exposures at 7.6 mT (100 repetitions). Occipital EEG spectral power was computed in the alpha range (8-12 Hz, reportedly the most sensitive to MF exposure in the literature) with/without exposure. Brain functional activation was studied using fMRI blood oxygen level-dependent (BOLD, inversely correlated with EEG alpha power) maps. No significant effects were detected on occipital EEG alpha power during or post-exposure for any exposure condition. Consistent with EEG results, no effects were observed on fMRI BOLD maps in any brain region. Our results suggest that acute exposure (2-10 s) to 60 Hz MF from 3 to 7.6 mT (30,000 to 76,000 times higher than average public exposure levels for 60 Hz MF) does not induce detectable changes in EEG or BOLD signals. Combined with previous findings in which effects were observed on the BOLD signal after 1 h exposure to 3 mT, 60 Hz MF, this suggests that MF exposure in the low mT range (<10 mT) might require prolonged durations of exposure to induce detectable effects. Bioelectromagnetics. 38:425-435, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Rho GTPases at the crossroad of signaling networks in mammals: impact of Rho-GTPases on microtubule organization and dynamics.

PubMed

Wojnacki, José; Quassollo, Gonzalo; Marzolo, María-Paz; Cáceres, Alfredo

2014-01-01

Microtubule (MT) organization and dynamics downstream of external cues is crucial for maintaining cellular architecture and the generation of cell asymmetries. In interphase cells RhoA, Rac, and Cdc42, conspicuous members of the family of small Rho GTPases, have major roles in modulating MT stability, and hence polarized cell behaviors. However, MTs are not mere targets of Rho GTPases, but also serve as signaling platforms coupling MT dynamics to Rho GTPase activation in a variety of cellular conditions. In this article, we review some of the key studies describing the reciprocal relationship between small Rho-GTPases and MTs during migration and polarization.

Activity-dependent rapid local RhoA synthesis is required for hippocampal synaptic plasticity.

PubMed

Briz, Victor; Zhu, Guoqi; Wang, Yubin; Liu, Yan; Avetisyan, Mariam; Bi, Xiaoning; Baudry, Michel

2015-02-04

Dendritic protein synthesis and actin cytoskeleton reorganization are important events required for the consolidation of hippocampal LTP and memory. However, the temporal and spatial relationships between these two processes remain unclear. Here, we report that treatment of adult rat hippocampal slices with BDNF or with tetraethylammonium (TEA), which induces a chemical form of LTP, produces a rapid and transient increase in RhoA protein levels. Changes in RhoA were restricted to dendritic spines of CA3 and CA1 and require de novo protein synthesis regulated by mammalian target of rapamycin (mTOR). BDNF-mediated stimulation of RhoA activity, cofilin phosphorylation, and actin polymerization were completely suppressed by protein synthesis inhibitors. Furthermore, intrahippocampal injections of RhoA antisense oligodeoxynucleotides inhibited theta burst stimulation (TBS)-induced RhoA upregulation in dendritic spines and prevented LTP consolidation. Addition of calpain inhibitors after BDNF or TEA treatment maintained RhoA levels elevated and prolonged the effects of BDNF and TEA on actin polymerization. Finally, the use of isoform-selective calpain inhibitors revealed that calpain-2 was involved in RhoA synthesis, whereas calpain-1 mediated RhoA degradation. Overall, this mechanism provides a novel link between dendritic protein synthesis and reorganization of the actin cytoskeleton in hippocampal dendritic spines during LTP consolidation. Copyright © 2015 the authors 0270-6474/15/352269-14$15.00/0.

Measurement of CP-Violating Asymmetries in B0 to (rho pi)0 Using a Time-Dependent Dalitz Plot Analysis

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

Wu, J.

We present the preliminary measurement of CP-violating asymmetries in B{sup 0} {yields} ({rho}{pi}){sup 0} {yields} {pi}{sup +}{pi}{sup -}{pi}{sup 0} decays using a time-dependent Dalitz plot analysis. The results are obtained from a data sample of 213 million {Upsilon}(4S) {yields} B{bar B} decays, collected by the BABAR detector at the PEP-II asymmetric-energy B Factory at SLAC. This analysis extends the narrow-rho quasi-two-body approximation used in the previous analysis, by taking into account the interference between the rho resonances of the three charges. We measure 16 coefficients of the bilinear form factor terms occurring in the time-dependent decay rate of the B{supmore » 0} meson with the use of a maximum-likelihood fit. We derive the physically relevant quantities from these coefficients. We measure the direct CP-violation parameters A{sub {rho}{pi}} = -0.088 {+-} 0.049 {+-} 0.013 and C = 0.34 {+-} 0.11 {+-} 0.05, where the first errors are statistical and the second systematic. For the mixing-induced CP-violation parameter we find S = -0.10 {+-} 0.14 {+-} 0.04, and for the dilution and strong phase shift parameters respectively, we obtain {Delta}C = 0.15 {+-} 0.11 {+-} 0.03 and {Delta}S = 0.22 {+-} 0.15 {+-} 0.03. For the angle alpha of the Unitarity Triangle we measure (113{sub -17}{sup +27} {+-} 6){sup o}, while only a weak constraint is achieved at the significance level of more than two standard deviations. Finally, for the relative strong phase {delta}{sub {+-}} between the B{sup 0} {yields} {rho}{sup -}{pi}{sup +} and B{sup 0} {yields} {rho}{sup +}{pi}{sup -} transitions we find (-67{sub -31}{sup +28} {+-} 7) deg, with a similarly weak constraint at two standard deviations and beyond.« less

Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid.

PubMed

Andres, Devon; Keyser, Brian M; Petrali, John; Benton, Betty; Hubbard, Kyle S; McNutt, Patrick M; Ray, Radharaman

2013-04-18

Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells. We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG. Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity.

Ginsenoside Rb1 inhibits autophagy through regulation of Rho/ROCK and PI3K/mTOR pathways in a pressure-overload heart failure rat model.

PubMed

Yang, Tianrui; Miao, Yunbo; Zhang, Tong; Mu, Ninghui; Ruan, Libo; Duan, Jinlan; Zhu, Ying; Zhang, Rongping

2018-06-01

This study was designed to explore the relationship between ginsenoside Rb1 (Grb1) and high-load heart failure (HF) in rats. The parameters of cardiac systolic function (left ventricular posterior wall thickness (LVPWT), left ventricular internal diastolic diameter (LVID), fraction shortening (FS) and mitral valves (MVs)) of rat hearts in each group were inspected by echocardiogram. The expressions of rat myocardial contractile proteins, autophagy-related proteins and the activation of Rho/ROCK and PI3K/mTOR pathways were detected by Western blot. LVPWT, FS, MVs and the expression of myocardial contractile proteins α-MHC, apoptosis-related proteins Bcl-2 and signalling pathway involved proteins pAkt and mTOR were significantly reduced in the HF, HF+5 mg/kg Grb1 (HF+Grb1-5) and HF+Grb1+arachidonic acid (AA) groups with LVID, β-MHC, cell apoptosis, cell autophagy and Rho/ROCK significantly increased compared with the control group, of which the tendency was contrary to the HF+20 mg/kg Grb1 (HF+Grb1-20) group compared with the HF group (P < 0.05). In the HF+Grb1+AA group, there was no significant change in the above indexes compared with the HF group. The results indicated that Grb1 can exert anti-HF function by inhibiting cardiomyocyte autophagy of rats through regulation of Rho/ROCK and PI3K/mTOR pathways. © 2018 Royal Pharmaceutical Society.

Macrophage differentiation induced by PMA is mediated by activation of RhoA/ROCK signaling.

PubMed

Yang, Lifeng; Dai, Fan; Tang, Lian; Le, Yulan; Yao, Wenjuan

2017-01-01

In order to investigate the effects of RhoA/ROCK signaling in macrophage differentiation, we used 100 ng/mL PMA to induce macrophage differentiation from U937 cells in vitro. The observation of cell morphology and the expression of CD68 and SR-A were performed to confirm the differentiation induced by PMA. Western blot analysis showed that the expression of ROCK1 and ROCK2 and the phosphorylation of MYPT1 were significantly increased after PMA treatment. Pulldown assay showed that the activation of RhoA was obviously enhanced when U937 cells were treated with PMA. In order to further demonstrate whether RhoA/ROCK signaling could mediate the macrophage differentiation induced by PMA, we successfully suppressed the expression of RhoA, ROCK1 and ROCK2 by performing siRNA technology in U937 cells, respectively. The macrophage differentiation and the expression of CD68 and SR-A were significantly inhibited by the suppression of RhoA, ROCK1 or ROCK2 in PMA-induced U937 cells, indicating that the macrophage differentiation induced by PMA is associated with RhoA/ROCK signaling pathway. In addition, we pretreated U937 cells with Y27632 (ROCK inhibitor, 20 μM) for 30 min and then observed the macrophage differentiation induced by PMA. The result illustrated that Y27632 pretreatment obviously inhibited PMA-induced differentiation and the expression of CD68 and SR-A. In conclusion, the activation of RhoA/ROCK signaling is responsible for the macrophage differentiation induced by PMA.

17Beta-hydroxysteroid dehydrogenase (17beta-HSD) in scleractinian corals and zooxanthellae.

PubMed

Blomquist, Charles H; Lima, P H; Tarrant, A M; Atkinson, M J; Atkinson, S

2006-04-01

Steroid metabolism studies have yielded evidence of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in corals. This project was undertaken to clarify whether there are multiple isoforms of 17beta-HSD, whether activity levels vary seasonally, and if zooxanthellae contribute to activity. 17Beta-HSD activity was characterized in zooxanthellate and azooxanthellate coral fragments collected in summer and winter and in zooxanthellae cultured from Montipora capitata. More specifically, 17beta-HSD activity was characterized with regard to steroid substrate and inhibitor specificity, coenzyme specificity, and Michaelis constants for estradiol (E2) and NADP+. Six samples each of M. capitata and Tubastrea coccinea (three summers, three winters) were assayed with E2 and NADP+. Specific activity levels (pmol/mg protein) varied 10-fold among M. capitata samples and 6-fold among T. coccinea samples. There was overlap of activity levels between summer and winter samples. NADP+/NAD+ activity ratios varied from 1.6 to 22.2 for M. capatita, 2.3 to 3.8 for T. coccinea and 0.7 to 1.1 for zooxanthellae. Coumestrol was the most inhibitory of the steroids and phytoestrogens tested. Our data confirm that corals and zooxanthellae contain 17beta-HSD and are consistent with the presence of more than one isoform of the enzyme.

Upregulated STAT3 and RhoA signaling in colorectal cancer (CRC) regulate the invasion and migration of CRC cells.

PubMed

Zhang, G-Y; Yang, W-H; Chen, Z

2016-05-01

We aimed to reveal the expression and activation of signal transducers and activators of transcription 3 (STAT3) and RhoA/Rho-associated coiled-coil forming kinase 1 (ROCK1) signaling in CRC tissues, and to investigate the regulatory role of STAT3 and RhoA signaling in the invasion and migration of colorectal cancer cells. We examined the expression of STAT3, RhoA and ROCK1 in CRC tissues with real-time PCR and Western blotting methods. And then we examined the interaction between STAT3 and RhoA/ROCK1 signaling in CRC HT-29 cells with gain-of-function and loss-of-function strategies. In addition, we determined the regulation by STAT3 and RhoA/ROCK1 on the invasion and migration of CRC HT-29 cells. Our study demonstrated a significant upregulation of RhoA and ROCK1 expression and STAT3-Y705 phosphorylation in 32 CRC specimens, compared to the 17 normal CRC tissues. Further study demonstrated there was a coordination between STAT3 and RhoA/Rock signaling in the HT-29 cells. Moreover, STAT3 knockdown or RhoA knockdown significantly repressed the migration and invasion in HT-29 cells and vice versa. STAT3 and RhoA signaling regulate the invasion and migration of CRC cells, implying the orchestrated and oncogenic roles of STAT3 and RhoA/ROCK1 signaling in CRC.

Expression Loss and Revivification of RhoB Gene in Ovary Carcinoma Carcinogenesis and Development

PubMed Central

Liu, Yingwei; Song, Na; Ren, Kexing; Meng, Shenglan; Xie, Yao; Long, Qida; Chen, Xiancheng; Zhao, Xia

2013-01-01

RhoB, a member of small GTPases belonging to the Ras protein superfamily, might have a suppressive activity in cancer progression. Here, expression of RhoB gene was evaluated in human benign, borderline and malignant ovary tumors by immunostaining, with normal ovary tissue as control. Malignant tumors were assessed according to Federation Internationale de Gynecologie Obstetrique (FIGO) guidelines and classified in stage I-IV. Revivification of RhoB gene was investigated by analyzing the effect of histone deacetylase (HDAC) inhibitor trichostatin (TSA) and methyltransferase inhibitor 5-azacytidine (5-Aza) on ovarian cancer cells via RT-PCR and western blot. Apoptosis of ovary cancer cells was detected using flowcytometry and fluorescence microscopy. Subsequently, RhoB expression is detected in normal ovary epithelium, borderline tumors, and decreases significantly or lost in the majority of ovarian cancer specimen (P<0.05). RhoB expression decreases significantly from stage II (71.4%) to stage III (43.5%) to stage IV (18.2%, P<0.05). TSA can both significantly revive the RhoB gene and mediate apoptosis of ovarian cancer cells, but 5-Aza couldn’t. Interference into Revivification of RhoB gene results in reduction of ovary carcinoma cell apoptosis. It is proposed that loss of RhoB expression occurs frequently in ovary carcinogenesis and progression and its expression could be regulated by histone deacetylation but not by promoter hypermethylation, which may serve as a prospective gene treatment target for the patients with ovarian malignancy not responding to standard therapies. PMID:24223801

RhoA/Rho-kinase signaling: a therapeutic target in pulmonary hypertension.

PubMed

Barman, Scott A; Zhu, Shu; White, Richard E

2009-01-01

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive elevation of pulmonary arterial pressure and vascular resistance due to pulmonary vasoconstriction and vessel remodeling as well as inflammation. Rho-kinases (ROCKs) are one of the best-described effectors of the small G-protein RhoA, and ROCKs are involved in a variety of cellular functions including muscle cell contraction, proliferation and vascular inflammation through inhibition of myosin light chain phosphatase and activation of downstream mediators. A plethora of evidence in animal models suggests that heightened RhoA/ROCK signaling is important in the pathogenesis of pulmonary hypertension by causing enhanced constriction and remodeling of the pulmonary vasculature. Both animal and clinical studies suggest that ROCK inhibitors are effective for treatment of severe PAH with minimal risk, which supports the premise that ROCKs are important therapeutic targets in pulmonary hypertension and that ROCK inhibitors are a promising new class of drugs for this devastating disease.

The proteinase-activated receptor-2 mediates phagocytosis in a Rho-dependent manner in human keratinocytes.

PubMed

Scott, Glynis; Leopardi, Sonya; Parker, Lorelle; Babiarz, Laura; Seiberg, Miri; Han, Rujiing

2003-09-01

Recent work shows that the G-protein-coupled receptor proteinase activated receptor-2 activates signals that stimulate melanosome uptake in keratinocytes in vivo and in vitro. The Rho family of GTP-binding proteins is involved in cytoskeletal remodeling during phagocytosis. We show that proteinase-activated receptor-2 mediated phagocytosis in human keratinocytes is Rho dependent and that proteinase-activated receptor-2 signals to activate Rho. In contrast, Rho activity did not affect either proteinase-activated receptor-2 activity or mRNA and protein levels. We explored the signaling mechanisms of proteinase-activated receptor-2 mediated Rho activation in human keratinocytes and show that activation of proteinase-activated receptor-2, either through specific proteinase-activated receptor-2 activating peptides or through trypsinization, elevates cAMP in keratinocytes. Proteinase-activated receptor-2 mediated Rho activation was pertussis toxin insensitive and independent of the protein kinase A signaling pathway. These data are the first to show that proteinase-activated receptor-2 mediated phagocytosis is Rho dependent and that proteinase-activated receptor-2 signals to Rho and cAMP in keratinocytes. Because phagocytosis of melanosomes is recognized as an important mechanism for melanosome transfer to keratinocytes, these results suggest that Rho is a critical signaling intermediate in melanosome uptake in keratinocytes.

MIP-2 causes differential activation of RhoA in mouse aortic versus pulmonary artery endothelial cells

PubMed Central

Moldobaeva, Aigul; Baek, Amy; Wagner, Elizabeth M.

2008-01-01

Previously, we have shown that endothelial cell chemotaxis to the proangiogenic chemokine MIP-2 (macrophage inflammatory protein-2), is much greater in mouse aortic endothelial cells (EC) than pulmonary arterial endothelial cells (PA EC). This was true despite the observation that both cell types display comparable levels of the ligand receptor, CXCR2 (8). Since the systemic arterial circulation is proangiogenic in the adult lung and the pulmonary circulation is relatively resistant to neovascularization, we questioned whether the observed functional heterogeneity is related to inherent differences in cell signaling cascades of the two EC subtypes. Specifically, we measured activation of Rac1 and RhoA, both thought to be involved in EC cell migration. Rac1 showed inconsistent and minimal changes in both cell types after MIP-2 treatment (p>0.05). However, activated RhoA was increased upon exposure to MIP-2 only in aortic EC (61% increase; p<0.05). Decreased RhoA activation after treatment of aortic EC with specific siRNA for RhoA resulted in a functional decrease in EC chemotaxis to MIP-2 (17% increase; p<0.05). Additionally, increased RhoA activation in PA EC with adenoviral infection of RhoA caused an increase in PA EC chemotaxis to MIP-2 (46% increase; p<0.05). Inhibition of RhoA activity with the Rho kinase inhibitor, Y27632 blocked aortic EC chemotaxis and stress fiber formation. Thus, RhoA activation is increased after MIP-2 treatment in mouse aortic endothelial cells but not in pulmonary artery endothelial cells. We conclude that RhoA is part of a signaling pathway essential for aortic cell migration after CXCR2 ligation. This result provides one explanation for the difference in chemotaxis observed in these two endothelial subtypes that express similar levels of CXCR2. PMID:17662312

CD147 modulates the differentiation of T-helper 17 cells in patients with rheumatoid arthritis.

PubMed

Yang, Hui; Wang, Jian; Li, Yu; Yin, Zhen-Jie; Lv, Ting-Ting; Zhu, Ping; Zhang, Yan

2017-01-01

The role of CD147 in regulation of rheumatoid arthritis (RA) is not fully elucidated. The aim of this study was to investigate the effect of cell-to-cell contact of activated CD14 + monocytes with CD4 + T cells, and the modulatory role of CD147 on T-helper 17 (Th17) cells differentiation in patients with RA. Twenty confirmed active RA patients and twenty normal controls were enrolled. CD4 + T cells and CD14 + monocytes were purified by magnetic beads cell sorting. Cells were cultured under different conditions in CD4 + T cells alone, direct cell-to-cell contact co-culture of CD4 + and CD14 + cells, or indirect transwell co-culture of CD4 + /CD14 + cells in response to LPS and anti-CD3 stimulation with or without anti-CD147 antibody pretreatments. The proportion of IL-17-producing CD4 + T cells (defined as Th17 cells) was determined by flow cytometry. The levels of interleukin (IL)-17, IL-6, and IL-1β in the supernatants of cultured cells were measured by ELISA. The optimal condition for in vitro induction of Th17 cells differentiation was co-stimulation with 0.1 μg/mL of LPS and 100 ng/mL of anti-CD3 for 3 days under direct cell-to-cell contact co-culture of CD4 + and CD14 + cells. Anti-CD147 antibody reduced the proportion of Th17 cells, and also inhibited the productions of IL-17, IL-6, and IL-1β in PBMC culture from RA patients. The current results revealed that Th17 differentiation required cell-to-cell contact with activated monocytes. CD147 promoted the differentiation of Th17 cells by regulation of cytokine production, which provided the evidence for pathogenesis and potential therapeutic targets for RA. © 2016 APMIS. Published by John Wiley & Sons Ltd.

7 CFR 774.17 - Loan application.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 7 2010-01-01 2010-01-01 false Loan application. 774.17 Section 774.17 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.17 Loan application. A complete...

RhoA GTPase inhibition organizes contraction during epithelial morphogenesis

PubMed Central

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

2016-01-01

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

Two Molecular Clouds near M17

NASA Astrophysics Data System (ADS)

Wilson, T. L.; Hanson, M. M.; Muders, D.

2003-06-01

We present fully sampled images in the C18O J=2-1 line extending over 13'×23', made with the Heinrich Hertz Telescope (HHT) on Mount Graham, AZ. The HHT has a resolution of 35" at the line frequency. This region includes two molecular clouds. Cloud A, to the north, is more compact, while cloud B is to the west of the H II region M17. Cloud B contains the well-known source M17SW. In C18O we find 13 maxima in cloud A and 39 in cloud B. Sixteen sources in cloud B are in M17SW, mapped previously with higher resolution. In cloud B, sources outside M17SW have line widths comparable to those in M17SW. In comparison, cloud A has lower C18O line intensities and smaller line widths but comparable densities and sizes. Maps of the cores of these clouds were also obtained in the J=5-4 line of CS, which traces higher H2 densities. Our images of the cores of clouds A and B show that for VLSR<=20 km s-1, the peaks of the CS emission are shifted closer to the H II region than the C18O maxima, so higher densities are found toward the H II region. Our CS data give additional support to the already strong evidence that M17SW and nearby regions are heated and compressed by the H II region. Our data show that cloud A has a smaller interaction with the H II region. We surmise that M17SW was an initially denser region, and the turn-on of the H II region will make this the next region of massive star formation. Outside of M17SW, the only other obvious star formation region may be in cloud A, since there is an intense millimeter dust continuum peak found by Henning et al. (1998) but no corresponding C18O maximum. If the CO/H2 ratio is constant, the dust must have a temperature of ~100 K or the H2 density is greater than 106 cm-3 or both to reconcile the C18O and dust data. Alternatively, if the CO/H2 ratio is low, perhaps much of the CO is depleted.

Critical functions of RhoB in support of glioblastoma tumorigenesis

PubMed Central

Ma, Yufang; Gong, Yuanying; Cheng, Zhixiang; Loganathan, Sudan; Kao, Crystal; Sarkaria, Jann N.; Abel, Ty W.; Wang, Jialiang

2015-01-01

Background RhoB is a member of the Rho small GTPase family that regulates cytoskeletal dynamics and vesicle trafficking. The RhoB homologs, RhoA and RhoC, have been shown to promote cancer progression and metastasis. In contrast, the functions of RhoB in human cancers are context dependent. Although expression of RhoB inversely correlates with disease progression in several epithelial cancers, recent data suggest that RhoB may support malignant phenotypes in certain cancer types. Methods We assessed RhoB protein levels in glioma surgical specimens and patient-derived xenografts. The roles of RhoB in glioblastoma were determined by loss-of-function and gain-of-function assays in vitro and in vivo. The impact on p53 and STAT3 signaling was investigated. Results RhoB expression was similar in tumor specimens compared with normal neural tissues obtained from epilepsy surgery. RhoB was expressed in the vast majority of xenograft tumors and spheroid cultures. Knockdown of RhoB induced cell-cycle arrest and apoptosis and compromised in vivo tumorigenic potential. However, overexpression of wild-type RhoB or a constitutively active mutant (RhoB-V14) did not significantly affect cell growth, which suggests that RhoB is not a rate-limiting oncogenic factor and is consistent with the scarcity of RhoB mutations in human cancer. Knockdown of RhoB reduced basal STAT3 activity and impaired cytokine-induced STAT3 activation. In glioblastoma tumors retaining wild-type p53, depletion of RhoB also activated p53 and induced expression of p21CIP1/WAF1. Conclusions Our data suggest that RhoB belongs to an emerging class of “nononcogene addiction” factors that are essential for maintenance of malignant phenotypes in human cancers. PMID:25216671

The Roles of T Helper 1, T Helper 17 and Regulatory T Cells in the Pathogenesis of Sarcoidosis.

PubMed

Mortaz, Esmaeil; Rezayat, Fatemeh; Amani, Davar; Kiani, Arda; Garssen, Johan; Adcock, Ian M; Velayati, Aliakbar

2016-08-01

Sarcoidosis is a systemic granulomatous disorder of unidentified etiology, with a heterogeneous clinical presentation. It is characterized by a reduced delayed-type hypersensitivity to tuberculin and common antigens. The balance between Th1, Th17 and Regulatory T(Treg) cells controls T-cell proliferation and activation.The Th17/Treg ratio in the peripheral blood and bronchoalveolar lavage fluidis increased in patients with active sarcoidosis. Amplified IL-17A expression in granulomas and the presence of IL-17A+, IL-17A+IL-4+ and IL-17A+IFN-γ+ memory T helper cells in the circulation and BAL indicate Th17 cell involvement in granuloma induction and/or maintenance in sarcoidosis. Sarcoidosis should therefore be considered as a Th1/Th17 multisystem disorder and anti-IL-17/Th17 approaches that control and reduce IL-17Amay be an option, therefore, for the treatment of sarcoidosis.Here we provide a short overview as to the role of Th17 cells as critical cells in the pathogenesis of sarcoidosis.

17 CFR 240.17h-2T - Risk assessment reporting requirements for brokers and dealers.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Risk assessment reporting... Organizations § 240.17h-2T Risk assessment reporting requirements for brokers and dealers. (a) Reporting requirements of risk assessment information required to be maintained by section 240.17h-1T. (1) Every broker...

7 CFR 1215.17 - Research.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 10 2013-01-01 2013-01-01 false Research. 1215.17 Section 1215.17 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POPCORN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Popcorn Promotion, Research, and Consumer Information Order Definitions § 1215.17...

7 CFR 1215.17 - Research.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 10 2010-01-01 2010-01-01 false Research. 1215.17 Section 1215.17 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POPCORN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Popcorn Promotion, Research, and Consumer Information Order Definitions § 1215.17...

7 CFR 1215.17 - Research.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 10 2012-01-01 2012-01-01 false Research. 1215.17 Section 1215.17 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POPCORN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Popcorn Promotion, Research, and Consumer Information Order Definitions § 1215.17...

7 CFR 1215.17 - Research.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 10 2011-01-01 2011-01-01 false Research. 1215.17 Section 1215.17 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POPCORN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Popcorn Promotion, Research, and Consumer Information Order Definitions § 1215.17...

7 CFR 1215.17 - Research.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 10 2014-01-01 2014-01-01 false Research. 1215.17 Section 1215.17 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE POPCORN PROMOTION, RESEARCH, AND CONSUMER INFORMATION Popcorn Promotion, Research, and Consumer Information Order Definitions § 1215.17...

7 CFR 1218.17 - Promotion.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1218.17 Section 1218.17 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.17 Promotion...

WinRho: Rh immune globulin prepared by ion exchange for intravenous use.

PubMed Central

Bowman, J M; Friesen, A D; Pollock, J M; Taylor, W E

1980-01-01

An Rh immune globulin [Rh IgG] for intravenous use, WinRho, has been prepared by the Winnipeg Rh Institute by a modification of the ion-exchange column method of Hoppe and colleagues. When administered to Rh-negative male and nonpregnant female volunteers WinRho was found to be nonpyrogenic, nontoxic, safe and protective against Rh alloimmunization. In a clinical trial with 240 microgram given at about 28 weeks' gestation and 120 microgram given after delivery to Rh-negative women at risk of Rh immunization WinRho was effective in preventing Rh immunization. Of the 870 women carrying Rh-positive fetuses who were treated with WinRho during pregnancy and were not tested several months after delivery 14 would have shown evidence of Rh immunization by the time of delivery if WinRho had been ineffective; none showed such evidence. Of the 1122 women carrying Rh-positive fetuses who were retested 4 to 6 months after delivery 83 would have shown evidence of Rh immunization at that time if WinRho had been ineffective; only 1 showed such evidence. The efficiency of yield of anti-D with the modified method of production, the fct that it can be given intravenously (a route that causes the patient less discomfort and immediately results in high anti-D levels) and the lower levels of contaminating IgA and IgM make WinRho the preparation of choice for preventing Rh immunization. PMID:6161687

Mutation in WDR4 impairs tRNA m(7)G46 methylation and causes a distinct form of microcephalic primordial dwarfism.

PubMed

Shaheen, Ranad; Abdel-Salam, Ghada M H; Guy, Michael P; Alomar, Rana; Abdel-Hamid, Mohamed S; Afifi, Hanan H; Ismail, Samira I; Emam, Bayoumi A; Phizicky, Eric M; Alkuraya, Fowzan S

2015-09-28

Primordial dwarfism is a state of extreme prenatal and postnatal growth deficiency, and is characterized by marked clinical and genetic heterogeneity. Two presumably unrelated consanguineous families presented with an apparently novel form of primordial dwarfism in which severe growth deficiency is accompanied by distinct facial dysmorphism, brain malformation (microcephaly, agenesis of corpus callosum, and simplified gyration), and severe encephalopathy with seizures. Combined autozygome/exome analysis revealed a novel missense mutation in WDR4 as the likely causal variant. WDR4 is the human ortholog of the yeast Trm82, an essential component of the Trm8/Trm82 holoenzyme that effects a highly conserved and specific (m(7)G46) methylation of tRNA. The human mutation and the corresponding yeast mutation result in a significant reduction of m(7)G46 methylation of specific tRNA species, which provides a potential mechanism for primordial dwarfism associated with this lesion, since reduced m(7)G46 modification causes a growth deficiency phenotype in yeast. Our study expands the number of biological pathways underlying primordial dwarfism and adds to a growing list of human diseases linked to abnormal tRNA modification.

7 CFR 1206.17 - Promotion.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1206.17 Section 1206.17 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MANGO PROMOTION, RESEARCH, AND INFORMATION Mango Promotion, Research, and Information Order Definitions § 1206.17 Promotion. Promotion means...

Plasma IL-17A levels in patients with late-life depression.

PubMed

Saraykar, Smita; Cao, Bo; Barroso, Lucelia S; Pereira, Kelly S; Bertola, Laiss; Nicolau, Mariana; Ferreira, Jessica D; Dias, Natalia S; Vieira, Erica L; Teixeira, Antonio L; Silva, Ana Paula M; Diniz, Breno S

2018-01-01

A consistent body of research has confirmed that patients with major depressive disorder (MDD) have increased concentrations of pro-inflammatory cytokines, including IL-6, TNF-α, IL-1β, the soluble IL-2 receptor, and C-reactive protein, compared to controls; however, there is limited information on IL-17A in MDD. Moreover, information about IL-17A in older populations, i.e., patients with late-life depression (LLD), is conspicuously missing from the literature. The purpose of this study was to investigate the role of IL-17A in LLD. A convenience sample of 129 individuals, 74 with LLD and 55 non-depressed controls, were enrolled in this study. The Mann-Whitney U test was used to compare plasma IL-17A levels between LLD and controls subjects, and Spearman's rank order correlation was used to investigate correlation of these levels with clinical, neuropsychological, and cognitive assessments. Plasma IL-17A levels were not statistically different between LLD patients and controls (p = 0.94). Among all subjects (LLD + control), plasma IL-17A did not correlate significantly with depressive symptoms (rho = -0.009, p = 0.92) but a significant correlation was observed with cognitive assessments (rho = 0.22, p = 0.01). Our findings do not support an association between plasma IL-17A levels and LLD. Nevertheless, IL-17A may be associated with cognitive impairment in LLD patients. If this finding is confirmed in future longitudinal studies, modulation of the T-helper 17 cell (Th17) immune response may be a treatment target for cognitive impairment in this population.

Discovery of a novel and potent class of anti-HIV-1 maturation inhibitors with improved virology profile against gag polymorphisms.

PubMed

Tang, Jun; Jones, Stacey A; Jeffrey, Jerry L; Miranda, Sonia R; Galardi, Cristin M; Irlbeck, David M; Brown, Kevin W; McDanal, Charlene B; Johns, Brian A

2017-06-15

A new class of betulin-derived α-keto amides was identified as HIV-1 maturation inhibitors. Through lead optimization, GSK8999 was identified with IC 50 values of 17nM, 23nM, 25nM, and 8nM for wild type, Q369H, V370A, and T371A respectively. When tested in a panel of 62 HIV-1 isolates covering a diversity of CA-SP1 genotypes including A, AE, B, C, and G using a PBMC based assay, GSK8999 was potent against 57 of 62 isolates demonstrating an improvement over the first generation maturation inhibitor BVM. The data disclosed here also demonstrated that the new α-keto amide GSK8999 has a mechanism of action consistent with inhibition of the proteolytic cleavage of CA-SP1. Copyright © 2017 Elsevier Ltd. All rights reserved.

LETTER TO THE EDITOR: Cross sections of 6Li(t,d1)7Li*[0.478] and 6Li(t,p1)8Li*[0.981] nuclear reactions in the 0-2 MeV energy range

NASA Astrophysics Data System (ADS)

Voronchev, V. T.; Kukulin, V. I.

2000-12-01

An original extrapolation technique developed previously is modified and applied to study nuclear reactions in the 6Li + T system at energies E = 0-2 MeV. Cross sections of gamma-ray-producing reactions 6Li(t,d1)7Li*[0.478] and 6Li(t,p1)8Li*[0.981] with important diagnostic implications are calculated. The (t,d1) nuclear data found exceed those accepted elsewhere by 2.5-3.5 times at sub-barrier energies. The cross sections of the (t,p1) reaction are calculated for the first time.

Coupling constant for N*(1535)N{rho}

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

Xie Jujun; Graduate University of Chinese Academy of Sciences, Beijing 100049; Wilkin, Colin

2008-05-15

The value of the N*(1535)N{rho} coupling constant g{sub N*N{rho}} derived from the N*(1535){yields}N{rho}{yields}N{pi}{pi} decay is compared with that deduced from the radiative decay N*(1535){yields}N{gamma} using the vector-meson-dominance model. On the basis of an effective Lagrangian approach, we show that the values of g{sub N*N{rho}} extracted from the available experimental data on the two decays are consistent, though the error bars are rather large.

Identification and characterization of novel associations in the CASP8/ALS2CR12 region on chromosome 2 with breast cancer risk

PubMed Central

Lin, Wei-Yu; Camp, Nicola J.; Ghoussaini, Maya; Beesley, Jonathan; Michailidou, Kyriaki; Hopper, John L.; Apicella, Carmel; Southey, Melissa C.; Stone, Jennifer; Schmidt, Marjanka K.; Broeks, Annegien; Van't Veer, Laura J.; Th Rutgers, Emiel J.; Muir, Kenneth; Lophatananon, Artitaya; Stewart-Brown, Sarah; Siriwanarangsan, Pornthep; Fasching, Peter A.; Haeberle, Lothar; Ekici, Arif B.; Beckmann, Matthias W.; Peto, Julian; Dos-Santos-Silva, Isabel; Fletcher, Olivia; Johnson, Nichola; Bolla, Manjeet K.; Wang, Qin; Dennis, Joe; Sawyer, Elinor J.; Cheng, Timothy; Tomlinson, Ian; Kerin, Michael J.; Miller, Nicola; Marmé, Frederik; Surowy, Harald M.; Burwinkel, Barbara; Guénel, Pascal; Truong, Thérèse; Menegaux, Florence; Mulot, Claire; Bojesen, Stig E.; Nordestgaard, Børge G.; Nielsen, Sune F.; Flyger, Henrik; Benitez, Javier; Zamora, M. Pilar; Arias Perez, Jose Ignacio; Menéndez, Primitiva; González-Neira, Anna; Pita, Guillermo; Alonso, M. Rosario; Álvarez, Nuria; Herrero, Daniel; Anton-Culver, Hoda; Brenner, Hermann; Dieffenbach, Aida Karina; Arndt, Volker; Stegmaier, Christa; Meindl, Alfons; Lichtner, Peter; Schmutzler, Rita K.; Müller-Myhsok, Bertram; Brauch, Hiltrud; Brüning, Thomas; Ko, Yon-Dschun; Tessier, Daniel C.; Vincent, Daniel; Bacot, Francois; Nevanlinna, Heli; Aittomäki, Kristiina; Blomqvist, Carl; Khan, Sofia; Matsuo, Keitaro; Ito, Hidemi; Iwata, Hiroji; Horio, Akiyo; Bogdanova, Natalia V.; Antonenkova, Natalia N.; Dörk, Thilo; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M.; Wu, Anna H.; Tseng, Chiu-Chen; Van Den Berg, David; Stram, Daniel O.; Neven, Patrick; Wauters, Els; Wildiers, Hans; Lambrechts, Diether; Chang-Claude, Jenny; Rudolph, Anja; Seibold, Petra; Flesch-Janys, Dieter; Radice, Paolo; Peterlongo, Paolo; Manoukian, Siranoush; Bonanni, Bernardo; Couch, Fergus J.; Wang, Xianshu; Vachon, Celine; Purrington, Kristen; Giles, Graham G.; Milne, Roger L.; Mclean, Catriona; Haiman, Christopher A.; Henderson, Brian E.; Schumacher, Fredrick; Le Marchand, Loic; Simard, Jacques; Goldberg, Mark S.; Labrèche, France; Dumont, Martine; Teo, Soo Hwang; Yip, Cheng Har; Hassan, Norhashimah; Vithana, Eranga Nishanthie; Kristensen, Vessela; Zheng, Wei; Deming-Halverson, Sandra; Shrubsole, Martha J.; Long, Jirong; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Kauppila, Saila; Andrulis, Irene L.; Knight, Julia A.; Glendon, Gord; Tchatchou, Sandrine; Devilee, Peter; Tollenaar, Robert A.E.M.; Seynaeve, Caroline; Van Asperen, Christi J.; García-Closas, Montserrat; Figueroa, Jonine; Lissowska, Jolanta; Brinton, Louise; Czene, Kamila; Darabi, Hatef; Eriksson, Mikael; Brand, Judith S.; Hooning, Maartje J.; Hollestelle, Antoinette; Van Den Ouweland, Ans M.W.; Jager, Agnes; Li, Jingmei; Liu, Jianjun; Humphreys, Keith; Shu, Xiao-Ou; Lu, Wei; Gao, Yu-Tang; Cai, Hui; Cross, Simon S.; Reed, Malcolm W. R.; Blot, William; Signorello, Lisa B.; Cai, Qiuyin; Pharoah, Paul D.P.; Perkins, Barbara; Shah, Mitul; Blows, Fiona M.; Kang, Daehee; Yoo, Keun-Young; Noh, Dong-Young; Hartman, Mikael; Miao, Hui; Chia, Kee Seng; Putti, Thomas Choudary; Hamann, Ute; Luccarini, Craig; Baynes, Caroline; Ahmed, Shahana; Maranian, Mel; Healey, Catherine S.; Jakubowska, Anna; Lubinski, Jan; Jaworska-Bieniek, Katarzyna; Durda, Katarzyna; Sangrajrang, Suleeporn; Gaborieau, Valerie; Brennan, Paul; Mckay, James; Slager, Susan; Toland, Amanda E.; Yannoukakos, Drakoulis; Shen, Chen-Yang; Hsiung, Chia-Ni; Wu, Pei-Ei; Ding, Shian-ling; Ashworth, Alan; Jones, Michael; Orr, Nick; Swerdlow, Anthony J; Tsimiklis, Helen; Makalic, Enes; Schmidt, Daniel F.; Bui, Quang M.; Chanock, Stephen J.; Hunter, David J.; Hein, Rebecca; Dahmen, Norbert; Beckmann, Lars; Aaltonen, Kirsimari; Muranen, Taru A.; Heikkinen, Tuomas; Irwanto, Astrid; Rahman, Nazneen; Turnbull, Clare A.; Waisfisz, Quinten; Meijers-Heijboer, Hanne E. J.; Adank, Muriel A.; Van Der Luijt, Rob B.; Hall, Per; Chenevix-Trench, Georgia; Dunning, Alison; Easton, Douglas F.; Cox, Angela

2015-01-01

Previous studies have suggested that polymorphisms in CASP8 on chromosome 2 are associated with breast cancer risk. To clarify the role of CASP8 in breast cancer susceptibility, we carried out dense genotyping of this region in the Breast Cancer Association Consortium (BCAC). Single-nucleotide polymorphisms (SNPs) spanning a 1 Mb region around CASP8 were genotyped in 46 450 breast cancer cases and 42 600 controls of European origin from 41 studies participating in the BCAC as part of a custom genotyping array experiment (iCOGS). Missing genotypes and SNPs were imputed and, after quality exclusions, 501 typed and 1232 imputed SNPs were included in logistic regression models adjusting for study and ancestry principal components. The SNPs retained in the final model were investigated further in data from nine genome-wide association studies (GWAS) comprising in total 10 052 case and 12 575 control subjects. The most significant association signal observed in European subjects was for the imputed intronic SNP rs1830298 in ALS2CR12 (telomeric to CASP8), with per allele odds ratio and 95% confidence interval [OR (95% confidence interval, CI)] for the minor allele of 1.05 (1.03–1.07), P = 1 × 10−5. Three additional independent signals from intronic SNPs were identified, in CASP8 (rs36043647), ALS2CR11 (rs59278883) and CFLAR (rs7558475). The association with rs1830298 was replicated in the imputed results from the combined GWAS (P = 3 × 10−6), yielding a combined OR (95% CI) of 1.06 (1.04–1.08), P = 1 × 10−9. Analyses of gene expression associations in peripheral blood and normal breast tissue indicate that CASP8 might be the target gene, suggesting a mechanism involving apoptosis. PMID:25168388

Identification and characterization of novel associations in the CASP8/ALS2CR12 region on chromosome 2 with breast cancer risk.

PubMed

Lin, Wei-Yu; Camp, Nicola J; Ghoussaini, Maya; Beesley, Jonathan; Michailidou, Kyriaki; Hopper, John L; Apicella, Carmel; Southey, Melissa C; Stone, Jennifer; Schmidt, Marjanka K; Broeks, Annegien; Van't Veer, Laura J; Th Rutgers, Emiel J; Muir, Kenneth; Lophatananon, Artitaya; Stewart-Brown, Sarah; Siriwanarangsan, Pornthep; Fasching, Peter A; Haeberle, Lothar; Ekici, Arif B; Beckmann, Matthias W; Peto, Julian; Dos-Santos-Silva, Isabel; Fletcher, Olivia; Johnson, Nichola; Bolla, Manjeet K; Wang, Qin; Dennis, Joe; Sawyer, Elinor J; Cheng, Timothy; Tomlinson, Ian; Kerin, Michael J; Miller, Nicola; Marmé, Frederik; Surowy, Harald M; Burwinkel, Barbara; Guénel, Pascal; Truong, Thérèse; Menegaux, Florence; Mulot, Claire; Bojesen, Stig E; Nordestgaard, Børge G; Nielsen, Sune F; Flyger, Henrik; Benitez, Javier; Zamora, M Pilar; Arias Perez, Jose Ignacio; Menéndez, Primitiva; González-Neira, Anna; Pita, Guillermo; Alonso, M Rosario; Alvarez, Nuria; Herrero, Daniel; Anton-Culver, Hoda; Brenner, Hermann; Dieffenbach, Aida Karina; Arndt, Volker; Stegmaier, Christa; Meindl, Alfons; Lichtner, Peter; Schmutzler, Rita K; Müller-Myhsok, Bertram; Brauch, Hiltrud; Brüning, Thomas; Ko, Yon-Dschun; Tessier, Daniel C; Vincent, Daniel; Bacot, Francois; Nevanlinna, Heli; Aittomäki, Kristiina; Blomqvist, Carl; Khan, Sofia; Matsuo, Keitaro; Ito, Hidemi; Iwata, Hiroji; Horio, Akiyo; Bogdanova, Natalia V; Antonenkova, Natalia N; Dörk, Thilo; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M; Wu, Anna H; Tseng, Chiu-Chen; Van Den Berg, David; Stram, Daniel O; Neven, Patrick; Wauters, Els; Wildiers, Hans; Lambrechts, Diether; Chang-Claude, Jenny; Rudolph, Anja; Seibold, Petra; Flesch-Janys, Dieter; Radice, Paolo; Peterlongo, Paolo; Manoukian, Siranoush; Bonanni, Bernardo; Couch, Fergus J; Wang, Xianshu; Vachon, Celine; Purrington, Kristen; Giles, Graham G; Milne, Roger L; Mclean, Catriona; Haiman, Christopher A; Henderson, Brian E; Schumacher, Fredrick; Le Marchand, Loic; Simard, Jacques; Goldberg, Mark S; Labrèche, France; Dumont, Martine; Teo, Soo Hwang; Yip, Cheng Har; Hassan, Norhashimah; Vithana, Eranga Nishanthie; Kristensen, Vessela; Zheng, Wei; Deming-Halverson, Sandra; Shrubsole, Martha J; Long, Jirong; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Kauppila, Saila; Andrulis, Irene L; Knight, Julia A; Glendon, Gord; Tchatchou, Sandrine; Devilee, Peter; Tollenaar, Robert A E M; Seynaeve, Caroline; Van Asperen, Christi J; García-Closas, Montserrat; Figueroa, Jonine; Lissowska, Jolanta; Brinton, Louise; Czene, Kamila; Darabi, Hatef; Eriksson, Mikael; Brand, Judith S; Hooning, Maartje J; Hollestelle, Antoinette; Van Den Ouweland, Ans M W; Jager, Agnes; Li, Jingmei; Liu, Jianjun; Humphreys, Keith; Shu, Xiao-Ou; Lu, Wei; Gao, Yu-Tang; Cai, Hui; Cross, Simon S; Reed, Malcolm W R; Blot, William; Signorello, Lisa B; Cai, Qiuyin; Pharoah, Paul D P; Perkins, Barbara; Shah, Mitul; Blows, Fiona M; Kang, Daehee; Yoo, Keun-Young; Noh, Dong-Young; Hartman, Mikael; Miao, Hui; Chia, Kee Seng; Putti, Thomas Choudary; Hamann, Ute; Luccarini, Craig; Baynes, Caroline; Ahmed, Shahana; Maranian, Mel; Healey, Catherine S; Jakubowska, Anna; Lubinski, Jan; Jaworska-Bieniek, Katarzyna; Durda, Katarzyna; Sangrajrang, Suleeporn; Gaborieau, Valerie; Brennan, Paul; Mckay, James; Slager, Susan; Toland, Amanda E; Yannoukakos, Drakoulis; Shen, Chen-Yang; Hsiung, Chia-Ni; Wu, Pei-Ei; Ding, Shian-Ling; Ashworth, Alan; Jones, Michael; Orr, Nick; Swerdlow, Anthony J; Tsimiklis, Helen; Makalic, Enes; Schmidt, Daniel F; Bui, Quang M; Chanock, Stephen J; Hunter, David J; Hein, Rebecca; Dahmen, Norbert; Beckmann, Lars; Aaltonen, Kirsimari; Muranen, Taru A; Heikkinen, Tuomas; Irwanto, Astrid; Rahman, Nazneen; Turnbull, Clare A; Waisfisz, Quinten; Meijers-Heijboer, Hanne E J; Adank, Muriel A; Van Der Luijt, Rob B; Hall, Per; Chenevix-Trench, Georgia; Dunning, Alison; Easton, Douglas F; Cox, Angela

2015-01-01

Previous studies have suggested that polymorphisms in CASP8 on chromosome 2 are associated with breast cancer risk. To clarify the role of CASP8 in breast cancer susceptibility, we carried out dense genotyping of this region in the Breast Cancer Association Consortium (BCAC). Single-nucleotide polymorphisms (SNPs) spanning a 1 Mb region around CASP8 were genotyped in 46 450 breast cancer cases and 42 600 controls of European origin from 41 studies participating in the BCAC as part of a custom genotyping array experiment (iCOGS). Missing genotypes and SNPs were imputed and, after quality exclusions, 501 typed and 1232 imputed SNPs were included in logistic regression models adjusting for study and ancestry principal components. The SNPs retained in the final model were investigated further in data from nine genome-wide association studies (GWAS) comprising in total 10 052 case and 12 575 control subjects. The most significant association signal observed in European subjects was for the imputed intronic SNP rs1830298 in ALS2CR12 (telomeric to CASP8), with per allele odds ratio and 95% confidence interval [OR (95% confidence interval, CI)] for the minor allele of 1.05 (1.03-1.07), P = 1 × 10(-5). Three additional independent signals from intronic SNPs were identified, in CASP8 (rs36043647), ALS2CR11 (rs59278883) and CFLAR (rs7558475). The association with rs1830298 was replicated in the imputed results from the combined GWAS (P = 3 × 10(-6)), yielding a combined OR (95% CI) of 1.06 (1.04-1.08), P = 1 × 10(-9). Analyses of gene expression associations in peripheral blood and normal breast tissue indicate that CASP8 might be the target gene, suggesting a mechanism involving apoptosis. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

46 CFR 34.17-20 - Discharge outlets-T/ALL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Discharge outlets-T/ALL. 34.17-20 Section 34.17-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-20 Discharge outlets—T/ALL. (a) Discharge outlets shall be of an approved...

46 CFR 34.17-20 - Discharge outlets-T/ALL.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Discharge outlets-T/ALL. 34.17-20 Section 34.17-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-20 Discharge outlets—T/ALL. (a) Discharge outlets shall be of an approved...

CASP - An intervention by community volunteers to reduce suicidal behaviour among refugees.

PubMed

Vijayakumar, Lakshmi; Mohanraj, Rani; Kumar, Shuba; Jeyaseelan, Visalakshi; Sriram, Savitha; Shanmugam, Madhumathi

2017-11-01

Refugees are at risk of psychiatric morbidity because of forced migration, traumatic events and resettlement in unfamiliar environments. Many live in low- and middle-income countries (LAMIC) under stressful conditions contributing to increased suicide risk. This study assessed the feasibility of regular contact and use of safety planning cards (CASP) by community volunteers (CVs) in reducing suicidal behaviour among Sri Lankan refugees residing in camps in Tamil Nadu, South India. A household survey was carried out on consenting adults in two refugee camps - one intervention and one control - randomly selected using lottery method. The primary outcome was reduction in suicidal behaviour. Experience of trauma during war and migration, depression, post-traumatic stress and alcohol use were documented. Individuals scoring >16 on Centre for Epidemiological Studies Depression (CESD) or >30 on Post-traumatic Stress Disorder (PTSD) or with active/passive suicidal ideation or a history of previous suicidal attempts were considered as high risk. CVs were trained to deliver CASP intervention to high-risk individuals. Change from baseline to follow-up was computed for intervention and control groups, and the difference between changes in suicide rates was compared using proportion test. In total, 639 refugees from intervention and 664 from control camps participated. Of the 288 high-risk refugees in intervention camp, 139 completed the intervention. In the control camp, 187 were categorised as high risk. Prevalence of suicide attempts was 6.1%. Following intervention, differences between sites in changes in combined suicide (attempted suicides and suicides) rates per 100,000 per year were 519 (95% confidence interval (CI): 136-902; p < .01). CASP, an intervention involving contact by CVs and use of safety planning cards, is feasible to implement and can reduce suicidal behaviour among refugees. Its replication in more settings will enhance validity.

CASP Position Paper: Specific Learning Disabilities and Patterns of Strengths and Weaknesses

ERIC Educational Resources Information Center

Christo, Catherine; Ponzuric, Jenny

2017-01-01

California Association of School Psychologists (CASP) adopted a Position Paper in March, 2014 intended to support school psychologists in California in electing to use a process known as Patterns of Strengths and Weaknesses (PSW) as one of three methods specified in IDEA 2014 and California Code of Regulations, Title 5, to identify students being…

RhoA/Rho-kinase triggers epithelial-mesenchymal transition in mesothelial cells and contributes to the pathogenesis of dialysis-related peritoneal fibrosis

PubMed Central

Wang, Qinglian; Yang, Xiaowei; Xu, Ying; Shen, Zhenwei; Cheng, Hongxia; Cheng, Fajuan; Liu, Xiang; Wang, Rong

2018-01-01

Peritoneal fibrosis (PF) with associated peritoneal dysfunction is almost invariably observed in long-term peritoneal dialysis (PD) patients. Advanced glycation end products (AGEs) are pro-oxidant compounds produced in excess during the metabolism of glucose and are present in high levels in standard PD solutions. The GTPase RhoA has been implicated in PF, but its specific role remains poorly understood. Here, we studied the effects of RhoA/Rho-kinase signaling in AGEs-induced epithelial-mesenchymal transition (EMT) in human peritoneal mesothelial cells (HPMCs), and evaluated morphological and molecular changes in a rat model of PD-related PF. Activation of RhoA/Rho-kinase and activating protein-1 (AP-1) was assessed in HPMCs using pull-down and electrophoretic mobility shift assays, respectively, while expression of transforming growth factor-β, fibronectin, α-smooth muscle actin, vimentin, N-cadherin, and E-cadherin expression was assessed using immunohistochemistry and western blot. AGEs exposure activated Rho/Rho-kinase in HPMCs and upregulated EMT-related genes via AP-1. These changes were prevented by the Rho-kinase inhibitors fasudil and Y-27632, and by the AP-1 inhibitor curcumin. Importantly, fasudil normalized histopathological and molecular alterations and preserved peritoneal function in rats. These data support the therapeutic potential of Rho-kinase inhibitors in PD-related PF. PMID:29581852

7 CFR 923.17 - Container.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Container. 923.17 Section 923.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... COUNTIES IN WASHINGTON Order Regulating Handling Definitions § 923.17 Container. Container means a box, bag...

7 CFR 922.17 - Container.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Container. 922.17 Section 922.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... IN WASHINGTON Order Regulating Handling Definitions § 922.17 Container. Container means a box, bag...

Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid

PubMed Central

2013-01-01

Background Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells. Results We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG. Conclusion Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity. PMID:23597229

Oral-resident natural Th17 cells and γδ T cells control opportunistic Candida albicans infections.

PubMed

Conti, Heather R; Peterson, Alanna C; Brane, Lucas; Huppler, Anna R; Hernández-Santos, Nydiaris; Whibley, Natasha; Garg, Abhishek V; Simpson-Abelson, Michelle R; Gibson, Gregory A; Mamo, Anna J; Osborne, Lisa C; Bishu, Shrinivas; Ghilardi, Nico; Siebenlist, Ulrich; Watkins, Simon C; Artis, David; McGeachy, Mandy J; Gaffen, Sarah L

2014-09-22

Oropharyngeal candidiasis (OPC) is an opportunistic fungal infection caused by Candida albicans. OPC is frequent in HIV/AIDS, implicating adaptive immunity. Mice are naive to Candida, yet IL-17 is induced within 24 h of infection, and susceptibility is strongly dependent on IL-17R signaling. We sought to identify the source of IL-17 during the early innate response to candidiasis. We show that innate responses to Candida require an intact TCR, as SCID, IL-7Rα(-/-), and Rag1(-/-) mice were susceptible to OPC, and blockade of TCR signaling by cyclosporine induced susceptibility. Using fate-tracking IL-17 reporter mice, we found that IL-17 is produced within 1-2 d by tongue-resident populations of γδ T cells and CD3(+)CD4(+)CD44(hi)TCRβ(+)CCR6(+) natural Th17 (nTh17) cells, but not by TCR-deficient innate lymphoid cells (ILCs) or NK cells. These cells function redundantly, as TCR-β(-/-) and TCR-δ(-/-) mice were both resistant to OPC. Whereas γδ T cells were previously shown to produce IL-17 during dermal candidiasis and are known to mediate host defense at mucosal surfaces, nTh17 cells are poorly understood. The oral nTh17 population expanded rapidly after OPC, exhibited high TCR-β clonal diversity, and was absent in Rag1(-/-), IL-7Rα(-/-), and germ-free mice. These findings indicate that nTh17 and γδ T cells, but not ILCs, are key mucosal sentinels that control oral pathogens. © 2014 Conti et al.

Interventional loopless antenna at 7 T.

PubMed

Ertürk, Mehmet Arcan; El-Sharkawy, Abdel-Monem M; Bottomley, Paul A

2012-09-01

The loopless antenna magnetic resonance imaging detector is comprised of a tuned coaxial cable with an extended central conductor that can be fabricated at submillimeter diameters for interventional use in guidewires, catheters, or needles. Prior work up to 4.7 T suggests a near-quadratic gain in signal-to-noise ratio with field strength and safe operation at 3 T. Here, for the first time, the signal-to-noise ratio performance and radiofrequency safety of the loopless antenna are investigated both theoretically, using the electromagnetic method-of-moments, and experimentally in a standard 7 T human scanner. The results are compared with equivalent 3 T devices. An absolute signal-to-noise ratio gain of 5.7 ± 1.5-fold was realized at 7 T vs. 3 T: more than 20-fold higher than at 1.5 T. The effective field-of-view area also increased approximately 10-fold compared with 3 T. Testing in a saline gel phantom suggested that safe operation is possible with maximum local 1-g average specific absorption rates of <12 W kg(-1) and temperature increases of <1.9°C, normalized to a 4 W kg(-1) radiofrequency field exposure at 7 T. The antenna did not affect the power applied to the scanner's transmit coil. The signal-to-noise ratio gain enabled magnetic resonance imaging microscopy at 40-50 μm resolution in diseased human arterial specimens, offering the potential of high-resolution large-field-of-view or endoscopic magnetic resonance imaging for targeted intervention in focal disease. Copyright © 2011 Wiley Periodicals, Inc.

7 CFR 947.17 - Export.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Export. 947.17 Section 947.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and... Definitions § 947.17 Export. Export means shipment of potatoes beyond the boundaries of continental United...

7 CFR 948.17 - Export.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Export. 948.17 Section 948.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and... Regulating Handling Definitions § 948.17 Export. Export means the shipment of potatoes to any destination...

Propulsion Control and Health Management (PCHM) Technology for Flight Test on the C-17 T-1 Aircraft

NASA Technical Reports Server (NTRS)

Simon, Donald L.; Garg, Sanjay; Venti, Michael

2004-01-01

The C-I 7 T-l Globemaster III is an Air Force flight research vehicle located at Edwards Air Force Base. NASA Dryden and the C-17 System Program Office have entered into a Memorandum of Agreement to permit NASA the use of the C-I 7 T-I to conduct flight research on a mutually coordinated schedule. The C-17 Propulsion Control and Health Management (PCHM) Working Group was formed in order to foster discussion and coordinate planning amongst the various government agencies conducting PCHM research with a potential need for flight testing, and to communicate to the PCHM community the capabilities of the C-17 T-l aircraft to support such flight testing. This paper documents the output of this Working Group, including a summary of the candidate PCHM technologies identified and their associated benefits relative to NASA goals and objectives.

Inflammatory T helper 17 cells promote depression-like behavior in mice.

PubMed

Beurel, Eléonore; Harrington, Laurie E; Jope, Richard S

2013-04-01

Recognition of substantial immune-neural interactions is revising dogmas about their insular actions and revealing that immune-neural interactions can substantially impact central nervous system functions. The inflammatory cytokine interleukin-6 promotes susceptibility to depression and drives production of inflammatory T helper 17 (Th17) T cells, raising the hypothesis that in mouse models, Th17 cells promote susceptibility to depression-like behaviors. Behavioral characteristics were measured in male mice administered Th17 cells, CD4(+) cells, or vehicle and in retinoid-related orphan receptor-γT (RORγT)(+/GFP) mice or male mice treated with RORγT inhibitor or anti-interleukin-17A antibodies. Mouse brain Th17 cells were elevated by learned helplessness and chronic restraint stress, two common depression-like models. Th17 cell administration promoted learned helplessness in 89% of mice in a paradigm where no vehicle-treated mice developed learned helplessness, and impaired novelty suppressed feeding and social interaction behaviors. Mice deficient in the RORγT transcription factor necessary for Th17 cell production exhibited resistance to learned helplessness, identifying modulation of RORγT as a potential intervention. Treatment with the RORγT inhibitor SR1001, or anti-interleukin-17A antibodies to abrogate Th17 cell function, reduced Th17-dependent learned helplessness. These findings indicate that Th17 cells are increased in the brain during depression-like states, promote depression-like behaviors in mice, and specifically inhibiting the production or function of Th17 cells reduces vulnerability to depression-like behavior, suggesting antidepressant effects may be attained by targeting Th17 cells. Copyright © 2013 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

7 CFR 958.17 - Container.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Container. 958.17 Section 958.17 Agriculture... COUNTIES IN IDAHO, AND MALHEUR COUNTY, OREGON Order Regulating Handling Definitions § 958.17 Container. Container means a sack, box, bag, crate, hamper, basket, carton, package, or any other type of receptacle...

23 CFR 1.7 - Urban area boundaries.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 23 Highways 1 2014-04-01 2014-04-01 false Urban area boundaries. 1.7 Section 1.7 Highways FEDERAL HIGHWAY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION GENERAL MANAGEMENT AND ADMINISTRATION GENERAL § 1.7 Urban area boundaries. Boundaries of an urban area shall be submitted by the State highway department...

23 CFR 1.7 - Urban area boundaries.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 23 Highways 1 2013-04-01 2013-04-01 false Urban area boundaries. 1.7 Section 1.7 Highways FEDERAL HIGHWAY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION GENERAL MANAGEMENT AND ADMINISTRATION GENERAL § 1.7 Urban area boundaries. Boundaries of an urban area shall be submitted by the State highway department...

23 CFR 1.7 - Urban area boundaries.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 23 Highways 1 2011-04-01 2011-04-01 false Urban area boundaries. 1.7 Section 1.7 Highways FEDERAL HIGHWAY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION GENERAL MANAGEMENT AND ADMINISTRATION GENERAL § 1.7 Urban area boundaries. Boundaries of an urban area shall be submitted by the State highway department...

23 CFR 1.7 - Urban area boundaries.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 23 Highways 1 2010-04-01 2010-04-01 false Urban area boundaries. 1.7 Section 1.7 Highways FEDERAL HIGHWAY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION GENERAL MANAGEMENT AND ADMINISTRATION GENERAL § 1.7 Urban area boundaries. Boundaries of an urban area shall be submitted by the State highway department...

Isolation and characterization of a 66-kDa protein from rat liver plasma membrane with RhoA-stimulated phospholipase D activity.

PubMed

Dunkirk, Shawn G; Wallert, Mark A; Baumgartner, Matt L; Provost, Joseph J

2002-02-01

A 66-kDa molecular weight protein with phospholipase D activity was solubilized and partially purified from rat liver plasma membrane. The activity and regulation of this phospholipase D have been characterized. Immunoblot analyses indicated that the enzyme was distinct from hPLD1 and PLD2, but was recognized by an antibody to the 12 terminal amino acids of PLD1. PLD activity was stimulated by 1-100 microM Ca(2+) and Mg(2+) and displayed a pH optimum of 7.5. Activity was inhibited by both saturated and unsaturated fatty acids. This PLD was activated in an ATP-independent manner by the PKC isozymes alpha and betaII but not activated by other PKC isozymes. It was also stimulated by the small G-proteins RhoA and ARF. RhoA stimulated the greatest activation, followed by ARF and PKC(alpha). This enzyme was further activated in a synergistic manner when combinations of PKC(alpha) and RhoA or ARF were used. This enzyme displayed a greater response activation by RhoA than to activation by ARF. While a potential breakdown product of PLD1, activation by RhoA indicates that the PLD characterized here is distinct from the other PLDs cloned or isolated to date. Copyright 2002 Elsevier Science (USA).

7 CFR 225.17 - Procurement standards.

Code of Federal Regulations, 2010 CFR

2010-01-01

...) Procurements by public sponsors comply with applicable State or local laws and the standards set forth in 7 CFR part 3016; and (2) Procurements by private nonprofit sponsors comply with standards set forth in 7 CFR... 7 Agriculture 4 2010-01-01 2010-01-01 false Procurement standards. 225.17 Section 225.17...

An activating mutant of Rac1 that fails to interact with Rho GDP-dissociation inhibitor stimulates membrane ruffling in mammalian cells.

PubMed Central

Gandhi, Payal N; Gibson, Richard M; Tong, Xiaofeng; Miyoshi, Jun; Takai, Yoshimi; Konieczkowski, Martha; Sedor, John R; Wilson-Delfosse, Amy L

2004-01-01

Rac1, a member of the Rho family of small GTP-binding proteins, is involved in the regulation of the actin cytoskeleton via activation of lamellipodia and membrane ruffle formation. RhoGDI (Rho-family-specific GDP-dissociation inhibitor) forms a complex with Rho proteins in the cytosol of mammalian cells. It not only regulates guanine nucleotide binding to Rho proteins, but may also function as a molecular shuttle to carry Rho proteins from an inactive cytosolic pool to the membrane for activation. These studies tested if RhoGDI is necessary for the translocation of Rac1 from the cytosol to the plasma membrane for the formation of membrane ruffles. We describe a novel mutant of Rac1, R66E (Arg66-->Glu), that fails to bind RhoGDI. This RhoGDI-binding-defective mutation is combined with a Rac1-activating mutation G12V, resulting in a double-mutant [Rac1(G12V/R66E)] that fails to interact with RhoGDI in COS-7 cells, but remains constitutively activated. This double mutant stimulates membrane ruffling to a similar extent as that observed after epidermal growth factor treatment of non-transfected cells. To confirm that Rac1 can signal ruffle formation in the absence of interaction with RhoGDI, Rac1(G12V) was overexpressed in cultured mesangial cells derived from a RhoGDI knockout mouse. Rac1-mediated membrane ruffling was indistinguishable between the RhoGDI(-/-) and RhoGDI(+/+) cell lines. In both the COS-7 and cultured mesangial cells, Rac1(G12V) and Rac1(G12V/R66E) co-localize with membrane ruffles. These findings suggest that interaction with RhoGDI is not essential in the mechanism by which Rac1 translocates to the plasma membrane to stimulate ruffle formation. PMID:14629200

45 CFR 17.7 - Retractions or corrections.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false Retractions or corrections. 17.7 Section 17.7... INFORMATION TO NEWS MEDIA § 17.7 Retractions or corrections. Where the Assistant Secretary for Public Affairs... person named therein requests a retraction or correction, the Department shall issue a retraction or...

45 CFR 17.7 - Retractions or corrections.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 1 2014-10-01 2014-10-01 false Retractions or corrections. 17.7 Section 17.7... INFORMATION TO NEWS MEDIA § 17.7 Retractions or corrections. Where the Assistant Secretary for Public Affairs... person named therein requests a retraction or correction, the Department shall issue a retraction or...

45 CFR 17.7 - Retractions or corrections.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false Retractions or corrections. 17.7 Section 17.7... INFORMATION TO NEWS MEDIA § 17.7 Retractions or corrections. Where the Assistant Secretary for Public Affairs... person named therein requests a retraction or correction, the Department shall issue a retraction or...

45 CFR 17.7 - Retractions or corrections.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 45 Public Welfare 1 2011-10-01 2011-10-01 false Retractions or corrections. 17.7 Section 17.7... INFORMATION TO NEWS MEDIA § 17.7 Retractions or corrections. Where the Assistant Secretary for Public Affairs... person named therein requests a retraction or correction, the Department shall issue a retraction or...

45 CFR 17.7 - Retractions or corrections.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 1 2013-10-01 2013-10-01 false Retractions or corrections. 17.7 Section 17.7... INFORMATION TO NEWS MEDIA § 17.7 Retractions or corrections. Where the Assistant Secretary for Public Affairs... person named therein requests a retraction or correction, the Department shall issue a retraction or...

7 CFR 331.17 - Records.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 5 2011-01-01 2011-01-01 false Records. 331.17 Section 331.17 Agriculture Regulations... OF AGRICULTURE POSSESSION, USE, AND TRANSFER OF SELECT AGENTS AND TOXINS § 331.17 Records. (a) An individual or entity required to register under this part must maintain complete records relating to the...

7 CFR 250.17 - Reports.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 4 2010-01-01 2010-01-01 false Reports. 250.17 Section 250.17 Agriculture Regulations... TERRITORIES AND POSSESSIONS AND AREAS UNDER ITS JURISDICTION General Operating Provisions § 250.17 Reports. (a) Inventory reports and receipt of donated foods. Distributing agencies shall complete and submit to the FNSRO...

7 CFR 250.17 - Reports.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 4 2011-01-01 2011-01-01 false Reports. 250.17 Section 250.17 Agriculture Regulations... TERRITORIES AND POSSESSIONS AND AREAS UNDER ITS JURISDICTION General Operating Provisions § 250.17 Reports. (a) Inventory reports and receipt of donated foods. Distributing agencies shall complete and submit to the FNSRO...

7 CFR 250.17 - Reports.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 4 2014-01-01 2014-01-01 false Reports. 250.17 Section 250.17 Agriculture Regulations... TERRITORIES AND POSSESSIONS AND AREAS UNDER ITS JURISDICTION General Operating Provisions § 250.17 Reports. (a) Inventory reports and receipt of donated foods. Distributing agencies shall complete and submit to the FNSRO...

Iron oxide nanoparticles attenuate T helper 17 cell responses in vitro and in vivo.

PubMed

Hsiao, Yai-Ping; Shen, Chien-Chang; Huang, Chung-Hsiung; Lin, Yu-Chin; Jan, Tong-Rong

2018-05-01

Iron oxide nanoparticles (IONPs) have been shown to attenuate T helper (Th)1 and Th2 cell-mediated immunity in ovalbumin (OVA)-sensitized mice. The objective of this study is to investigate the effects of IONPs on the immune responses of Th17 cells, a subset of T cells involved in various inflammatory pathologies. For in vivo study, a murine model of delayed-type hypersensitivity (DTH) was employed. BALB/c mice received a single dose of IONPs (0.2-10 mg iron/kg) via the tail vein 1 h prior to ovalbumin (OVA) sensitization. Their footpads were subcutaneously challenged with OVA to induce DTH reactions. The expression of Th17 cell-related molecules in inflamed footpads were examined by immunohistochemistry. For in vitro study, OVA-primed splenocytes were directly exposed to IONPs (1-100 μg iron/mL), and then re-stimulated with OVA in culture. The expression of Th17 cell-related molecules were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. IONP administration attenuated the number of interleukin (IL)-6, IL-17, the transcription factor ROR-γ, and chemokine receptor 6 positive cells in OVA-challenged footpads, whereas the number of transforming growth factor-β, IL-23 and chemokine (C-C motif) ligand 20 positive cells was not altered. Direct exposure of OVA-primed splenocytes to IONPs suppressed the production of IL-6 and IL-17, and the mRNA expression of IL-17 and ROR-γt. These data indicate that exposure to IONPs attenuates Th17 cell responses in vivo and in vitro. Copyright © 2018. Published by Elsevier B.V.

Structure and extent of the giant molecular cloud near M17

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

Elmegreen, B.G.; Lada, C.J.; Dickinson, D.F.

1979-06-01

Carbon monoxide emission at ..nu../sub LSR/ = 20 +- 2 km s/sup -1/ is found to extend 4/sup 0/ (approx.170 pc) southwest of M17, and is studied in an attempt to understand the internal structure and dynamics of a giant molecular cloud complex. The region contains two primary clouds. The first has at least 2 x 10/sup 5/ M/sub sun/ of molecular gas and extends for 1./sup 0/8 (72 pc) parallel to, but below the galactic plane southwest of M17. The second, located above the plane approximately 2./sup 0/5 southwest of M17, is about 1./sup 0/7 in extent, but containsmore » considerably less molecular mass (> or approx. =3 x 10/sup 4/ M/sub sun/). Between these two clouds is a 1/sup 0/ long region of relatively low intensity, clumpy CO emission which appears to bridge the two main clouds. The molecular mass within this bridge is estimated to be 2 x 10/sup 4/ M/sub sun/. The cloud associated with M17 is itself divided into four discrete fragments of approximately equal mass (4 x 10/sup 4/ M/sub sun/). The /sup 12/CO and /sup 13/CO line widths are higher in these four fragments than they are between the fragments. OB star formation is active only in the northeastern two of these fragments. The /sup 13/CO line widths in the discrete fragments satisfy the virial theorem for the derived masses. (b) The /sup 13/CO velocity structure in the large complex containing M17 shows a gradual change from regularity in the northeast to irregularity and occasionally multipeaked profiles in the southwest. This change corresponds to a gradient in the degree of compactness and intensity of star formation in the four fragments. A massive (10/sup 5/ M/sub sun/) molecular cloud complex associated with M16, 2/sup 0/ north of M17, and the two clouds southwest of M17, form a pattern of equally spaced star-forming clouds whose positions alternate above and below the galactic plane. Patchy CO emission is found between these three objects. The entire region of molecular emission is approx.250 pc

Use of Synthetic Isoprenoids to Target Protein Prenylation and Rho GTPases in Breast Cancer Invasion

PubMed Central

Chen, Min; Knifley, Teresa; Subramanian, Thangaiah; Spielmann, H. Peter; O’Connor, Kathleen L.

2014-01-01

Dysregulation of Ras and Rho family small GTPases drives the invasion and metastasis of multiple cancers. For their biological functions, these GTPases require proper subcellular localization to cellular membranes, which is regulated by a series of post-translational modifications that result in either farnesylation or geranylgeranylation of the C-terminal CAAX motif. This concept provided the rationale for targeting farnesyltransferase (FTase) and geranylgeranyltransferases (GGTase) for cancer treatment. However, the resulting prenyl transferase inhibitors have not performed well in the clinic due to issues with alternative prenylation and toxicity. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called Prenyl Function Inhibitors (PFIs), which are farnesol or geranyl-geraniol analogs that act as alternate substrates for FTase or GGTase. Here, we test the ability of our lead PFIs, anilinogeraniol (AGOH) and anilinofarnesol (AFOH), to block the invasion of breast cancer cells. We found that AGOH treatment effectively decreased invasion of MDA-MB-231 cells in a two-dimensional (2D) invasion assay at 100 µM while it blocked invasive growth in three-dimensional (3D) culture model at as little as 20 µM. Notably, the effect of AGOH on 3D invasive growth was phenocopied by electroporation of cells with C3 exotransferase. To determine if RhoA and RhoC were direct targets of AGOH, we performed Rho activity assays in MDA-MB-231 and MDA-MB-468 cells and found that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation. Notably, the geranylgeraniol analog AFOH was more potent than AGOH in inhibiting RhoA and RhoC activation and invasive growth. Interestingly, neither AGOH nor AFOH impacted 3D growth of MCF10A cells. Collectively, this study demonstrates that AGOH and AFOH dramatically inhibit breast cancer invasion, at least in part by blocking Rho function, thus, suggesting that targeting prenylation by using

SUMO regulates proteasome-dependent degradation of FLASH/Casp8AP2

PubMed Central

Vennemann, Astrid; Hofmann, Thomas G.

2013-01-01

FLASH/Casp8AP2 is a huge multifunctional protein involved in multiple cellular processes, reaching from death receptor signaling to regulation of histone gene transcription and histone mRNA processing. Previous work has shown that FLASH localizes to Cajal bodies and promyelocytic leukemia (PML) bodies. However, the function of its nuclear body association remains unclear. Here we demonstrate that murine FLASH is covalently modified by SUMO at Lys residue 1792. Interestingly, ectopic expression of SUMO results in proteasome-dependent degradation of FLASH. A point mutant of FLASH with a mutated SUMO acceptor lysine residue, FLASHK1792R, is resistant to SUMO-induced degradation. Finally, we show that arsenic trioxide, a drug known to potentiate SUMO modification and degradation of PML, triggers recruitment of FLASH to PML bodies and concomitant loss of FLASH protein. Our data suggest that SUMO targets FLASH for proteasome-dependent degradation, which is associated with recruitment of FLASH to PML bodies. PMID:23673342

7 CFR 1794.17 - Mitigation.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 12 2013-01-01 2013-01-01 false Mitigation. 1794.17 Section 1794.17 Agriculture... § 1794.17 Mitigation. (a) General. In addition to complying with the requirements of 40 CFR 1502.14(f... (FONSI) and the Record of Decision (ROD). (b) Water and waste program. (1) Mitigation measures which...

7 CFR 1794.17 - Mitigation.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 12 2014-01-01 2013-01-01 true Mitigation. 1794.17 Section 1794.17 Agriculture... § 1794.17 Mitigation. (a) General. In addition to complying with the requirements of 40 CFR 1502.14(f... (FONSI) and the Record of Decision (ROD). (b) Water and waste program. (1) Mitigation measures which...

7 CFR 1794.17 - Mitigation.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 12 2011-01-01 2011-01-01 false Mitigation. 1794.17 Section 1794.17 Agriculture... § 1794.17 Mitigation. (a) General. In addition to complying with the requirements of 40 CFR 1502.14(f... (FONSI) and the Record of Decision (ROD). (b) Water and waste program. (1) Mitigation measures which...

7 CFR 1794.17 - Mitigation.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 12 2012-01-01 2012-01-01 false Mitigation. 1794.17 Section 1794.17 Agriculture... § 1794.17 Mitigation. (a) General. In addition to complying with the requirements of 40 CFR 1502.14(f... (FONSI) and the Record of Decision (ROD). (b) Water and waste program. (1) Mitigation measures which...

Transiting exoplanets from the CoRoT space mission. XVII. The hot Jupiter CoRoT-17b: a very old planet

NASA Astrophysics Data System (ADS)

Csizmadia, Sz.; Moutou, C.; Deleuil, M.; Cabrera, J.; Fridlund, M.; Gandolfi, D.; Aigrain, S.; Alonso, R.; Almenara, J.-M.; Auvergne, M.; Baglin, A.; Barge, P.; Bonomo, A. S.; Bordé, P.; Bouchy, F.; Bruntt, H.; Carone, L.; Carpano, S.; Cavarroc, C.; Cochran, W.; Deeg, H. J.; Díaz, R. F.; Dvorak, R.; Endl, M.; Erikson, A.; Ferraz-Mello, S.; Fruth, Th.; Gazzano, J.-C.; Gillon, M.; Guenther, E. W.; Guillot, T.; Hatzes, A.; Havel, M.; Hébrard, G.; Jehin, E.; Jorda, L.; Léger, A.; Llebaria, A.; Lammer, H.; Lovis, C.; MacQueen, P. J.; Mazeh, T.; Ollivier, M.; Pätzold, M.; Queloz, D.; Rauer, H.; Rouan, D.; Santerne, A.; Schneider, J.; Tingley, B.; Titz-Weider, R.; Wuchterl, G.

2011-07-01

We report on the discovery of a hot Jupiter-type exoplanet, CoRoT-17b, detected by the CoRoT satellite. It has a mass of 2.43 ± 0.30 MJup and a radius of 1.02 ± 0.07 RJup, while its mean density is 2.82 ± 0.38 g/cm3. CoRoT-17b is in a circular orbit with a period of 3.7681 ± 0.0003 days. The host star is an old (10.7 ± 1.0 Gyr) main-sequence star, which makes it an intriguing object for planetary evolution studies. The planet's internal composition is not well constrained and can range from pure H/He to one that can contain ~380 earth masses of heavier elements. The CoRoT space mission, launched on December 27th 2006, has been developed and is operated by CNES, with the contribution of Austria, Belgium, Brazil, ESA (RSSD and Science Programme), Germany and Spain. Part of the observations were obtained at the Canada-France-Hawaii Telescope (CFHT) which is operated by the National Research Council of Canada, the Institut National des Sciences de l'Univers of the Centre National de la Recherche Scientifique of France, and the University of Hawaii. Based on observations made with HARPS spectrograph on the 3.6-m European Organisation for Astronomical Research in the Southern Hemisphere telescope at La Silla Observatory, Chile (ESO program 184.C-0639). Based on observations made with the IAC80 telescope operated on the island of Tenerife by the Instituto de Astrofísica de Canarias in the Spanish Observatorio del Teide. Part of the data presented herein were obtained at the W.M. Keck Observatory, which is operated as a scientific partnership among the California Institute of Technology, the University of California and the National Aeronautics and Space Administration. The Observatory was made possible by the generous financial support of the W.M. Keck Foundation.

Observation of Effectiveness of Clinical Sterilization by CASP-80A Low-Temperature Plasma Sterilizer

NASA Astrophysics Data System (ADS)

Li, Si; Zhang, Yangde; Liu, Weidong

2006-09-01

The influence on the effectiveness of sterilization by low-temperature plasma sterilizer CASP-80A was investigated so as to provide a theoretical basis for reducing medical costs and achieving ideal sterilization effectiveness. To conduct the on-site simulation test, a clinical material sterilization test and a test of the influence of organic substance were conducted, the former by using the representative of Bacillus Stearothermophilus, preparing the bacteria-contaminated carrier through polytetrafluoroethylene (PTFE) simulated hose endoscopes, and the latter by using calf serum as the influence factor of the organic substance. The results show that the CASP-80A low-temperature plasma sterilizer could achieve effective sterilization by either the short-cycle or the long-cycle sterilization method depending on different materials, apparatus, and extent of contamination. The organic substances could influence the effectiveness of sterilization by the low-temperature plasma (H2O2) sterilizer.

Myc requires RhoA/SRF to reprogram glutamine metabolism.

PubMed

Haikala, Heidi M; Marques, Elsa; Turunen, Mikko; Klefström, Juha

2018-05-04

RhoA regulates actin cytoskeleton but recent evidence suggest a role for this conserved Rho GTPase also in other cellular processes, including transcriptional control of cell proliferation and survival. Interestingy, loss of RhoA is synthetic lethal with oncogenic Myc, a master transcription factor that turns on anabolic metabolism to promote cell growth in many cancers. We show evidence indicating that the synthetic lethal interaction between RhoA loss and Myc arises from deficiency in glutamine utilization, resulting from impaired co-regulation of glutaminase expression and anaplerosis by Myc and RhoA - serum response factor (SRF) pathway. The results suggest metabolic coordination between Myc and RhoA/SRF in sustaining cancer cell viability and indicate RhoA/SRF as a potential vulnerability in cancer cells for therapeutic targeting.

RhoA/rho kinase signaling reduces connexin43 expression in high glucose-treated glomerular mesangial cells with zonula occludens-1 involvement

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

Xie, Xi; Department of Pharmaceutical Engineering, Ocean College, Hainan University, Haikou 570228; Chen, Cheng

RhoA/Rho kinase (ROCK) signaling has been suggested to be involved in diabetic nephropathy (DN) pathogenesis. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. Both of them have been found to regulate nuclear factor kappa-B (NF-κB) activation in high glucose-treated glomerular mesangial cells (GMCs). The aim of this study was to investigate the relationship between RhoA/ROCK signaling and Cx43 in the DN pathogenesis. We found that upregulation of Cx43 expression inhibited NF-κB p65 nuclear translocation induced by RhoA/ROCK signaling in GMCs. Inhibition of RhoA/ROCK signaling attenuated the high glucose-induced decrease in Cx43. F-actin accumulation and anmore » enhanced interaction between zonula occludens-1 (ZO-1) and Cx43 were observed in high glucose-treated GMCs. ZO-1 depletion or disruption of F-actin formation also inhibited the reduction in Cx43 protein levels induced by high glucose. In conclusion, activated RhoA/ROCK signaling induces Cx43 degradation in GMCs cultured in high glucose, depending on F-actin regulation. Increased F-actin induced by RhoA/ROCK signaling promotes the association between ZO-1 and Cx43, which possibly triggered Cx43 endocytosis, a mechanism of NF-κB activation in high glucose-treated GMCs. - Highlights: • RhoA/ROCK signaling induces Cx43 degradation in GMCs. • F-actin and ZO-1 have functions in the regulation of Cx43 by RhoA/ROCK signaling. • We reveal the relationship between RhoA/ROCK and Cx43 in the activation of NF-κB.« less

Oral-resident natural Th17 cells and γδ T cells control opportunistic Candida albicans infections

PubMed Central

Conti, Heather R.; Peterson, Alanna C.; Brane, Lucas; Huppler, Anna R.; Hernández-Santos, Nydiaris; Whibley, Natasha; Garg, Abhishek V.; Simpson-Abelson, Michelle R.; Gibson, Gregory A.; Mamo, Anna J.; Osborne, Lisa C.; Bishu, Shrinivas; Ghilardi, Nico; Siebenlist, Ulrich; Watkins, Simon C.; Artis, David; McGeachy, Mandy J.

2014-01-01

Oropharyngeal candidiasis (OPC) is an opportunistic fungal infection caused by Candida albicans. OPC is frequent in HIV/AIDS, implicating adaptive immunity. Mice are naive to Candida, yet IL-17 is induced within 24 h of infection, and susceptibility is strongly dependent on IL-17R signaling. We sought to identify the source of IL-17 during the early innate response to candidiasis. We show that innate responses to Candida require an intact TCR, as SCID, IL-7Rα−/−, and Rag1−/− mice were susceptible to OPC, and blockade of TCR signaling by cyclosporine induced susceptibility. Using fate-tracking IL-17 reporter mice, we found that IL-17 is produced within 1–2 d by tongue-resident populations of γδ T cells and CD3+CD4+CD44hiTCRβ+CCR6+ natural Th17 (nTh17) cells, but not by TCR-deficient innate lymphoid cells (ILCs) or NK cells. These cells function redundantly, as TCR-β−/− and TCR-δ−/− mice were both resistant to OPC. Whereas γδ T cells were previously shown to produce IL-17 during dermal candidiasis and are known to mediate host defense at mucosal surfaces, nTh17 cells are poorly understood. The oral nTh17 population expanded rapidly after OPC, exhibited high TCR-β clonal diversity, and was absent in Rag1−/−, IL-7Rα−/−, and germ-free mice. These findings indicate that nTh17 and γδ T cells, but not ILCs, are key mucosal sentinels that control oral pathogens. PMID:25200028

17 CFR 240.17Ad-7 - Record retention.

Code of Federal Regulations, 2010 CFR

2010-04-01

... electronic or micrographic media and may be preserved in those formats for the time required by § 240.17Ad-7... substitute for the hard copy records required to be maintained pursuant to § 240.17Ad-6. (1) For purposes of..., in escrow with an independent third party and keep current a copy of the physical and logical format...

Subunit profiling and functional characteristics of acetylcholine receptors in GT1-7 cells.

PubMed

Arai, Yuki; Ishii, Hirotaka; Kobayashi, Makito; Ozawa, Hitoshi

2017-03-01

GnRH neurons form a final common pathway for the central regulation of reproduction. Although the involvement of acetylcholine in GnRH secretion has been reported, direct effects of acetylcholine and expression profiles of acetylcholine receptors (AChRs) still remain to be studied. Using immortalized GnRH neurons (GT1-7 cells), we analyzed molecular expression and functionality of AChRs. Expression of the mRNAs were identified in the order α7 > β2 = β1 ≧ α4 ≧ α5 = β4 = δ > α3 for nicotinic acetylcholine receptor (nAChR) subunits and m4 > m2 for muscarinic acetylcholine receptor (mAChR) subtypes. Furthermore, this study revealed that α7 nAChRs contributed to Ca 2+ influx and GnRH release and that m2 and m4 mAChRs inhibited forskolin-induced cAMP production and isobutylmethylxanthine-induced GnRH secretion. These findings demonstrate the molecular profiles of AChRs, which directly contribute to GnRH secretion in GT1-7 cells, and provide one possible regulatory action of acetylcholine in GnRH neurons.

Isochoric p-{rho}-T measurements on 1,1-difluoroethane (R152a) from 158 to 400 K and 1,1,1-trifluoroethane (R143a) from 166 to 400 K at pressures to 35 MPa

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

Magee, J.W.

1998-09-01

The p-{rho}-T relationships have been measured for 1,1-difluoroethane (R152a) and 1,1,1-trifluoroethane (R143a) by an isochoric method with gravimetric determinations of the amount of substance. Temperatures ranged from 158 to 400K for R152a and from 166 to 400 K for R143a, while pressures were up to 35 MPa. Measurements were conducted on compressed liquid samples. Determinations of saturated liquid densities were made by extrapolating each isochore to the vapor pressure, and determining the temperature and density at the intersection. Published p-{rho}-T data are in good agreement with this study. For the p-{rho}-T apparatus, the uncertainty of the temperature is {+-}0.03 K,more » and for pressure it is {+-}0.01% at p > 3 MPa and {+-}0.05% at p > 3 MPa and {+-}0.05% at p < 3MPa. The principal source of uncertainty is the cell volume ({approximately}28.5 cm{sup 3}), which has a standard uncertainty of {+-}0.003 cm{sup 3}. When all components of experimental uncertainty are considered, the expanded relative uncertainty (with a coverage factor k = 2 and thus a two-standard deviation estimate) of the density measurement is estimated to be {+-}0.05%.« less

A tip-localized RhoGAP controls cell polarity by globally inhibiting Rho GTPase at the cell apex.

PubMed

Hwang, Jae-Ung; Vernoud, Vanessa; Szumlanski, Amy; Nielsen, Erik; Yang, Zhenbiao

2008-12-23

Highly elongated eukaryotic cells (e.g., neuronal axons, fungal hyphae, and pollen tubes) are generated through continuous apically restricted growth (tip growth), which universally requires tip-localized Rho GTPases. We used the oscillating pollen tube as a model system to determine the function and regulation of Rho GTPases in tip growth. Our previous work showed that the spatiotemporal dynamics of the apical cap of the activated Rho-like GTPase from Plant 1 (ROP1) are critical for tip growth in pollen tubes. However, the underlying mechanism for the generation and maintenance of this dynamic apical cap is poorly understood. A screen for mutations that enhance ROP1-overexpression-induced depolarization of pollen-tube growth identified REN1 (ROP1 enhancer 1) in Arabidopsis, whose null mutations turn elongated pollen tubes into bulbous cells. REN1 encodes a novel Rho GTPase-activating protein (RhoGAP) required for restricting the ROP1 activity to the pollen-tube tip. REN1 was localized to exocytic vesicles accumulated in the pollen-tube apex, as well as to the apical plasma membrane at the site of ROP1 activation. The apical localization of REN1 and its function in controlling growth polarity was compromised by disruption of ROP1-dependent F-actin and vesicular trafficking, which indicates that REN1 targeting and function is regulated by ROP1 downstream signaling. Our findings suggest that the REN1 RhoGAP controls a negative-feedback-based global inhibition of ROP1. This function provides a critical self-organizing mechanism, by which ROP signaling is spatially limited to the growth site and temporally oscillates during continuous tip growth. Similar spatiotemporal control of Rho GTPase signaling may also play an important role in cell-polarity control in other systems, including tip growth in fungi and cell movement in animals.

High speed photoacoustic imaging with fast OPO laser at 1.7 μm (Conference Presentation)

NASA Astrophysics Data System (ADS)

Piao, Zhonglie; Teng, Ma; Li, Jiawen; Qu, Yueqiao; Yu, Mingyue; Shung, K. Kirk; Zhou, Qifa; Kim, Chang-Seok; Chen, Zhongping

2016-03-01

Acute cardiovascular events are mostly due to a blood clot or thrombus induced by the sudden rupture of vulnerable atherosclerotic plaques within coronary artery walls. Based on the high optical absorption contrast of the lipid rich plaques within the vessel wall, intravascular photoacoustic (IVPA) imaging at 1.7 μm spectral band has shown promising capabilities for detecting of lipid composition, but the translation of the technology for in vivo application is limited by the slow imaging speed. In this work, we will present a high speed integrated IVPA/US imaging system with a 500 Hz optical parametric oscillator laser at 1725 nm (5 nm linewidth). A miniature catheter with 1.0 mm outer diameter was designed with a polished 200 μm multimode fiber and an ultrasound transducer with 45 MHz center frequency. Two optical illumination methods by gradient-index (GRIN) lens and ball lens are introduced and compared for higher spatial resolution. At 1725 nm, atherosclerotic rabbit abdominal aorta was imaged at two frame per second, which is more than one order of magnitude faster than previous reported IVPA imaging. Furthermore, by wide tuning range of the laser wavelength from 1680 nm to 1770 nm, spectroscopic photoacoustic analysis of lipid-mimicking phantom and an human atherosclerotic artery was performed ex vivo.

17 CFR 240.17Ad-7 - Record retention.

Code of Federal Regulations, 2013 CFR

2013-04-01

...) The records required by § 240.17Ad-6(a)(1), (3)(i), (6) or (11) shall be maintained for a period of not less than two years, the first six months in an easily accessible place. (b) The records required by § 240.17Ad-6(a) (2), (3)(ii), (4), (5) or (7) shall be maintained for a period of not less than...

17 CFR 240.17Ad-7 - Record retention.

Code of Federal Regulations, 2012 CFR

2012-04-01

...) The records required by § 240.17Ad-6(a)(1), (3)(i), (6) or (11) shall be maintained for a period of not less than two years, the first six months in an easily accessible place. (b) The records required by § 240.17Ad-6(a) (2), (3)(ii), (4), (5) or (7) shall be maintained for a period of not less than...

17 CFR 240.17Ad-7 - Record retention.

Code of Federal Regulations, 2011 CFR

2011-04-01

...) The records required by § 240.17Ad-6(a)(1), (3)(i), (6) or (11) shall be maintained for a period of not less than two years, the first six months in an easily accessible place. (b) The records required by § 240.17Ad-6(a) (2), (3)(ii), (4), (5) or (7) shall be maintained for a period of not less than...

17 CFR 240.17Ad-7 - Record retention.

Code of Federal Regulations, 2014 CFR

2014-04-01

.... (a) The records required by § 240.17Ad-6(a)(1), (3)(i), (6) or (11) shall be maintained for a period of not less than two years, the first six months in an easily accessible place. (b) The records required by § 240.17Ad-6(a) (2), (3)(ii), (4), (5) or (7) shall be maintained for a period of not less than...

Evidence for an angiotensin-(1-7) neuropeptidase expressed in the brain medulla and CSF of sheep.

PubMed

Marshall, Allyson C; Pirro, Nancy T; Rose, James C; Diz, Debra I; Chappell, Mark C

2014-07-01

Angiotensin-(1-7) [Ang-(1-7)] is an alternative product of the brain renin-angiotensin system that exhibits central actions to lower blood pressure and improve baroreflex sensitivity. We previously identified a peptidase that metabolizes Ang-(1-7) to the inactive metabolite product Ang-(1-4) in CSF of adult sheep. This study purified the peptidase 1445-fold from sheep brain medulla and characterized this activity. The peptidase was sensitive to the chelating agents o-phenanthroline and EDTA, as well as the mercury compound p-chloromercuribenzoic acid (PCMB). Selective inhibitors to angiotensin-converting enzyme, neprilysin, neurolysin, and thimet oligopeptidase did not attenuate activity; however, the metallopeptidase agent JMV-390 was a potent inhibitor of Ang-(1-7) hydrolysis (Ki = 0.8 nM). Kinetic studies using (125) I-labeled Ang-(1-7), Ang II, and Ang I revealed comparable apparent Km values (2.6, 2.8, and 4.3 μM, respectively), but a higher apparent Vmax for Ang-(1-7) (72 vs. 30 and 6 nmol/min/mg, respectively; p < 0.01). HPLC analysis of the activity confirmed the processing of unlabeled Ang-(1-7) to Ang-(1-4) by the peptidase, but revealed < 5% hydrolysis of Ang II or Ang I, and no hydrolysis of neurotensin, bradykinin or apelin-13. The unique characteristics of the purified neuropeptidase may portend a novel pathway to influence actions of Ang-(1-7) within the brain. Angiotensin-(1-7) actions are mediated by the AT7 /Mas receptor and include reduced blood pressure, decreased oxidative stress, enhanced baroreflex sensitivity, and increased nitric oxide (NO). Ang-(1-7) is directly formed from Ang I by neprilysin (NEP). We identify a new pathway for Ang-(1-7) metabolism in the brain distinct from angiotensin-converting enzyme-dependent hydrolysis. The Ang-(1-7) endopeptidase (A7-EP) degrades the peptide to Ang-(1-4) and may influence central Ang-(1-7) tone. © 2014 International Society for Neurochemistry.

Reclassification of strains MAFF 303099T and R7A into Mesorhizobiumjaponicum sp. nov.

PubMed

Martínez-Hidalgo, Pilar; Ramírez-Bahena, Martha Helena; Flores-Félix, José David; Igual, José M; Sanjuán, Juan; León-Barrios, Milagros; Peix, Alvaro; Velázquez, Encarna

2016-12-01

In this work we revise the taxonomic status of the Lotus-nodulating strains MAFF 303099T and R7A isolated in Japan and New Zealand, respectively. Their 16S rRNA gene sequences are identical and show 98.0, 99.7, 99.8 and 99.9 % similarity values with respect to Mesorhizobium loti NZP 2213T, M. jarvisii ATCC 33669T, M. huakuii USDA 4779T (=CCBAU 2609T) and M. erdmanii USDA 3471T, respectively. The analysis of recA and glnII gene sequeces showed that M. jarvisii ATCC 33669T and M. huakuii USDA 4779T (=CCBAU 2609T) are the most closely related strains to MAFF 303099T and R7A, with similarity values suggesting that these two strains belong to a different species for which MAFF 303099T is selected as the type strain. The DNA-DNA relatedness values between strain MAFF 303099T and its closest phylogenetic relatives ranged from 53 to 60 % in average. Strains MAFF 303099T and R7A presented slight differences in the proportions of C18 : 1ω7c 11-methyl and C19 : 0 cyclo ω8c fatty acids with respect to M. jarvisii ATCC 33669T and M. huakuii USDA 4779T, and also in several phenotypic characteristics. Therefore, we propose the reclassification of these two strains into a novel species named Mesorhizobium japonicum sp. nov., with the type strain being MAFF 303099T (=LMG 29417T=CECT 9101T).

7 CFR 1209.17 - Promotion.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 10 2010-01-01 2010-01-01 false Promotion. 1209.17 Section 1209.17 Agriculture... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE MUSHROOM PROMOTION, RESEARCH, AND CONSUMER INFORMATION ORDER Mushroom Promotion, Research, and Consumer Information Order Definitions § 1209...

FRankenstein becomes a cyborg: the automatic recombination and realignment of fold recognition models in CASP6.

PubMed

Kosinski, Jan; Gajda, Michal J; Cymerman, Iwona A; Kurowski, Michal A; Pawlowski, Marcin; Boniecki, Michal; Obarska, Agnieszka; Papaj, Grzegorz; Sroczynska-Obuchowicz, Paulina; Tkaczuk, Karolina L; Sniezynska, Paulina; Sasin, Joanna M; Augustyn, Anna; Bujnicki, Janusz M; Feder, Marcin

2005-01-01

In the course of CASP6, we generated models for all targets using a new version of the "FRankenstein's monster approach." Previously (in CASP5) we were able to build many very accurate full-atom models by selection and recombination of well-folded fragments obtained from crude fold recognition (FR) results, followed by optimization of the sequence-structure fit and assessment of alternative alignments on the structural level. This procedure was however very arduous, as most of the steps required extensive visual and manual input from the human modeler. Now, we have automated the most tedious steps, such as superposition of alternative models, extraction of best-scoring fragments, and construction of a hybrid "monster" structure, as well as generation of alternative alignments in the regions that remain poorly scored in the refined hybrid model. We have also included the ROSETTA method to construct those parts of the target for which no reasonable structures were generated by FR methods (such as long insertions and terminal extensions). The analysis of successes and failures of the current version of the FRankenstein approach in modeling of CASP6 targets reveals that the considerably streamlined and automated method performs almost as well as the initial, mostly manual version, which suggests that it may be a useful tool for accurate protein structure prediction even in the hands of nonexperts. 2005 Wiley-Liss, Inc.

A comparison of QuantStudio™ 3D Digital PCR and ARMS-PCR for measuring plasma EGFR T790M mutations of NSCLC patients.

PubMed

Feng, Qin; Gai, Fei; Sang, Yaxiong; Zhang, Jie; Wang, Ping; Wang, Yue; Liu, Bing; Lin, Dongmei; Yu, Yang; Fang, Jian

2018-01-01

The AURA3 clinical trial has shown that advanced non-small cell lung cancer (NSCLC) patients with EGFR T790M mutations in circulating tumor DNA (ctDNA) could benefit from osimertinib. The aim of this study was to assess the usefulness of QuantStudio™ 3D Digital PCR System platform for the detection of plasma EGFR T790M mutations in NSCLC patients, and compare the performances of 3D Digital PCR and ARMS-PCR. A total of 119 Chinese patients were enrolled in this study. Mutant allele frequency of plasma EGFR T790M was detected by 3D Digital PCR, then 25 selected samples were verified by ARMS-PCR and four of them were verified by next generation sequencing (NGS). In total, 52.94% (69/119) had EGFR T790M mutations detected by 3D Digital PCR. In 69 positive samples, the median mutant allele frequency (AF) was 1.09% and three cases presented low concentration (AF <0.1%). Limited by the amount of plasma DNA, 17 samples (AF <2.5%) and eight samples (T790M-) were selected for verification by ARMS-PCR. Four of those samples were verified by NGS as a third verification method. Among the selected 17 positive cases, ten samples presented mutant allele frequency <0.5%, and seven samples presented intermediate mutant allele frequency (0.5% AF 2.5%). However, only three samples (3/17) were identified as positive by ARMS-PCR, namely, P6 (AF =1.09%), P7 (AF =2.09%), and P8 (AF =2.21%). It is worth mentioning that sample P9 (AF =2.05%, analyzed by 3D Digital PCR) was identified as T790M- by ARMS-PCR. Four samples were identified as T790M+ by both NGS and 3D Digital PCR, and typically three samples (3/4) presented at a low ratio (AF <0.5%). Our study demonstrated that 3D Digital PCR is a novel method with high sensitivity and specificity to detect EGFR T790M mutation in plasma.

7 CFR 773.11-773.17 - [Reserved

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 7 2010-01-01 2010-01-01 false [Reserved] 773.11-773.17 Section 773.11-773.17 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS SPECIAL APPLE LOAN PROGRAM §§ 773.11-773.17 [Reserved] ...

7 CFR 773.11-773.17 - [Reserved

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 7 2014-01-01 2014-01-01 false [Reserved] 773.11-773.17 Section 773.11-773.17 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS SPECIAL APPLE LOAN PROGRAM §§ 773.11-773.17 [Reserved] ...

7 CFR 773.11-773.17 - [Reserved

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 7 2011-01-01 2011-01-01 false [Reserved] 773.11-773.17 Section 773.11-773.17 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS SPECIAL APPLE LOAN PROGRAM §§ 773.11-773.17 [Reserved] ...

7 CFR 773.11-773.17 - [Reserved

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 7 2012-01-01 2012-01-01 false [Reserved] 773.11-773.17 Section 773.11-773.17 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS SPECIAL APPLE LOAN PROGRAM §§ 773.11-773.17 [Reserved] ...

7 CFR 773.11-773.17 - [Reserved

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 7 2013-01-01 2013-01-01 false [Reserved] 773.11-773.17 Section 773.11-773.17 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS SPECIAL APPLE LOAN PROGRAM §§ 773.11-773.17 [Reserved] ...

Contribution of Rho kinase to the early phase of the calcium-contraction coupling in airway smooth muscle.

PubMed

Mbikou, Prisca; Fajmut, Ales; Brumen, Milan; Roux, Etienne

2011-02-01

We investigated theoretically and experimentally the role of Rho kinase (RhoK) in Ca(2+)-contraction coupling in rat airways. Isometric contraction was measured on tracheal, extrapulmonary and intrapulmonary bronchial rings. Intracellular [Ca(2+)] was recorded in freshly isolated tracheal myocytes. Stimulation by carbachol (0.3 and 10 μm) and 50 mm external KCl induced a short-time, Hill-shaped contraction obtained within 90 s, followed by a sustained or an additional delayed contraction. Responses of [Ca(2+)](i) to acetylcholine consisted in a fast peak followed by a plateau and, in 42% of the cells, superimposed Ca(2+) oscillations. The RhoK inhibitor Y27632 (10 μm) did not alter the [Ca(2+)](i) response. Whatever the agonist, Y27632 did not modify the basal tension but decreased the amplitude of the short-duration response, without altering the additional delayed contraction. The Myosin Light Chain Phosphatase (MLCP) inhibitor calyculin A increased the basal tension and abolished the effect of RhoK. KN93 (Ca(2+)-calmodulin-dependent protein kinase II inhibitor) and DIDS (inhibitor of Ca(2+)-activated Cl(-) channels) had no influence on the RhoK effect. We built a theoretical model of Ca(2+)-dependent active/inactive RhoK ratio and subsequent RhoK-dependent MLCP inactivation, which was further coupled with a four-state model of the contractile apparatus and Ca(2+)-dependent MLCK activation. The model explains the time course of the short-duration contraction and the role of RhoK by Ca(2+)-dependent activation of MLCK and RhoK, which inactivates MLCP. Oscillatory and non-oscillatory [Ca(2+)](i) responses result in a non-oscillatory contraction, the amplitude of which is encoded by the plateau value and oscillation frequency. In conclusion, Ca(2+)-dependent but CaMK II-independent RhoK activation contributes to the early phase of the contractile response via MLCP inhibition.

Role of Melatonin in the Regulation of Differentiation of T Cells Producing Interleukin-17 (Th17).

PubMed

Kuklina, E M; Glebezdina, N S; Nekrasova, I V

2016-03-01

We studied the ability of melatonin in physiological and pharmacological concentrations to induce and/or regulate differentiation of T cells producing IL-17 (Th17). This hormone produced the opposite effect on CD4+T cells, which depended on their activation status. Melatonin induced the synthesis of IL-17A by intact T cells, but had little effect on activated cells. Melatonin in high (pharmacological) concentration decreased the intracellular expression of this cytokine under conditions of polyclonal activation. Melatonin had a dose-depended effect. Taking into the fact that Th17 cells play an important role in the immune defense, it can be suggested that the regulation of their activity by melatonin contributes to this process.

Silibinin-Induced Apoptosis and Downregulation of MicroRNA-21 and MicroRNA-155 in MCF-7 Human Breast Cancer Cells

PubMed Central

Zadeh, Masoud Maleki; Ranji, Najmeh; Majidi, Mohammad; Falahi, Fahimeh

2016-01-01

Purpose MicroRNAs (miRNAs) have received much attention owing to their aberrant expression in various stages of cancer. In many biological processes, miRNAs negatively regulate gene expression, and may be useful in therapeutic strategies. The present study evaluated the effects of silibinin (silybin), a natural flavonoid, on miRNA expression and attempted to elucidate therapeutic targets in MCF-7 breast cancer cells. Methods The rates of cell proliferation and apoptosis were determined in silibinin-treated and untreated MCF-7 cells. Furthermore, the expression levels of miR-21 and miR-155 were measured in MCF-7 cells after incubation with silibinin (100 µg/mL), and the putative targets of the miRNAs within the apoptotic pathways were predicted using bioinformatic approaches. The expression levels of some of these targets were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results Silibinin induced apoptosis in MCF-7 cells in a dose- and time-dependent manner. qRT-PCR analysis revealed a decrease in miR-21 and miR-155 expression levels in silibinin-treated cells relative to the levels in the untreated cells. Potential miR-21 and miR-155 targets within the apoptotic pathways, such as CASP-9, BID, APAF-1, CASP-3, CASP-8, and PDCD4, were predicted by in silico analysis. qRT-PCR analysis showed upregulation of some of these potential targets including caspase-9 (CASP-9) and BID after silibinin treatment for 48 hours. Conclusion Our results suggest a correlation between the expression of miR-21 and miR-155, and MCF-7 cell proliferation. The antiproliferative activity of silibinin may partly be attributable to the downregulation of miR-21 and miR-155, and the upregulation of their apoptotic targets. Furthermore, the upregulation of CASP-9 and BID indicates that silibinin induces apoptosis through both the extrinsic and intrinsic pathways. PMID:27066095

Silibinin-Induced Apoptosis and Downregulation of MicroRNA-21 and MicroRNA-155 in MCF-7 Human Breast Cancer Cells.

PubMed

Zadeh, Masoud Maleki; Motamed, Nasrin; Ranji, Najmeh; Majidi, Mohammad; Falahi, Fahimeh

2016-03-01

MicroRNAs (miRNAs) have received much attention owing to their aberrant expression in various stages of cancer. In many biological processes, miRNAs negatively regulate gene expression, and may be useful in therapeutic strategies. The present study evaluated the effects of silibinin (silybin), a natural flavonoid, on miRNA expression and attempted to elucidate therapeutic targets in MCF-7 breast cancer cells. The rates of cell proliferation and apoptosis were determined in silibinin-treated and untreated MCF-7 cells. Furthermore, the expression levels of miR-21 and miR-155 were measured in MCF-7 cells after incubation with silibinin (100 µg/mL), and the putative targets of the miRNAs within the apoptotic pathways were predicted using bioinformatic approaches. The expression levels of some of these targets were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Silibinin induced apoptosis in MCF-7 cells in a dose- and time-dependent manner. qRT-PCR analysis revealed a decrease in miR-21 and miR-155 expression levels in silibinin-treated cells relative to the levels in the untreated cells. Potential miR-21 and miR-155 targets within the apoptotic pathways, such as CASP-9, BID, APAF-1, CASP-3, CASP-8, and PDCD4, were predicted by in silico analysis. qRT-PCR analysis showed upregulation of some of these potential targets including caspase-9 (CASP-9) and BID after silibinin treatment for 48 hours. Our results suggest a correlation between the expression of miR-21 and miR-155, and MCF-7 cell proliferation. The antiproliferative activity of silibinin may partly be attributable to the downregulation of miR-21 and miR-155, and the upregulation of their apoptotic targets. Furthermore, the upregulation of CASP-9 and BID indicates that silibinin induces apoptosis through both the extrinsic and intrinsic pathways.

Comparing localized and nonlocalized dynamic 31P magnetic resonance spectroscopy in exercising muscle at 7T

PubMed Central

Meyerspeer, Martin; Robinson, Simon; Nabuurs, Christine I; Scheenen, Tom; Schoisengeier, Adrian; Unger, Ewald; Kemp, Graham J; Moser, Ewald

2012-01-01

By improving spatial and anatomical specificity, localized spectroscopy can enhance the power and accuracy of the quantitative analysis of cellular metabolism and bioenergetics. Localized and nonlocalized dynamic 31P magnetic resonance spectroscopy using a surface coil was compared during aerobic exercise and recovery of human calf muscle. For localization, a short echo time single-voxel magnetic resonance spectroscopy sequence with adiabatic refocusing (semi-LASER) was applied, enabling the quantification of phosphocreatine, inorganic phosphate, and pH value in a single muscle (medial gastrocnemius) in single shots (TR = 6 s). All measurements were performed in a 7 T whole body scanner with a nonmagnetic ergometer. From a series of equal exercise bouts we conclude that: (a) with localization, measured phosphocreatine declines in exercise to a lower value (79 ± 7% cf. 53 ± 10%, P = 0.002), (b) phosphocreatine recovery shows shorter half time (t1/2 = 34 ± 7 s cf. t1/2 = 42 ± 7 s, nonsignificant) and initial postexercise phosphocreatine resynthesis rate is significantly higher (32 ± 5 mM/min cf. 17 ± 4 mM/min, P = 0.001) and (c) in contrast to nonlocalized 31P magnetic resonance spectroscopy, no splitting of the inorganic phosphate peak is observed during exercise or recovery, just an increase in line width during exercise. This confirms the absence of contaminating signals originating from weaker-exercising muscle, while an observed inorganic phosphate line broadening most probably reflects variations across fibers in a single muscle. Magn Reson Med, 2012. © 2012 Wiley Periodicals, Inc. PMID:22334374

Evidence for B{sup 0}{yields}{rho}{sup 0}{rho}{sup 0} Decays and Implications for the Cabibbo-Kobayashi-Maskawa Angle {alpha}

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

Aubert, B.; Bona, M.; Boutigny, D.

2007-03-16

We search for the decays B{sup 0}{yields}{rho}{sup 0}{rho}{sup 0}, B{sup 0}{yields}{rho}{sup 0}f{sub 0}(980), and B{sup 0}{yields}f{sub 0}(980)f{sub 0}(980) in a sample of about 384x10{sup 6} {upsilon}(4S){yields}BB decays collected with the BABAR detector at the PEP-II asymmetric-energy e{sup +}e{sup -} collider at Stanford Linear Accelerator Center. We find evidence for B{sup 0}{yields}{rho}{sup 0}{rho}{sup 0} with 3.5{sigma} significance and measure the branching fraction B=(1.07{+-}0.33{+-}0.19)x10{sup -6} and longitudinal polarization fraction f{sub L}=0.87{+-}0.13{+-}0.04, where the first uncertainty is statistical, and the second is systematic. The uncertainty on the Cabibbo-Kobayashi-Maskawa quark-mixing matrix unitarity angle {alpha} due to penguin contributions in B{yields}{rho}{rho} decays is 18 deg.more » at the 1{sigma} level. We also set upper limits on the B{sup 0}{yields}{rho}{sup 0}f{sub 0}(980) and B{sup 0}{yields}f{sub 0}(980)f{sub 0}(980) decay rates.« less

7 CFR 989.17 - Acquire.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 8 2011-01-01 2011-01-01 false Acquire. 989.17 Section 989.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE RAISINS PRODUCED FROM GRAPES GROWN IN...

7 CFR 989.17 - Acquire.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 8 2014-01-01 2014-01-01 false Acquire. 989.17 Section 989.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE RAISINS PRODUCED FROM GRAPES GROWN IN...

7 CFR 989.17 - Acquire.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 8 2013-01-01 2013-01-01 false Acquire. 989.17 Section 989.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE RAISINS PRODUCED FROM GRAPES GROWN IN...

7 CFR 989.17 - Acquire.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Acquire. 989.17 Section 989.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE RAISINS PRODUCED FROM GRAPES GROWN IN...

7 CFR 989.17 - Acquire.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 8 2012-01-01 2012-01-01 false Acquire. 989.17 Section 989.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE RAISINS PRODUCED FROM GRAPES GROWN IN...

7 CFR 57.17 - Nondiscrimination.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 3 2011-01-01 2011-01-01 false Nondiscrimination. 57.17 Section 57.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards..., political beliefs, sexual orientation, or marital or family status. [69 FR 57166, Sept. 24, 2004] ...

7 CFR 57.17 - Nondiscrimination.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Nondiscrimination. 57.17 Section 57.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards..., political beliefs, sexual orientation, or marital or family status. [69 FR 57166, Sept. 24, 2004] ...

7 CFR 57.17 - Nondiscrimination.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 3 2012-01-01 2012-01-01 false Nondiscrimination. 57.17 Section 57.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards..., political beliefs, sexual orientation, or marital or family status. [69 FR 57166, Sept. 24, 2004] ...

7 CFR 57.17 - Nondiscrimination.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 3 2013-01-01 2013-01-01 false Nondiscrimination. 57.17 Section 57.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards..., political beliefs, sexual orientation, or marital or family status. [69 FR 57166, Sept. 24, 2004] ...

7 CFR 57.17 - Nondiscrimination.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 3 2014-01-01 2014-01-01 false Nondiscrimination. 57.17 Section 57.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards..., political beliefs, sexual orientation, or marital or family status. [69 FR 57166, Sept. 24, 2004] ...

7 CFR 993.17 - Ton.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Ton. 993.17 Section 993.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE DRIED PRUNES PRODUCED IN CALIFORNIA Order...

7 CFR 993.17 - Ton.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 8 2012-01-01 2012-01-01 false Ton. 993.17 Section 993.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE DRIED PRUNES PRODUCED IN CALIFORNIA Order...

7 CFR 993.17 - Ton.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 8 2013-01-01 2013-01-01 false Ton. 993.17 Section 993.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE DRIED PRUNES PRODUCED IN CALIFORNIA Order...

7 CFR 993.17 - Ton.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 8 2014-01-01 2014-01-01 false Ton. 993.17 Section 993.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE DRIED PRUNES PRODUCED IN CALIFORNIA Order...

7 CFR 993.17 - Ton.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 8 2011-01-01 2011-01-01 false Ton. 993.17 Section 993.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE DRIED PRUNES PRODUCED IN CALIFORNIA Order...

7 CFR 966.17 - District.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false District. 966.17 Section 966.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE TOMATOES GROWN IN FLORIDA Order...

7 CFR 1001.17 - [Reserved

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 9 2010-01-01 2009-01-01 true [Reserved] 1001.17 Section 1001.17 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE NORTHEAST MARKETING AREA Order Regulating...

7 CFR 945.17 - District.