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Sample records for active ar variants

  1. Differential regulation of metabolic pathways by androgen receptor (AR) and its constitutively active splice variant, AR-V7, in prostate cancer cells

    PubMed Central

    Shafi, Ayesha A.; Putluri, Vasanta; Arnold, James M.; Tsouko, Efrosini; Maity, Suman; Roberts, Justin M.; Coarfa, Cristian; Frigo, Daniel E.; Putluri, Nagireddy; Sreekumar, Arun; Weigel, Nancy L.

    2015-01-01

    Metastatic prostate cancer (PCa) is primarily an androgen-dependent disease, which is treated with androgen deprivation therapy (ADT). Tumors usually develop resistance (castration-resistant PCa [CRPC]), but remain androgen receptor (AR) dependent. Numerous mechanisms for AR-dependent resistance have been identified including expression of constitutively active AR splice variants lacking the hormone-binding domain. Recent clinical studies show that expression of the best-characterized AR variant, AR-V7, correlates with resistance to ADT and poor outcome. Whether AR-V7 is simply a constitutively active substitute for AR or has novel gene targets that cause unique downstream changes is unresolved. Several studies have shown that AR activation alters cell metabolism. Using LNCaP cells with inducible expression of AR-V7 as a model system, we found that AR-V7 stimulated growth, migration, and glycolysis measured by ECAR (extracellular acidification rate) similar to AR. However, further analyses using metabolomics and metabolic flux assays revealed several differences. Whereas AR increased citrate levels, AR-V7 reduced citrate mirroring metabolic shifts observed in CRPC patients. Flux analyses indicate that the low citrate is a result of enhanced utilization rather than a failure to synthesize citrate. Moreover, flux assays suggested that compared to AR, AR-V7 exhibits increased dependence on glutaminolysis and reductive carboxylation to produce some of the TCA (tricarboxylic acid cycle) metabolites. These findings suggest that these unique actions represent potential therapeutic targets. PMID:26378018

  2. CUDC-101, a Novel Inhibitor of Full-Length Androgen Receptor (flAR) and Androgen Receptor Variant 7 (AR-V7) Activity: Mechanism of Action and In Vivo Efficacy.

    PubMed

    Sun, Huiying; Mediwala, Sanjay N; Szafran, Adam T; Mancini, Michael A; Marcelli, Marco

    2016-06-01

    Castration-resistant prostate cancer (CRPC) is an androgen receptor (AR)-dependent disease expected to cause the death of more than 27,000 Americans in 2015. There are only a few available treatments for CRPC, making the discovery of new drugs an urgent need. We report that CUDC-101 (an inhibitor od HER2/NEU, EGFR and HDAC) inhibits both the full length AR (flAR) and the AR variant AR-V7. This observation prompted experiments to discover which of the known activities of CUDC-101 is responsible for the inhibition of flAR/AR-V7 signaling. We used pharmacologic and genetic approaches, and found that the effect of CUDC-101 on flAR and AR-V7 was duplicated only by other HDAC inhibitors, or by silencing the HDAC isoforms HDAC5 and HDAC10. We observed that CUDC-101 treatment or AR-V7 silencing by RNAi equally reduced transcription of the AR-V7 target gene, PSA, without affecting viability of 22Rv1 cells. However, when cellular proliferation was used as an end point, CUDC-101 was more effective than AR-V7 silencing, raising the prospect that CUDC-101 has additional targets beside AR-V7. In support of this, we found that CUDC-101 increased the expression of the cyclin-dependent kinase inhibitor p21, and decreased that of the oncogene HER2/NEU. To determine if CUDC-101 reduces growth in a xenograft model of prostate cancer, this drug was given for 14 days to castrated male SCID mice inoculated with 22Rv1 cells. Compared to vehicle, CUDC-101 reduced xenograft growth in a statistically significant way, and without macroscopic side effects. These studies demonstrate that CUDC-101 inhibits wtAR and AR-V7 activity and growth of 22Rv1 cells in vitro and in vivo. These effects result from the ability of CUDC-101 to target not only HDAC signaling, which was associated with decreased flAR and AR-V7 activity, but multiple additional oncogenic pathways. These observations raise the possibility that treatment of CRPC may be achieved by using similarly multi-targeted approaches. PMID

  3. Regulation of androgen receptor splice variant AR3 by PCGEM1

    PubMed Central

    Zhang, Ziqiang; Zhou, Nanjiang; Huang, Jianguo; Ho, Tsui-Ting; Zhu, Zhuxian; Qiu, Zhongmin; Zhou, Xinchun; Bai, Chunxue; Wu, Fangting; Xu, Min; Mo, Yin-Yuan

    2016-01-01

    The androgen receptor (AR) is required for prostate development and is also a major driver of prostate cancer pathogenesis. Thus androgen deprivation therapy (ADT) is the mainstay of treatment for advanced prostate cancer. However, castration resistance due to expression of constitutively active AR splice variants is a significant challenge to prostate cancer therapy; little is known why effectiveness of ADT can only last for a relatively short time. In the present study, we show that PCGEM1 interacts with splicing factors heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and U2AF65, as determined by RNA precipitation and Western blot, suggesting a role for PCGEM1 in alternative splicing. In support of this possibility, PCGEM1 is correlated with AR3, a predominant and clinically important form of AR splice variants in prostate cancer. Moreover, androgen deprivation (AD) induces PCGEM1 and causes its accumulation in nuclear speckles. Finally, we show that the AD-induced PCGEM1 regulates the competition between hnRNP A1 and U2AF65 for AR pre-mRNA. AD promotes PCGEM1 to interact with both hnRNP A1 and U2AF65 with different consequences. While the interaction of PCGEM1 with hnRNP A1 suppresses AR3 by exon skipping, its interaction with U2AF65 promotes AR3 by exonization. Together, we demonstrate an AD-mediated AR3 expression involving PCGEM1 and splicing factors. PMID:26848868

  4. The RNA-binding protein Sam68 regulates expression and transcription function of the androgen receptor splice variant AR-V7

    PubMed Central

    Stockley, Jacqueline; Markert, Elke; Zhou, Yan; Robson, Craig N.; Elliott, David J.; Lindberg, Johan; Leung, Hing Y.; Rajan, Prabhakar

    2015-01-01

    Castration-resistant (CR) prostate cancer (PCa) partly arises due to persistence of androgen receptor (AR) transcriptional activity in the absence of cognate ligand. An emerging mechanism underlying the CRPCa phenotype and predicting response to therapy is the expression of the constitutively-active AR-V7 splice variant generated by AR cryptic exon 3b inclusion. Here, we explore the role of the RNA-binding protein (RBP) Sam68 (encoded by KHDRBS1), which is over-expressed in clinical PCa, on AR-V7 expression and transcription function. Using a minigene reporter, we show that Sam68 controls expression of exon 3b resulting in an increase in endogenous AR-V7 mRNA and protein expression in RNA-binding-dependent manner. We identify a novel protein-protein interaction between Sam68 and AR-V7 mediated by a common domain shared with full-length AR, and observe these proteins in the cell nucleoplasm. Using a luciferase reporter, we demonstrate that Sam68 co-activates ligand-independent AR-V7 transcriptional activity in an RNA-binding-independent manner, and controls expression of the endogenous AR-V7-specific gene target UBE2C. Our data suggest that Sam68 has separable effects on the regulation of AR-V7 expression and transcriptional activity, through its RNA-binding capacity. Sam68 and other RBPs may control expression of AR-V7 and other splice variants as well as their downstream functions in CRPCa. PMID:26310125

  5. The RNA-binding protein Sam68 regulates expression and transcription function of the androgen receptor splice variant AR-V7.

    PubMed

    Stockley, Jacqueline; Markert, Elke; Zhou, Yan; Robson, Craig N; Elliott, David J; Lindberg, Johan; Leung, Hing Y; Rajan, Prabhakar

    2015-01-01

    Castration-resistant (CR) prostate cancer (PCa) partly arises due to persistence of androgen receptor (AR) transcriptional activity in the absence of cognate ligand. An emerging mechanism underlying the CRPCa phenotype and predicting response to therapy is the expression of the constitutively-active AR-V7 splice variant generated by AR cryptic exon 3b inclusion. Here, we explore the role of the RNA-binding protein (RBP) Sam68 (encoded by KHDRBS1), which is over-expressed in clinical PCa, on AR-V7 expression and transcription function. Using a minigene reporter, we show that Sam68 controls expression of exon 3b resulting in an increase in endogenous AR-V7 mRNA and protein expression in RNA-binding-dependent manner. We identify a novel protein-protein interaction between Sam68 and AR-V7 mediated by a common domain shared with full-length AR, and observe these proteins in the cell nucleoplasm. Using a luciferase reporter, we demonstrate that Sam68 co-activates ligand-independent AR-V7 transcriptional activity in an RNA-binding-independent manner, and controls expression of the endogenous AR-V7-specific gene target UBE2C. Our data suggest that Sam68 has separable effects on the regulation of AR-V7 expression and transcriptional activity, through its RNA-binding capacity. Sam68 and other RBPs may control expression of AR-V7 and other splice variants as well as their downstream functions in CRPCa. PMID:26310125

  6. An imaging agent to detect androgen receptor and its active splice variants in prostate cancer

    PubMed Central

    Imamura, Yusuke; Tien, Amy H.; Pan, Jinhe; Leung, Jacky K.; Banuelos, Carmen A.; Jian, Kunzhong; Wang, Jun; Mawji, Nasrin R.; Fernandez, Javier Garcia; Lin, Kuo-Shyan; Andersen, Raymond J.; Sadar, Marianne D.

    2016-01-01

    Constitutively active splice variants of androgen receptor (AR-Vs) lacking ligand-binding domain (LBD) are a mechanism of resistance to androgen receptor LBD–targeted (AR LBD–targeted) therapies for metastatic castration-resistant prostate cancer (CRPC). There is a strong unmet clinical need to identify prostate cancer patients with AR-V–positive lesions to determine whether they will benefit from further AR LBD–targeting therapies or should receive taxanes or investigational drugs like EPI-506 or galeterone. Both EPI-506 (NCT02606123) and galeterone (NCT02438007) are in clinical trials and are proposed to have efficacy against lesions that are positive for AR-Vs. AR activation function-1 (AF-1) is common to the N-terminal domains of full-length AR and AR-Vs. Here, we provide proof of concept for developing imaging compounds that directly bind AR AF-1 to detect both AR-Vs and full-length AR. 123I-EPI-002 had specific binding to AR AF-1, which enabled direct visualization of CRPC xenografts that express full-length AR and AR-Vs. Our findings highlight the potential of 123I-EPI-002 as an imaging agent for the detection of full-length AR and AR-Vs in CRPC. PMID:27525313

  7. FOXA1 regulates androgen receptor variant activity in models of castrate-resistant prostate cancer

    PubMed Central

    Nakjang, Sirintra; Chaytor, Lewis; Grey, James; Robson, Craig N.; Gaughan, Luke

    2015-01-01

    Retention of androgen receptor (AR) signalling in castrate-resistant prostate cancer (CRPC) highlights the requirement for the development of more effective AR targeting therapies. A key mechanism of resistance to anti-androgens is through expression of constitutively active AR variants (AR-Vs) that are refractory to next-generation therapies, including Enzalutamide and Abiraterone. By maintaining an androgenic gene signature, AR-Vs drive tumour survival and progression in castrate conditions. Critically, however, our understanding of the mechanics of AR-V-driven transcription is limited, particularly with respect to dependency on pioneer factor function. Here we show that depletion of FOXA1 in the CWR22Rv1 CRPC cell line abrogates the oncogenic potential of AR-Vs. Gene expression profiling reveals that approximately 41% of the AR-V transcriptome requires FOXA1 and that depletion of FOXA1 attenuates AR-V binding at a sub-set of analysed co-regulated genes. Interestingly, AR-V levels are elevated in cells depleted of FOXA1 as a consequence of attenuated negative feedback on the AR gene, but is insufficient to maintain cell growth as evidenced by marked anti-proliferative effects in FOXA1 knockdown cells. In all, our data suggests that AR-Vs are dependent on FOXA1 for sustaining a pro-proliferative gene signature and agents targeting FOXA1 may represent novel therapeutic options for CRPC patients. PMID:26336819

  8. Droplet Digital PCR Based Androgen Receptor Variant 7 (AR-V7) Detection from Prostate Cancer Patient Blood Biopsies

    PubMed Central

    Ma, Yafeng; Luk, Alison; Young, Francis P.; Lynch, David; Chua, Wei; Balakrishnar, Bavanthi; de Souza, Paul; Becker, Therese M.

    2016-01-01

    Androgen receptor splice variant V7 (AR-V7) was recently identified as a valuable predictive biomarker in metastatic castrate-resistant prostate cancer. Here, we report a new, sensitive and accurate screen for AR-V7 mRNA expression directly from circulating tumor cells (CTCs): We combined EpCAM-based immunomagnetic CTC isolation using the IsoFlux microfluidic platform with droplet digital polymerase chain reaction (ddPCR) to analyze total AR and AR-V7 expression from prostate cancer patients CTCs. We demonstrate that AR-V7 is reliably detectable in enriched CTC samples with as little as five CTCs, even considering tumor heterogeneity, and confirm detection of AR-V7 in CTC samples from advanced prostate cancer (PCa) patients with AR-V7 detection limited to castrate resistant disease status in our sample set. Sensitive molecular analyses of circulating tumor cells (CTCs) or circulating tumor nucleic acids present exciting strategies to detect biomarkers, such as AR-V7 from non-invasive blood samples, so-called blood biopsies. PMID:27527157

  9. Nitrite Reductase Activity in Engineered Azurin Variants.

    PubMed

    Berry, Steven M; Strange, Jacob N; Bladholm, Erika L; Khatiwada, Balabhadra; Hedstrom, Christine G; Sauer, Alexandra M

    2016-05-01

    Nitrite reductase (NiR) activity was examined in a series of dicopper P.a. azurin variants in which a surface binding copper site was added through site-directed mutagenesis. Four variants were synthesized with copper binding motifs inspired by the catalytic type 2 copper binding sites found in the native noncoupled dinuclear copper enzymes nitrite reductase and peptidylglycine α-hydroxylating monooxygenase. The four azurin variants, denoted Az-NiR, Az-NiR3His, Az-PHM, and Az-PHM3His, maintained the azurin electron transfer copper center, with the second designed copper site located over 13 Å away and consisting of mutations Asn10His,Gln14Asp,Asn16His-azurin, Asn10His,Gln14His,Asn16His-azurin, Gln8Met,Gln14His,Asn16His-azurin, and Gln8His,Gln14His,Asn16His-azurin, respectively. UV-visible absorption spectroscopy, EPR spectroscopy, and electrochemistry of the sites demonstrate copper binding as well as interaction with small exogenous ligands. The nitrite reduction activity of the variants was determined, including the catalytic Michaelis-Menten parameters. The variants showed activity (0.34-0.59 min(-1)) that was slower than that of native NiRs but comparable to that of other model systems. There were small variations in activity of the four variants that correlated with the number of histidines in the added copper site. Catalysis was found to be reversible, with nitrite produced from NO. Reactions starting with reduced azurin variants demonstrated that electrons from both copper centers were used to reduce nitrite, although steady-state catalysis required the T2 copper center and did not require the T1 center. Finally, experiments separating rates of enzyme reduction from rates of reoxidation by nitrite demonstrated that the reaction with nitrite was rate limiting during catalysis. PMID:27055058

  10. Real-time optical recording of beta1-adrenergic receptor activation reveals supersensitivity of the Arg389 variant to carvedilol.

    PubMed

    Rochais, Francesca; Vilardaga, Jean-Pierre; Nikolaev, Viacheslav O; Bünemann, Moritz; Lohse, Martin J; Engelhardt, Stefan

    2007-01-01

    Antagonists of beta-adrenergic receptors (beta-ARs) have become a main therapeutic regimen for the treatment of heart failure even though the mechanisms of their beneficial effects are still poorly understood. Here, we used fluorescent resonance energy transfer-based (FRET-based) approaches to directly monitor activation of the beta(1)-AR and downstream signaling. While the commonly used beta-AR antagonists metoprolol, bisoprolol, and carvedilol displayed varying degrees of inverse agonism on the Gly389 variant of the receptor (i.e., actively switching off the beta(1)-AR), surprisingly, only carvedilol showed very specific and marked inverse agonist effects on the more frequent Arg389 variant. These specific effects of carvedilol on the Arg389 variant of the beta(1)-AR were also seen for control of beating frequency in rat cardiac myocytes expressing the 2 receptor variants. This FRET sensor permitted direct observation of activation of the beta(1)-AR in living cells in real time. It revealed that beta(1)-AR variants dramatically differ in their responses to diverse beta blockers, with possible consequences for their clinical use. PMID:17200720

  11. The role of mitochondrial fusion and StAR phosphorylation in the regulation of StAR activity and steroidogenesis.

    PubMed

    Castillo, Ana F; Orlando, Ulises; Helfenberger, Katia E; Poderoso, Cecilia; Podesta, Ernesto J

    2015-06-15

    The steroidogenic acute regulatory (StAR) protein regulates the rate-limiting step in steroidogenesis, i.e. the delivery of cholesterol from the outer (OMM) to the inner (IMM) mitochondrial membrane. StAR is a 37-kDa protein with an N-terminal mitochondrial targeting sequence that is cleaved off during mitochondrial import to yield 30-kDa intramitochondrial StAR. StAR acts exclusively on the OMM and its activity is proportional to how long it remains on the OMM. However, the precise fashion and the molecular mechanism in which StAR remains on the OMM have not been elucidated yet. In this work we will discuss the role of mitochondrial fusion and StAR phosphorylation by the extracellular signal-regulated kinases 1/2 (ERK1/2) as part of the mechanism that regulates StAR retention on the OMM and activity. PMID:25540920

  12. 40Ar/39Ar constraints on the activity of the Temsamane extensional detachment (eastern Rif, Morocco)

    NASA Astrophysics Data System (ADS)

    Jabaloy Sánchez, A.; Booth-Rea, G.; Azdimousa, A.; Asebriy, L.; Vázquez-Vílchez, M.; Martínez-Martínez, J. M.; Gabites, J.

    2012-04-01

    The subducted North Maghrebian passive margin was exhumed by an upper crustal brittle-ductile extensional detachment and brittle low-angle normal faults in a continental subduction transform setting. The Temsamane detachment in the eastern Rif is defined by a ductile shear zone approximately 100 m thick with a low-angle ramp geometry that cuts down into the Temsamane fold-nappe stack. The shear zone shows southwestward kinematics and separates epizone metapelites of the Temsamane units below from the epizone to diagenetic rocks of the Tanger-Ketama-Aknoul units above. To the east, the detachment becomes brittle, branching into a listric-fan that cuts through 10-6 Ma sediments and volcanoclastics in the Tres Forcas cape. New 40Ar/39Ar radiometric ages on amphiboles and micas from the footwall of the Temsamane detachment indicate that the metamorphic peak was reached in the footwall (Temsamane units) at ca. 21 Ma, producing the amphibolite epidote facies in the Ras Afrou Unit. The cooling of the footwall rocks below the 325 °C occurred between the 16 and 13 Ma, while apatite fission track ages indicate that the cooling below the 120 °C occurred at ca. 11 Ma. The 40Ar/39Ar radiometric ages on amphiboles and micas of the metamorphic klippes over the Temsamene units (Ait-Amrâne massif) indicates that the Jurassic marbles of the Tanger-Ketama Unit reached their metamorphic peak at ca. 80 Ma, in agreement with previously published K/Ar ages in micas. The rocks of the Tanger-Ketama Unit cooled below the 120 °C between 17.0 ± 2.4 Ma and 13.9 ± 1.8 Ma. We interpret the increase of cooling rates of the footwall rocks between 15-13 Ma and 11 Ma as due to the activity of the Temsamane detachment fault. Thus, both the North Maghrebian and the South Iberian subducted passive margins were exhumed in the Betic and Rif branches of the Gibraltar arc by SW-directed brittle-ductile detachments during the Late Miocene in an oblique collisional setting.

  13. Partners in crime: deregulation of AR activity and androgen synthesis in prostate cancer

    PubMed Central

    Knudsen, Karen E.; Penning, Trevor

    2010-01-01

    Prostate cancer remains a leading cause of cancer death, as there are no durable means to treat advanced disease. Treatment of non-organ confined prostate cancer hinges on its androgen dependence. First line therapeutic strategies suppress androgen receptor (AR) activity, via androgen ablation and direct AR antagonists. While initially effective, incurable, "castration-resistant" tumors arise due to resurgent AR activity. Alterations of AR and/or associated regulatory networks are known to restore receptor activity and support resultant therapy-resistant tumor progression. However, recent evidence also reveals an unexpected contribution of AR ligand, wherein alterations in pathways controlling androgen synthesis support castrate-resistant AR activity. Herein, mechanisms underlying the lethal pairing of AR deregulation and aberrant androgen synthesis in prostate cancer progression will be discussed. PMID:20138542

  14. Alteration of natural (37)Ar activity concentration in the subsurface by gas transport and water infiltration.

    PubMed

    Guillon, Sophie; Sun, Yunwei; Purtschert, Roland; Raghoo, Lauren; Pili, Eric; Carrigan, Charles R

    2016-05-01

    High (37)Ar activity concentration in soil gas is proposed as a key evidence for the detection of underground nuclear explosion by the Comprehensive Nuclear Test-Ban Treaty. However, such a detection is challenged by the natural background of (37)Ar in the subsurface, mainly due to Ca activation by cosmic rays. A better understanding and improved capability to predict (37)Ar activity concentration in the subsurface and its spatial and temporal variability is thus required. A numerical model integrating (37)Ar production and transport in the subsurface is developed, including variable soil water content and water infiltration at the surface. A parameterized equation for (37)Ar production in the first 15 m below the surface is studied, taking into account the major production reactions and the moderation effect of soil water content. Using sensitivity analysis and uncertainty quantification, a realistic and comprehensive probability distribution of natural (37)Ar activity concentrations in soil gas is proposed, including the effects of water infiltration. Site location and soil composition are identified as the parameters allowing for a most effective reduction of the possible range of (37)Ar activity concentrations. The influence of soil water content on (37)Ar production is shown to be negligible to first order, while (37)Ar activity concentration in soil gas and its temporal variability appear to be strongly influenced by transient water infiltration events. These results will be used as a basis for practical CTBTO concepts of operation during an OSI. PMID:26939033

  15. Alternatively Spliced Androgen Receptor Variants

    PubMed Central

    Dehm, Scott M.; Tindall, Donald J.

    2011-01-01

    Alternative splicing is an important mechanism for increasing functional diversity from a limited set of genes. De-regulation of this process is common in diverse pathologic conditions. The androgen receptor (AR) is a steroid receptor transcription factor with functions critical for normal male development as well as the growth and survival of normal and cancerous prostate tissue. Studies of AR function in androgen insensitivity syndrome (AIS) and prostate cancer (PCa) have demonstrated loss-of-function AR alterations in AIS, and gain-of-function AR alterations in PCa. Over the past two decades, AR gene alterations have been identified in various individuals with AIS, which disrupt normal AR splicing patterns and yield dysfunctional AR protein variants. More recently, altered AR splicing patterns have been identified as a mechanism of PCa progression and resistance to androgen-depletion therapy. Several studies have described the synthesis of alternatively spliced transcripts encoding truncated AR isoforms that lack the ligand-binding domain, which is the ultimate target of androgen depletion. Many of these truncated AR isoforms function as constitutively active, ligand-independent transcription factors that can support androgen-independent expression of AR target genes, as well as the androgen-independent growth of PCa cells. In this review, we will summarize the various alternatively spliced AR variants that have been discovered, with a focus on their role and origin in the pathologic conditions of AIS and PCa. PMID:21778211

  16. Stromal TGF-β signaling induces AR activation in prostate cancer

    PubMed Central

    Yang, Feng; Chen, Yizhen; Shen, Tao; Guo, Dan; Dakhova, Olga; Ittmann, Michael M.; Creighton, Chad J.; Zhang, Yiqun; Dang, Truong D.; Rowley, David R.

    2014-01-01

    AR signaling is essential for the growth and survival of prostate cancer (PCa), including most of the lethal castration-resistant PCa (CRPC). We previously reported that TGF-β signaling in prostate stroma promotes prostate tumor angiogenesis and growth. By using a PCa/stroma co-culture model, here we show that stromal TGF-β signaling induces comprehensive morphology changes of PCa LNCaP cells. Furthermore, it induces AR activation in LNCaP cells in the absence of significant levels of androgen, as evidenced by induction of several AR target genes including PSA, TMPRSS2, and KLK4. SD-208, a TGF-β receptor 1 specific inhibitor, blocks this TGF-β induced biology. Importantly, stromal TGF-β signaling together with DHT induce robust activation of AR. MDV3100 effectively blocks DHT-induced, but not stromal TGF-β signaling induced AR activation in LNCaP cells, indicating that stromal TGF-β signaling induces both ligand-dependent and ligand-independent AR activation in PCa. TGF-β induces the expression of several growth factors and cytokines in prostate stromal cells, including IL-6, and BMP-6. Interestingly, BMP-6 and IL-6 together induces robust AR activation in these co-cultures, and neutralizing antibodies against BMP-6 and IL-6 attenuate this action. Altogether, our study strongly suggests tumor stromal microenvironment induced AR activation as a direct mechanism of CRPC. PMID:25333263

  17. Biogeochemical cyclic activity of bacterial arsB in arsenic-contaminated mines.

    PubMed

    Chang, Jin-Soo; Ren, Xianghao; Kim, Kyoung-Woong

    2008-01-01

    Biogeochemical cyclic activity of the ars (arsenic resistance system) operon is arsB influx/efflux encoded by the ecological of Pseudomonas putida. This suggests that studying arsenite-oxidizing bacteria may lead to a better understanding of molecular geomicrobiology, which can be applied to the bioremediation of arsenic-contaminated mines. This is the first report in which multiple arsB-binding mechanisms have been used on indigenous bacteria. In ArsB (strains OS-5; ABB83931; OS-19; ABB04282 and RW-28; ABB88574), there are ten putative enzyme, Histidine (His) 131, His 133, His 137, Arginine (Arg) 135, Arg 137, Arg 161, Trptohan (Trp) 142, Trp 164, Trp 166, and Trp 171, which are each located in different regions of the partial sequence. The adenosine triphosphate (ATP)-binding cassette transports, binding affinities and associating ratable constants show that As-binding is comparatively insensitive to the location of the residues within the moderately stable alpha-helical structure. The alpha-helical structures in ArsB-permease and anion permease arsB have been shown to import/export arsenic in P. putida. We proposed that arsB residues, His 131, His 133, His 137, Arg 135, Arg 137, Arg 161, Trp 142, Trp 164, Trp 166, and Trp 171 are required for arsenic binding and activation of arsA/arsB or arsAB. This arsB influx/efflux pum-ping is important, and the effect in arsenic species change and mobility in mine soil has got a significantly ecological role because it allows arsenic oxidizing/reducing bacteria to control biogeochemical cycle of abandoned mines. PMID:19202875

  18. Cellulase variants with improved expression, activity and stability, and use thereof

    SciTech Connect

    Aehle, Wolfgang; Bott, Richard R; Bower, Benjamin; Caspi, Jonathan; Estell, David A; Goedegebuur, Frits; Hommes, Ronaldus W.J.; Kaper, Thijs; Kelemen, Bradley; Kralj, Slavko; Van Lieshout, Johan; Nikolaev, Igor; Van Stigt Thans, Sander; Wallace, Louise; Vogtentanz, Gudrun; Sandgren, Mats

    2014-03-25

    The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having improved expression, activity and/or stability. Also described are nucleic acids encoding the cellulase variants, compositions comprising the cellulase variants, and methods of use thereof.

  19. Variants in RBP4 and AR genes modulate age at onset in familial amyloid polyneuropathy (FAP ATTRV30M).

    PubMed

    Santos, Diana; Coelho, Teresa; Alves-Ferreira, Miguel; Sequeiros, Jorge; Mendonça, Denisa; Alonso, Isabel; Lemos, Carolina; Sousa, Alda

    2016-05-01

    Familial amyloid polyneuropathy (FAP) ATTRV30M is a neurodegenerative disorder due to point mutations in the transthyretin gene, with V30M being the commonest. FAP ATTRV30M shows a wide variation in age at onset (AO) between clusters, families and generations. Portuguese patients also show remarkable AO differences between genders. Genes found to be associated with FAP ATTRV30M pathways may act as AO modifiers. Our aim was to further explore the role of APCS and RBP4 genes and to study for the first time the involvement of sex-linked genetic modifiers - AR and HSD17B1 genes - in AO variation in Portuguese families. We collected DNA from a sample of 318 patients, currently under follow-up. A total of 18 tagging SNPs from APCS, RBP4, AR and HSD17B1 and 5 additional SNPs from APCS and RBP4 previously studied were genotyped. To account for nonindependency of AO between members of the same family, we used generalized estimating equations (GEEs). We found that APCS and RBP4 were associated with late AO. In addition, rs11187545 of the RBP4 was associated with an early AO. For the AR, in the male group three SNPs were associated with an early AO, whereas in the female group four were associated with both an early and later AO. These results strengthened the role of APCS and RBP4 genes and revealed for the first time the contribution of AR genes as an AO modifier in both males and females. These findings may have important implications in genetic counseling and for new therapeutic strategies. PMID:26286643

  20. Curcumin down-regulates AR gene expression and activation in prostate cancer cell lines.

    PubMed

    Nakamura, Keiichiro; Yasunaga, Yutaka; Segawa, Takehiko; Ko, Daejin; Moul, Judd W; Srivastava, Shiv; Rhim, Johng S

    2002-10-01

    Curcumin, traditionally used as a seasoning spice in Indian cuisine, has been reported to decrease the proliferation potential of prostate cancer cells, by a mechanism that is not fully understood. In the current study, we have evaluated the effects of curcumin in cell growth, activation of signal transduction, and transforming activities of both androgen-dependent and independent cell lines. Prostate cancer cell lines, LNCaP and PC-3, were treated with curcumin and its effects were further analyzed on signal transduction and expression of androgen receptor (AR) and AR-related cofactors using transient transfection assay and Western blotting. Our results show that curcumin down-regulates transactivation and expression of AR, activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB), and CREB (cAMP response element-binding protein)-binding protein (CBP). Curcumin also inhibited the transforming activities of both cell lines as evidenced by the reduced colony forming ability in soft agar. The results obtained here demonstrate that curcumin has a potential therapeutic effect on prostate cancer cells through down-regulation of AR and AR-related cofactors (AP-1, NF-kappaB and CBP). PMID:12239622

  1. Measurement of the activity of an artificial neutrino source based on {sup 37}Ar

    SciTech Connect

    Abdurashitov, D. N.; Veretenkin, E. P.; Gavrin, V. N.; Gorbachev, V. V.; Ibragimova, T. V.; Kalikhov, A. V.; Mirmov, I. N. Shikhin, A. A.; Yants, V. E.; Barsanov, V. I.; Dzhanelidze, A. A.; Zlokazov, S. B.; Markov, S. Yu.; Shakirov, Z. N.; Cleveland, B. T.

    2007-02-15

    The activity of an artificial neutrino source based on {sup 37}Ar was measured by a specially developed method of directly counting {sup 37}Ar decays in a proportional counter. This source was used to irradiate the target of the SAGE radiochemical gallium-germanium neutrino telescope at the Baksan Neutrino Observatory (Institute for Nuclear Research, Russian Academy of Sciences, Moscow), whereupon the measurements were performed at the Institute of Reactor Materials (Zarechny, Sverdlovsk oblast, Russia). The method used to prepare gaseous samples for measurements in proportional counters and the counting procedure are described. The measured activity of the {sup 37}Ar neutrino source is 405.1 {+-} 3.7 kCi (corrected for decays that occurred within the period between the instant of activity measurement and the commencement of the irradiation of Ga target at 04:00 Moscow time, 30.04.2004)

  2. The novel HDAC inhibitor AR-42-induced anti-colon cancer cell activity is associated with ceramide production

    SciTech Connect

    Xu, Weihong; Xu, Bin; Yao, Yiting; Yu, Xiaoling; Shen, Jie

    2015-08-07

    In the current study, we investigated the potential activity of AR-42, a novel histone deacetylase (HDAC) inhibitor, against colon cancer cells. Our in vitro results showed that AR-42 induced ceramide production, exerted potent anti-proliferative and pro-apoptotic activities in established (SW-620 and HCT-116 lines) and primary human colon cancer cells. Exogenously-added sphingosine 1-phosphate (S1P) suppressed AR-42-induced activity, yet a cell-permeable ceramide (C4) facilitated AR-42-induced cytotoxicity against colon cancer cells. In addition, AR-42-induced ceramide production and anti-colon cancer cell activity were inhibited by the ceramide synthase inhibitor fumonisin B1, but were exacerbated by PDMP, which is a ceramide glucosylation inhibitor. In vivo, oral administration of a single dose of AR-42 dramatically inhibited SW-620 xenograft growth in severe combined immunodeficient (SCID) mice, without inducing overt toxicities. Together, these results show that AR-42 dramatically inhibits colon cancer cell proliferation in vitro and in vivo, and ceramide production might be the key mechanism responsible for its actions. - Highlights: • AR-42 is anti-proliferative against primary/established colon cancer cells. • AR-42 induces significant apoptotic death in primary/established colon cancer cells. • Ceramide production mediates AR-42-induced cytotoxicity in colon cancer cells. • AR-42 oral administration potently inhibits SW-620 xenograft growth in SCID mice.

  3. Pacific Northwest USDA-ARS Jointed Goatgrass Research and Extension Activities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The USDA-ARS weed scientists have conducted research and extension activities on six research projects funded by the National Jointed Goatgrass Research Program (NJGGRP). This poster reviews the objectives and major research findings from these federally funded projects. Most of these projects wer...

  4. Identification of Androgen Receptor Splice Variants in the Pten Deficient Murine Prostate Cancer Model.

    PubMed

    Liang, Mengmeng; Adisetiyo, Helty; Li, Xiuqing; Liu, Xiuqing; Liu, Ren; Gill, Parkash; Roy-Burman, Pradip; Jones, Jeremy O; Mulholland, David J

    2015-01-01

    Androgen receptor (AR) variants are associated with resistance to anti androgen therapy both in human prostate cancer cell lines and clinical samples. These observations support the hypothesis that AR isoform accumulation is a consequence of selective therapeutic pressure on the full length AR. The Pten deficient prostate cancer model proceeds with well-defined kinetics including progression to castration resistant prostate cancer (CRPC). While surgical castration and enzalutamide treatments yield an initial therapeutic response, Pten-/-epithelia continue to proliferate yielding locally invasive primary tumor pathology. That most epithelium remains AR positive, but ligand independent, suggests the presence of oncogenic AR variants. To address this hypothesis, we have used a panel of recently described Pten-/- tumor cell lines derived from both from hormone intact (E4, E8) and castrated Pten mutants (cE1, cE2) followed by RACE PCR to identify and characterize three novel truncated, amino terminus containing AR variants (mAR-Va, b, c). Variants appear not only conserved throughout progression but are correlated with nearly complete loss of full length AR (AR-FL) at castrate androgen levels. The overexpression of variants leads to enhanced transcriptional activity of AR while knock down studies show reduced transcriptional output. Collectively, the identification of truncated AR variants in the conditional PTEN deletion model supports a role for maintaining the CRPC phenotype and provides further therapeutic applications of this preclinical model. PMID:26196517

  5. Expression of androgen receptor splice variants in clinical breast cancers

    PubMed Central

    Dvinge, Heidi; Tarulli, Gerard A.; Hanson, Adrienne R.; Ryan, Natalie K.; Pickering, Marie A.; Birrell, Stephen N.; Hu, Dong Gui; Mackenzie, Peter I.; Russell, Roslin; Caldas, Carlos; Raj, Ganesh V.; Dehm, Scott M.; Plymate, Stephen R.; Bradley, Robert K.; Tilley, Wayne D.; Selth, Luke A.

    2015-01-01

    The importance of androgen receptor (AR) signaling is increasingly being recognized in breast cancer, which has elicited clinical trials aimed at assessing the efficacy of androgen deprivation therapy (ADT) for metastatic disease. In prostate cancer, resistance to ADT is frequently associated with the emergence of androgen-independent splice variants of the AR (AR variants, AR-Vs) that lack the LBD and are constitutively active. Women with breast cancer may be prone to a similar phenomenon. Herein, we show that in addition to the prototypical transcript, the AR gene produces a diverse range of AR-V transcripts in primary breast tumors. The most frequently and highly expressed variant was AR-V7 (exons 1/2/3/CE3), which was detectable at the mRNA level in > 50% of all breast cancers and at the protein level in a subset of ERα-negative tumors. Functionally, AR-V7 is a constitutively active and ADT-resistant transcription factor that promotes growth and regulates a transcriptional program distinct from AR in ERα-negative breast cancer cells. Importantly, we provide ex vivo evidence that AR-V7 is upregulated by the AR antagonist enzalutamide in primary breast tumors. These findings have implications for treatment response in the ongoing clinical trials of ADT in breast cancer. PMID:26554309

  6. Expression of androgen receptor splice variants in clinical breast cancers.

    PubMed

    Hickey, Theresa E; Irvine, Connie M; Dvinge, Heidi; Tarulli, Gerard A; Hanson, Adrienne R; Ryan, Natalie K; Pickering, Marie A; Birrell, Stephen N; Hu, Dong Gui; Mackenzie, Peter I; Russell, Roslin; Caldas, Carlos; Raj, Ganesh V; Dehm, Scott M; Plymate, Stephen R; Bradley, Robert K; Tilley, Wayne D; Selth, Luke A

    2015-12-29

    The importance of androgen receptor (AR) signaling is increasingly being recognized in breast cancer, which has elicited clinical trials aimed at assessing the efficacy of androgen deprivation therapy (ADT) for metastatic disease. In prostate cancer, resistance to ADT is frequently associated with the emergence of androgen-independent splice variants of the AR (AR variants, AR-Vs) that lack the LBD and are constitutively active. Women with breast cancer may be prone to a similar phenomenon. Herein, we show that in addition to the prototypical transcript, the AR gene produces a diverse range of AR-V transcripts in primary breast tumors. The most frequently and highly expressed variant was AR-V7 (exons 1/2/3/CE3), which was detectable at the mRNA level in > 50% of all breast cancers and at the protein level in a subset of ERα-negative tumors. Functionally, AR-V7 is a constitutively active and ADT-resistant transcription factor that promotes growth and regulates a transcriptional program distinct from AR in ERα-negative breast cancer cells. Importantly, we provide ex vivo evidence that AR-V7 is upregulated by the AR antagonist enzalutamide in primary breast tumors. These findings have implications for treatment response in the ongoing clinical trials of ADT in breast cancer. PMID:26554309

  7. 40Ar/39Ar geochronology of the Neogene-Quaternary Harrat Al-Madinah intercontinental volcanic field, Saudi Arabia: Implications for duration and migration of volcanic activity

    NASA Astrophysics Data System (ADS)

    Moufti, M. R.; Moghazi, A. M.; Ali, K. A.

    2013-01-01

    New 40Ar/39Ar ages, based on incremental heating techniques for groundmass separates of 25 samples, are presented for the Harrat Al-Madinah volcanic field, part of Harrat Rahat in the north western part of the Arabian plate. This area is an active volcanic field characterized by the occurrence of two historical eruptions approximately in 641 and 1256 AD. Field investigations of the main volcanic landforms indicate dominantly monogenetic strombolian eruptions, in addition to local more explosive eruptions. The lavas consist mainly of olivine basalt and hawaiite flows with minor evolved rocks of mugearite, benmoreite, and trachyte that occur mainly as domes, tuff cones and occasionally as lava flows. Previous K/Ar dating shows that the Harrat Al-Madinah lava flows and associated domes comprise seven units spanning an age range of ca. 1.7 Ma-Recent. The new 40Ar/39Ar age determinations confirm, to a great extent, the previously obtained K/Ar ages in the sense that no major systematic biases were found in the general stratigraphy of the different flow units. However, the 40Ar/39Ar plateau ages show that volcanism in this area began in the Neogene (˜10 Ma) and continued to Recent, with the most voluminous eruptions occurring in the Quaternary. Neogene volcanism occurred in at least three pulses around 10, 5 and 2 Ma, whereas Quaternary volcanism produced at least seven units reflecting lava flow emplacement in the time period of 1.90 Ma-Recent. Thus, the whole duration of volcanic activity in the Harrat Al-Madinah (10 Ma-Recent) appears much longer than that previously identified. The longevity of volcanism in the same part of the moving Arabian plate and absence of evidence for uni-directional migration of volcanic activity indicate that there is no fixed plume beneath this region. The NNW-trending distribution of the volcanic vents is parallel to the Red Sea, and suggests their origin is related to periodic extensional episodes along the reactivated Red Sea fault

  8. 40Ar/39Ar dating of tuff vents in the Campi Flegrei caldera (southern Italy): Toward a new chronostratigraphic reconstruction of the Holocene volcanic activity

    USGS Publications Warehouse

    Fedele, L.; Insinga, D.D.; Calvert, A.T.; Morra, V.; Perrotta, A.; Scarpati, C.

    2011-01-01

    The Campi Flegrei hosts numerous monogenetic vents inferred to be younger than the 15 ka Neapolitan Yellow Tuff. Sanidine crystals from the three young Campi Flegrei vents of Fondi di Baia, Bacoli and Nisida were dated using 40Ar/39Ar geochronology. These vents, together with several other young edifices, occur roughly along the inner border of the Campi Flegrei caldera, suggesting that the volcanic conduits are controlled by caldera-bounding faults. Plateau ages of ∼9.6 ka (Fondi di Baia), ∼8.6 ka (Bacoli) and ∼3.9 ka (Nisida) indicate eruptive activity during intervals previously interpreted as quiescent. A critical revision, involving calendar age correction of literature 14C data and available 40Ar/39Ar age data, is presented. A new reference chronostratigraphic framework for Holocene Phlegrean activity, which significantly differs from the previously adopted ones, is proposed. This has important implications for understanding the Campi Flegrei eruptive history and, ultimately, for the evaluation of related volcanic risk and hazard, for which the inferred history of its recent activity is generally taken into account.

  9. Constitutively active UVR8 photoreceptor variant in Arabidopsis.

    PubMed

    Heijde, Marc; Binkert, Melanie; Yin, Ruohe; Ares-Orpel, Florence; Rizzini, Luca; Van De Slijke, Eveline; Persiau, Geert; Nolf, Jonah; Gevaert, Kris; De Jaeger, Geert; Ulm, Roman

    2013-12-10

    Arabidopsis thaliana UV RESISTANCE LOCUS 8 (UVR8) is a UV-B photoreceptor that initiates photomorphogenic responses underlying acclimation and UV-B tolerance in plants. UVR8 is a homodimer in its ground state, and UV-B exposure results in its instantaneous monomerization followed by interaction with CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), a major factor in UV-B signaling. UV-B photoreception by UVR8 is based on intrinsic tryptophan aromatic amino acid residues, with tryptophan-285 as the main chromophore. We generated transgenic plants expressing UVR8 with a single amino acid change of tryptophan-285 to alanine. UVR8(W285A) appears monomeric and shows UV-B-independent interaction with COP1. Phenotypically, the plants expressing UVR8(W285A) exhibit constitutive photomorphogenesis associated with constitutive activation of target genes, elevated levels of anthocyanins, and enhanced, acclimation-independent UV-B tolerance. Moreover, we have identified COP1, REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 and 2 (RUP1 and RUP2), and the SUPPRESSOR OF PHYA-105 (SPA) family as proteins copurifying with UVR8(W285A). Whereas COP1, RUP1, and RUP2 are known to directly interact with UVR8, we show that SPA1 interacts with UVR8 indirectly through COP1. We conclude that UVR8(W285A) is a constitutively active UVR8 photoreceptor variant in Arabidopsis, as is consistent with the crucial importance of monomer formation and COP1 binding for UVR8 activity. PMID:24277841

  10. Substrate dependent in vitro antifungal activity of Bacillus sp strain AR2

    PubMed Central

    2014-01-01

    Background Biosurfactants are a structurally diverse group of secondary metabolites with lots of potential to serve mankind. Depending upon the structure and composition they may exhibit properties that make them suitable for a particular application. Structural and compositional diversity of biosurfactant is unambiguously substrate dependent. The present study investigates the qualitative and quantitative effect of different water soluble carbon source on the biosurfactant produced by Bacillus amylofaciens strain AR2. Results Strain AR2 produced lipopeptide type biosurfactant while growing on water soluble carbon sources. Maximum biosurfactant production was observed in the sucrose supplemented minimal salt medium (MSM). Strain AR2 exhibited carbon source dependent surface tension reduction in the range of 30-37 mN/m, critical micelle concentration (CMC) in the range 80-110 mg/l and emulsification index (EI24 kerosene) in the range of 32-66%. In dextrose, sucrose and glycerol supplemented MSM, strain AR2 produced lipopeptides as a mixture of surfactin, iturin and fengycin. However, in the presence of maltose, lactose and sorbitol only iturin was produced. This substrate dependent compositional variation in the lipopeptides significantly influenced antifungal activity. Lipopeptides produced by strain AR2 while growing on sucrose and dextrose based MSM was observed to be most efficient as an antifungal agent. Conclusions These results suggest that carbon source provided for the growth and biosurfactant production not only influences the yield but also the type of biosurfactant. Sucrose is the most suitable carbon source for production of lipopeptide biosurfactant with antifungal activity. PMID:24885467

  11. Recent findings of green tea extract AR25 (Exolise) and its activity for the treatment of obesity.

    PubMed

    Chantre, P; Lairon, D

    2002-01-01

    The green tea extract AR25 is an 80% ethanolic dry extract standardized at 25% catechins expressed as epigallocatechin gallate (EGCG). In vitro, green tea extract AR25 exerts a direct inhibition of gastric and pancreatic lipases and a stimulation of thermogenesis. In an open study, the effects of extract AR25 were evaluated in moderately obese patients. After 3 months, body weight was decreased by 4.6% and waist circumference by 4.48%. These results suggest the green tea extract AR25 to be a natural product for the treatment of obesity, which exerts its activity by several ways: inhibition of lipases and stimulation of thermogenesis. PMID:11924761

  12. A novel prostate cancer therapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variants

    PubMed Central

    Sun, Feng; Indran, Inthrani R.; Zhang, Zhi Wei; Tan, M.H.Eileen; Li, Yu; Lim, Z.L.Ryan; Hua, Rui; Yang, Chong; Soon, Fen-Fen; Li, Jun; Xu, H.Eric; Cheung, Edwin; Yong, Eu-Leong

    2015-01-01

    Persistent androgen receptor (AR) signaling is the key driving force behind progression and development of castration-resistant prostate cancer (CRPC). In many patients, AR COOH-terminal truncated splice variants (ARvs) play a critical role in contributing to the resistance against androgen depletion therapy. Unfortunately, clinically used antiandrogens like bicalutamide (BIC) and enzalutamide (MDV), which target the ligand binding domain, have failed to suppress these AR variants. Here, we report for the first time that a natural prenylflavonoid, icaritin (ICT), can co-target both persistent AR and ARvs. ICT was found to inhibit transcription of key AR-regulated genes, such as KLK3 [prostate-specific antigen (PSA)] and ARvs-regulated genes, such as UBE2C and induce apoptosis in AR-positive prostate cancer (PC) cells. Mechanistically, ICT promoted the degradation of both AR and ARvs by binding to arylhydrocarbon-receptor (AhR) to mediate ubiquitin-proteasomal degradation. Therefore, ICT impaired AR transactivation in PC cells. Knockdown of AhR gene restored AR stability and partially prevented ICT-induced growth suppression. In clinically relevant murine models orthotopically implanted with androgen-sensitive and CRPC cells, ICT was able to target AR and ARvs, to inhibit AR signaling and tumor growth with no apparent toxicity. Our results provide a mechanistic framework for the development of ICT, as a novel lead compound for AR-positive PC therapeutics, especially for those bearing AR splice variants. PMID:25908644

  13. Alternatively Spliced Telomerase Reverse Transcriptase Variants Lacking Telomerase Activity Stimulate Cell Proliferation

    PubMed Central

    Hrdličková, Radmila; Nehyba, Jiří

    2012-01-01

    Eight human and six chicken novel alternatively spliced (AS) variants of telomerase reverse transcriptase (TERT) were identified, including a human variant (Δ4-13) containing an in-frame deletion which removed exons 4 through 13, encoding the catalytic domain of telomerase. This variant was expressed in telomerase-negative normal cells and tissues as well as in transformed telomerase-positive cell lines and cells which employ an alternative method to maintain telomere length. The overexpression of the Δ4-13 variant significantly elevated the proliferation rates of several cell types without enhancing telomerase activity, while decreasing the endogenous expression of this variant by use of small interfering RNA (siRNA) technology reduced cell proliferation. The expression of the Δ4-13 variant stimulated Wnt signaling. In chicken cells, AS TERT variants containing internal deletions or insertions that eliminated or reduced telomerase activity also enhanced cell proliferation. This is the first report that naturally occurring AS TERT variants which lack telomerase activity stimulate cell proliferation. PMID:22907755

  14. Ionic liquid activated Bacillus subtilis lipase A variants through cooperative surface substitutions.

    PubMed

    Zhao, Jing; Jia, Ning; Jaeger, Karl-Erich; Bocola, Marco; Schwaneberg, Ulrich

    2015-10-01

    The interest in performing enzyme-catalyzed reactions in amphiphilic systems, e.g., imidazolium-based ionic liquids (ILs) or surfactants, has been increased over the past decades. Directed protein evolution has been successful in tailoring enzymes for desired properties. Herein, nine IL-resistant Bacillus subtilis lipase A variants, particularly an IL-activated variant M1 (M134N/N138S/L140S), were identified by directed evolution. For instance, variant M2 (M134R/L140S) showed almost doubled specific activity (16.9 vs. 9.4 U/mg) and resistance (233% vs. 111%) at 9 vol% 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([C4 mim][TfO]) compared with wild-type. The specific activities and resistance of purified individual single and double variants have been studied in five different IL-aqueous mixtures. The re-activation of lipase variant M1 (not wild-type) at high IL concentration was attributed to the cooperative effect of three surface substitutions (M134N, N138S, L140S) near the substrate-binding cleft. The presence of IL/substrate clusters under assay conditions was likely related to the re-activation effect. This study provides first example of IL-activated lipase variant generated by protein engineering, and helps to better understand the protein-IL interaction. PMID:25899108

  15. Titanium spallation cross sections between 30 and 584 MeV and Ar-39 activities on the moon

    NASA Technical Reports Server (NTRS)

    Steinbrunn, F.; Fireman, E. L.

    1974-01-01

    The production cross sections of Ar-39 for Ti spallation at 45-, 319-, 433-, and 584-MeV proton energies were measured to be 0.37 + or - 09, 12.4 + or - 3.7, 9.1 + or - 2.7, and 17.8 + or - 6.2 mb, respectively. Normalized Ar-39 production rates and activities are also derived for protons above 40 Mev and for three differential proton spectra. It is concluded that even for samples of high-Ti content, Ti spallation by solar protons below 200-MeV energy does not contribute significantly to their Ar-39 radioactivity.

  16. Tephrochronology of the Mont-Dore volcanic Massif (Massif Central, France): new 40Ar/39Ar constraints on the Late Pliocene and Early Pleistocene activity

    NASA Astrophysics Data System (ADS)

    Nomade, Sébastien; Pastre, Jean-François; Nehlig, Pierre; Guillou, Hervé; Scao, Vincent; Scaillet, Stéphane

    2014-03-01

    The Mont-Dore Massif (500 km2), the youngest stratovolcano of the French Massif Central, consists of two volcanic edifices: the Guéry and the Sancy. To improve our knowledge of the oldest explosive stages of the Mont-Dore Massif, we studied 40Ar/39Ar-dated (through single-grain laser and step-heating experiments) 11 pyroclastic units from the Guéry stratovolcano. We demonstrate that the explosive history of the Guéry can be divided into four cycles of explosive eruption activity between 3.09 and 1.46 Ma (G.I to G.IV). We have also ascertained that deposits associated with the 3.1-3.0-Ma rhyolitic activity, which includes the 5-km3 "Grande Nappe" ignimbrite, are not recorded in the central part of the Mont-Dore Massif. All the pyroclastites found in the left bank of the Dordogne River belong to a later explosive phase (2.86-2.58 Ma, G.II) and were channelled down into valleys or topographic lows where they are currently nested. This later activity also gave rise to most of the volcanic products in the Perrier Plateau (30 km east of the Mont-Dore Massif); three quarters of the volcano-sedimentary sequence (up to 100 m thick) was emplaced within less than 20 ky, associated with several flank collapses in the northeastern part of the Guéry. The age of the "Fournet flora" (2.69 ± 0.01 Ma) found within an ash bed belonging to G.II suggests that temperate forests already existed in the French Massif Central before the Pliocene/Pleistocene boundary. The Guéry's third explosive eruption activity cycle (G.III) lasted between 2.36 and 1.91 Ma. It encompassed the Guéry Lake and Morangie pumice and ash deposits, as well as seven other important events recorded as centimetric ash beds some 60 to 100 km southeast of the Massif in the Velay region. We propose a general tephrochronology for the Mont-Dore stratovolcano covering the last 3.1 My. This chronology is based on 44 40Ar/39Ar-dated events belonging to eight explosive eruption cycles each lasting between 100 and 200

  17. Directed evolution and characterization of atrazine chlorohydrolase variants with enhanced activity.

    PubMed

    Wang, Y; Li, X; Chen, X; Chen, D

    2013-10-01

    Atrazine chlorohydrolase (AtzA, EC 3.8.1.8) has attracted widespread interests as it catalyzes conversion of toxic atrazine to nontoxic hydroxyatrazine and can be used in the biodegradation of atrazine. To facilitate this application, a Haematococcus pluvialis-based method was applied to screen AtzA variants from a random mutagenesis library. Eight variants with enhanced enzyme activity were obtained. They showed 2.7- to 5.0-fold increase in specific activity compared with the wild type. Sequencing revealed that the two most active variants contained single substitution at Val12 and Leu395, respectively, while several improved variants contained substitutions at the four sites of Met315, His399, Asn429, and Val466 simultaneously, indicating that these residues contribute to the enzyme activity of AtzA. Kinetic analysis showed that five variants decreased the Km value 0.6- to 0.9-fold, whereas all the variants increased the catalytic efficiency (kcat/Km value) 2.5- to 4.1-fold compared to the wild type. The modeled three-dimensional structure showed that AtzA is comprised of a typical (β/α)8 domain of the amidohydrolase superfamily and a dual β-sheet domain. An iron ion and five ligand-binding residues are located in the β-barrel core of the (β/α)8 domain. Some substituted residues are involved in hydrogen bond formation in the (β/α)8-neighboring β-sheet. PMID:24237144

  18. Estimation of (41)Ar activity concentration and release rate from the TRIGA Mark-II research reactor.

    PubMed

    Hoq, M Ajijul; Soner, M A Malek; Rahman, A; Salam, M A; Islam, S M A

    2016-03-01

    The BAEC TRIGA research reactor (BTRR) is the only nuclear reactor in Bangladesh. Bangladesh Atomic Energy Regulatory Authority (BAERA) regulations require that nuclear reactor licensees undertake all reasonable precautions to protect the environment and the health and safety of persons, including identifying, controlling and monitoring the release of nuclear substances to the environment. The primary activation product of interest in terms of airborne release from the reactor is (41)Ar. (41)Ar is a noble gas readily released from the reactor stacks and most has not decayed by the time it moves offsite with normal wind speed. Initially (41)Ar is produced from irradiation of dissolved air in the primary water which eventually transfers into the air in the reactor bay. In this study, the airborne radioisotope (41)Ar generation concentration, ground level concentration and release rate from the BTRR bay region are evaluated theoretically during the normal reactor operation condition by several governing equations. This theoretical calculation eventually minimizes the doubt about radiological safety to determine the radiation level for (41)Ar activity whether it is below the permissible limit or not. Results show that the estimated activity for (41)Ar is well below the maximum permissible concentration limit set by the regulatory body, which is an assurance for the reactor operating personnel and general public. Thus the analysis performed within this paper is so much effective in the sense of ensuring radiological safety for working personnel and the environment. PMID:26736180

  19. The human 5-HT7 serotonin receptor splice variants: constitutive activity and inverse agonist effects

    PubMed Central

    Krobert, Kurt A; Levy, Finn Olav

    2002-01-01

    Using membranes from stably or transiently transfected HEK293 cells cultured in 5-HT-free medium and expressing the recombinant human 5-HT7 receptor splice variants (h5-HT7(a), h5-HT7(b) and h5-HT7(d)), we compared their abilities to constitutively activate adenylyl cyclase (AC).All h5-HT7 splice variants elevated basal and forskolin-stimulated AC. The basal AC activity was reduced by the 5-HT7 antagonist methiothepin and this effect was blocked by mesulergine (neutral 5-HT7 antagonist) indicating that the inhibitory effect of methiothepin is inverse agonism at the 5-HT7 receptor.Receptor density correlated poorly with constitutive AC activity in stable clonal cell lines and transiently transfected cells. Mean constitutive AC activity as a percentage of forskolin-stimulated AC was significantly higher for the h5-HT7(b) splice variant compared to the h5-HT7(a) and h5-HT7(d) splice variants but only in stable cell lines.All eight 5-HT antagonists tested inhibited constitutive AC activity of all splice variants in a concentration-dependent manner. No differences in inverse agonist potencies (pIC50) were observed between the splice variants. The rank order of potencies was in agreement and highly correlated with antagonist potencies (pKb) determined by antagonism of 5-HT-stimulated AC activity (methiothepin>metergoline>mesulergine⩾clozapine⩾spiperone⩾ritanserin>methysergide>ketanserin).The efficacy of inverse agonism was not receptor level dependent and varied for several 5-HT antagonists between membrane preparations of transiently and stably transfected cells.It is concluded that the h5-HT7 splice variants display similar constitutive activity and inverse agonist properties. PMID:11906971

  20. Active control of divertor asymmetry on EAST by localized D2 and Ar puffing

    NASA Astrophysics Data System (ADS)

    Wang, Dongsheng; Guo, Houyang; Wang, Huiqian; Luo, Guangnan; Wu, Zhenwei; Wu, Jinhua; Gao, Wei; Wang, Liang; Li, Qiang; East Team

    2011-03-01

    The divertor asymmetry in particle and power fluxes has been investigated on the EAST superconducting tokamak [S. Wu and EAST Team, Fusion Eng. Des. 82, 463 (2007)] for both single null (SN) and double null (DN) divertor configurations. D2 and Ar puffing at various divertor locations has also been explored as an active means to reduce peak target heat load and control divertor asymmetry. For SN, peak heat load on the outer divertor target is 2-3 times that on the inner divertor target under typical ohmic plasma conditions. DN operation leads to a stronger in-out asymmetry favoring the outer divertor. D2 and Ar puffing promotes partial detachment near the strike points, greatly reducing peak target heat load (over 50%), while the far-SOL divertor plasma remains attached. What is remarkable is that the particle flux is even increased away from the strike points when the B×∇B drift is directed toward the divertor target, thus facilitating particle removal.

  1. Active control of divertor asymmetry on EAST by localized D{sub 2} and Ar puffing

    SciTech Connect

    Wang Dongsheng; Luo Guangnan; Guo Houyang; Wang Huiqian; Wu Zhenwei; Wu Jinhua; Gao Wei; Wang Liang; Li Qiang

    2011-03-15

    The divertor asymmetry in particle and power fluxes has been investigated on the EAST superconducting tokamak [S. Wu and EAST Team, Fusion Eng. Des. 82, 463 (2007)] for both single null (SN) and double null (DN) divertor configurations. D{sub 2} and Ar puffing at various divertor locations has also been explored as an active means to reduce peak target heat load and control divertor asymmetry. For SN, peak heat load on the outer divertor target is 2-3 times that on the inner divertor target under typical ohmic plasma conditions. DN operation leads to a stronger in-out asymmetry favoring the outer divertor. D{sub 2} and Ar puffing promotes partial detachment near the strike points, greatly reducing peak target heat load (over 50%), while the far-SOL divertor plasma remains attached. What is remarkable is that the particle flux is even increased away from the strike points when the Bx{nabla}B drift is directed toward the divertor target, thus facilitating particle removal.

  2. Structural variants of yeast prions show conformer-specific requirements for chaperone activity

    PubMed Central

    Stein, Kevin C.; True, Heather L.

    2016-01-01

    Summary Molecular chaperones monitor protein homeostasis and defend against the misfolding and aggregation of proteins that is associated with protein conformational disorders. In these diseases, a variety of different aggregate structures can form. These are called prion strains, or variants, in prion diseases, and cause variation in disease pathogenesis. Here, we use variants of the yeast prions [RNQ+] and [PSI+] to explore the interactions of chaperones with distinct aggregate structures. We found that prion variants show striking variation in their relationship with Hsp40s. Specifically, the yeast Hsp40 Sis1, and its human ortholog Hdj1, had differential capacities to process prion variants, suggesting that Hsp40 selectivity has likely changed through evolution. We further show that such selectivity involves different domains of Sis1, with some prion conformers having a greater dependence on particular Hsp40 domains. Moreover, [PSI+] variants were more sensitive to certain alterations in Hsp70 activity as compared to [RNQ+] variants. Collectively, our data indicate that distinct chaperone machinery is required, or has differential capacity, to process different aggregate structures. Elucidating the intricacies of chaperone-client interactions, and how these are altered by particular client structures, will be crucial to understanding how this system can go awry in disease and contribute to pathological variation. PMID:25060529

  3. Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants?

    PubMed Central

    Lovelock, Paul K; Spurdle, Amanda B; Mok, Myth TS; Farrugia, Daniel J; Lakhani, Sunil R; Healey, Sue; Arnold, Stephen; Buchanan, Daniel; Investigators, kConFab; Couch, Fergus J; Henderson, Beric R; Goldgar, David E; Tavtigian, Sean V; Chenevix-Trench, Georgia; Brown, Melissa A

    2007-01-01

    Introduction Many of the DNA sequence variants identified in the breast cancer susceptibility gene BRCA1 remain unclassified in terms of their potential pathogenicity. Both multifactorial likelihood analysis and functional approaches have been proposed as a means to elucidate likely clinical significance of such variants, but analysis of the comparative value of these methods for classifying all sequence variants has been limited. Methods We have compared the results from multifactorial likelihood analysis with those from several functional analyses for the four BRCA1 sequence variants A1708E, G1738R, R1699Q, and A1708V. Results Our results show that multifactorial likelihood analysis, which incorporates sequence conservation, co-inheritance, segregation, and tumour immunohistochemical analysis, may improve classification of variants. For A1708E, previously shown to be functionally compromised, analysis of oestrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumour expression data significantly strengthened the prediction of pathogenicity, giving a posterior probability of pathogenicity of 99%. For G1738R, shown to be functionally defective in this study, immunohistochemistry analysis confirmed previous findings of inconsistent 'BRCA1-like' phenotypes for the two tumours studied, and the posterior probability for this variant was 96%. The posterior probabilities of R1699Q and A1708V were 54% and 69%, respectively, only moderately suggestive of increased risk. Interestingly, results from functional analyses suggest that both of these variants have only partial functional activity. R1699Q was defective in foci formation in response to DNA damage and displayed intermediate transcriptional transactivation activity but showed no evidence for centrosome amplification. In contrast, A1708V displayed an intermediate transcriptional transactivation activity and a normal foci formation response in response to DNA damage but induced centrosome amplification. Conclusion

  4. Androgen receptor splice variant 7 (AR-V7) and drug efficacy in castration-resistant prostate cancer: Biomarker for treatment selection exclusion or inclusion?

    PubMed

    Leibrand, Crystal R; Price, Douglas K; Figg, William D

    2016-05-01

    Currently there are no molecular biomarkers used to help guide treatment selection for those patients with castration-resistant prostate cancer. A recent study published in JAMA Oncology (Antonarakis et al.) presents evidence supporting the potential use of androgen receptor splice variant 7 as a biomarker for optimal treatment selection in this population. PMID:26985596

  5. Innate immune activity conditions the effect of regulatory variants upon monocyte gene expression.

    PubMed

    Fairfax, Benjamin P; Humburg, Peter; Makino, Seiko; Naranbhai, Vivek; Wong, Daniel; Lau, Evelyn; Jostins, Luke; Plant, Katharine; Andrews, Robert; McGee, Chris; Knight, Julian C

    2014-03-01

    To systematically investigate the impact of immune stimulation upon regulatory variant activity, we exposed primary monocytes from 432 healthy Europeans to interferon-γ (IFN-γ) or differing durations of lipopolysaccharide and mapped expression quantitative trait loci (eQTLs). More than half of cis-eQTLs identified, involving hundreds of genes and associated pathways, are detected specifically in stimulated monocytes. Induced innate immune activity reveals multiple master regulatory trans-eQTLs including the major histocompatibility complex (MHC), coding variants altering enzyme and receptor function, an IFN-β cytokine network showing temporal specificity, and an interferon regulatory factor 2 (IRF2) transcription factor-modulated network. Induced eQTL are significantly enriched for genome-wide association study loci, identifying context-specific associations to putative causal genes including CARD9, ATM, and IRF8. Thus, applying pathophysiologically relevant immune stimuli assists resolution of functional genetic variants. PMID:24604202

  6. Innate Immune Activity Conditions the Effect of Regulatory Variants upon Monocyte Gene Expression

    PubMed Central

    Fairfax, Benjamin P.; Naranbhai, Vivek; Wong, Daniel; Lau, Evelyn; Jostins, Luke; Plant, Katharine; Andrews, Robert; McGee, Chris; Knight, Julian C.

    2014-01-01

    To systematically investigate the impact of immune stimulation upon regulatory variant activity, we exposed primary monocytes from 432 healthy Europeans to interferon-γ (IFN-γ) or differing durations of lipopolysaccharide and mapped expression quantitative trait loci (eQTLs). More than half of cis-eQTLs identified, involving hundreds of genes and associated pathways, are detected specifically in stimulated monocytes. Induced innate immune activity reveals multiple master regulatory trans-eQTLs including the major histocompatibility complex (MHC), coding variants altering enzyme and receptor function, an IFN-β cytokine network showing temporal specificity, and an interferon regulatory factor 2 (IRF2) transcription factor–modulated network. Induced eQTL are significantly enriched for genome-wide association study loci, identifying context-specific associations to putative causal genes including CARD9, ATM, and IRF8. Thus, applying pathophysiologically relevant immune stimuli assists resolution of functional genetic variants. PMID:24604202

  7. Second-Generation HSP90 Inhibitor Onalespib Blocks mRNA Splicing of Androgen Receptor Variant 7 in Prostate Cancer Cells.

    PubMed

    Ferraldeschi, Roberta; Welti, Jonathan; Powers, Marissa V; Yuan, Wei; Smyth, Tomoko; Seed, George; Riisnaes, Ruth; Hedayat, Somaieh; Wang, Hannah; Crespo, Mateus; Nava Rodrigues, Daniel; Figueiredo, Ines; Miranda, Susana; Carreira, Suzanne; Lyons, John F; Sharp, Swee; Plymate, Stephen R; Attard, Gerhardt; Wallis, Nicola; Workman, Paul; de Bono, Johann S

    2016-05-01

    Resistance to available hormone therapies in prostate cancer has been associated with alternative splicing of androgen receptor (AR) and specifically, the expression of truncated and constitutively active AR variant 7 (AR-V7). The transcriptional activity of steroid receptors, including AR, is dependent on interactions with the HSP90 chaperone machinery, but it is unclear whether HSP90 modulates the activity or expression of AR variants. Here, we investigated the effects of HSP90 inhibition on AR-V7 in prostate cancer cell lines endogenously expressing this variant. We demonstrate that AR-V7 and full-length AR (AR-FL) were depleted upon inhibition of HSP90. However, the mechanisms underlying AR-V7 depletion differed from those for AR-FL. Whereas HSP90 inhibition destabilized AR-FL and induced its proteasomal degradation, AR-V7 protein exhibited higher stability than AR-FL and did not require HSP90 chaperone activity. Instead, HSP90 inhibition resulted in the reduction of AR-V7 mRNA levels but did not affect total AR transcript levels, indicating that HSP90 inhibition disrupted AR-V7 splicing. Bioinformatic analyses of transcriptome-wide RNA sequencing data confirmed that the second-generation HSP90 inhibitor onalespib altered the splicing of at least 557 genes in prostate cancer cells, including AR. These findings indicate that the effects of HSP90 inhibition on mRNA splicing may prove beneficial in prostate cancers expressing AR-V7, supporting further clinical investigation of HSP90 inhibitors in malignancies no longer responsive to androgen deprivation. Cancer Res; 76(9); 2731-42. ©2016 AACR. PMID:27197266

  8. Cellulase variants

    SciTech Connect

    Blazej, Robert; Toriello, Nicholas; Emrich, Charles; Cohen, Richard N.; Koppel, Nitzan

    2015-07-14

    This invention provides novel variant cellulolytic enzymes having improved activity and/or stability. In certain embodiments the variant cellulotyic enzymes comprise a glycoside hydrolase with or comprising a substitution at one or more positions corresponding to one or more of residues F64, A226, and/or E246 in Thermobifida fusca Cel9A enzyme. In certain embodiments the glycoside hydrolase is a variant of a family 9 glycoside hydrolase. In certain embodiments the glycoside hydrolase is a variant of a theme B family 9 glycoside hydrolase.

  9. Juvenile GM2 gangliosidosis (AMB variant): inability to activate hexosaminidase A by activator protein.

    PubMed Central

    Inui, K; Grebner, E E; Jackson, L G; Wenger, D A

    1983-01-01

    Two sibling from a consanguineous Puerto Rican marriage were found to have a juvenile-onset type of lipidosis first noted at age 2 1/2 by expressing difficulties with motor function and developmental delay. They continued to deteriorate, showing muscle atrophy, spasticity, and loss of speech, and death occurred at ages 7 and 8. Examination of the brains from these patients revealed that the concentration of GM2 ganglioside was about 56% of the total gangliosides. Hexosaminidase and percent hexosaminidase A (HEX A) and other lysosomal enzymes were normal in cultured skin fibroblasts, liver, and brain. The concentration of the activator protein required for the enzymatic hydrolysis of GM2 ganglioside was in high normal levels in the brain of the patient available. However, the HEX A from the patient's brain and liver as well as from skin fibroblast lysates could not be activated to hydrolyze GM2 ganglioside by the activator protein from a control or himself. The HEX A from a control could be activated by the activator protein from controls or this patient. These patients appear to have a defect in HEX A, which does not affect it heat stability, electrophoretic migration, and activity toward fluorogenic substrates, but may affect the binding of the activator protein required for GM2 ganglioside hydrolysis. We propose to call these patients the AMB variant of GM2 gangliosidosis to denote the mutation in HEX A but with normal levels of HEX A and B with synthetic substrates. This is to distinguish these patients from those missing the activator protein and normal HEX A and B levels. Images Fig. 1 Fig. 2 PMID:6224417

  10. The geology and 40Ar/ 39Ar geochronology of magmatic activity and related mineralization in the Nevados del Famatina mining district, La Rioja province, Argentina

    NASA Astrophysics Data System (ADS)

    Losada-Calderón, A. J.; McBride, S. L.; Bloom, M. S.

    1994-01-01

    The Nevados del Famatina mining district (NFMD) is located in La Rioja province, Argentina. This district contains porphyry-style mineralization (Nevados del Famatina) and high sulfidation veins (La Mejicana). The stratigraphic column in the NFMD begins with Cambrian siltstones which were metamorphosed during the Late Ordovician - Early Silurian and intruded by Late Ordovician-Silurian granitic rocks. These units were covered by Upper Paleozoic and Tertiary continental sedimentary rocks which are intercalated with and overlain by dacitic-rhyodacitic porphyritic rocks (Mogote Formation) emplaced during the Pliocene. All these units are covered by Pleistocene sediments and Quaternary alluvial and colluvial deposits. Magmatic activity and related mineralization in the NFMD have been dated by the 40Ar/ 39Ar technique. Step heating studies of orthoclase and biotite phenocrysts from the Mogote Formation in the NFMD suggest that the igneous rocks were emplaced around 5.0±0.3 Ma ago. However, plateau ages of biotite from the outer carapace of the subjacent granodioritic magma chamber and of muscovite from quartz-sericite alteration at both Nevados del Famatina and La Mejicana are around 3.8±0.2 Ma. Emplacement of the shallow stocks is separated from cooling of the outer carapace of the subjacent granodioritic magma chamber to temperatures below 350° C by a time span of approximately 1 Ma. During this interval, a convective hydrothermal system was established proximal to the granodioritic magma chamber, which resulted in porphyry molybdenumcoppergold mineralization adjacent to the igneous rocks and more distal high sulfidation veins located in fault zones.

  11. Extraction of Desired Signal Based on AR Model with Its Application to Atrial Activity Estimation in Atrial Fibrillation

    NASA Astrophysics Data System (ADS)

    Wang, Gang; Rao, Ni-ni; Shepherd, Simon J.; Beggs, Clive B.

    2008-12-01

    The use of electrocardiograms (ECGs) to diagnose and analyse atrial fibrillation (AF) has received much attention recently. When studying AF, it is important to isolate the atrial activity (AA) component of the ECG plot. We present a new autoregressive (AR) model for semiblind source extraction of the AA signal. Previous researchers showed that one could extract a signal with the smallest normalized mean square prediction error (MSPE) as the first output from linear mixtures by minimizing the MSPE. However the extracted signal will be not always the desired one even if the AR model parameters of one source signal are known. We introduce a new cost function, which caters for the specific AR model parameters, to extract the desired source. Through theoretical analysis and simulation we demonstrate that this algorithm can extract any desired signal from mixtures provided that its AR parameters are first obtained. We use this approach to extract the AA signal from 12-lead surface ECG signals for hearts undergoing AF. In our methodology we roughly estimated the AR parameters from the fibrillatory wave segment in the V1 lead, and then used this algorithm to extract the AA signal. We validate our approach using real-world ECG data.

  12. No first ionization potential fractionation in the active stars AR Piscium and AY Ceti

    NASA Astrophysics Data System (ADS)

    Sanz-Forcada, J.; Affer, L.; Micela, G.

    2009-10-01

    Context: The comparison of coronal and photospheric abundances in cool stars is an essential question to resolve. In the Sun an enhancement of the elements with low first ionization potential (FIP) is observed in the corona with respect to the photosphere. Stars with high levels of activity seem to show a depletion of elements with low FIP when compared to solar standard values; however, the few cases of active stars in which photospheric values are available for comparison lead to confusing results, and an enlargement of the sample is mandatory for solving this longstanding problem. Aims: We calculate in this paper the photospheric and coronal abundances of two well known active binary systems, AR Psc and AY Cet, to get further insight into the complications of coronal abundances. Methods: Coronal abundances of 9 elements were calculated by means of the reconstruction of a detailed emission measure distribution, using a line-based method that considers the lines from different elements separately. Photospheric abundances of 8 elements were calculated using high-resolution optical spectra of the stars. Results: The results once again show a lack of any FIP-related effect in the coronal abundances of the stars. The presence of metal abundance depletion (MAD) or inverse FIP effects in some stars could stem from a mistaken comparison to solar photospheric values or from a deficient calculation of photospheric abundances in fast-rotating stars. Conclusions: The lack of FIP fractionation seems to confirm that Alfvén waves combined with pondermotive forces are dominant in the corona of active stars. Tables 2 and 3 are only available in electronic form at http://www.aanda.org

  13. Titanium spallation cross sections between 30 and 584 MeV and Ar-39 activities on the moon

    NASA Technical Reports Server (NTRS)

    Steinburnn, F.; Fireman, E. L.

    1974-01-01

    The production cross sections of Ar39 for Ti spallation at 45-, 319-, 433-, and 584-MeV proton energies were measured to be 0.37 + or - 0.09, 12.4 + or - 3.7, 9.1 + or - 2.7, and 17.8 + or - 6.2 mb, respectively. Normalized Ar39 production rates and activities are also derived for protons above 40 MeV and for three differential proton spectra of the type approximately E(- alpha). It is concluded that, even for samples of high-Ti content, Ti spallation by solar protons below 200-MeV energy does not contribute significantly to their Ar39 radioactivity.

  14. Protective LRRK2 R1398H Variant Enhances GTPase and Wnt Signaling Activity

    PubMed Central

    Nixon-Abell, Jonathon; Berwick, Daniel C.; Grannó, Simone; Spain, Victoria A.; Blackstone, Craig; Harvey, Kirsten

    2016-01-01

    Mutations in LRRK2 are a common cause of familial and idiopathic Parkinson’s disease (PD). Recently, the LRRK2 GTPase domain R1398H variant was suggested in genetic studies to confer protection against PD but mechanistic data supporting this is lacking. Here, we present evidence that R1398H affects GTPase function, axon outgrowth, and Wnt signaling in a manner opposite to pathogenic LRRK2 mutations. LRRK2 R1398H GTPase domain dimerization and GTP hydrolysis were increased whereas GTP binding was reduced, leading to a decrease in active GTP-bound LRRK2. This protective variant also increased axon length of primary cortical neurones in comparison to wild-type LRRK2, whereas the R1441G LRRK2 pathogenic mutant decreased axon outgrowth. Importantly, R1398H enhanced the stimulatory effect of LRRK2 on canonical Wnt signaling whereas the G2385R risk variant, in accordance with all previously tested pathogenic LRRK2 mutants, had the opposite effect. Molecular modeling placed R1398H in close proximity to PD-causing mutations suggesting that this protective LRRK2 variant, like familial mutations, affects intramolecular RocCOR domain interactions. Thus, our data suggest that R1398H LRRK2 is a bona fide protective variant. The opposite effects of protective versus PD associated LRRK2 variants on GTPase function and canonical Wnt signaling activity also suggests that regulation of these two basic signaling mechanisms is important for neuronal function. We conclude that LRRK2 mediated Wnt signaling and GTPase function are fundamental in conferring disease susceptibility and have clear implications for therapeutic target identification. PMID:27013965

  15. AR-V7 and prostate cancer: The watershed for treatment selection?

    PubMed

    Ciccarese, Chiara; Santoni, Matteo; Brunelli, Matteo; Buti, Sebastiano; Modena, Alessandra; Nabissi, Massimo; Artibani, Walter; Martignoni, Guido; Montironi, Rodolfo; Tortora, Giampaolo; Massari, Francesco

    2016-02-01

    The androgen receptor (AR) plays a key role in progression to metastatic castration-resistant prostate cancer (mCRPC). Despite the recent progress in targeting persistent AR activity with the next-generation hormonal therapies (abiraterone acetate and enzalutamide), resistance to these agents limits therapeutic efficacy for many patients. Several explanations for response and/or resistance to abiraterone acetate and enzalutamide are emerging, but growing interest is focusing on importance of AR splice variants (AR-Vs) and in particular of AR-V7. Increasing evidences highlight the concept that variant expression could be used as a potential predictive biomarker and a therapeutic target in advanced prostate cancer. Therefore, understanding the mechanisms of treatment resistance or sensitivity can help to achieve a more effective management of mCRPC, increasing clinical outcomes and representing a promising and engaging area of prostate cancer research. PMID:26827690

  16. PROBA2/SWAP Observations of the Unusual Activity of AR12192

    NASA Astrophysics Data System (ADS)

    Seaton, Daniel B.; West, Matthew J.

    2015-04-01

    AR 12192 was the most productive active region of the present solar cycle in terms of flares, but it exhibited many unusual properties. It announced its presence on 14 October 2014 with an eruption that led to the formation of perhaps the largest post-eruptive loop system seen in the solar corona in solar cycle 24. Later this region produced a series of large M- and X-class flares, most of which were essentially confined flares, not associated with any coronal mass ejection. During its second passage across the solar disk, it was associated with a huge region of open field that was observed extending to heights as great as 3 solar-radii in the EUV, some of the largest heights at which any EUV structure has been observed. We discuss our observations of this region with the SWAP EUV imager on board the PROBA2 spacecraft and the implications of the many unusual events and structures associated with this region on our understanding of the mechanisms, such as magnetic reconnection, that drive coronal dynamics, in particular those involved in the onset of flares and eruptions.

  17. A Computational Methodology to Screen Activities of Enzyme Variants

    PubMed Central

    Hediger, Martin R.; De Vico, Luca; Svendsen, Allan; Besenmatter, Werner; Jensen, Jan H.

    2012-01-01

    We present a fast computational method to efficiently screen enzyme activity. In the presented method, the effect of mutations on the barrier height of an enzyme-catalysed reaction can be computed within 24 hours on roughly 10 processors. The methodology is based on the PM6 and MOZYME methods as implemented in MOPAC2009, and is tested on the first step of the amide hydrolysis reaction catalyzed by the Candida Antarctica lipase B (CalB) enzyme. The barrier heights are estimated using adiabatic mapping and shown to give barrier heights to within 3 kcal/mol of B3LYP/6-31G(d)//RHF/3-21G results for a small model system. Relatively strict convergence criteria (0.5 kcal/(molÅ)), long NDDO cutoff distances within the MOZYME method (15 Å) and single point evaluations using conventional PM6 are needed for reliable results. The generation of mutant structures and subsequent setup of the semiempirical calculations are automated so that the effect on barrier heights can be estimated for hundreds of mutants in a matter of weeks using high performance computing. PMID:23284627

  18. Bond activation with an apparently benign ethynyl dithiocarbamate Ar-C≡C-S-C(S)NR2.

    PubMed

    Ung, Gaël; Frey, Guido D; Schoeller, Wolfgang W; Bertrand, Guy

    2011-10-10

    The hedgehog molecule: A simple ethynyl dithiocarbamate [Ar-C≡C-S-C(S)NR(2)] is able to cleave a broad range of enthalpically strong σ bonds and to activate carbon dioxide and elemental sulfur. Depending on the substrate, the bond activation process involves either the existence of an equilibrium with the nonobservable mesoionic carbene isomer or the cooperation of the nucleophilic carbon-carbon triple bond and the electrophilic CS carbon atom. PMID:23210141

  19. CALCULATING SEPARATE MAGNETIC FREE ENERGY ESTIMATES FOR ACTIVE REGIONS PRODUCING MULTIPLE FLARES: NOAA AR11158

    SciTech Connect

    Tarr, Lucas; Longcope, Dana; Millhouse, Margaret

    2013-06-10

    It is well known that photospheric flux emergence is an important process for stressing coronal fields and storing magnetic free energy, which may then be released during a flare. The Helioseismic and Magnetic Imager (HMI) on board the Solar Dynamics Observatory (SDO) captured the entire emergence of NOAA AR 11158. This region emerged as two distinct bipoles, possibly connected underneath the photosphere, yet characterized by different photospheric field evolutions and fluxes. The combined active region complex produced 15 GOES C-class, two M-class, and the X2.2 Valentine's Day Flare during the four days after initial emergence on 2011 February 12. The M and X class flares are of particular interest because they are nonhomologous, involving different subregions of the active region. We use a Magnetic Charge Topology together with the Minimum Current Corona model of the coronal field to model field evolution of the complex. Combining this with observations of flare ribbons in the 1600 A channel of the Atmospheric Imaging Assembly on board SDO, we propose a minimization algorithm for estimating the amount of reconnected flux and resulting drop in magnetic free energy during a flare. For the M6.6, M2.2, and X2.2 flares, we find a flux exchange of 4.2 Multiplication-Sign 10{sup 20} Mx, 2.0 Multiplication-Sign 10{sup 20} Mx, and 21.0 Multiplication-Sign 10{sup 20} Mx, respectively, resulting in free energy drops of 3.89 Multiplication-Sign 10{sup 30} erg, 2.62 Multiplication-Sign 10{sup 30} erg, and 1.68 Multiplication-Sign 10{sup 32} erg.

  20. Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β2AR.

    PubMed

    Lamichhane, Rajan; Liu, Jeffrey J; Pljevaljcic, Goran; White, Kate L; van der Schans, Edwin; Katritch, Vsevolod; Stevens, Raymond C; Wüthrich, Kurt; Millar, David P

    2015-11-17

    Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β2-adrenergic receptor (β2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs. PMID:26578769

  1. Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity.

    PubMed

    Nguyen, Hien Anh; Su, Ying; Lavie, Arnon

    2016-08-19

    Current FDA-approved l-asparaginases also possess significant l-glutaminase activity, which correlates with many of the toxic side effects of these drugs. Therefore, l-asparaginases with reduced l-glutaminase activity are predicted to be safer. We exploited our recently described structures of the Erwinia chrysanthemi l-asparaginase (ErA) to inform the design of mutants with diminished ability to hydrolyze l-glutamine. Structural analysis of these variants provides insight into the molecular basis for the increased l-asparagine specificity. A primary role is attributed to the E63Q mutation that acts to hinder the correct positioning of l-glutamine but not l-asparagine. The substitution of Ser-254 with either an asparagine or a glutamine increases the l-asparagine specificity but only when combined with the E63Q mutation. The A31I mutation reduces the substrate Km value; this is a key property to allow the required therapeutic l-asparagine depletion. Significantly, an ultra-low l-glutaminase ErA variant maintained its cell killing ability. By diminishing the l-glutaminase activity of these highly active l-asparaginases, our engineered ErA variants hold promise as l-asparaginases with fewer side effects. PMID:27354283

  2. High throughput microscopy identifies bisphenol AP, a bisphenol A analog, as a novel AR down-regulator

    PubMed Central

    Stossi, Fabio; Dandekar, Radhika D.; Bolt, Michael J.; Newberg, Justin Y.; Mancini, Maureen G.; Kaushik, Akash K.; Putluri, Vasanta; Sreekumar, Arun; Mancini, Michael A.

    2016-01-01

    Prostate cancer remains a deadly disease especially when patients become resistant to drugs that target the Androgen Receptor (AR) ligand binding domain. At this stage, patients develop recurring castrate-resistant prostate cancers (CRPCs). Interestingly, CRPC tumors maintain dependency on AR for growth; moreover, in CRPCs, constitutively active AR splice variants (e.g., AR-V7) begin to be expressed at higher levels. These splice variants lack the ligand binding domain and are rendered insensitive to current endocrine therapies. Thus, it is of paramount importance to understand what regulates the expression of AR and its splice variants to identify new therapeutic strategies in CRPCs. Here, we used high throughput microscopy and quantitative image analysis to evaluate effects of selected endocrine disruptors on AR levels in multiple breast and prostate cancer cell lines. Bisphenol AP (BPAP), which is used in chemical and medical industries, was identified as a down-regulator of both full length AR and the AR-V7 splice variant. We validated its activity by performing time-course, dose-response, Western blot and qPCR analyses. BPAP also reduced the percent of cells in S phase, which was accompanied by a ~60% loss in cell numbers and colony formation in anchorage-independent growth assays. Moreover, it affected mitochondria size and cell metabolism. In conclusion, our high content analysis-based screening platform was used to classify the effect of compounds on endogenous ARs, and identified BPAP as being capable of causing AR (both full-length and variants) down-regulation, cell cycle arrest and metabolic alterations in CRPC cell lines. PMID:26918604

  3. An anthrax toxin variant with an improved activity in tumor targeting

    PubMed Central

    Wein, Alexander N.; Peters, Diane E.; Valivullah, Zaheer; Hoover, Benjamin J.; Tatineni, Aparna; Ma, Qian; Fattah, Rasem; Bugge, Thomas H.; Leppla, Stephen H.; Liu, Shihui

    2015-01-01

    Anthrax lethal toxin (LT) is an A-B type toxin secreted by Bacillus anthracis, consisting of the cellular binding moiety, protective antigen (PA), and the catalytic moiety, lethal factor (LF). To target cells, PA binds to cell-surface receptors and is then proteolytically processed forming a LF-binding competent PA oligomer where each LF binding site is comprised of three subsites on two adjacent PA monomers. We previously generated PA-U2-R200A, a urokinase-activated PA variant with LF-binding subsite II residue Arg200 mutated to Ala, and PA-L1-I210A, a matrix metalloproteinase-activated PA variant with subsite III residue Ile210 mutated to Ala. PA-U2-R200A and PA-L1-I210A displayed reduced cytotoxicity when used singly. However, when combined, they formed LF-binding competent heterogeneous oligomers by intermolecular complementation, and achieved high specificity in tumor targeting. Nevertheless, each of these proteins, in particular PA-L1-I210A, retained residual LF-binding ability. In this work, we screened a library containing all possible amino acid substitutions for LF-binding site to find variants with activity strictly dependent upon intermolecular complementation. PA-I207R was identified as an excellent replacement for the original clockwise-side variant, PA-I210A. Consequently, the new combination of PA-L1-I207R and PA-U2-R200A showed potent anti-tumor activity and low toxicity, exceeding the performance of the original combination, and warranting further investigation. PMID:26584669

  4. Comparative toxicity and efficacy of engineered anthrax lethal toxin variants with broad anti-tumor activities

    SciTech Connect

    Peters, Diane E.; Hoover, Benjamin; Cloud, Loretta Grey; Liu, Shihui; Molinolo, Alfredo A.; Leppla, Stephen H.; Bugge, Thomas H.

    2014-09-01

    We have previously designed and characterized versions of anthrax lethal toxin that are selectively cytotoxic in the tumor microenvironment and which display broad and potent anti-tumor activities in vivo. Here, we have performed the first direct comparison of the safety and efficacy of three engineered anthrax lethal toxin variants requiring activation by either matrix-metalloproteinases (MMPs), urokinase plasminogen activator (uPA) or co-localized MMP/uPA activities. C57BL/6J mice were challenged with six doses of engineered toxins via intraperitoneal (I.P.) or intravenous (I.V.) dose routes to determine the maximum tolerated dose for six administrations (MTD6) and dose-limiting toxicities. Efficacy was evaluated using the B16-BL6 syngraft model of melanoma; mice bearing established tumors were treated with six I.P. doses of toxin and tumor measurements and immunohistochemistry, paired with terminal blood work, were used to elaborate upon the anti-tumor mechanism and relative efficacy of each variant. We found that MMP-, uPA- and dual MMP/uPA-activated anthrax lethal toxins exhibited the same dose-limiting toxicity; dose-dependent GI toxicity. In terms of efficacy, all three toxins significantly reduced primary B16-BL6 tumor burden, ranging from 32% to 87% reduction, and they also delayed disease progression as evidenced by dose-dependent normalization of blood work values. While target organ toxicity and effective doses were similar amongst the variants, the dual MMP/uPA-activated anthrax lethal toxin exhibited the highest I.P. MTD6 and was 1.5–3-fold better tolerated than the single MMP- and uPA-activated toxins. Overall, we demonstrate that this dual MMP/uPA-activated anthrax lethal toxin can be administered safely and is highly effective in a preclinical model of melanoma. This modified bacterial cytotoxin is thus a promising candidate for further clinical development and evaluation for use in treating human cancers. - Highlights: • Toxicity and anti

  5. A functional variant of elafin with improved anti-inflammatory activity for pulmonary inflammation.

    PubMed

    Small, Donna M; Zani, Marie-Louise; Quinn, Derek J; Dallet-Choisy, Sandrine; Glasgow, Arlene M A; O'Kane, Cecilia; McAuley, Danny F; McNally, Paul; Weldon, Sinéad; Moreau, Thierry; Taggart, Clifford C

    2015-01-01

    Elafin is a serine protease inhibitor produced by epithelial and immune cells with anti-inflammatory properties. Research has shown that dysregulated protease activity may elicit proteolytic cleavage of elafin, thereby impairing the innate immune function of the protein. The aim of this study was to generate variants of elafin (GG- and QQ-elafin) that exhibit increased protease resistance while retaining the biological properties of wild-type (WT) elafin. Similar to WT-elafin, GG- and QQ-elafin variants retained antiprotease activity and susceptibility to transglutaminase-mediated fibronectin cross-linking. However, in contrast to WT-elafin, GG- and QQ-elafin displayed significantly enhanced resistance to degradation when incubated with bronchoalveolar lavage fluid from patients with cystic fibrosis. Intriguingly, both variants, particularly GG-elafin, demonstrated improved lipopolysaccharide (LPS) neutralization properties in vitro. In addition, GG-elafin showed improved anti-inflammatory activity in a mouse model of LPS-induced acute lung inflammation. Inflammatory cell infiltration into the lung was reduced in lungs of mice treated with GG-elafin, predominantly neutrophilic infiltration. A reduction in MCP-1 levels in GG-elafin treated mice compared to the LPS alone treatment group was also demonstrated. GG-elafin showed increased functionality when compared to WT-elafin and may be of future therapeutic relevance in the treatment of lung diseases characterized by a protease burden. PMID:25189740

  6. Potential role of TCF7L2 gene variants on cardiac sympathetic/parasympathetic activity

    PubMed Central

    Boccardi, Virginia; Ambrosino, Immacolata; Papa, Michela; Fiore, Daniela; Rizzo, Maria Rosaria; Paolisso, Giuseppe; Barbieri, Michelangela

    2010-01-01

    Variants in transcription factor 7-like 2 (266096218TCF7L2266096218USuser266096218Gene names have been italicized per house style. Please check and confirm whether there are other instances that need to be italicized or instances where italics have been inappropriately applied.) gene have been found strongly associated with an increased risk of type 2 diabetes, as well as with an impairment of glucagon-like peptide-1 (GLP-1) signalling chain. In rats, stimulation of central GLP-1 receptors increases heart rate and activates autonomic regulatory neurons. We aimed to evaluate the potential role of TCF7L2 gene polymorphisms on sympathovagal response in relation to changes in plasma insulin and/or GLP-1 concentration after glucose ingestion. Genotyping was performed for rs12255372 and rs7903146 TCF7L2 gene variants in 250 non-related healthy volunteers (mean age 27±3 years). Consistent with previous reports, both single-nucleotide polymorphisms were in strong linkage disequilibrium (D′=0.87, r2=0.76). A subset of 167 patients underwent an oral glucose tolerance test while a continuous recording of heart rate variability was performed. At baseline, no differences in fasting plasma insulin, in GLP-1 levels and in LF/HF (low frequency/high frequency) ratio between the three genotypes were found. Along with glucose ingestion TT subjects had lower INSAUC (insulin area under curve), as well as higher LF/HFAUC (LF/HF area under curve) values. No difference in GLP-1AUC (GLP-1 area under curve) between TCF7L2 gene variants was found. A multivariate analysis including multiple covariates showed that only INSAUC, GLP-1AUC and TCF7L2 gene variants were independently associated with LF/HFAUC. In conclusion, TT genotype of rs12255372 and rs7903146 TCF7L2 gene variants is associated with lower insulin secretion and higher cardiosympathetic activity. Moreover, such effect is independent of GLP-1 and insulin plasma concentrations suggesting a potential role of such gene variants in

  7. An international land-biosphere model benchmarking activity for the IPCC Fifth Assessment Report (AR5)

    SciTech Connect

    Hoffman, Forrest M; Randerson, James T; Thornton, Peter E; Bonan, Gordon; Erickson III, David J; Fung, Inez

    2009-12-01

    The need to capture important climate feedbacks in general circulation models (GCMs) has resulted in efforts to include atmospheric chemistry and land and ocean biogeochemistry into the next generation of production climate models, called Earth System Models (ESMs). While many terrestrial and ocean carbon models have been coupled to GCMs, recent work has shown that such models can yield a wide range of results (Friedlingstein et al., 2006). This work suggests that a more rigorous set of global offline and partially coupled experiments, along with detailed analyses of processes and comparisons with measurements, are needed. The Carbon-Land Model Intercomparison Project (C-LAMP) was designed to meet this need by providing a simulation protocol and model performance metrics based upon comparisons against best-available satellite- and ground-based measurements (Hoffman et al., 2007). Recently, a similar effort in Europe, called the International Land Model Benchmark (ILAMB) Project, was begun to assess the performance of European land surface models. These two projects will now serve as prototypes for a proposed international land-biosphere model benchmarking activity for those models participating in the IPCC Fifth Assessment Report (AR5). Initially used for model validation for terrestrial biogeochemistry models in the NCAR Community Land Model (CLM), C-LAMP incorporates a simulation protocol for both offline and partially coupled simulations using a prescribed historical trajectory of atmospheric CO2 concentrations. Models are confronted with data through comparisons against AmeriFlux site measurements, MODIS satellite observations, NOAA Globalview flask records, TRANSCOM inversions, and Free Air CO2 Enrichment (FACE) site measurements. Both sets of experiments have been performed using two different terrestrial biogeochemistry modules coupled to the CLM version 3 in the Community Climate System Model version 3 (CCSM3): the CASA model of Fung, et al., and the carbon

  8. An International Land-Biosphere Model Benchmarking Activity for the IPCC Fifth Assessment Report (AR5)

    NASA Astrophysics Data System (ADS)

    Hoffman, F. M.; Randerson, J. T.; Thornton, P. E.; Bonan, G. B.; Brooks, B. J.; Erickson, D. J.; Fung, I.

    2009-12-01

    The need to capture important climate feedbacks in general circulation models (GCMs) has resulted in efforts to include atmospheric chemistry and land and ocean biogeochemistry into the next generation of production climate models, called Earth System Models (ESMs). While many terrestrial and ocean carbon models have been coupled to GCMs, recent work has shown that such models can yield a wide range of results (Friedlingstein et al., 2006). This work suggests that a more rigorous set of global offline and partially coupled experiments, along with detailed analyses of processes and comparisons with measurements, are needed. The Carbon-Land Model Intercomparison Project (C-LAMP) was designed to meet this need by providing a simulation protocol and model performance metrics based upon comparisons against best-available satellite- and ground-based measurements (Hoffman et al., 2007). Recently, a similar effort in Europe, called the International Land Model Benchmark (ILAMB) Project, was begun to assess the performance of European land surface models. These two projects will now serve as prototypes for a proposed international land-biosphere model benchmarking activity for those models participating in the IPCC Fifth Assessment Report (AR5). Initially used for model validation for terrestrial biogeochemistry models in the NCAR Community Land Model (CLM), C-LAMP incorporates a simulation protocol for both offline and partially coupled simulations using a prescribed historical trajectory of atmospheric CO2 concentrations. Models are confronted with data through comparisons against AmeriFlux site measurements, MODIS satellite observations, NOAA Globalview flask records, TRANSCOM inversions, and Free Air CO2 Enrichment (FACE) site measurements. Both sets of experiments have been performed using two different terrestrial biogeochemistry modules coupled to the CLM version 3 in the Community Climate System Model version 3 (CCSM3): the CASA model of Fung, et al., and the carbon

  9. USDA-ARS extension activities in medical, veterinary and urban entomology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Within the USDA Agricultural Research Service (USDA-ARS), National Program 104 conducts research on veterinary, medical, and urban entomology. The goal of this program is to develop more effective methods of preventing or suppressing insects, ticks, and mites that affect animal and human well-being....

  10. Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity.

    PubMed

    Markovski, Monica; Bohrhunter, Jessica L; Lupoli, Tania J; Uehara, Tsuyoshi; Walker, Suzanne; Kahne, Daniel E; Bernhardt, Thomas G

    2016-04-26

    To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a of Escherichia coli require the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b-LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed. PMID:27071112

  11. Neuromuscular Activity of Upper and Lower Limbs during two Backstroke Swimming Start Variants

    PubMed Central

    De Jesus, Karla; De Jesus, Kelly; Medeiros, Alexandre I. A.; Gonçalves, Pedro; Figueiredo, Pedro; Fernandes, Ricardo J.; Vilas-Boas, João Paulo

    2015-01-01

    A proficient start is decisive in sprint competitive swimming events and requires swimmers’ to exert maximal forces in a short period to complete the task successfully. The aim of this study was to compare the electromyographic (EMG) activity in-between the backstroke start with feet positioned parallel and partially emerged performed with the hands on the highest horizontal and on the vertical handgrip at hands-off, take-off, flight and entry start phases. EMG comparisons between starting variants were supported by upper and lower limb joint angles at starting position and 15 m start time data. Following a four-week start training to familiarize participants with each start variant, 10 male competitive backstroke swimmers performed randomly six 15 m maximal trials, being three of each start variant. Surface EMG of Biceps Brachii, Triceps Brachii, Rectus Femoris, Biceps Femoris, Gastrocnemius Medialis and Tibialis Anterior was recorded and processed using the time integral EMG (iEMG). Eight video cameras (four surface and four underwater) were used to determine backstroke start phases and joint angles at starting position. EMG, joint angles and temporal parameters have not evidenced changes due to the different handgrips. Nevertheless, clear differences were observed in both variants for upper and lower limb muscles activity among starting phases (e.g. Biceps Brachii at take-off vs. flight phase, 15.17% ± 2.76% and 22.38% ± 4.25%; 14.24% ± 7.11% and 25.90% ± 8.65%, for variant with hands horizontal and vertically positioned, respectively). It was concluded that different handgrips did not affect EMG, kinematics and temporal profile in backstroke start. Despite coaches might plan similar strength training for both start variants, further attention should be given on the selection of proper exercises to maximize the contribution of relevant muscles at different starting phases. Key points An effective swim start component (from the starting signal until the

  12. Neuromuscular Activity of Upper and Lower Limbs during two Backstroke Swimming Start Variants.

    PubMed

    De Jesus, Karla; De Jesus, Kelly; Medeiros, Alexandre I A; Gonçalves, Pedro; Figueiredo, Pedro; Fernandes, Ricardo J; Vilas-Boas, João Paulo

    2015-09-01

    A proficient start is decisive in sprint competitive swimming events and requires swimmers' to exert maximal forces in a short period to complete the task successfully. The aim of this study was to compare the electromyographic (EMG) activity in-between the backstroke start with feet positioned parallel and partially emerged performed with the hands on the highest horizontal and on the vertical handgrip at hands-off, take-off, flight and entry start phases. EMG comparisons between starting variants were supported by upper and lower limb joint angles at starting position and 15 m start time data. Following a four-week start training to familiarize participants with each start variant, 10 male competitive backstroke swimmers performed randomly six 15 m maximal trials, being three of each start variant. Surface EMG of Biceps Brachii, Triceps Brachii, Rectus Femoris, Biceps Femoris, Gastrocnemius Medialis and Tibialis Anterior was recorded and processed using the time integral EMG (iEMG). Eight video cameras (four surface and four underwater) were used to determine backstroke start phases and joint angles at starting position. EMG, joint angles and temporal parameters have not evidenced changes due to the different handgrips. Nevertheless, clear differences were observed in both variants for upper and lower limb muscles activity among starting phases (e.g. Biceps Brachii at take-off vs. flight phase, 15.17% ± 2.76% and 22.38% ± 4.25%; 14.24% ± 7.11% and 25.90% ± 8.65%, for variant with hands horizontal and vertically positioned, respectively). It was concluded that different handgrips did not affect EMG, kinematics and temporal profile in backstroke start. Despite coaches might plan similar strength training for both start variants, further attention should be given on the selection of proper exercises to maximize the contribution of relevant muscles at different starting phases. Key pointsAn effective swim start component (from the starting signal until the

  13. Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity.

    PubMed

    van der Veen, Bart A; Potocki-Véronèse, Gabrielle; Albenne, Cécile; Joucla, Gilles; Monsan, Pierre; Remaud-Simeon, Magali

    2004-02-27

    Amylosucrase is a glucosyltransferase belonging to family 13 of glycoside hydrolases and catalyses the formation of an amylose-type polymer from sucrose. Its potential use as an industrial tool for the synthesis or the modification of polysaccharides, however, is limited by its low catalytic efficiency on sucrose alone, its low stability, and its side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling, and selective screening (directed evolution) was started, in order to generate more efficient variants of the enzyme. A convenient zero background expression cloning strategy was developed. Mutant gene libraries were generated by error-prone polymerase chain reaction (PCR), using Taq polymerase with unbalanced dNTPs or Mutazyme trade mark, followed by recombination of the PCR products by DNA shuffling. A selection method was developed to allow only the growth of amylosucrase active clones on solid mineral medium containing sucrose as the sole carbon source. Automated protocols were designed to screen amylosucrase activity from mini-cultures using dinitrosalicylic acid staining of reducing sugars and iodine staining of amylose-like polymer. A pilot experiment using the described mutagenesis, selection, and screening methods yielded two variants with significantly increased activity (five-fold under the screening conditions). Sequence analysis of these variants revealed mutations in amino acid residues which would not be considered for rational design of improved amylosucrase variants. A method for the characterisation of amylosucrase action on sucrose, consisting of accurate measurement of glucose and fructose concentrations, was introduced. This allows discrimination between hydrolysis and transglucosylation, enabling a more detailed comparison between wild-type and mutant enzymes. PMID:14988004

  14. Identifying candidate causal variants responsible for altered activity of the ABCB1 multidrug resistance gene.

    PubMed

    Soranzo, Nicole; Cavalleri, Gianpiero L; Weale, Michael E; Wood, Nicholas W; Depondt, Chantal; Marguerie, Richard; Sisodiya, Sanjay M; Goldstein, David B

    2004-07-01

    The difficulty of fine localizing the polymorphisms responsible for genotype-phenotype correlations is emerging as an important constraint in the implementation and interpretation of genetic association studies, and calls for the definition of protocols for the follow-up of associated variants. One recent example is the 3435C>T polymorphism in the multidrug transporter gene ABCB1, associated with protein expression and activity, and with several clinical conditions. Available data suggest that 3435C>T may not directly cause altered transport activity, but may be associated with one or more causal variants in the poorly characterized stretch of linkage disequilibrium (LD) surrounding it. Here we describe a strategy for the follow-up of reported associations, including a Bayesian formalization of the associated interval concept previously described by Goldstein. We focus on the region of high LD around 3435C>T to compile an exhaustive list of variants by (1) using a relatively coarse set of marker typings to assess the pattern of LD, and (2) resequencing derived and ancestral chromosomes at 3435C>T through the associated interval. We identified three intronic sites that are strongly associated with the 3435C>T polymorphism. One of them is associated with multidrug resistance in patients with epilepsy (chi2 = 3.78, P = 0.052), and sits within a stretch of significant evolutionary conservation. We argue that these variants represent additional candidates for influencing multidrug resistance due to P-glycoprotein activity, with the IVS 26+80 T>C being the best candidate among the three intronic sites. Finally, we describe a set of six haplotype tagging single-nucleotide polymorphisms that represent common ABCB1 variation surrounding 3435C>T in Europeans. PMID:15197162

  15. A New Trick of an Old Molecule: Androgen Receptor Splice Variants Taking the Stage?!

    PubMed Central

    Guo, Zhiyong; Qiu, Yun

    2011-01-01

    Prostate cancer is the second leading cause of cancer-related death in American men. Although most prostate cancers are initially androgen-dependent and respond to androgen ablation therapy, majority of them eventually relapse and progress into incurable castration-resistant (or hormone refractory) prostate cancer. The underlying mechanisms are the focus of intensive investigation for development of more effective treatment. Mounting evidence from both clinical and basic research has demonstrated that the activity of the androgen receptor (AR) is still required for castration-resistant prostate cancer. Multiple mechanisms by which AR is re-activated under androgen-depleted conditions may be involved in the development of castration resistance. The recent identification of AR splicing variants may add another layer of complexity in AR biology. The present review summarizes recent progress in study of AR splicing variants in prostate cancer. PMID:21750650

  16. Characterization of self-generated variants in Pseudoalteromonas lipolytica biofilm with increased antifouling activities.

    PubMed

    Zeng, Zhenshun; Guo, Xing-Pan; Li, Baiyuan; Wang, Pengxia; Cai, Xingsheng; Tian, Xinpeng; Zhang, Si; Yang, Jin-Long; Wang, Xiaoxue

    2015-12-01

    Pseudoalteromonas is widespread in various marine environments, and most strains can affect invertebrate larval settlement and metamorphosis by forming biofilms. However, the impact and the molecular basis of population diversification occurring in Pseudoalteromonas biofilms are poorly understood. Here, we show that morphological diversification is prevalent in Pseudoalteromonas species during biofilm formation. Two types of genetic variants, wrinkled (frequency of 12±5%) and translucent (frequency of 5±3%), were found in Pseudoalteromonas lipolytica biofilms. The inducing activities of biofilms formed by the two variants on larval settlement and metamorphosis of the mussel Mytilus coruscus were significantly decreased, suggesting strong antifouling activities. Using whole-genome re-sequencing combined with genetic manipulation, two genes were identified to be responsible for the morphology alternations. A nonsense mutation in AT00_08765 led to a wrinkled morphology due to the overproduction of cellulose, whereas a point mutation in AT00_17125 led to a translucent morphology via a reduction in capsular polysaccharide production. Taken together, the results suggest that the microbial behavior on larval settlement and metamorphosis in marine environment could be affected by the self-generated variants generated during the formation of marine biofilms, thereby rendering potential application in biocontrol of marine biofouling. PMID:26264135

  17. Functional analysis of ars gene cluster of Pannonibacter indicus strain HT23(T) (DSM 23407(T)) and identification of a proline residue essential for arsenate reductase activity.

    PubMed

    Bandyopadhyay, Saumya; Das, Subrata K

    2016-04-01

    Arsenic is a naturally occurring ubiquitous highly toxic metalloid. In this study, we have identified ars gene cluster in Pannonibacter indicus strain HT23(T) (DSM 23407(T)), responsible for reduction of toxic pentavalent arsenate. The ars gene cluster is comprised of four non-overlapping open reading frames (ORFs) encoding a transcriptional regulator (ArsR), a low molecular weight protein tyrosine phosphatases (LMW-PTPase) with hypothetical function, an arsenite efflux pump (Acr3), and an arsenate reductase (ArsC). Heterologous expression of arsenic inducible ars gene cluster conferred arsenic resistance to Escherichia coli ∆ars mutant strain AW3110. The recombinant ArsC was purified and assayed. Site-directed mutagenesis was employed to ascertain the role of specific amino acids in ArsC catalysis. Pro94X (X = Ala, Arg, Cys, and His) amino acid substitutions led to enzyme inactivation. Circular dichroism spectra analysis suggested Pro94 as an essential amino acid for enzyme catalytic activity as it is indispensable for optimum protein folding in P. indicus Grx-coupled ArsC. PMID:26915994

  18. The importance of non-nuclear AR signaling in prostate cancer progression and therapeutic resistance.

    PubMed

    Zarif, Jelani C; Miranti, Cindy K

    2016-05-01

    The androgen receptor (AR) remains the major oncogenic driver of prostate cancer, as evidenced by the efficacy of androgen deprivation therapy (ADT) in naïve patients, and the continued effectiveness of second generation ADTs in castration resistant disease. However, current ADTs are limited to interfering with AR ligand binding, either through suppression of androgen production or the use of competitive antagonists. Recent studies demonstrate 1) the expression of constitutively active AR splice variants that no longer depend on androgen, and 2) the ability of AR to signal in the cytoplasm independently of its transcriptional activity (non-genomic); thus highlighting the need to consider other ways to target AR. Herein, we review canonical AR signaling, but focus on AR non-genomic signaling, some of its downstream targets and how these effectors contribute to prostate cancer cell behavior. The goals of this review are to 1) re-highlight the continued importance of AR in prostate cancer as the primary driver, 2) discuss the limitations in continuing to use ligand binding as the sole targeting mechanism, 3) discuss the implications of AR non-genomic signaling in cancer progression and therapeutic resistance, and 4) address the need to consider non-genomic AR signaling mechanisms and pathways as a viable targeting strategy in combination with current therapies. PMID:26829214

  19. 40Ar/39Ar laser fusion and K-Ar ages from Lathrop Wells, Nevada, and Cima, California. The age of the latest volcanic activity in the Yucca Mountain area

    USGS Publications Warehouse

    Turrin, Brent D.; Champion, Duane E.

    1991-01-01

    K-Ar and 40Ar/39Ar ages from the Lathrop Wells volcanic center, Nevada, and from the Cima volcanic field, California, indicate that the recently reported 20-ka age estimate for the Lathrop Wells volcanic center is incorrect. Instead an age of 119??11 to 141??10 ka is indicated for the Lathrop Wells volcanic center. This age corrected is concordant with the ages determined by two independent isotopic geochronometric techniques and with the stratigraphy of surficial deposits in the Yucca Mountain region. In addition, paleomagnetic data and radiometric age data indicate only two volcanic events at the Lathrop Wells volcanic center that are probably closely linked in time, not as many as five as recently reported.

  20. {sup 40}Ar/{sup 39}Ar laser fusion and K-Ar ages from Lathrop Wells, Nevada, and Cima, California: The age of the latest volcanic activity in the Yucca Mountain area

    SciTech Connect

    Turrin, B.D. |; Champion, D.E.

    1991-05-01

    K-Ar and {sup 40}Ar/{sup 39}Ar ages from the Lathrop Wells volcanic center, Nevada, and from the Cima volcanic field, California, indicate that the recently reported 20-ka age estimate for the Lathrop Wells volcanic center is incorrect. Instead, an age of 119 {plus_minus} 11 to 141 {plus_minus} 10 ka is indicated for the Lathrop Wells volcanic center. This age corrected is concordant with the ages determined by two independent isotopic geochronometric techniques and with the stratigraphy of surficial deposits in the Yucca Mountain region. In addition, paleomagnetic data and radiometric age data indicate only two volcanic events at the Lathrop Wells volcanic center that are probably closely linked in time, not as many as five as recently reported. 32 refs., 2 figs., 2 tabs.

  1. APOL1 kidney disease risk variants cause cytotoxicity by depleting cellular potassium and inducing stress-activated protein kinases

    PubMed Central

    Olabisi, Opeyemi A.; Zhang, Jia-Yue; VerPlank, Lynn; Zahler, Nathan; DiBartolo, Salvatore; Heneghan, John F.; Schlöndorff, Johannes S.; Suh, Jung Hee; Yan, Paul; Alper, Seth L.; Friedman, David J.; Pollak, Martin R.

    2016-01-01

    Two specific genetic variants of the apolipoprotein L1 (APOL1) gene are responsible for the high rate of kidney disease in people of recent African ancestry. Expression in cultured cells of these APOL1 risk variants, commonly referred to as G1 and G2, results in significant cytotoxicity. The underlying mechanism of this cytotoxicity is poorly understood. We hypothesized that this cytotoxicity is mediated by APOL1 risk variant-induced dysregulation of intracellular signaling relevant for cell survival. To test this hypothesis, we conditionally expressed WT human APOL1 (G0), the APOL1 G1 variant, or the APOL1 G2 variant in human embryonic kidney cells (T-REx-293) using a tetracycline-mediated (Tet-On) system. We found that expression of either G1 or G2 APOL1 variants increased apparent cell swelling and cell death compared with G0-expressing cells. These manifestations of cytotoxicity were preceded by G1 or G2 APOL1-induced net efflux of intracellular potassium as measured by X-ray fluorescence, resulting in the activation of stress-activated protein kinases (SAPKs), p38 MAPK, and JNK. Prevention of net K+ efflux inhibited activation of these SAPKs by APOL1 G1 or G2. Furthermore, inhibition of SAPK signaling and inhibition of net K+ efflux abrogated cytotoxicity associated with expression of APOL1 risk variants. These findings in cell culture raise the possibility that nephrotoxicity of APOL1 risk variants may be mediated by APOL1 risk variant-induced net loss of intracellular K+ and subsequent induction of stress-activated protein kinase pathways. PMID:26699492

  2. Rs6295 promoter variants of the serotonin type 1A receptor are differentially activated by c-Jun in vitro and correlate to transcript levels in human epileptic brain tissue.

    PubMed

    Pernhorst, Katharina; van Loo, Karen M J; von Lehe, Marec; Priebe, Lutz; Cichon, Sven; Herms, Stefan; Hoffmann, Per; Helmstaedter, Christoph; Sander, Thomas; Schoch, Susanne; Becker, Albert J

    2013-03-01

    Many brain disorders, including epilepsy, migraine and depression, manifest with episodic symptoms that may last for various time intervals. Transient alterations of neuronal function such as related to serotonin homeostasis generally underlie this phenomenon. Several nucleotide polymorphisms (SNPs) in gene promoters associated with these diseases have been described. For obvious reasons, their regulatory roles on gene expression particularly in human brain tissue remain largely enigmatic. The rs6295 G-/C-allelic variant is located in the promoter region of the human HTR1a gene, encoding the G-protein-coupled receptor for 5-hydroxytryptamine (5HT1AR). In addition to reported transcriptional repressor binding, our bioinformatic analyses predicted a reduced binding affinity of the transcription factor (TF) c-Jun for the G-allele. In vitro luciferase transfection assays revealed c-Jun to (a) activate the rs6295 C- significantly stronger than the G-allelic variant and (b) antagonize efficiently the repressive effect of Hes5 on the promoter. The G-allele of rs6295 is known to be associated with aspects of major depression and migraine. In order to address a potential role of rs6295 variants in human brain tissue, we have isolated DNA and mRNA from fresh frozen hippocampal tissue of pharmacoresistant temporal lobe epilepsy (TLE) patients (n=140) after epilepsy surgery for seizure control. We carried out SNP genotyping studies and mRNA analyses in order to determine HTR1a mRNA expression in human hippocampal samples stratified according to the rs6295 allelic variant. The mRNA expression of HTR1a was significantly more abundant in hippocampal mRNA of TLE patients homozygous for the rs6295 C-allele as compared to those with the GG-genotype. These data may point to a novel, i.e., rs6295 allelic variant and c-Jun dependent transcriptional 5HT1AR 'receptoropathy'. PMID:23333373

  3. Mitochondria-associated endoplasmic reticulum membrane (MAM) regulates steroidogenic activity via steroidogenic acute regulatory protein (StAR)-voltage-dependent anion channel 2 (VDAC2) interaction.

    PubMed

    Prasad, Manoj; Kaur, Jasmeet; Pawlak, Kevin J; Bose, Mahuya; Whittal, Randy M; Bose, Himangshu S

    2015-01-30

    Steroid hormones are essential for carbohydrate metabolism, stress management, and reproduction and are synthesized from cholesterol in mitochondria of adrenal glands and gonads/ovaries. In acute stress or hormonal stimulation, steroidogenic acute regulatory protein (StAR) transports substrate cholesterol into the mitochondria for steroidogenesis by an unknown mechanism. Here, we report for the first time that StAR interacts with voltage-dependent anion channel 2 (VDAC2) at the mitochondria-associated endoplasmic reticulum membrane (MAM) prior to its translocation to the mitochondrial matrix. In the MAM, StAR interacts with mitochondrial proteins Tom22 and VDAC2. However, Tom22 knockdown by siRNA had no effect on pregnenolone synthesis. In the absence of VDAC2, StAR was expressed but not processed into the mitochondria as a mature 30-kDa protein. VDAC2 interacted with StAR via its C-terminal 20 amino acids and N-terminal amino acids 221-229, regulating the mitochondrial processing of StAR into the mature protein. In the absence of VDAC2, StAR could not enter the mitochondria or interact with MAM-associated proteins, and therefore steroidogenesis was inhibited. Furthermore, the N terminus was not essential for StAR activity, and the N-terminal deletion mutant continued to interact with VDAC2. The endoplasmic reticulum-targeting prolactin signal sequence did not affect StAR association with the MAM and thus its mitochondrial targeting. Therefore, VDAC2 controls StAR processing and activity, and MAM is thus a central location for initiating mitochondrial steroidogenesis. PMID:25505173

  4. Mitochondria-associated Endoplasmic Reticulum Membrane (MAM) Regulates Steroidogenic Activity via Steroidogenic Acute Regulatory Protein (StAR)-Voltage-dependent Anion Channel 2 (VDAC2) Interaction*

    PubMed Central

    Prasad, Manoj; Kaur, Jasmeet; Pawlak, Kevin J.; Bose, Mahuya; Whittal, Randy M.; Bose, Himangshu S.

    2015-01-01

    Steroid hormones are essential for carbohydrate metabolism, stress management, and reproduction and are synthesized from cholesterol in mitochondria of adrenal glands and gonads/ovaries. In acute stress or hormonal stimulation, steroidogenic acute regulatory protein (StAR) transports substrate cholesterol into the mitochondria for steroidogenesis by an unknown mechanism. Here, we report for the first time that StAR interacts with voltage-dependent anion channel 2 (VDAC2) at the mitochondria-associated endoplasmic reticulum membrane (MAM) prior to its translocation to the mitochondrial matrix. In the MAM, StAR interacts with mitochondrial proteins Tom22 and VDAC2. However, Tom22 knockdown by siRNA had no effect on pregnenolone synthesis. In the absence of VDAC2, StAR was expressed but not processed into the mitochondria as a mature 30-kDa protein. VDAC2 interacted with StAR via its C-terminal 20 amino acids and N-terminal amino acids 221–229, regulating the mitochondrial processing of StAR into the mature protein. In the absence of VDAC2, StAR could not enter the mitochondria or interact with MAM-associated proteins, and therefore steroidogenesis was inhibited. Furthermore, the N terminus was not essential for StAR activity, and the N-terminal deletion mutant continued to interact with VDAC2. The endoplasmic reticulum-targeting prolactin signal sequence did not affect StAR association with the MAM and thus its mitochondrial targeting. Therefore, VDAC2 controls StAR processing and activity, and MAM is thus a central location for initiating mitochondrial steroidogenesis. PMID:25505173

  5. Amyloidogenic amyloid-β-peptide variants induce microbial agglutination and exert antimicrobial activity.

    PubMed

    Spitzer, Philipp; Condic, Mateja; Herrmann, Martin; Oberstein, Timo Jan; Scharin-Mehlmann, Marina; Gilbert, Daniel F; Friedrich, Oliver; Grömer, Teja; Kornhuber, Johannes; Lang, Roland; Maler, Juan Manuel

    2016-01-01

    Amyloid-β (Aβ) peptides are the main components of the plaques found in the brains of patients with Alzheimer's disease. However, Aβ peptides are also detectable in secretory compartments and peripheral blood contains a complex mixture of more than 40 different modified and/or N- and C-terminally truncated Aβ peptides. Recently, anti-infective properties of Aβ peptides have been reported. Here, we investigated the interaction of Aβ peptides of different lengths with various bacterial strains and the yeast Candida albicans. The amyloidogenic peptides Aβ1-42, Aβ2-42, and Aβ3p-42 but not the non-amyloidogenic peptides Aβ1-40 and Aβ2-40 bound to microbial surfaces. As observed by immunocytochemistry, scanning electron microscopy and Gram staining, treatment of several bacterial strains and Candida albicans with Aβ peptide variants ending at position 42 (Aβx-42) caused the formation of large agglutinates. These aggregates were not detected after incubation with Aβx-40. Furthermore, Aβx-42 exerted an antimicrobial activity on all tested pathogens, killing up to 80% of microorganisms within 6 h. Aβ1-40 only had a moderate antimicrobial activity against C. albicans. Agglutination of Aβ1-42 was accelerated in the presence of microorganisms. These data demonstrate that the amyloidogenic Aβx-42 variants have antimicrobial activity and may therefore act as antimicrobial peptides in the immune system. PMID:27624303

  6. Comparative toxicity and efficacy of engineered anthrax lethal toxin variants with broad anti-tumor activities.

    PubMed

    Peters, Diane E; Hoover, Benjamin; Cloud, Loretta Grey; Liu, Shihui; Molinolo, Alfredo A; Leppla, Stephen H; Bugge, Thomas H

    2014-09-01

    We have previously designed and characterized versions of anthrax lethal toxin that are selectively cytotoxic in the tumor microenvironment and which display broad and potent anti-tumor activities in vivo. Here, we have performed the first direct comparison of the safety and efficacy of three engineered anthrax lethal toxin variants requiring activation by either matrix-metalloproteinases (MMPs), urokinase plasminogen activator (uPA) or co-localized MMP/uPA activities. C57BL/6J mice were challenged with six doses of engineered toxins via intraperitoneal (I.P.) or intravenous (I.V.) dose routes to determine the maximum tolerated dose for six administrations (MTD6) and dose-limiting toxicities. Efficacy was evaluated using the B16-BL6 syngraft model of melanoma; mice bearing established tumors were treated with six I.P. doses of toxin and tumor measurements and immunohistochemistry, paired with terminal blood work, were used to elaborate upon the anti-tumor mechanism and relative efficacy of each variant. We found that MMP-, uPA- and dual MMP/uPA-activated anthrax lethal toxins exhibited the same dose-limiting toxicity; dose-dependent GI toxicity. In terms of efficacy, all three toxins significantly reduced primary B16-BL6 tumor burden, ranging from 32% to 87% reduction, and they also delayed disease progression as evidenced by dose-dependent normalization of blood work values. While target organ toxicity and effective doses were similar amongst the variants, the dual MMP/uPA-activated anthrax lethal toxin exhibited the highest I.P. MTD6 and was 1.5-3-fold better tolerated than the single MMP- and uPA-activated toxins. Overall, we demonstrate that this dual MMP/uPA-activated anthrax lethal toxin can be administered safely and is highly effective in a preclinical model of melanoma. This modified bacterial cytotoxin is thus a promising candidate for further clinical development and evaluation for use in treating human cancers. PMID:24971906

  7. Galeterone and VNPT55 induce proteasomal degradation of AR/AR-V7, induce significant apoptosis via cytochrome c release and suppress growth of castration resistant prostate cancer xenografts in vivo

    PubMed Central

    Kwegyir-Afful, Andrew K.; Ramalingam, Senthilmurugan; Purushottamachar, Puranik; Ramamurthy, Vidya P.; Njar, Vincent C.O.

    2015-01-01

    Galeterone (Gal) is a first-in-class multi-target oral small molecule that will soon enter pivotal phase III clinical trials in castration resistant prostate cancer (CRPC) patients. Gal disrupts androgen receptor (AR) signaling via inhibition of CYP17, AR antagonism and AR degradation. Resistance to current therapy is attributed to up-regulation of full-length AR (fAR), splice variants AR (AR-Vs) and AR mutations. The effects of gal and VNPT55 were analyzed on f-AR and AR-Vs (AR-V7/ARv567es) in LNCaP, CWR22Rv1 and DU145 (transfected with AR-Vs) human PC cells in vitro and CRPC tumor xenografts. Galeterone/VNPT55 decreased fAR/AR-V7 mRNA levels and implicates Mdm2/CHIP enhanced ubiquitination of posttranslational modified receptors, targeting them for proteasomal degradation. Gal and VNPT55 also induced significant apoptosis in PC cells via increased Bax/Bcl2 ratio, cytochrome-c release with concomitant cleavage of caspase 3 and PARP. More importantly, gal and VNPT55 exhibited strong in vivo anti-CRPC activities, with no apparent host toxicities. This study demonstrate that gal and VNPT55 utilize cell-based mechanisms to deplete both fAR and AR-Vs. Importantly, the preclinical activity profiles, including profound apoptotic induction and inhibition of CRPC xenografts suggest that these agents offer considerable promise as new therapeutics for patients with CRPC and those resistant to current therapy. PMID:26196320

  8. Stimulation of StAR expression by cAMP is controlled by inhibition of highly inducible SIK1 via CRTC2, a co-activator of CREB.

    PubMed

    Lee, Jinwoo; Tong, Tiegang; Takemori, Hiroshi; Jefcoate, Colin

    2015-06-15

    In mouse steroidogenic cells the activation of cholesterol metabolism is mediated by steroidogenic acute regulatory protein (StAR). Here, we visualized a coordinated regulation of StAR transcription, splicing and post-transcriptional processing, which are synchronized by salt inducible kinase (SIK1) and CREB-regulated transcription coactivator (CRTC2). To detect primary RNA (pRNA), spliced primary RNA (Sp-RNA) and mRNA in single cells, we generated probe sets by using fluorescence in situ hybridization (FISH). These methods allowed us to address the nature of StAR gene expression and to visualize protein-nucleic acid interactions through direct detection. We show that SIK1 represses StAR expression in Y1 adrenal and MA10 testis cells through inhibition of processing mediated by CRTC2. Digital image analysis matches qPCR analyses of the total cell culture. Evidence is presented for spatially separate accumulation of StAR pRNA and Sp-RNA at the gene loci in the nucleus. These findings establish that cAMP, SIK and CRTC mediate StAR expression through activation of individual StAR gene loci. PMID:25662274

  9. Bacillus cereus AR156 activates PAMP-triggered immunity and induces a systemic acquired resistance through a NPR1-and SA-dependent signaling pathway.

    PubMed

    Niu, Dongdong; Wang, Xiujuan; Wang, Yanru; Song, Xiaoou; Wang, Jiansheng; Guo, Jianhua; Zhao, Hongwei

    2016-01-01

    Induced resistance responses play a potent role in plant defense system against pathogen attack. Bacillus cereus AR156 is a plant growth promoting rhizobacterium (PGPR) that installs induced systemic resistance (ISR) to Pseudomonas syringae pv. tomato (Pst) in Arabidopsis. Here, we show that AR156 leaf infiltration enhances disease resistance in Arabidopsis through the activation of a systemic acquired resistance (SAR). PR1 protein expression and reactive oxygen species (ROS) burst are strongly induced in plants treated with AR156 and inoculated with Pst than that in plants inoculated with Pst only. Moreover, AR156 can trigger SAR in jar1 or ein2 mutants, but not in the NahG transgenic and NPR1 mutant plants. Our results indicate that AR156-induced SAR depends on SA-signaling pathway and NPR1, but not JA and ET. Also, AR156-treated plants are able to rapidly activate MAPK signaling and FRK1 gene expression, which are involved in pathogen associated molecular pattern (PAMP)-triggered immunity (PTI). Altogether, our results indicate that AR156 can induce SAR by the SA-signaling pathways in an NPR1-dependent manner and involves multiple PTI components. PMID:26616055

  10. Novel Stably Transfected Human Reporter Cell Line AIZ-AR as a Tool for an Assessment of Human Androgen Receptor Transcriptional Activity

    PubMed Central

    Bartonkova, Iveta; Novotna, Aneta; Dvorak, Zdenek

    2015-01-01

    Androgen receptor plays multiple physiological and pathological roles in human organism. In the current paper, we describe construction and characterization of a novel stably transfected human reporter cell line AIZ-AR for assessment of transcriptional activity of human androgen receptor. Cell line AIZ-AR is derived from human prostate carcinoma epithelial cell line 22Rv1 that was transfected with reporter plasmid containing 3 copies of androgen response regions (ARRs) followed by a single copy of androgen response element (ARE) from the promoter region of human prostate specific antigen (PSA) gene. AIZ-AR cells remained fully functional for more than 60 days and over 25 passages in the culture and even after cryopreservation. Time-course analyses showed that AIZ-AR cells allow detection of AR ligands as soon as after 8 hours of the treatment. We performed dose-response analyses with 23 steroids in 96-well plate format. We observed activation of AR by androgens, but not by estrogens and mineralocorticoids. Some glucocorticoids and progesterone also induced luciferase, but their potencies were 2-3 orders of magnitude weaker as compared to androgens. Taken together, we have developed a rapid, sensitive, selective, high-throughput and reproducible tool for detection of human AR ligands, with potential use in pharmacological and environmental applications. PMID:25811655

  11. Enhanced expression of Rubisco activase splicing variants differentially affects Rubisco activity during low temperature treatment in Lolium perenne.

    PubMed

    Jurczyk, Barbara; Pociecha, Ewa; Grzesiak, Maciej; Kalita, Katarzyna; Rapacz, Marcin

    2016-07-01

    Alternative splicing of the Rubisco activase gene was shown to be a point for optimization of photosynthetic carbon assimilation. It can be expected to be a stress-regulated event that depends on plant freezing tolerance. The aim of the study was to examine the relationships among Rubisco activity, the expression of two Rubisco activase splicing variants and photoacclimation to low temperature. The experiment was performed on two Lolium perenne genotypes with contrasting levels of freezing tolerance. The study investigated the effect of pre-hardening (15°C) and cold acclimation (4°C) on net photosynthesis, photosystem II photochemical activity, Rubisco activity and the expression of two splicing variants of the Rubisco activase gene. The results showed an induction of Rubisco activity at both 15°C and 4°C only in a highly freezing-tolerant genotype. The enhanced Rubisco activity after pre-hardening corresponded to increased expression of the splicing variant representing the large isoform, while the increase in Rubisco activity during cold acclimation was due to the activation of both transcript variants. These boosts in Rubisco activity also corresponded to an activation of non-photochemical mechanism of photoacclimation induced at low temperature exclusively in the highly freezing-tolerant genotype. In conclusion, enhanced expression of Rubisco activase splicing variants caused an increase in Rubisco activity during pre-hardening and cold acclimation in the more freezing-tolerant Lolium perenne genotype. The induction of the transcript variant representing the large isoform may be an important element of increasing the carbon assimilation rate supporting the photochemical mechanism of photosynthetic acclimation to cold. PMID:27152456

  12. A metrological approach to measuring 40Ar* concentrations in K-Ar and 40Ar/39Ar mineral standards

    NASA Astrophysics Data System (ADS)

    Morgan, Leah E.; Postma, Onno; Kuiper, Klaudia F.; Mark, Darren F.; van der Plas, Wim; Davidson, Stuart; Perkin, Michael; Villa, Igor M.; Wijbrans, Jan R.

    2011-10-01

    In geochronology, isotopic ages are determined from the ratio of parent and daughter nuclide concentrations in minerals. For dating of geological material using the K-Ar system, the simultaneous determination of 40Ar and 40K concentrations on the same aliquot is not possible. Therefore, a widely used variant, the 40Ar/39Ar technique, involves the production of 39Ar from 39K by neutron bombardment and the reliance on indirect natural calibrators of the neutron flux, referred to as "mineral standards." Many mineral standards still in use rely on decades-old determinations of 40Ar concentrations; resulting uncertainties, both systematic and analytical, impede the determination of higher accuracy ages using the K-Ar decay system. We discuss the theoretical approach and technical design of a gas delivery system which emits metrologically traceable amounts of 40Ar and will allow for the sensitivity calibration of noble gas mass spectrometers. The design of this system is based on a rigorous assessment of the uncertainty budget and detailed tests of a prototype system. A number of obstacles and proposed resolutions are discussed along with the selection of components and their integration into a pipette system.

  13. Variants of the yeast MAPK Mpk1 are fully functional independently of activation loop phosphorylation.

    PubMed

    Goshen-Lago, Tal; Goldberg-Carp, Anat; Melamed, Dganit; Darlyuk-Saadon, Ilona; Bai, Chen; Ahn, Natalie G; Admon, Arie; Engelberg, David

    2016-09-01

    MAP kinases of the ERK family are conserved from yeast to humans. Their catalytic activity is dependent on dual phosphorylation of their activation loop's TEY motif, catalyzed by MAPK kinases (MEKs). Here we studied variants of Mpk1, a yeast orthologue of Erk, which is essential for cell wall integrity. Cells lacking MPK1, or the genes encoding the relevant MEKs, MKK1 and MKK2, do not proliferate under cell wall stress, imposed, for example, by caffeine. Mutants of Mpk1, Mpk1(Y268C) and Mpk1(Y268A), function independently of Mkk1 and Mkk2. We show that these variants are phosphorylated at their activation loop in mkk1∆mkk2∆ and mkk1∆mkk2∆pbs2∆ste7∆ cells, suggesting that they autophosphorylate. However, strikingly, when Y268C/A mutations were combined with the kinase-dead mutation, K54R, or mutations at the TEY motif, T190A+Y192F, the resulting proteins still allowed mkk1∆mkk2∆ cells to proliferate under caffeine stress. Mutating the equivalent residue, Tyr-280/Tyr-261, in Erk1/Erk2 significantly impaired Erk1/2's catalytic activity. This study describes the first case in which a MAPK, Erk/Mpk1, imposes a phenotype via a mechanism that is independent of TEY phosphorylation and an unusual case in which an equivalent mutation in a highly conserved domain of yeast and mammalian Erks causes an opposite effect. PMID:27413009

  14. Association of low-activity MAOA allelic variants with violent crime in incarcerated offenders

    PubMed Central

    Stetler, Dean A.; Davis, Chad; Leavitt, Kathryn; Schriger, Ilana; Benson, Katie; Bhakta, Samir; Wang, Lam Chee; Oben, Cynthia; Watters, Matthew; Haghnegahdar, Tara; Bortolato, Marco

    2015-01-01

    The main enzyme for serotonin degradation, monoamine oxidase (MAO) A, has recently emerged as a key biological factor in the predisposition to impulsive aggression. Male carriers of low-activity variants of the main functional polymorphism of the MAOA gene (MAOA-uVNTR) have been shown to exhibit a greater proclivity to engage in violent acts. Thus, we hypothesized that low-activity MAOA-uVNTR alleles may be associated with a higher risk for criminal violence among male offenders. To test this possibility, we analyzed the MAOA-uVNTR variants of violent (n=49) and non-violent (n=40) male Caucasian and African-American convicts in a correctional facility. All participants were also tested with the Childhood Trauma Questionnaire (CTQ), Barratt Impulsivity Scale (BIS-11) and Buss-Perry Aggression Questionnaire (BPAQ) to assess their levels of childhood trauma exposure, impulsivity and aggression, respectively. Our results revealed a robust (P<0.0001) association between low-activity MAOA-uVNTR alleles and violent crime. This association was replicated in the group of Caucasian violent offenders (P<0.01), but reached only a marginal trend (P=0.08) in their African American counterparts. While violent crime charges were not associated with CTQ, BIS-11 and BPAQ scores, carriers of low-activity alleles exhibited a mild, yet significant (P<0.05) increase in BIS-11 total and attentional-impulsiveness scores. In summary, these findings support the role of MAOA gene as a prominent genetic determinant for criminal violence. Further studies are required to confirm these results in larger samples of inmates and evaluate potential interactions between MAOA alleles and environmental vulnerability factors. PMID:25082653

  15. Structural Characterization of Human 8-Oxoguanine DNA Glycosylase Variants Bearing Active Site Mutations

    SciTech Connect

    Radom,C.; Banerjee, A.; Verdine, G.

    2007-01-01

    The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5 to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is 'hardwired'. Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F{sup *149}) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F{sup *292}) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.

  16. Association of low-activity MAOA allelic variants with violent crime in incarcerated offenders.

    PubMed

    Stetler, Dean A; Davis, Chad; Leavitt, Kathryn; Schriger, Ilana; Benson, Katie; Bhakta, Samir; Wang, Lam Chee; Oben, Cynthia; Watters, Matthew; Haghnegahdar, Tara; Bortolato, Marco

    2014-11-01

    The main enzyme for serotonin degradation, monoamine oxidase (MAO) A, has recently emerged as a key biological factor in the predisposition to impulsive aggression. Male carriers of low-activity variants of the main functional polymorphism of the MAOA gene (MAOA-uVNTR) have been shown to exhibit a greater proclivity to engage in violent acts. Thus, we hypothesized that low-activity MAOA-uVNTR alleles may be associated with a higher risk for criminal violence among male offenders. To test this possibility, we analyzed the MAOA-uVNTR variants of violent (n = 49) and non-violent (n = 40) male Caucasian and African-American convicts in a correctional facility. All participants were also tested with the Childhood Trauma Questionnaire (CTQ), Barratt Impulsivity Scale (BIS-11) and Buss-Perry Aggression Questionnaire (BPAQ) to assess their levels of childhood trauma exposure, impulsivity and aggression, respectively. Our results revealed a robust (P < 0.0001) association between low-activity MAOA-uVNTR alleles and violent crime. This association was replicated in the group of Caucasian violent offenders (P < 0.01), but reached only a marginal trend (P = 0.08) in their African American counterparts. While violent crime charges were not associated with CTQ, BIS-11 and BPAQ scores, carriers of low-activity alleles exhibited a mild, yet significant (P < 0.05) increase in BIS-11 total and attentional-impulsiveness scores. In summary, these findings support the role of MAOA gene as a prominent genetic determinant for criminal violence. Further studies are required to confirm these results in larger samples of inmates and evaluate potential interactions between MAOA alleles and environmental vulnerability factors. PMID:25082653

  17. Joint Identification of Genetic Variants for Physical Activity in Korean Population

    PubMed Central

    Kim, Jayoun; Kim, Jaehee; Min, Haesook; Oh, Sohee; Kim, Yeonjung; Lee, Andy H.; Park, Taesung

    2014-01-01

    There has been limited research on genome-wide association with physical activity (PA). This study ascertained genetic associations between PA and 344,893 single nucleotide polymorphism (SNP) markers in 8842 Korean samples. PA data were obtained from a validated questionnaire that included information on PA intensity and duration. Metabolic equivalent of tasks were calculated to estimate the total daily PA level for each individual. In addition to single- and multiple-SNP association tests, a pathway enrichment analysis was performed to identify the biological significance of SNP markers. Although no significant SNP was found at genome-wide significance level via single-SNP association tests, 59 genetic variants mapped to 76 genes were identified via a multiple SNP approach using a bootstrap selection stability measure. Pathway analysis for these 59 variants showed that maturity onset diabetes of the young (MODY) was enriched. Joint identification of SNPs could enable the identification of multiple SNPs with good predictive power for PA and a pathway enriched for PA. PMID:25026172

  18. Protein profiling and histone deacetylation activities in somaclonal variants of oil palm (Elaeis guineensis Jacq.).

    PubMed

    Yaacob, Jamilah Syafawati; Loh, Hwei-San; Mat Taha, Rosna

    2013-01-01

    Mantled fruits as a result of somaclonal variation are often observed from the oil palm plantlets regenerated via tissue culture. The mantling of fruits with finger-like and thick outer coating phenotypes significantly reduces the seed size and oil content, posing a threat to oil palm planters, and may jeopardize the economic growth of countries that depend particularly on oil palm plantation. The molecular aspects of the occurrence of somaclonal variations are yet to be known, possibly due to gene repression such as DNA methylation, histone methylation and histone deacetylation. Histone deacetylases (HDACs), involved in eukaryotic gene regulation by catalyzing the acetyl groups are removal from lysine residues on histone, hence transcriptionally repress gene expression. This paper described the total protein polymorphism profiles of somaclonal variants of oil palm and the effects of histone deacetylation on this phenomenon. Parallel to the different phenotypes, the protein polymorphism profiles of the mantled samples (leaves, fruits, and florets) and the phenotypically normal samples were proven to be different. Higher HDAC activity was found in mantled leaf samples than in the phenotypically normal leaf samples, leading to a preliminary conclusion that histone deacetylation suppressed gene expression and contributed to the development of somaclonal variants. PMID:23844406

  19. Assessing the volcanic hazard for Rome: 40Ar/39Ar and In-SAR constraints on the most recent eruptive activity and present-day uplift at Colli Albani Volcanic District

    NASA Astrophysics Data System (ADS)

    Marra, F.; Gaeta, M.; Giaccio, B.; Jicha, B. R.; Palladino, D. M.; Polcari, M.; Sottili, G.; Taddeucci, J.; Florindo, F.; Stramondo, S.

    2016-07-01

    We present new 40Ar/39Ar data which allow us to refine the recurrence time for the most recent eruptive activity occurred at Colli Albani Volcanic District (CAVD) and constrain its geographic area. Time elapsed since the last eruption (36 kyr) overruns the recurrence time (31 kyr) in the last 100 kyr. New interferometric synthetic aperture radar data, covering the years 1993-2010, reveal ongoing inflation with maximum uplift rates (>2 mm/yr) in the area hosting the most recent (<200 ka) vents, suggesting that the observed uplift might be caused by magma injection within the youngest plumbing system. Finally, we frame the present deformation within the structural pattern of the area of Rome, characterized by 50 m of regional uplift since 200 ka and by geologic evidence for a recent (<2000 years) switch of the local stress-field, highlighting that the precursors of a new phase of volcanic activity are likely occurring at the CAVD.

  20. The activation energy for nanocrystalline diamond films deposited from an Ar/H2/CH4 hot-filament reactor.

    PubMed

    Barbosa, D C; Melo, L L; Trava-Airoldi, V J; Corat, E J

    2009-06-01

    In this work we have investigated the effect of substrate temperature on the growth rate and properties of nanocrystalline diamond thin films deposited by hot filament chemical vapor deposition (HFCVD). Mixtures of 0.5 vol% CH4 and 25 vol% H2 balanced with Ar at a pressure of 50 Torr and typical deposition time of 12 h. We present the measurement of the activation energy by accurately controlling the substrate temperature independently of other CVD parameters. Growth rates have been measured in the temperature range from 550 to 800 degrees C. Characterization techniques have involved Raman spectroscopy, high resolution X-ray difractometry and scanning electron microscopy. We also present a comparison with most activation energy for micro and nanocrystalline diamond determinations in the literature and propose that there is a common trend in most observations. The result obtained can be an evidence that the growth mechanism of NCD in HFCVD reactors is very similar to MCD growth. PMID:19504946

  1. Substrate-specific modulation of CYP3A4 activity by genetic variants of cytochrome P450 oxidoreductase (POR)

    PubMed Central

    Agrawal, Vishal; Choi, Ji Ha; Giacomini, Kathleen M.; Miller, Walter L.

    2010-01-01

    Objectives CYP3A4 receives electrons from P450 oxidoreductase (POR) to metabolize about 50% of clinically used drugs. There is substantial inter-individual variation in CYP3A4 catalytic activity that is not explained by CYP3A4 genetic variants. CYP3A4 is flexible and distensible, permitting it to accommodate substrates varying in shape and size. To elucidate mechanisms of variability in CYP3A4 catalysis, we examined the effects of genetic variants of POR, and explored the possibility that substrate-induced conformational changes in CYP3A4 differentially affect the ability of POR variants to support catalysis. Methods We expressed human CYP3A4 and four POR variants (Q153R, A287P, R457H, A503V) in bacteria, reconstituted them in vitro and measured the Michaelis constant and maximum velocity with testosterone, midazolam, quinidine and erythromycin as substrates. Results POR A287P and R457H had low activity with all substrates; Q153R had 76–94% of wild type (WT) activity with midazolam and erythromycin, but 129–150% activity with testosterone and quinidine. The A503V polymorphism reduced CYP3A4 activity to 61–77% of wild type with testosterone and midazolam, but had nearly wild type activity with quinidine and erythromycin. Conclusion POR variants affect CYP3A4 activities. The impact of a POR variant on catalysis by CYP3A4 is substrate-specific, probably due to substrate-induced conformational changes in CYP3A4. PMID:20697309

  2. THE KINEMATICS AND PLASMA PROPERTIES OF A SOLAR SURGE TRIGGERED BY CHROMOSPHERIC ACTIVITY IN AR11271

    SciTech Connect

    Kayshap, P.; Srivastava, Abhishek K.; Murawski, K.

    2013-01-20

    We observe a solar surge in NOAA AR11271 using the Solar Dynamics Observatory (SDO) Atmospheric Imaging Assembly 304 A image data on 2011 August 25. The surge rises vertically from its origin up to a height of Almost-Equal-To 65 Mm with a terminal velocity of Almost-Equal-To 100 km s{sup -1}, and thereafter falls and fades gradually. The total lifetime of the surge was Almost-Equal-To 20 minutes. We also measure the temperature and density distribution of the observed surge during its maximum rise and find an average temperature and a density of 2.0 MK and 4.1 Multiplication-Sign 10{sup 9} cm{sup -3}, respectively. The temperature map shows the expansion and mixing of cool plasma lagging behind the hot coronal plasma along the surge. Because SDO/HMI temporal image data do not show any detectable evidence of significant photospheric magnetic field cancellation for the formation of the observed surge, we infer that it is probably driven by magnetic-reconnection-generated thermal energy in the lower chromosphere. The radiance (and thus the mass density) oscillations near the base of the surge are also evident, which may be the most likely signature of its formation by a reconnection-generated pulse. In support of the present observational baseline of the triggering of the surge due to chromospheric heating, we devise a numerical model with conceivable implementation of the VAL-C atmosphere and a thermal pulse as an initial trigger. We find that the pulse steepens into a slow shock at higher altitudes which triggers plasma perturbations exhibiting the observed features of the surge, e.g., terminal velocity, height, width, lifetime, and heated fine structures near its base.

  3. Crystal Structure of an Activated Variant of Small Heat Shock Protein Hsp16.5

    SciTech Connect

    Mchaourab, Hassane S.; Lin, Yi-Lun; Spiller, Benjamin W.

    2013-04-17

    How does the sequence of a single small heat shock protein (sHSP) assemble into oligomers of different sizes? To gain insight into the underlying structural mechanism, we determined the crystal structure of an engineered variant of Methanocaldococcus jannaschii Hsp16.5 wherein a 14 amino acid peptide from human heat shock protein 27 (Hsp27) was inserted at the junction of the N-terminal region and the {alpha}-crystallin domain. In response to this insertion, the oligomer shell expands from 24 to 48 subunits while maintaining octahedral symmetry. Oligomer rearrangement does not alter the fold of the conserved {alpha}-crystallin domain nor does it disturb the interface holding the dimeric building block together. Rather, the flexible C-terminal tail of Hsp16.5 changes its orientation relative to the {alpha}-crystallin domain which enables alternative packing of dimers. This change in orientation preserves a peptide-in-groove interaction of the C-terminal tail with an adjacent {beta}-sandwich, thereby holding the assembly together. The interior of the expanded oligomer, where substrates presumably bind, retains its predominantly nonpolar character relative to the outside surface. New large windows in the outer shell provide increased access to these substrate-binding regions, thus accounting for the higher affinity of this variant to substrates. Oligomer polydispersity regulates sHSPs chaperone activity in vitro and has been implicated in their physiological roles. The structural mechanism of Hsp16.5 oligomer flexibility revealed here, which is likely to be highly conserved across the sHSP superfamily, explains the relationship between oligomer expansion observed in disease-linked mutants and changes in chaperone activity.

  4. Anti-candidal activity of genetically engineered histatin variants with multiple functional domains.

    PubMed

    Oppenheim, Frank G; Helmerhorst, Eva J; Lendenmann, Urs; Offner, Gwynneth D

    2012-01-01

    The human bodily defense system includes a wide variety of innate antimicrobial proteins. Histatins are small molecular weight proteins produced by the human salivary glands that exhibit antifungal and antibacterial activities. While evolutionarily old salivary proteins such as mucins and proline-rich proteins contain large regions of tandem repeats, relatively young proteins like histatins do not contain such repeated domains. Anticipating that domain duplications have a functional advantage, we genetically engineered variants of histatin 3 with one, two, three, or four copies of the functional domain by PCR and splice overlap. The resulting proteins, designated reHst3 1-mer, reHist3 2-mer, reHis3 3-mer and reHist3 4-mer, exhibited molecular weights of 4,062, 5,919, 7,777, and 9,634 Da, respectively. The biological activities of these constructs were evaluated in fungicidal assays toward Candida albicans blastoconidia and germinated cells. The antifungal activities per mole of protein increased concomitantly with the number of functional domains present. This increase, however, was higher than could be anticipated from the molar concentration of functional domains present in the constructs. The demonstrated increase in antifungal activity may provide an evolutionary explanation why such domain multiplication is a frequent event in human salivary proteins. PMID:23251551

  5. STEREO Observing AR903

    NASA Technical Reports Server (NTRS)

    2006-01-01

    A close up of loops in a magnetic active region. These loops, observed by STEREO's SECCHI/EUVI telescope, are at a million degrees K. This powerful active region, AR903, observed here on Dec. 4, 2006, produced a series of intense flares, particle storms, and coronal mass ejections over the next few days.

  6. Androgen receptor variant-driven prostate cancer: clinical implications and therapeutic targeting.

    PubMed

    Antonarakis, E S; Armstrong, A J; Dehm, S M; Luo, J

    2016-09-01

    While there are myriad mechanisms of primary and acquired resistance to conventional and next-generation hormonal therapies in prostate cancer, the potential role of androgen receptor splice variants (AR-Vs) has recently gained momentum. AR-Vs are abnormally truncated isoforms of the androgen receptor (AR) protein that lack the COOH-terminal domain but retain the NH2-terminal domain and DNA-binding domain and are thus constitutively active even in the absence of ligands. Although multiple preclinical studies have previously implicated AR-Vs in the development of castration resistance as well as resistance to abiraterone and enzalutamide, recent technological advances have made it possible to reliably detect and quantify AR-Vs from human clinical tumor specimens including blood samples. Initial clinical studies have now shown that certain AR-Vs, in particular AR-V7, may be associated with resistance to abiraterone and enzalutamide but not taxane chemotherapies when detected in circulating tumor cells. Efforts are now underway to clinically validate AR-V7 as a relevant treatment-selection biomarker in the context of other key genomic aberrations in men with metastatic castration-resistant prostate cancer. Additional efforts are underway to therapeutically target both AR and AR-Vs either directly or indirectly. Whether AR-Vs represent drivers of castration-resistant prostate cancer, or whether they are simply passenger events associated with aggressive disease or clonal heterogeneity, will ultimately be answered only through these types of clinical trials. PMID:27184811

  7. Larvicidal and Biting Deterrent Activity of Essential Oils of Curcuma longa, Ar-turmerone, and Curcuminoids Against Aedes aegypti and Anopheles quadrimaculatus (Culicidae: Diptera).

    PubMed

    Ali, Abbas; Wang, Yan-Hong; Khan, Ikhlas A

    2015-09-01

    Essential oils and extract of Curcuma longa, ar-turmerone, and curcuminoids were evaluated for their larvicidal and deterrent activity against mosquitoes. Ar-turmerone and curcuminoids constituted 36.9, 24.9 and 50.6% of rhizome oil, leaf oil, and rhizome extract, respectively. Ar-turmerone was the major compound of the rhizome oil (36.9%) and leaf oil (24.9%). The ethanolic extract had 15.4% ar-turmerone with 6.6% bisdesmethoxycurcumin, 6.1% desmethoxycurcumin, and 22.6% curcumin. In in vitro studies, essential oils of the leaf (biting deterrence index [BDI] = 0.98), rhizome (BDI = 0.98), and rhizome ethanolic extract (BDI = 0.96) at 10 µg/cm(2) showed biting deterrent activity similar to DEET at 25 nmol/cm(2) against Aedes aegypti L. Among the pure compounds, ar-turmerone (BDI = 1.15) showed the biting deterrent activity higher than DEET at 25 nmol/cm(2) whereas the activity of other compounds was lower than DEET. In Anopheles quadrimaculatus Say, only ar-turmerone showed deterrent activity similar to DEET. In dose-response bioassay, ar-turmerone showed significantly higher biting deterrence than DEET at all the dosages. Ar-turmerone, at 15 nmol/cm(2), showed activity similar to DEET at 25 nmol/cm(2) and activity at 5 nmol/cm(2) was similar to DEET at 20 and 15 nmol/cm(2). Leaf essential oil with LC(50) values of 1.8 and 8.9 ppm against larvae of An. quadrimaculatus and Ae. aegypti, respectively, showed highest toxicity followed by rhizome oil and ethanolic extract. Among the pure compounds, ar-turmerone with LC(50) values of 2.8 and 2.5 ppm against larvae of An. quadrimaculatus and Ae. aegypti, respectively, was most toxic followed by bisdesmethoxycurcumin, curcumin, and desmethoxycurcumin. PMID:26336212

  8. A TNF Variant that Associates with Susceptibility to Musculoskeletal Disease Modulates Thyroid Hormone Receptor Binding to Control Promoter Activation

    PubMed Central

    Kiss-Toth, Endre; Harlock, Edward; Lath, Darren; Quertermous, Thomas; Wilkinson, J. Mark

    2013-01-01

    Tumor necrosis factor (TNF) is a powerful pro-inflammatory cytokine and immuno-regulatory molecule, and modulates susceptibility to musculoskeletal diseases. Several meta-analyses and replicated association studies have implicated the minor ‘A’ variant within the TNF promoter single nucleotide polymorphism (SNP) rs361525 (-238A/G) as a risk allele in joint related disorders, including psoriatic and juvenile idiopathic arthritis, and osteolysis after joint arthroplasty. Here we characterized the effect of this variant on TNF promoter function. A transcriptional reporter, encoding the -238A variant of the TNF promoter, resulted in 2.2 to 2.8 times greater transcriptional activation versus the ‘G’ variant in murine macrophages when stimulated with pro-inflammatory stimuli. Bioinformatic analysis predicted a putative binding site for thyroid hormone receptor (TR) for the -238A but not the -238G allele. Overexpression of TR-α induced promoter expression 1.8-fold in the presence of the ‘A’ allele only. TR-α expression both potentiated and sensitized the -238A response to LPS or a titanium particulate stimulus, whilst siRNA knockdown of either THRA or THRB impaired transcriptional activation for the -238A variant only. This effect was independent of receptor-ligand binding of triiodothyronine. Immunohistochemical analysis of osteolysis interface membranes from patients undergoing revision surgery confirmed expression of TR-α within osteoclast nuclei at the resorption surface. The ‘A’ allele at rs361525 confers increased transcriptional activation of the TNF promoter and influences susceptibility to several arthritic conditions. This effect is modulated, at least in part, by binding of TR, which both sensitizes and potentiates transcriptional activation of the ‘A’ variant independent of its endogenous ligand. PMID:24069456

  9. Insights from Zebrafish and Mouse Models on the Activity and Safety of Ar-Turmerone as a Potential Drug Candidate for the Treatment of Epilepsy

    PubMed Central

    Orellana-Paucar, Adriana Monserrath; Afrikanova, Tatiana; Thomas, Joice; Aibuldinov, Yelaman K.; Dehaen, Wim; de Witte, Peter A. M.; Esguerra, Camila V.

    2013-01-01

    In a previous study, we uncovered the anticonvulsant properties of turmeric oil and its sesquiterpenoids (ar-turmerone, α-, β-turmerone and α-atlantone) in both zebrafish and mouse models of chemically-induced seizures using pentylenetetrazole (PTZ). In this follow-up study, we aimed at evaluating the anticonvulsant activity of ar-turmerone further. A more in-depth anticonvulsant evaluation of ar-turmerone was therefore carried out in the i.v. PTZ and 6-Hz mouse models. The potential toxic effects of ar-turmerone were evaluated using the beam walking test to assess mouse motor function and balance. In addition, determination of the concentration-time profile of ar-turmerone was carried out for a more extended evaluation of its bioavailability in the mouse brain. Ar-turmerone displayed anticonvulsant properties in both acute seizure models in mice and modulated the expression patterns of two seizure-related genes (c-fos and brain-derived neurotrophic factor [bdnf]) in zebrafish. Importantly, no effects on motor function and balance were observed in mice after treatment with ar-turmerone even after administering a dose 500-fold higher than the effective dose in the 6-Hz model. In addition, quantification of its concentration in mouse brains revealed rapid absorption after i.p. administration, capacity to cross the BBB and long-term brain residence. Hence, our results provide additional information on the anticonvulsant properties of ar-turmerone and support further evaluation towards elucidating its mechanism of action, bioavailability, toxicity and potential clinical application. PMID:24349101

  10. Insights from zebrafish and mouse models on the activity and safety of ar-turmerone as a potential drug candidate for the treatment of epilepsy.

    PubMed

    Orellana-Paucar, Adriana Monserrath; Afrikanova, Tatiana; Thomas, Joice; Aibuldinov, Yelaman K; Dehaen, Wim; de Witte, Peter A M; Esguerra, Camila V

    2013-01-01

    In a previous study, we uncovered the anticonvulsant properties of turmeric oil and its sesquiterpenoids (ar-turmerone, α-, β-turmerone and α-atlantone) in both zebrafish and mouse models of chemically-induced seizures using pentylenetetrazole (PTZ). In this follow-up study, we aimed at evaluating the anticonvulsant activity of ar-turmerone further. A more in-depth anticonvulsant evaluation of ar-turmerone was therefore carried out in the i.v. PTZ and 6-Hz mouse models. The potential toxic effects of ar-turmerone were evaluated using the beam walking test to assess mouse motor function and balance. In addition, determination of the concentration-time profile of ar-turmerone was carried out for a more extended evaluation of its bioavailability in the mouse brain. Ar-turmerone displayed anticonvulsant properties in both acute seizure models in mice and modulated the expression patterns of two seizure-related genes (c-fos and brain-derived neurotrophic factor [bdnf]) in zebrafish. Importantly, no effects on motor function and balance were observed in mice after treatment with ar-turmerone even after administering a dose 500-fold higher than the effective dose in the 6-Hz model. In addition, quantification of its concentration in mouse brains revealed rapid absorption after i.p. administration, capacity to cross the BBB and long-term brain residence. Hence, our results provide additional information on the anticonvulsant properties of ar-turmerone and support further evaluation towards elucidating its mechanism of action, bioavailability, toxicity and potential clinical application. PMID:24349101

  11. Affective mentalizing and brain activity at rest in the behavioral variant of frontotemporal dementia

    PubMed Central

    Caminiti, Silvia P.; Canessa, Nicola; Cerami, Chiara; Dodich, Alessandra; Crespi, Chiara; Iannaccone, Sandro; Marcone, Alessandra; Falini, Andrea; Cappa, Stefano F.

    2015-01-01

    Background bvFTD patients display an impairment in the attribution of cognitive and affective states to others, reflecting GM atrophy in brain regions associated with social cognition, such as amygdala, superior temporal cortex and posterior insula. Distinctive patterns of abnormal brain functioning at rest have been reported in bvFTD, but their relationship with defective attribution of affective states has not been investigated. Objective To investigate the relationship among resting-state brain activity, gray matter (GM) atrophy and the attribution of mental states in the behavioral variant of fronto-temporal degeneration (bvFTD). Methods We compared 12 bvFTD patients with 30 age- and education-matched healthy controls on a) performance in a task requiring the attribution of affective vs. cognitive mental states; b) metrics of resting-state activity in known functional networks; and c) the relationship between task-performances and resting-state metrics. In addition, we assessed a connection between abnormal resting-state metrics and GM atrophy. Results Compared with controls, bvFTD patients showed a reduction of intra-network coherent activity in several components, as well as decreased strength of activation in networks related to attentional processing. Anomalous resting-state activity involved networks which also displayed a significant reduction of GM density. In patients, compared with controls, higher affective mentalizing performance correlated with stronger functional connectivity between medial prefrontal sectors of the default-mode and attentional/performance monitoring networks, as well as with increased coherent activity in components of the executive, sensorimotor and fronto-limbic networks. Conclusions Some of the observed effects may reflect specific compensatory mechanisms for the atrophic changes involving regions in charge of affective mentalizing. The analysis of specific resting-state networks thus highlights an intermediate level of

  12. Peptide-Mediated Specific Immobilization of Catalytically Active Cytochrome P450 BM3 Variant.

    PubMed

    Zernia, Sarah; Ott, Florian; Bellmann-Sickert, Kathrin; Frank, Ronny; Klenner, Marcus; Jahnke, Heinz-Georg; Prager, Andrea; Abel, Bernd; Robitzki, Andrea; Beck-Sickinger, Annette G

    2016-04-20

    Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is an interesting target for biotechnological applications, because of its vast substrate variety combined with high P450 monooxygenase activity. The low stability in vitro could be overcome by immobilization on surfaces. Here we describe a novel method for immobilization on metal surfaces by using selectively binding peptides. A P450 BM3 triple mutant (3M-P450BM3: A74G, F87V, L188Q) was purified as protein thioester and ligated to indium tin oxide or gold binding peptides (BP) named HighSP-BP and Cys-BP, respectively. The ligation products were characterized by Western Blot and tryptic digestion combined with mass spectrometry, and displayed high affinity binding on the depicted surfaces. Next, we could demonstrate by benzyloxyresorufin O-dealkylation assay (BROD assay) that the activity of immobilized ligation products is higher than for the soluble form. The study provides a new tool for selective modification and immobilization of P450 variants. PMID:26967204

  13. New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits

    PubMed Central

    Osakada, Fumitaka; Mori, Takuma; Cetin, Ali H.; Marshel, James H.; Virgen, Beatriz; Callaway, Edward M.

    2011-01-01

    SUMMARY Glycoprotein-deleted (ΔG) rabies virus is a powerful tool for studies of neural circuit structure. Here we describe the development and demonstrate the utility of new resources that allow experiments directly investigating relationships between the structure and function of neural circuits. New methods and reagents allowed efficient production of twelve novel ΔG rabies variants from plasmid DNA. These new rabies viruses express useful neuroscience tools, including: the Ca++ indicator GCaMP3, for monitoring activity; Channelrhodopsin-2, for photoactivation; allatostatin receptor, for inactivation by ligand application; rtTA, ERT2CreERT2, or FLPo, for control of gene expression. These new tools allow neurons targeted based on their connectivity, to have their function assayed or their activity or gene expression manipulated. Combining these tools with in vivo imaging and optogenetic methods, and/or inducible gene expression in transgenic mice, will facilitate experiments investigating neural circuit development, plasticity, and function that have not been possible with existing reagents. PMID:21867879

  14. Calibration and validation of the soil moisture active passive mission with USDA-ARS experimental watersheds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Soil Moisture Active Passive Mission (SMAP) is a new NASA mission scheduled for 2014 that will provide a number of soil moisture and freeze/thaw products. The soil moisture products will span spatial resolutions from 3 to 36 km. Key to the validation and calibration of the satellite products are...

  15. Initial validation of the Soil Moisture Active Passive mission using USDA-ARS watersheds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Soil Moisture Active Passive (SMAP) Mission was launched in January 2015 to measure global surface soil moisture. The calibration and validation program of SMAP relies upon an international cooperative of in situ networks to provide ground truth references across a variety of landscapes. The U...

  16. Validation of the Soil Moisture Active Passive mission using USDA-ARS experimental watersheds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The calibration and validation program of the Soil Moisture Active Passive mission (SMAP) relies upon an international cooperative of in situ networks to provide ground truth references across a variety of landscapes. The USDA Agricultural Research Service operates several experimental watersheds wh...

  17. Combined experimental and modeling studies of microwave activated CH4/H2/Ar plasmas for microcrystalline, nanocrystalline, and ultrananocrystalline diamond deposition

    NASA Astrophysics Data System (ADS)

    Richley, James C.; Fox, Oliver J. L.; Ashfold, Michael N. R.; Mankelevich, Yuri A.

    2011-03-01

    A comprehensive study of microwave (MW) activated CH4/H2/Ar plasmas used for diamond chemical vapor deposition is reported, focusing particularly on the effects of gross variations in the H2/Ar ratio in the input gas mixture (from H2/Ar mole fraction ratios of > 10:1, through to ˜1:99). Absolute column densities of C2(a) and CH(X) radicals and of H(n = 2) atoms have been determined by cavity ringdown spectroscopy, as functions of height (z) above a substrate and of process conditions (CH4, H2, and Ar input mole fractions, total pressure, p, and input microwave power, P). Optical emission spectroscopy has also been used to explore the relative densities of electronically excited H atoms, and CH, C2, and C3 radicals, as functions of these same process conditions. These experimental data are complemented by extensive 2D (r, z) modeling of the plasma chemistry, which provides a quantitative rationale for all of the experimental observations. Progressive replacement of H2 by Ar (at constant p and P) leads to an expanded plasma volume. Under H2-rich conditions, > 90% of the input MW power is absorbed through rovibrational excitation of H2. Reducing the H2 content (as in an Ar-rich plasma) leads to a reduction in the absorbed power density; the plasma necessarily expands in order to accommodate a given input power. The average power density in an Ar-rich plasma is much lower than that in an H2-rich plasma operating at the same p and P. Progressive replacement of H2 by Ar is shown also to result in an increased electron temperature, an increased [H]/[H2] number density ratio, but little change in the maximum gas temperature in the plasma core (which is consistently ˜3000 K). Given the increased [H]/[H2] ratio, the fast H-shifting (CyHx + H ↔ CyHx-1 + H2; y = 1-3) reactions ensure that the core of Ar-rich plasma contains much higher relative abundances of "product" species like C atoms, and C2, and C3 radicals. The effects of Ar dilution on the absorbed power

  18. Combined experimental and modeling studies of microwave activated CH{sub 4}/H{sub 2}/Ar plasmas for microcrystalline, nanocrystalline, and ultrananocrystalline diamond deposition

    SciTech Connect

    Richley, James C.; Fox, Oliver J. L.; Ashfold, Michael N. R.; Mankelevich, Yuri A.

    2011-03-15

    A comprehensive study of microwave (MW) activated CH{sub 4}/H{sub 2}/Ar plasmas used for diamond chemical vapor deposition is reported, focusing particularly on the effects of gross variations in the H{sub 2}/Ar ratio in the input gas mixture (from H{sub 2}/Ar mole fraction ratios of > 10:1, through to {approx}1:99). Absolute column densities of C{sub 2}(a) and CH(X) radicals and of H(n = 2) atoms have been determined by cavity ringdown spectroscopy, as functions of height (z) above a substrate and of process conditions (CH{sub 4}, H{sub 2}, and Ar input mole fractions, total pressure, p, and input microwave power, P). Optical emission spectroscopy has also been used to explore the relative densities of electronically excited H atoms, and CH, C{sub 2}, and C{sub 3} radicals, as functions of these same process conditions. These experimental data are complemented by extensive 2D (r, z) modeling of the plasma chemistry, which provides a quantitative rationale for all of the experimental observations. Progressive replacement of H{sub 2} by Ar (at constant p and P) leads to an expanded plasma volume. Under H{sub 2}-rich conditions, > 90% of the input MW power is absorbed through rovibrational excitation of H{sub 2}. Reducing the H{sub 2} content (as in an Ar-rich plasma) leads to a reduction in the absorbed power density; the plasma necessarily expands in order to accommodate a given input power. The average power density in an Ar-rich plasma is much lower than that in an H{sub 2}-rich plasma operating at the same p and P. Progressive replacement of H{sub 2} by Ar is shown also to result in an increased electron temperature, an increased [H]/[H{sub 2}] number density ratio, but little change in the maximum gas temperature in the plasma core (which is consistently {approx}3000 K). Given the increased [H]/[H{sub 2}] ratio, the fast H-shifting (C{sub y}H{sub x} + H {r_reversible} C{sub y}H{sub x-1} + H{sub 2}; y = 1-3) reactions ensure that the core of Ar-rich plasma

  19. ARS Biodiesel Research Initiatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biodiesel activities within ARS are concerned with the production, quality, and properties of this alternative fuel from agriculturally derived fats and oils. Currently, in the absence of tax incentives, biodiesel production when using refined fats and oils and conventional alkali transesterificati...

  20. DECONSTRUCTING ACTIVE REGION AR10961 USING STEREO, HINODE, TRACE, AND SOHO

    SciTech Connect

    Noglik, Jane B.; Walsh, Robert W.; Marsh, M. S.; Maclean, Rhona C.

    2009-10-01

    Active region 10961 was observed over a five-day period (2007 July 2-6) by instrumentation on-board STEREO, Hinode, TRACE, and SOHO. As it progressed from Sun's center to the solar limb, a comprehensive analysis of the extreme ultraviolet, X-ray, and magnetic field data reveals clearly observable changes in the global nature of the region. Temperature analyses undertaken using STEREO Extreme Ultraviolet Imager double filter ratios and X-ray imaging telescope single and combined filter ratios demonstrate an overall cooling of the region from between 1.6-3.0 MK to 1.0-2.0 MK over the five days. Similarly, Hinode Extreme Ultraviolet Imaging Spectrograph density measurements show a corresponding increase in density of 27%. Moss, cool (1 MK) outer loop areas, and hotter core loop regions were examined and compared with potential magnetic field extrapolations from SOHO Michelson Doppler Imager magnetogram data. In particular, it was found that the potential field model was able to predict the structure of the hotter X-ray loops and that the larger cool loops seen in 171 A images appeared to follow the separatrix surfaces. The reasons behind the high-density moss regions only observed on one side of the active region are examined further.

  1. Validation of the Soil Moisture Active Passive mission using USDA-ARS experimental watersheds

    NASA Astrophysics Data System (ADS)

    Cosh, M. H.; Jackson, T. J.; Bindlish, R.; Colliander, A.; Kim, S.; Das, N. N.; Yueh, S. H.; Bosch, D. D.; Goodrich, D. C.; Prueger, J. H.; Starks, P. J.; Livingston, S.; Seyfried, M. S.; Coopersmith, E. J.

    2015-12-01

    The calibration and validation program of the Soil Moisture Active Passive mission (SMAP) relies upon an international cooperative of in situ networks to provide ground truth references across a variety of landscapes. The USDA Agricultural Research Service operates several experimental watersheds which contribute to the validation of SMAP soil moisture products. These watersheds consist of a network of in situ sensors that measure soil moisture at a variety of depths including the 5 cm depth, which is critical for satellite validation. Comparisons of the in situ network estimates to the satellite products are ongoing, but initial results have shown strong correlation between satellite estimates and in situ soil moisture measurements once scaling functions were applied. The scaling methodologies for the in situ networks are being reviewed and evaluated. Results from the Little Washita, Fort Cobb, St. Joseph's and Little River Experimental Watersheds show good agreement between the satellite products and in situ measurements. Walnut Gulch results show high accuracy, although with the caveat that these domains are semi-arid with a substantially lower dynamic range. The South Fork Watershed is examined more closely for its detailed scaling function development as well as an apparent bias between satellite and in situ values.

  2. Time-variant fMRI activity in the brainstem and higher structures in response to acupuncture.

    PubMed

    Napadow, Vitaly; Dhond, Rupali; Park, Kyungmo; Kim, Jieun; Makris, Nikos; Kwong, Kenneth K; Harris, Richard E; Purdon, Patrick L; Kettner, Norman; Hui, Kathleen K S

    2009-08-01

    Acupuncture modulation of activity in the human brainstem is not well known. This structure is plagued by physiological artifact in neuroimaging experiments. In addition, most studies have used short (<15 min) block designs, which miss delayed responses following longer duration stimulation. We used brainstem-focused cardiac-gated fMRI and evaluated time-variant brain response to longer duration (>30 min) stimulation with verum (VA, electro-stimulation at acupoint ST-36) or sham point (SPA, non-acupoint electro-stimulation) acupuncture. Our results provide evidence that acupuncture modulates brainstem nuclei important to endogenous monoaminergic and opioidergic systems. Specifically, VA modulated activity in the substantia nigra (SN), nucleus raphe magnus, locus ceruleus, nucleus cuneiformis, and periaqueductal gray (PAG). Activation in the ventrolateral PAG was greater for VA compared to SPA. Linearly decreasing time-variant activation, suggesting classical habituation, was found in response to both VA and SPA in sensorimotor (SII, posterior insula, premotor cortex) brain regions. However, VA also produced linearly time-variant activity in limbic regions (amygdala, hippocampus, and SN), which was bimodal and not likely habituation--consisting of activation in early blocks, and deactivation by the end of the run. Thus, acupuncture induces different brain response early, compared to 20-30 min after stimulation. We attribute the fMRI differences between VA and SPA to more varied and stronger psychophysical response induced by VA. Our study demonstrates that acupuncture modulation of brainstem structures can be studied non-invasively in humans, allowing for comparison to animal studies. Our protocol also demonstrates a fMRI approach to study habituation and other time-variant phenomena over longer time durations. PMID:19345268

  3. Ars insulator identified in sea urchin possesses an activity to ensure the transgene expression in mouse cells.

    PubMed

    Tajima, Shoji; Shinohara, Keiko; Fukumoto, Maiko; Zaitsu, Reiko; Miyagawa, Junichi; Hino, Shinjiro; Fan, Jun; Akasaka, Koji; Matsuoka, Masao

    2006-04-01

    Sea urchin arylsulfatase (Ars) gene locus has features of an insulator, i.e., blocking of enhancer and promoter interaction, and protection of a transgene against positional effects [Akasaka et al. (1999) Cell. Mol. Biol. 45, 555-565]. To examine the effect of Ars insulator on long-term expression of a transgene, the insulator was inserted into LTR of retrovirus vector harboring hrGFP gene as a reporter, and then introduced into mouse myoblast cells. The isolated clones transduced with the reporter gene with or without Ars insulator were cultured for more than 20 wk in the absence of a selection reagent, and the expression of hrGFP was periodically determined. Expression of hrGFP in four clones transduced with the reporter gene without Ars insulator was completely silenced after 20 wk of culture. On the other hand, hrGFP was expressed in all clones with Ars insulator inserted in one of the two different orientations. Histone H3 deacetylation and DNA methylation of the 5'LTR promoter region, signs for heterochromatin and silencing, were suppressed in the clones that were expressing hrGFP. Ars insulator is effective in maintaining a transgene in mouse cells in an orientation-dependent manner, and will be a useful tool to ensure stable expression of a transgene. PMID:16672271

  4. Molecular Insights into the Dynamics of Pharmacogenetically Important N-Terminal Variants of the Human β2-Adrenergic Receptor

    PubMed Central

    Sengupta, Durba; Joshi, Manali

    2014-01-01

    The human β2-adrenergic receptor (β2AR), a member of the G-protein coupled receptor (GPCR) family, is expressed in bronchial smooth muscle cells. Upon activation by agonists, β2AR causes bronchodilation and relief in asthma patients. The N-terminal polymorphism of β2AR at the 16th position, Arg16Gly, has warranted a lot of attention since it is linked to variations in response to albuterol (agonist) treatment. Although the β2AR is one of the well-studied GPCRs, the N-terminus which harbors this mutation, is absent in all available experimental structures. The goal of this work was to study the molecular level differences between the N-terminal variants using structural modeling and atomistic molecular dynamics simulations. Our simulations reveal that the N-terminal region of the Arg variant shows greater dynamics than the Gly variant, leading to differential placement. Further, the position and dynamics of the N-terminal region, further, affects the ligand binding-site accessibility. Interestingly, long-range effects are also seen at the ligand binding site, which is marginally larger in the Gly as compared to the Arg variant resulting in the preferential docking of albuterol to the Gly variant. This study thus reveals key differences between the variants providing a molecular framework towards understanding the variable drug response in asthma patients. PMID:25501358

  5. Characterization of Antibacterial and Hemolytic Activity of Synthetic Pandinin 2 Variants and Their Inhibition against Mycobacterium tuberculosis

    PubMed Central

    Rodríguez, Alexis; Villegas, Elba; Montoya-Rosales, Alejandra; Rivas-Santiago, Bruno; Corzo, Gerardo

    2014-01-01

    The contention and treatment of Mycobacterium tuberculosis and other bacteria that cause infectious diseases require the use of new type of antibiotics. Pandinin 2 (Pin2) is a scorpion venom antimicrobial peptide highly hemolytic that has a central proline residue. This residue forms a structural “kink” linked to its pore-forming activity towards human erythrocytes. In this work, the residue Pro14 of Pin2 was both substituted and flanked using glycine residues (P14G and P14GPG) based on the low hemolytic activities of antimicrobial peptides with structural motifs Gly and GlyProGly such as magainin 2 and ponericin G1, respectively. The two Pin2 variants showed antimicrobial activity against E. coli, S. aureus, and M. tuberculosis. However, Pin2 [GPG] was less hemolytic (30%) than that of Pin2 [G] variant. In addition, based on the primary structure of Pin2 [G] and Pin2 [GPG], two short peptide variants were designed and chemically synthesized keeping attention to their physicochemical properties such as hydrophobicity and propensity to adopt alpha-helical conformations. The aim to design these two short antimicrobial peptides was to avoid the drawback cost associated to the synthesis of peptides with large sequences. The short Pin2 variants named Pin2 [14] and Pin2 [17] showed antibiotic activity against E. coli and M. tuberculosis. Besides, Pin2 [14] presented only 25% of hemolysis toward human erythrocytes at concentrations as high as 100 µM, while the peptide Pin2 [17] did not show any hemolytic effect at the same concentration. Furthermore, these short antimicrobial peptides had better activity at molar concentrations against multidrug resistance M. tuberculosis than that of the conventional antibiotics ethambutol, isoniazid and rifampicin. Therefore, Pin2 [14] and Pin2 [17] have the potential to be used as an alternative antibiotics and anti-tuberculosis agents with reduced hemolytic effects. PMID:25019413

  6. Altered Secretory Activity of APE1/Ref-1 D148E Variants Identified in Human Patients With Bladder Cancer

    PubMed Central

    2016-01-01

    Purpose: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox modulation. Recently, serum and urinary APE1/Ref-1 levels were reported to be increased in patients with bladder cancer. Genetic variations of APE/Ref-1 are associated with the risk of cancer. However, the effect of APE1/Ref-1 variants on its secretory activity is yet unknown. Methods: APE1/Ref-1 variants were evaluated by DNA sequencing analysis of reverse transcription polymerase chain reaction products in coding DNA sequences (CDS) of APE1/Ref-1 in bladder tissue samples from patients with bladder cancer (n=10). Secretory activity of APE1/Ref-1 variants was evaluated with immunoblot and enzyme-linked immunosorbent assay of the culture medium supernatants. Results: Four different substitution mutants (D148E, I64V/D148E, W67R/D148E, and E86G/D148E) of APE1/Ref-1 were identified in bladder cancer specimens. However, deletion mutants of APE1/Ref-1 CDS were not found. The secretory activity of the APE1/Ref-1 variants (D148E, I64V/D148E, and E86G/D148E) was increased compared to that of wild type APE1/Ref-1. Furthermore, the secretory activity in basal or hyperacetylated conditions was much higher than that in APE1/Ref-1 D148E-transfected HEK293 cells. Conclusions: Taken together, our data suggest that the increased secretory activity of D148E might contribute to increased serum levels of APE1/Ref-1 in patients with bladder cancer. PMID:27230458

  7. Bacillus cereus AR156 primes induced systemic resistance by suppressing miR825/825* and activating defense-related genes in Arabidopsis.

    PubMed

    Niu, Dongdong; Xia, Jing; Jiang, Chunhao; Qi, Beibei; Ling, Xiaoyu; Lin, Siyuan; Zhang, Weixiong; Guo, Jianhua; Jin, Hailing; Zhao, Hongwei

    2016-04-01

    Small RNAs play an important role in plant immune responses. However, their regulatory function in induced systemic resistance (ISR) is nascent. Bacillus cereus AR156 is a plant growth-promoting rhizobacterium that induces ISR in Arabidopsis against bacterial infection. Here, by comparing small RNA profiles of Pseudomonas syringae pv. tomato (Pst) DC3000-infected Arabidopsis with and without AR156 pretreatment, we identified a group of Arabidopsis microRNAs (miRNAs) that are differentially regulated by AR156 pretreatment. miR825 and miR825* are two miRNA generated from a single miRNA gene. Northern blot analysis indicated that they were significantly downregulated in Pst DC3000-infected plants pretreated with AR156, in contrast to the plants without AR156 pretreatment. miR825 targets two ubiquitin-protein ligases, while miR825* targets toll-interleukin-like receptor (TIR)-nucleotide binding site (NBS) and leucine-rich repeat (LRR) type resistance (R) genes. The expression of these target genes negatively correlated with the expression of miR825 and miR825*. Moreover, transgenic plants showing reduced expression of miR825 and miR825* displayed enhanced resistance to Pst DC3000 infection, whereas transgenic plants overexpressing miR825 and miR825* were more susceptible. Taken together, our data indicates that Bacillus cereus AR156 pretreatment primes ISR to Pst infection by suppressing miR825 and miR825* and activating the defense related genes they targeted. PMID:26526683

  8. A comparison study: Direct wafer bonding of SiC–SiC by standard surface-activated bonding and modified surface-activated bonding with Si-containing Ar ion beam

    NASA Astrophysics Data System (ADS)

    Mu, Fengwen; Iguchi, Kenichi; Nakazawa, Haruo; Takahashi, Yoshikazu; Fujino, Masahisa; He, Ran; Suga, Tadatomo

    2016-08-01

    In this study, the results of direct wafer bonding of SiC–SiC at room temperature by standard surface-activated bonding (SAB) and modified SAB with a Si-containing Ar ion beam were compared, in terms of bonding energy, interface structure and composition, and the effects of rapid thermal annealing (RTA) at 1273 K in Ar gas. Compared with that obtained by the standard SAB, the bonding interface obtained by the modified SAB with a Si-containing Ar ion beam is ∼30% stronger and almost completely recrystallized without oxidation during RTA, which should be due to the in situ Si compensation during surface activation by the Si-containing Ar ion beam.

  9. The TLQP-21 Peptide Activates the G-protein-coupled receptor C3aR1 via a Folding-upon-Binding Mechanism

    PubMed Central

    Severini, Cinzia; Gopinath, Tata; Braun, Patrick D.; Sassano, Maria F.; Gurney, Allison; Roth, Bryan L.; Vulchanova, Lucy; Possenti, Roberta; Veglia, Gianluigi; Bartolomucci, Alessandro

    2014-01-01

    SUMMARY TLQP-21, a VGF-encoded peptide is emerging as a novel target for obesity-associated disorders. TLQP-21 is found in the sympathetic nerve terminals in the adipose tissue and targets the G-protein-coupled-receptor (GPCR) Complement-3a-Receptor1 (C3aR1). So far, the mechanisms of TLQP-21-induced receptor activation remained unexplored. Here, we report that TLQP-21 is intrinsically disordered and undergoes a disorder-to-order transition, adopting an α-helical conformation, upon targeting cells expressing the C3aR1. We determined that the hot spots for TLQP-21 are located at the C-terminus, with mutations in the last four amino acids progressively reducing the bioactivity and, a single site mutation (R21A) or C-terminal amidation abolishing its function completely. Interestingly, the human TLQP-21 sequence carrying a S20A substitution activates the human C3aR1 receptor with lower potency compared to the rodent sequence. These studies reveal the mechanism of action of TLQP-21 and provide molecular templates for designing agonists and antagonists to modulate C3aR1 functions. PMID:25456411

  10. The TLQP-21 peptide activates the G-protein-coupled receptor C3aR1 via a folding-upon-binding mechanism.

    PubMed

    Cero, Cheryl; Vostrikov, Vitaly V; Verardi, Raffaello; Severini, Cinzia; Gopinath, Tata; Braun, Patrick D; Sassano, Maria F; Gurney, Allison; Roth, Bryan L; Vulchanova, Lucy; Possenti, Roberta; Veglia, Gianluigi; Bartolomucci, Alessandro

    2014-12-01

    TLQP-21, a VGF-encoded peptide is emerging as a novel target for obesity-associated disorders. TLQP-21 is found in the sympathetic nerve terminals in the adipose tissue and targets the G-protein-coupled receptor complement-3a receptor1 (C3aR1). The mechanisms of TLQP-21-induced receptor activation remain unexplored. Here, we report that TLQP-21 is intrinsically disordered and undergoes a disorder-to-order transition, adopting an α-helical conformation upon targeting cells expressing the C3aR1. We determined that the hot spots for TLQP-21 are located at the C terminus, with mutations in the last four amino acids progressively reducing the bioactivity and, a single site mutation (R21A) or C-terminal amidation abolishing its function completely. Additionally, the human TLQP-21 sequence carrying a S20A substitution activates the human C3aR1 receptor with lower potency compared to the rodent sequence. These studies reveal the mechanism of action of TLQP-21 and provide molecular templates for designing agonists and antagonists to modulate C3aR1 functions. PMID:25456411

  11. Discovery of 34 g ,mCl (p,γ ) 35Ar resonances activated at classical nova temperatures

    NASA Astrophysics Data System (ADS)

    Fry, C.; Wrede, C.; Bishop, S.; Brown, B. A.; Chen, A. A.; Faestermann, T.; Hertenberger, R.; Parikh, A.; Pérez-Loureiro, D.; Wirth, H.-F.; García, A.; Ortez, R.

    2015-01-01

    Background: The thermonuclear 34 g ,mCl (p,γ ) 35Ar reaction rates are unknown due to a lack of experimental nuclear physics data. Uncertainties in these rates translate to uncertainties in 34S production in models of classical novae on oxygen-neon white dwarfs. 34S abundances have the potential to aid in the classification of presolar grains. Purpose: Determine resonance energies for the 34 g ,mCl (p,γ ) 35Ar reactions within the region of astrophysical interest for classical novae to a precision of a few keV as an essential first step toward constraining their thermonuclear reaction rates. Method: 35Ar excited states were populated by the 36Ar (d,t ) 35Ar reaction at E (d )=22 MeV and reaction products were momentum analyzed by a high resolution quadrupole-dipole-dipole-dipole (Q3D) magnetic spectrograph. Results: Seventeen new 35Ar levels have been detected at a statistically significant level in the region Ex≈5.9 -6.7 MeV (Er<800 keV ) and their excitation energies have been determined to typical uncertainties of 3 keV. The uncertainties for five previously known levels have also been reduced substantially. The measured level density was compared to those calculated using the WBMB Hamiltonian within the s d -p f model space. Conclusions: Most of the resonances in the region of astrophysical interest have likely been discovered and their energies have been determined, but the resonance strengths are still unknown, and experimentally constraining the 34 g ,mCl (p,γ ) 35Ar reaction rates will require further experiments.

  12. Ar-39-Ar-40 ages of four ureilites

    NASA Technical Reports Server (NTRS)

    Bogard, D. D.; Garrison, D. H.

    1994-01-01

    Ureilites Novo Urei, Havero, and Kenna show strong evidence of one or more Ar-40 degassing events in the time period of 3.3-4.1 Ga ago. These ages may be compared to current interpretations of ureilite chronology. These include the suggestion of metasomatic activity on the parent body 3.7 Ga ago that reset some Sm-Nd ages and the suggestion that ureilites have experienced terrestrial contamination of several trace elements (including Pb and LREE), which makes suspect ages younger than approximately 4.5 Ga. Because the K-Ar chronometer can be sensitive to metamorphic events, we made Ar-39-Ar-40 determinations on bulk samples (0.12-0.14 g each) of four ureilites. The Ar-39-Ar-40 age spectra and K/Ca ratios as a function of cumulative Ar release from stepwise temperature extractions for the four ureilites analyzed are shown. Because Ar-39-Ar-40 ages shown by low and high temperature extractions may be suspect, we examined the intermediate temperature extractions. Although interpretation of these spectra is obviously uncertain, we believe that the most recent times of Ar degassing can be roughly inferred. These times are approximately 3.3 Ga for Havero, 3.3-3.7 Ga for Novo Urei, and approximately 4.1 Ga for Kenna, for which Ar degassing may not have been complete. The indication of Ar-39-Ar-40 degassing ages of 3.3-4.1 Ga for three ureilites that also contain an enhanced LREE component and (excepting Havero) produce a 3.74 Ga Sm-Nd age, suggests that both chronometers may have responded to the same parent body event. On the other hand, it is also possible that the Ar data reflect one or more separate events that did not strongly affect the Sm-Nd system, a situation that commonly occurs in eucrites. Thus the existence of reset Ar ages does not require similarly reset Sm-Nd ages.

  13. Interactions of Genetic Variants with Physical Activity are Associated with Blood Pressure in Chinese: The GenSalt Study

    PubMed Central

    Montasser, May E.; Gu, Donfeng; Chen, Jing; Shimmin, Lawrence C.; Gu, Charles; Kelly, Tanika N.; Jaquish, Cashell E.; Rice, Treva; Rao, DC; Cao, Jie; Chen, Jichun; Liu, De-pei; Whelton, Paul; He, Jiang; Hixson, James E.

    2016-01-01

    Background Blood pressure (BP) homeostasis involves complex interactions among genetic and non-genetic factors, providing major challenges to dissection of the genetic components that influence BP and hypertension. In this study, we examine the effects of interaction of genetic variants with physical activity on BP in a relatively genetically homogenous cohort of rural Chinese villagers. Methods Generalized estimating equations analysis was used to test for associations of systolic blood pressure (SBP) and diastolic blood pressure (DBP) with variants in 24 genes in BP pathways (196 SNPs) among 3,142 Chinese participants divided according to physical activity (active versus inactive groups). Results In the physically active group, 2 SNPs in NR3C2 were significantly associated with lower SBP, and a SNP in SCNN1B was significantly associated with lower SBP and DBP. In the physically inactive group, a SNP in APLNR was associated with lower SBP, a SNP in GNB3 was associated with higher SBP and DBP, and a SNP in BDKRB2 was associated with lower DBP. Cumulative effects in carriers of minor alleles of these SNPs showed reductions of SBP and DBP as large as 8 and 5 mmHg, respectively, in the active individuals compared to inactive individuals carrying the same number of minor alleles. Conclusions We found that physical activity modifies the effects of genetic variants on BP. However, our results also show that active individuals with specific genotypes always have lower BP than inactive individuals with the same genotypes, demonstrating the overall beneficial effects of physical activity on blood pressure. PMID:21654856

  14. Inversed relationship between CD44 variant and c-Myc due to oxidative stress-induced canonical Wnt activation

    SciTech Connect

    Yoshida, Go J. Saya, Hideyuki

    2014-01-10

    Highlights: •CD44 variant8–10 and c-Myc are inversely expressed in gastric cancer cells. •Redox-stress enhances c-Myc expression via canonical Wnt signal. •CD44v, but not CD44 standard, suppresses redox stress-induced Wnt activation. •CD44v expression promotes both transcription and proteasome degradation of c-Myc. •Inversed expression pattern between CD44v and c-Myc is often recognized in vivo. -- Abstract: Cancer stem-like cells express high amount of CD44 variant8-10 which protects cancer cells from redox stress. We have demonstrated by immunohistochemical analysis and Western blotting, and reverse-transcription polymerase chain reaction, that CD44 variant8-10 and c-Myc tend to show the inversed expression manner in gastric cancer cells. That is attributable to the oxidative stress-induced canonical Wnt activation, and furthermore, the up-regulation of the downstream molecules, one of which is oncogenic c-Myc, is not easily to occur in CD44 variant-positive cancer cells. We have also found out that CD44v8-10 expression is associated with the turn-over of the c-Myc with the experiments using gastric cancer cell lines. This cannot be simply explained by the model of oxidative stress-induced Wnt activation. CD44v8-10-positive cancer cells are enriched at the invasive front. Tumor tissue at the invasive area is considered to be composed of heterogeneous cellular population; dormant cancer stem-like cells with CD44v8-10 {sup high}/ Fbw7 {sup high}/ c-Myc {sup low} and proliferative cancer stem-like cells with CD44v8-10 {sup high}/ Fbw7 {sup low}/ c-Myc {sup high}.

  15. Complement C5a-C5aR interaction enhances MAPK signaling pathway activities to mediate renal injury in trichloroethylene sensitized BALB/c mice.

    PubMed

    Zhang, Jia-xiang; Zha, Wan-sheng; Ye, Liang-ping; Wang, Feng; Wang, Hui; Shen, Tong; Wu, Chang-hao; Zhu, Qi-xing

    2016-02-01

    We have previously shown complement activation as a possible mechanism for trichloroethylene (TCE) sensitization, leading to multi-organ damage including the kidneys. In particular, excessive deposition of C5 and C5b-9-the membrane attack complex, which can generate significant tissue damage, was observed in the kidney tissue after TCE sensitization. The present study tested the hypothesis that anaphylatoxin C5a binding to its receptor C5aR mediates renal injury in TCE-sensitized BALB/c mice. BALB/c mice were sensitized through skin challenge with TCE, with or without pretreatment by the C5aR antagonist W54011. Kidney histopathology and the renal functional test were performed to assess renal injury, and immunohistochemistry and fluorescent labeling were carried out to assess C5a and C5aR expressions. TCE sensitization up-regulated C5a and C5aR expressions in kidney tissue, generated inflammatory infiltration, renal tubule damage, glomerular hypercellularity and impaired renal function. Antagonist pretreatment blocked C5a binding to C5aR and attenuated TCE-induced tissue damage and renal dysfunction. TCE sensitization also caused the deposition of major pro-inflammatory cytokines IL-2, TNF-α and IFN-γ in the kidney tissue (P < 0.05); this was accompanied by increased expression of P-p38, P-ERK and P-JNK proteins (P < 0.05). Pretreatment with the C5aR antagonist attenuated the increase of expression of P-p38, P-ERK and P-JNK proteins (P < 0.05) and also consistently reduced the TCE sensitization-induced increase of IL-2, TNF-α and IFN-γ (P < 0.05). These data identify C5a binding to C5aR, MAP kinase activation, and inflammatory cytokine release as a novel mechanism for complement-mediated renal injury by sensitization with TCE or other environmental chemicals. PMID:26095957

  16. Expression, purification and activity assay of a patchoulol synthase cDNA variant fused to thioredoxin in Escherichia coli.

    PubMed

    Hartwig, S; Frister, T; Alemdar, S; Li, Z; Krings, U; Berger, R G; Scheper, T; Beutel, S

    2014-05-01

    Probing a cDNA library extracted from Pogostemon cablin (Indian Patchouli) with gene specific primers, a variant of patchoulol synthase PTS (GenBank acc. No.: AY508730) was amplified, cloned, and sequenced. The amino acid sequence deduced from the cloned cDNA exhibited a sequence variation of 3.4% compared to the annotated sequence. The enzyme variant tended to form inclusion bodies when expressed in Escherichia coli. The coding sequence was fused to the T7-tag, His-tag and to thioredoxin. Constructs were expressed in three different E. coli expression strains, with several strain/construct combinations yielding soluble enzyme. By fusion to thioredoxin and careful codon optimization of the eukaryotic sequence, soluble expression could be improved on average by 42% in comparison to an unoptimized, His-tagged construct. The thioredoxin-fused protein was successfully purified using a one-step Co(2+)-IMAC purification procedure. Bioactivity assays using prepared farnesyl diphosphate (FDP) in milliliter-scale batch reactions, showed activity of the fused enzyme even with thioredoxin attached. The product spectrum of the enzyme was compared to patchouli oil standards by GC-MS and the main products were identified. Interestingly, the variant showed a shift in product spectrum with germacrene A being the most abundant product instead of patchouli alcohol. In silico structural modeling shows a possible chemical and structural change in the active site of the enzyme, which might be responsible for the shift in product composition. PMID:24576659

  17. CD and MCD Studies of the Effects of Component B Variant Binding on the Biferrous Active Site of Methane Monooxygenase†

    PubMed Central

    Mitić, Nataša; Schwartz, Jennifer K.; Brazeau, Brian J.; Lipscomb, John D.; Solomon, Edward I.

    2008-01-01

    The multi-component soluble form of methane monooxygenase (sMMO) catalyzes the oxidation of methane through activation of O2 at a non-heme biferrous center in the hydroxylase component, MMOH. Reactivity is limited without binding of the sMMO effector protein, MMOB. Past studies show that mutations of specific MMOB surface residues cause large changes in rates of individual steps in the MMOH reaction cycle. To define the structural and mechanistic bases for these observations, CD, MCD, and VTVH MCD spectroscopies coupled with Ligand Field calculations are used to elucidate changes occurring near and at the MMOH biferrous cluster upon binding of MMOB and the MMOB variants. Perturbations to both the CD and MCD are observed upon binding wild-type MMOB and the MMOB variant that similarly increases O2 reactivity. MMOB variants that do not greatly increase O2 reactivity fail to cause one or both of these changes. LF calculations indicate that reorientation of the terminal glutamate on Fe2 reproduces the spectral perturbations in MCD. Although this structural change allows O2 to bridge the diiron site and shifts the redox active orbitals for good overlap, it is not sufficient for enhanced O2 reactivity of the enzyme. Binding of the T111Y-MMOB variant to MMOH induces the MCD, but not CD changes, and causes only a small increase in reactivity. Thus, both the geometric rearrangement at Fe2 (observed in MCD) coupled with a more global conformational change that may control O2 access (probed by CD), induced by MMOB binding, are critical factors in the reactivity of sMMO. PMID:18627173

  18. Ar-Ar_Redux: rigorous error propagation of 40Ar/39Ar data, including covariances

    NASA Astrophysics Data System (ADS)

    Vermeesch, P.

    2015-12-01

    Rigorous data reduction and error propagation algorithms are needed to realise Earthtime's objective to improve the interlaboratory accuracy of 40Ar/39Ar dating to better than 1% and thereby facilitate the comparison and combination of the K-Ar and U-Pb chronometers. Ar-Ar_Redux is a new data reduction protocol and software program for 40Ar/39Ar geochronology which takes into account two previously underappreciated aspects of the method: 1. 40Ar/39Ar measurements are compositional dataIn its simplest form, the 40Ar/39Ar age equation can be written as: t = log(1+J [40Ar/39Ar-298.5636Ar/39Ar])/λ = log(1 + JR)/λ Where λ is the 40K decay constant and J is the irradiation parameter. The age t does not depend on the absolute abundances of the three argon isotopes but only on their relative ratios. Thus, the 36Ar, 39Ar and 40Ar abundances can be normalised to unity and plotted on a ternary diagram or 'simplex'. Argon isotopic data are therefore subject to the peculiar mathematics of 'compositional data', sensu Aitchison (1986, The Statistical Analysis of Compositional Data, Chapman & Hall). 2. Correlated errors are pervasive throughout the 40Ar/39Ar methodCurrent data reduction protocols for 40Ar/39Ar geochronology propagate the age uncertainty as follows: σ2(t) = [J2 σ2(R) + R2 σ2(J)] / [λ2 (1 + R J)], which implies zero covariance between R and J. In reality, however, significant error correlations are found in every step of the 40Ar/39Ar data acquisition and processing, in both single and multi collector instruments, during blank, interference and decay corrections, age calculation etc. Ar-Ar_Redux revisits every aspect of the 40Ar/39Ar method by casting the raw mass spectrometer data into a contingency table of logratios, which automatically keeps track of all covariances in a compositional context. Application of the method to real data reveals strong correlations (r2 of up to 0.9) between age measurements within a single irradiation batch. Propertly taking

  19. Characterization of Escherichia coli thioredoxin variants mimicking the active-sites of other thiol/disulfide oxidoreductases.

    PubMed Central

    Mössner, E.; Huber-Wunderlich, M.; Glockshuber, R.

    1998-01-01

    Thiol/disulfide oxidoreductases like thioredoxin, glutaredoxin, DsbA, or protein disulfide isomerase (PDI) share the thioredoxin fold and a catalytic disulfide bond with the sequence Cys-Xaa-Xaa-Cys (Xaa corresponds to any amino acid). Despite their structural similarities, the enzymes have very different redox properties, which is reflected by a 100,000-fold difference in the equilibrium constant (K(eq)) with glutathione between the most oxidizing member, DsbA, and the most reducing member, thioredoxin. Here we present a systematic study on a series of variants of thioredoxin from Escherichia coli, in which the Xaa-Xaa dipeptide was exchanged by that of glutaredoxin, PDI, and DsbA. Like the corresponding natural enzymes, all thioredoxin variants proved to be stronger oxidants than the wild-type, with the order wild-type < PDI-type < DsbA-type < glutaredoxin-type. The most oxidizing, glutaredoxin-like variant has a 420-fold decreased value of K(eq), corresponding to an increase in redox potential by 75 mV. While oxidized wild-type thioredoxin is more stable than the reduced form (delta deltaG(ox/red) = 16.9 kJ/mol), both redox forms have almost the same stability in the variants. The pH-dependence of the reactivity with the alkylating agent iodoacetamide proved to be the best method to determine the pKa value of thioredoxin's nucleophilic active-site thiol (Cys32). A pKa of 7.1 was measured for Cys32 in the reduced wild-type. All variants showed a lowered pKa of Cys32, with the lowest value of 5.9 for the glutaredoxin-like variant. A correlation of redox potential and the Cys32 pKa value could be established on a quantitative level. However, the predicted correlation between the measured delta deltaG(ox/red) values and Cys32 pKa values was only qualitative. PMID:9605329

  20. Combinatorial active-site variants confer sustained clavulanate resistance in BlaC β-lactamase from Mycobacterium tuberculosis

    PubMed Central

    Egesborg, Philippe; Carlettini, Hélène; Volpato, Jordan P; Doucet, Nicolas

    2015-01-01

    Bacterial resistance to β-lactam antibiotics is a global issue threatening the success of infectious disease treatments worldwide. Mycobacterium tuberculosis has been particularly resilient to β-lactam treatment, primarily due to the chromosomally encoded BlaC β-lactamase, a broad-spectrum hydrolase that renders ineffective the vast majority of relevant β-lactam compounds currently in use. Recent laboratory and clinical studies have nevertheless shown that specific β-lactam–BlaC inhibitor combinations can be used to inhibit the growth of extensively drug-resistant strains of M. tuberculosis, effectively offering new tools for combined treatment regimens against resistant strains. In the present work, we performed combinatorial active-site replacements in BlaC to demonstrate that specific inhibitor-resistant (IRT) substitutions at positions 69, 130, 220, and/or 234 can act synergistically to yield active-site variants with several thousand fold greater in vitro resistance to clavulanate, the most common clinical β-lactamase inhibitor. While most single and double variants remain sensitive to clavulanate, double mutants R220S-K234R and S130G-K234R are substantially less affected by time-dependent clavulanate inactivation, showing residual β-lactam hydrolytic activities of 46% and 83% after 24 h incubation with a clinically relevant inhibitor concentration (5 μg/ml, 25 µM). These results demonstrate that active-site alterations in BlaC yield resistant variants that remain active and stable over prolonged bacterial generation times compatible with mycobacterial proliferation. These results also emphasize the formidable adaptive potential of inhibitor-resistant substitutions in β-lactamases, potentially casting a shadow on specific β-lactam–BlaC inhibitor combination treatments against M. tuberculosis. PMID:25492589

  1. Ionic liquid and deep eutectic solvent-activated CelA2 variants generated by directed evolution.

    PubMed

    Lehmann, Christian; Bocola, Marco; Streit, Wolfgang R; Martinez, Ronny; Schwaneberg, Ulrich

    2014-06-01

    Chemoenzymatic cellulose degradation is one of the key steps for the production of biomass-based fuels under mild conditions. An effective cellulose degradation process requires diverse physico-chemical dissolution of the biomass prior to enzymatic degradation. In recent years, "green" solvents, such as ionic liquids and, more recently, deep eutectic liquids, have been proposed as suitable alternatives for biomass dissolution by homogenous catalysis. In this manuscript, a directed evolution campaign of an ionic liquid tolerant β-1,4-endoglucanase (CelA2) was performed in order to increase its performance in the presence of choline chloride/glycerol (ChCl:Gly) or 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), as a first step to identify residues which govern ionic strength resistance and obtaining insights for employing cellulases on the long run in homogenous catalysis of lignocellulose degradation. After mutant library screening, variant M4 (His288Phe, Ser300Arg) was identified, showing a dramatically reduced activity in potassium phosphate buffer and an increased activity in the presence of ChCl:Gly or [BMIM]Cl. Further characterization showed that the CelA2 variant M4 is activated in the presence of these solvents, representing a first report of an engineered enzyme with an ionic strength activity switch. Structural analysis revealed that Arg300 could be a key residue for the ionic strength activation through a salt bridge with the neighboring Asp287. Experimental and computational results suggest that the salt bridge Asp287-Arg300 generates a nearly inactive CelA2 variant and activity is regained when ChCl:Gly or [BMIM]Cl are supplemented (~5-fold increase from 0.64 to 3.37 μM 4-MU/h with the addition ChCl:Gly and ~23-fold increase from 3.84 to 89.21 μM 4-pNP/h with the addition of [BMIM]Cl). Molecular dynamic simulations further suggest that the salt bridge between Asp287 and Arg300 in variant M4 (His288Phe, Ser300Arg) modulates the observed salt

  2. Paleomagnetic and 40Ar/39Ar results from the Grant intrusive breccia and coparison to the Permian Downeys Bluff Sill; evidence for Permian igneous activity at Hicks Dome, southern Illinois Basin

    USGS Publications Warehouse

    Reynolds, Richard L.; Goldhaber, Martin B.; Snee, Lawrence W.

    1997-01-01

    Igneous processes at Hicks dome, a structural upwarp at lat 37.5 degrees N., long 88.4 degrees W. in the southern part of the Illinois Basin, may have thermally affected regional basinal fluid flow and may have provided fluorine for the formation of the Illinois-Kentucky Fluorspar district. The timing of both igneous activity and mineralization is poorly known. For this reason, we have dated an intrusive breccia at Hicks dome, the Grant intrusion, using 40Ar/39Ar geochronometric and paleomagnetic methods. Concordant plateau dates, giving Permian ages, were obtained from amphibole (272.1+or-0.7 [1 sigma] Ma) and phlogopite (272.7+or-0.7 [1 sigma] Ma). After alternating-field (AF) demagnetization, specimens that contain titanomagnetite-bearing igneous rock fragments give a mean remanent direction of declination (D)=168.4 degrees; inclination (I)=-8 degrees; alpha 95=8.6 degrees; number of specimens (N)=10; this direction yields a virtual geomagnetic pole (VGP) at lat 54.8 degrees N., long 119.0 degrees E., delta p=4.4 degrees, delta m=8.7 degrees, near the late Paleozoic part of the North American apparent pole wander path. A nearly identical magnetization was found for the nearby Downeys Bluff sill (previously dated at about 275+or-24 Ma by the Rb-Sr method), in southern Illinois. Both AF and thermal demagnetization isolated shallow, southeasterly remanent directions carried by magnetite in the sill and from pyrrhotite in the baked contact of the Upper Mississippian Downeys Bluff Limestone: D=158.6 degrees; I=-11.8 degrees; alpha 95=3.8 degrees; N=15, yielding a VGP at lat 53.0 degrees N., long 128.7 degrees E., delta p=2.0 degrees, delta m=3.9 degrees. The paleomagnetic results, isotopic dates, and petrographic evidence thus favor the acquisition of thermal remanent magnetization by the Grant breccia and the Downeys Bluff sill during the Permian. The isotopic dates record rapid cooling from temperatures greater than 550 degrees C to less than 300 degrees C (the

  3. Central Activation of the A1 Adenosine Receptor (A1AR) Induces a Hypothermic, Torpor-Like State in the Rat

    PubMed Central

    Madden, Christopher J.; Morrison, Shaun F.

    2013-01-01

    Since central activation of A1 adenosine receptors (A1ARs) plays an important role in the induction of the hypothermic and hypometabolic torpid state in hibernating mammals, we investigated the potential for the A1AR agonist N6-cyclohexyladenosine to induce a hypothermic, torpor-like state in the (nonhibernating) rat. Core and brown adipose tissue temperatures, EEG, heart rate, and arterial pressure were recorded in free-behaving rats, and c-fos expression in the brain was analyzed, following central administration of N6-cyclohexyladenosine. Additionally, we recorded the sympathetic nerve activity to brown adipose tissue; expiratory CO2 and skin, core, and brown adipose tissue temperatures; and shivering EMGs in anesthetized rats following central and localized, nucleus of the solitary tract, administration of N6-cyclohexyladenosine. In rats exposed to a cool (15°C) ambient temperature, central A1AR stimulation produced a torpor-like state similar to that in hibernating species and characterized by a marked fall in body temperature due to an inhibition of brown adipose tissue and shivering thermogenesis that is mediated by neurons in the nucleus of the solitary tract. During the induced hypothermia, EEG amplitude and heart rate were markedly reduced. Skipped heartbeats and transient bradycardias occurring during the hypothermia were vagally mediated since they were eliminated by systemic muscarinic receptor blockade. These findings demonstrate that a deeply hypothermic, torpor-like state can be pharmacologically induced in a nonhibernating mammal and that recovery of normothermic homeostasis ensues upon rewarming. These results support the potential for central activation of A1ARs to be used in the induction of a hypothermic, therapeutically beneficial state in humans. PMID:24005302

  4. The 420K LEKTI variant alters LEKTI proteolytic activation and results in protease deregulation: implications for atopic dermatitis.

    PubMed

    Fortugno, Paola; Furio, Laetitia; Teson, Massimo; Berretti, Matteo; El Hachem, May; Zambruno, Giovanna; Hovnanian, Alain; D'Alessio, Marina

    2012-10-01

    Lymphoepithelial Kazal-type related inhibitor (LEKTI) is a multidomain serine protease inhibitor which plays a central role in skin permeability barrier and allergy. Loss-of-function mutations in the LEKTI encoding gene SPINK5 cause Netherton syndrome, a rare and severe genetic skin disease with a profound skin barrier defect and atopic manifestations. Several studies also reported genetic association between the multifactorial disease atopic dermatitis (AD) and a frequent and non-conservative LEKTI variant, E420K, in different populations. Here, we provide evidence that the 420K variant impacts on LEKTI function by increasing the likelihood of furin-dependent LEKTI precursor cleavage within the linker region D6-D7. This results in the reversal of the cleavage priorities for LEKTI proteolytic activation and prevents the formation of the LEKTI fragment D6D9 known to display the strongest inhibitory activity against kallikrein (KLK) 5-mediated desmoglein-1 (DSG1) degradation. Using in situ and gel zymographies, we show that the modification of the subtle balance in LEKTI inhibitory fragments leads to enhanced KLK5, KLK7 and elastase-2 (ELA-2) activities in 420KK epidermis. By immunohistochemistry and western blot analyses, we found that increased epidermal protease activity correlates with reduced DSG1 protein expression and accelerated profilaggrin proteolysis. All changes determined by the presence of residue 420K within the LEKTI sequence likely contribute to defective skin barrier permeability. Remarkably, LEKTI 420KK epidermis displays an increased expression of the proallergic cytokine thymic stromal lymphopoietin (TSLP). This is the first functional evidence supporting association studies which identified the 420K LEKTI variant as a predisposing factor to AD, in combination with other genetic and environmental factors. PMID:22730493

  5. A Rb1 promoter variant with reduced activity contributes to osteosarcoma susceptibility in irradiated mice

    PubMed Central

    2014-01-01

    Background Syndromic forms of osteosarcoma (OS) account for less than 10% of all recorded cases of this malignancy. An individual OS predisposition is also possible by the inheritance of low penetrance alleles of tumor susceptibility genes, usually without evidence of a syndromic condition. Genetic variants involved in such a non-syndromic form of tumor predisposition are difficult to identify, given the low incidence of osteosarcoma cases and the genetic heterogeneity of patients. We recently mapped a major OS susceptibility QTL to mouse chromosome 14 by comparing alpha-radiation induced osteosarcoma in mouse strains which differ in their tumor susceptibility. Methods Tumor-specific allelic losses in murine osteosacoma were mapped along chromosome 14 using microsatellite markers and SNP allelotyping. Candidate gene search in the mapped interval was refined using PosMed data mining and mRNA expression analysis in normal osteoblasts. A strain-specific promoter variant in Rb1 was tested for its influence on mRNA expression using reporter assay. Results A common Rb1 allele derived from the BALB/cHeNhg strain was identified as the major determinant of radiation-induced OS risk at this locus. Increased OS-risk is linked with a hexanucleotide deletion in the promoter region which is predicted to change WT1 and SP1 transcription factor-binding sites. Both in-vitro reporter and in-vivo expression assays confirmed an approx. 1.5 fold reduced gene expression by this promoter variant. Concordantly, the 50% reduction in Rb1 expression in mice bearing a conditional hemizygous Rb1 deletion causes a significant rise of OS incidence following alpha-irradiation. Conclusion This is the first experimental demonstration of a functional and genetic link between reduced Rb1 expression from a common promoter variant and increased tumor risk after radiation exposure. We propose that a reduced Rb1 expression by common variants in regulatory regions can modify the risk for a malignant

  6. Application of a combined effect compartment and binding model for gastric acid inhibition of AR-HO47108: a potassium competitive acid blocker, and its active metabolite AR-HO47116 in the dog.

    PubMed

    Abelö, Angela; Andersson, Magdalena; Holmberg, Ann Aurell; Karlsson, Mats O

    2006-10-01

    The effect of AR-HO47108, a potassium competitive acid blocker, and its active metabolite AR-HO47116 was studied in Heidenhain pouch dogs following administration of single oral and intravenous doses of the two compounds. The histamine-stimulated acid secretion was measured in different periods after dose up to 24h. All data obtained in the different studies was pooled and analyzed by non-linear mixed effects modelling. It was found that there is a delay between the plasma concentration-time peak and the maximum inhibitory effect and that the effect persisted longer than anticipated from the plasma concentration half-lives of the compounds. In addition, it was found that the peak effect was reached earlier at higher doses. The effect data was well described by a combined effect compartment and binding model and both distribution to the biophase i.e. the canaliculus of the parietal cell and a rate limiting binding interaction between drug and enzyme appear to contribute to the observed delay. In addition, a secretion rate dependent washout from the biophase may contribute. Furthermore, because the parent compound and metabolite bind to the same enzyme, the effect is determined by competition between the two for the same enzyme. The metabolite was found to be less potent than the parent compound, with Kd values of the combined model of 125 and 11.2 nM for the metabolite and parent compound, respectively. However, the metabolite is generated in high concentrations that rapidly exceed the concentration of parent compound after oral administration of parent compound, and this together with its longer plasma half-life will make its contribution to the overall effect increase with time and slightly prolong the duration of the effect. PMID:16831536

  7. Combined GSTM1-Null, GSTT1-Active, GSTA1 Low-Activity and GSTP1-Variant Genotype Is Associated with Increased Risk of Clear Cell Renal Cell Carcinoma

    PubMed Central

    Coric, Vesna M.; Simic, Tatjana P.; Pekmezovic, Tatjana D.; Basta-Jovanovic, Gordana M.; Savic Radojevic, Ana R.; Radojevic-Skodric, Sanja M.; Matic, Marija G.; Dragicevic, Dejan P.; Radic, Tanja M.; Bogdanovic, Ljiljana M.; Dzamic, Zoran M.; Pljesa-Ercegovac, Marija S.

    2016-01-01

    The aim of this study was to evaluate specific glutathione S-transferase (GST) gene variants as determinants of risk in patients with clear cell renal cell carcinoma (cRCC), independently or simultaneously with established RCC risk factors, as well as to discern whether phenotype changes reflect genotype-associated risk. GSTA1, GSTM1, GSTP1 and GSTT1 genotypes were determined in 199 cRCC patients and 274 matched controls. Benzo(a)pyrene diolepoxide (BPDE)-DNA adducts were determined in DNA samples obtained from cRCC patients by ELISA method. Significant association between GST genotype and risk of cRCC development was found for the GSTM1-null and GSTP1-variant genotype (p = 0.02 and p<0.001, respectively). Furthermore, 22% of all recruited cRCC patients were carriers of combined GSTM1-null, GSTT1-active, GSTA1-low activity and GSTP1-variant genotype, exhibiting 9.32-fold elevated cRCC risk compared to the reference genotype combination (p = 0.04). Significant association between GST genotype and cRCC risk in smokers was found only for the GSTP1 genotype, while GSTM1-null/GSTP1-variant/GSTA1 low-activity genotype combination was present in 94% of smokers with cRCC, increasing the risk of cRCC up to 7.57 (p = 0.02). Furthermore, cRCC smokers with GSTM1-null genotype had significantly higher concentration of BPDE-DNA adducts in comparison with GSTM1-active cRCC smokers (p = 0.05). GSTM1, GSTT1, GSTA1 and GSTP1 polymorphisms might be associated with the risk of cRCC, with special emphasis on GSTM1-null and GSTP1-variant genotypes. Combined GSTM1-null, GSTT1-active, GSTA1 low activity and GSTP1-variant genotypes might be considered as “risk-carrying genotype combination” in cRCC. PMID:27500405

  8. Molecular determinants of ATP-sensitive potassium channel MgATPase activity: diabetes risk variants and diazoxide sensitivity.

    PubMed

    Fatehi, Mohammad; Carter, Chris R J; Youssef, Nermeen; Hunter, Beth E; Holt, Andrew; Light, Peter E

    2015-01-01

    ATP-sensitive K(+) (KATP) channels play an important role in insulin secretion. KATP channels possess intrinsic MgATPase activity that is important in regulating channel activity in response to metabolic changes, although the precise structural determinants are not clearly understood. Furthermore, the sulfonylurea receptor 1 (SUR1) S1369A diabetes risk variant increases MgATPase activity, but the molecular mechanisms remain to be determined. Therefore, we hypothesized that residue-residue interactions between 1369 and 1372, predicted from in silico modelling, influence MgATPase activity, as well as sensitivity to the clinically used drug diazoxide that is known to increase MgATPase activity. We employed a point mutagenic approach with patch-clamp and direct biochemical assays to determine interaction between residues 1369 and 1372. Mutations in residues 1369 and 1372 predicted to decrease the residue interaction elicited a significant increase in MgATPase activity, whereas mutations predicted to possess similar residue interactions to wild-type (WT) channels elicited no alterations in MgATPase activity. In contrast, mutations that were predicted to increase residue interactions resulted in significant decreases in MgATPase activity. We also determined that a single S1369K substitution in SUR1 caused MgATPase activity and diazoxide pharmacological profiles to resemble those of channels containing the SUR2A subunit isoform. Our results provide evidence, at the single residue level, for a molecular mechanism that may underlie the association of the S1369A variant with type 2 diabetes. We also show a single amino acid difference can account for the markedly different diazoxide sensitivities between channels containing either the SUR1 or SUR2A subunit isoforms. PMID:26181369

  9. Molecular determinants of ATP-sensitive potassium channel MgATPase activity: diabetes risk variants and diazoxide sensitivity

    PubMed Central

    Fatehi, Mohammad; Carter, Chris R.J.; Youssef, Nermeen; Hunter, Beth E.; Holt, Andrew; Light, Peter E.

    2015-01-01

    ATP-sensitive K+ (KATP) channels play an important role in insulin secretion. KATP channels possess intrinsic MgATPase activity that is important in regulating channel activity in response to metabolic changes, although the precise structural determinants are not clearly understood. Furthermore, the sulfonylurea receptor 1 (SUR1) S1369A diabetes risk variant increases MgATPase activity, but the molecular mechanisms remain to be determined. Therefore, we hypothesized that residue–residue interactions between 1369 and 1372, predicted from in silico modelling, influence MgATPase activity, as well as sensitivity to the clinically used drug diazoxide that is known to increase MgATPase activity. We employed a point mutagenic approach with patch-clamp and direct biochemical assays to determine interaction between residues 1369 and 1372. Mutations in residues 1369 and 1372 predicted to decrease the residue interaction elicited a significant increase in MgATPase activity, whereas mutations predicted to possess similar residue interactions to wild-type (WT) channels elicited no alterations in MgATPase activity. In contrast, mutations that were predicted to increase residue interactions resulted in significant decreases in MgATPase activity. We also determined that a single S1369K substitution in SUR1 caused MgATPase activity and diazoxide pharmacological profiles to resemble those of channels containing the SUR2A subunit isoform. Our results provide evidence, at the single residue level, for a molecular mechanism that may underlie the association of the S1369A variant with type 2 diabetes. We also show a single amino acid difference can account for the markedly different diazoxide sensitivities between channels containing either the SUR1 or SUR2A subunit isoforms. PMID:26181369

  10. Synergistic Effect of Simple Sugars and Carboxymethyl Cellulose on the Production of a Cellulolytic Cocktail from Bacillus sp. AR03 and Enzyme Activity Characterization.

    PubMed

    Manfredi, Adriana P; Pisa, José H; Valdeón, Daniel H; Perotti, Nora I; Martínez, María A

    2016-04-01

    A cellulase-producing bacterium isolated from pulp and paper feedstock, Bacillus sp. AR03, was evaluated by means of a factorial design showing that peptone and carbohydrates were the main variables affecting enzyme production. Simple sugars, individually and combined with carboxymethyl cellulose (CMC), were further examined for their influence on cellulase production by strain AR03. Most of the mono and disaccharides assayed presented a synergistic effect with CMC. As a result, a peptone-based broth supplemented with 10 g/L sucrose and 10 g/L CMC maximized enzyme production after 96 h of cultivation. This medium was used to produce endoglucanases in a 1-L stirred tank reactor in batch mode at 30 °C, which reduced the fermentation period to 48 h and reaching 3.12 ± 0.02 IU/mL of enzyme activity. Bacillus sp. AR03 endoglucanases showed an optimum temperature of 60 °C and a pH of 6.0 with a wide range of pH stability. Furthermore, presence of 10 mM Mn(2+) and 5 mM Co(2+) produced an increase of enzyme activity (246.7 and 183.7 %, respectively), and remarkable tolerance to NaCl, Tween 80, and EDTA was also observed. According to our results, the properties of the cellulolytic cocktail from Bacillus sp. AR03 offer promising features in view of potential biorefinery applications. PMID:26797928

  11. Metabolism of chlorobiphenyls by a variant biphenyl dioxygenase exhibiting enhanced activity toward dibenzofuran

    SciTech Connect

    Viger, Jean-Francois; Mohammadi, Mahmood; Barriault, Diane; Sylvestre, Michel

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Burkholderia xenovorans LB400 biphenyl dioxygenase (BphAE{sub LB400}) metabolizes PCBs. Black-Right-Pointing-Pointer Asn338Gln/Leu409Phe double mutation speeds up electron transfer of enzyme reaction. Black-Right-Pointing-Pointer We tested how the mutations affect the PCB-degrading abilities of BphAE{sub LB400} variants. Black-Right-Pointing-Pointer The same mutations also broaden the PCB substrate range of BphAE{sub LB400} variants. -- Abstract: The biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE{sub LB400}) catalyzes the dihydroxylation of biphenyl and of several polychlorinated biphenyls (PCBs) but it poorly oxidizes dibenzofuran. In this work we showed that BphAE{sub RR41}, a variant which was previously found to metabolize dibenzofuran more efficiently than its parent BphAE{sub LB400}, metabolized a broader range of PCBs than BphAE{sub LB400}. Hence, BphAE{sub RR41} was able to metabolize 2,6,2 Prime ,6 Prime -, 3,4,3 Prime ,5 Prime - and 2,4,3 Prime ,4 Prime -tetrachlorobiphenyl that BphAE{sub LB400} is unable to metabolize. BphAE{sub RR41} was obtained by changing Thr335Phe336Asn338Ile341Leu409 of BphAE{sub LB400} to Ala335Met336Gln338Val341Phe409. Site-directed mutagenesis was used to create combinations of each substitution, in order to assess their individual contributions. Data show that the same Asn338Glu/Leu409Phe substitution that enhanced the ability to metabolize dibenzofuran resulted in a broadening of the PCB substrates range of the enzyme. The role of these substitutions on regiospecificities toward selected PCBs is also discussed.

  12. macroH2A1 Histone Variants Are Depleted on Active Genes but Concentrated on the Inactive X Chromosome†

    PubMed Central

    Changolkar, Lakshmi N.; Pehrson, John R.

    2006-01-01

    Using a novel thiol affinity chromatography approach to purify macroH2A1-containing chromatin fragments, we examined the distribution of macroH2A1 histone variants in mouse liver chromatin. We found that macroH2A1 was depleted on the transcribed regions of active genes. This depletion was observed on all of the 20 active genes that we probed, with only one site showing a small amount of enrichment. In contrast, macroH2A1 was concentrated on the inactive X chromosome, consistent with our previous immunofluorescence studies. This preferential localization was seen on genes that are active in liver, genes that are inactive in liver, and intergenic regions but was absent from four regions that escape X inactivation. These results support the hypothesis that macroH2As function as transcriptional repressors. Also consistent with this hypothesis is our finding that the heterochromatin protein HP1β copurifies with the macroH2A1-containing chromatin fragments. This study presents the first detailed examination of the distribution of macroH2A1 variants on specific sequences. Our results indicate that macroH2As have complex distribution patterns that are influenced by both local factors and long-range mechanisms. PMID:16738309

  13. The road to the first, fully active and more stable human insulin variant with an additional disulfide bond.

    PubMed

    Vinther, Tine N; Kjeldsen, Thomas B; Jensen, Knud J; Hubálek, František

    2015-11-01

    Insulin, a small peptide hormone, is crucial in maintaining blood glucose homeostasis. The stability and activity of the protein is directed by an intricate system involving disulfide bonds to stabilize the active monomeric species and by their non-covalent oligomerization. All known insulin variants in vertebrates consist of two peptide chains and have six cysteine residues, which form three disulfide bonds, two of them link the two chains and a third is an intra-chain bond in the A-chain. This classical insulin fold appears to have been conserved over half a billion years of evolution. We addressed the question whether a human insulin variant with four disulfide bonds could exist and be fully functional. In this review, we give an overview of the road to engineering four-disulfide bonded insulin analogs. During our journey, we discovered several active four disulfide bonded insulin analogs with markedly improved stability and gained insights into the instability of analogs with seven cysteine residues, importance of dimerization for stability, insulin fibril formation process, and the conformation of insulin binding to its receptor. Our results also open the way for new strategies in the development of insulin biopharmaceuticals. PMID:26382042

  14. Detection of hormonal anabolic compounds in calf urine and unverified growth-promoting preparations: application of the AR-LUX bioassay for screening and determination of androgenic activity.

    PubMed

    Blankvoort, Barry M G; Aarts, Jac M M J G; Schilt, Robert; Geerdink, Peter; Spenkelink, Bert; Rodenburg, Richard J T

    2003-11-01

    Despite a ban by the European Union, the use of anabolic steroids and repartitioning agents in cattle is still occasionally observed. Due to continuing improvements in analytical techniques, very low detection limits for individual compounds have been achieved. In response to these developments, cocktails composed of several steroids have been applied, thus hampering detection due to lower levels of the individual compounds. Bioassays capable of measuring the integrated effect of cocktails might therefore provide valuable additional tools in controlling the use of illegal anabolics. We investigated the feasibility of using the AR-LUX assay to detect the presence in cattle urine of growth promoters that exert their effects via androgen response elements (AREs). The AR-LUX assay is based on a human cell line featuring a luciferase reporter gene under transcriptional control of an authenticated ARE. Several column purification and liquid/liquid extraction methods were investigated to optimize the efficiency of anabolic compounds extraction and minimize cytotoxic effects of the urine matrix. The AR-LUX assay was found to be applicable to the detection of anabolic steroids excreted in urine samples with a discriminatory power similar to that of GC-MS analysis. Finally, some liquid products probably destined for growth-promoting purposes confiscated outside the Netherlands were analyzed. Although common chemical-analytical methods did not detect any anabolic steroids in these samples, the presence of compounds activating ARE-mediated gene expression was clearly established. PMID:14700232

  15. Validating optical emission spectroscopy as a diagnostic of microwave activated CH{sub 4}/Ar/H{sub 2} plasmas used for diamond chemical vapor deposition

    SciTech Connect

    Ma Jie; Ashfold, Michael N. R.; Mankelevich, Yuri A.

    2009-02-15

    Spatially resolved optical emission spectroscopy (OES) has been used to investigate the gas phase chemistry and composition in a microwave activated CH{sub 4}/Ar/H{sub 2} plasma operating at moderate power densities ({approx}30 W cm{sup -3}) and pressures ({<=}175 Torr) during chemical vapor deposition of polycrystalline diamond. Several tracer species are monitored in order to gain information about the plasma. Relative concentrations of ground state H (n=1) atoms have been determined by actinometry, and the validity of this method have been demonstrated for the present experimental conditions. Electronically excited H (n=3 and 4) atoms, Ar (4p) atoms, and C{sub 2} and CH radicals have been studied also, by monitoring their emissions as functions of process parameters (Ar and CH{sub 4} flow rates, input power, and pressure) and of distance above the substrate. These various species exhibit distinctive behaviors, reflecting their different formation mechanisms. Relative trends identified by OES are found to be in very good agreement with those revealed by complementary absolute absorption measurements (using cavity ring down spectroscopy) and with the results of complementary two-dimensional modeling of the plasma chemistry prevailing within this reactor.

  16. The biological activity of chernozems in the Central Caucasus Mountains (Terskii variant of altitudinal zonality), Kabardino-Balkaria

    NASA Astrophysics Data System (ADS)

    Gedgafova, F. V.; Uligova, T. S.; Gorobtsova, O. N.; Tembotov, R. Kh.

    2015-12-01

    Some parameters of the biological activity (humus content; activity of hydrolytic enzymes invertase, phosphatase, urease; and the intensity of carbon dioxide emission) were studied in the chernozems of agrocenoses and native biogeocenoses in the foothills of the Caucasus Mountains representing the Terskii variant of the altitudinal zonality. The statistically significant differences were revealed between the relevant characteristics of the soils of the agrocenoses and of the native biogeocenoses. The integral index of the ecological-biological state of the soils was used to estimate changes in the biological activity of the arable chernozems. The 40-60% decrease of this index in the cultivated chernozems testified to their degradation with a decrease in fertility and the disturbance of ecological functions as compared to these characteristics in the virgin chernozems.

  17. PLTP activity inversely correlates with CAAD: effects of PON1 enzyme activity and genetic variants on PLTP activity1[S

    PubMed Central

    Kim, Daniel Seung; Burt, Amber A.; Ranchalis, Jane E.; Vuletic, Simona; Vaisar, Tomas; Li, Wan-Fen; Rosenthal, Elisabeth A.; Dong, Weijiang; Eintracht, Jason F.; Motulsky, Arno G.; Brunzell, John D.; Albers, John J.; Furlong, Clement E.; Jarvik, Gail P.

    2015-01-01

    Recent studies have failed to demonstrate a causal cardioprotective effect of HDL cholesterol levels, shifting focus to the functional aspects of HDL. Phospholipid transfer protein (PLTP) is an HDL-associated protein involved in reverse cholesterol transport. This study sought to determine the genetic and nongenetic predictors of plasma PLTP activity (PLTPa), and separately, to determine whether PLTPa predicted carotid artery disease (CAAD). PLTPa was measured in 1,115 European ancestry participants from a case-control study of CAAD. A multivariate logistic regression model was used to elucidate the relationship between PLTPa and CAAD. Separately, a stepwise linear regression determined the nongenetic clinical and laboratory characteristics that best predicted PLTPa. A final stepwise regression considering both nongenetic and genetic variables identified the combination of covariates that explained maximal PLTPa variance. PLTPa was significantly associated with CAAD (7.90 × 10−9), with a 9% decrease in odds of CAAD per 1 unit increase in PLTPa (odds ratio = 0.91). Triglyceride levels (P = 0.0042), diabetes (P = 7.28 × 10−5), paraoxonase 1 (PON1) activity (P = 0.019), statin use (P = 0.026), PLTP SNP rs4810479 (P = 6.38 × 10−7), and PCIF1 SNP rs181914932 (P = 0.041) were all significantly associated with PLTPa. PLTPa is significantly inversely correlated with CAAD. Furthermore, we report a novel association between PLTPa and PON1 activity, a known predictor of CAAD. PMID:26009633

  18. CORSiCA Atmospheric Observatory: Intense activities in 2012 in the frame of HyMeX and ChArMEx

    NASA Astrophysics Data System (ADS)

    Lambert, Dominique

    2013-04-01

    In the western Mediterranean basin, Corsica Island is at a strategic location for atmospheric studies in the framework of the Mediterranean projects HyMeX (Hydrological Mediterranean Experiment; http://www.hymex.org/) and ChArMEx (the Chemistry-Aerososl MEditerranean Experiment; http://charmex.lsce.ipsl.fr/) developped under the umbrella of the programme MISTRALS (Mediterranean Integrated Studies at Regional and Local Scales; http://www.mistrals-home.org). The development of a multi-sites instrumented platform located on this island is the core of the project CORSiCA (Corsican Observatory for Research and Studies on Climate and Atmosphere-ocean environment). CORSiCA is operated in the framework of the HyMEx and ChArMEx Long Observation Period (LOP), Enhanced Observation Period (EOP) and Special Observation Periods (SOP). In the present communication we will focus on the scientific objectives and describe the diversity of observations that have been performed by many institutes in Corsica in the frame of 2012 campaigns including HyMeX Special Observation Period 1 in autumn, ChArMEx pre-campaign and VESSAER in summer. Results from these campaigns will be illustrated and projects for future campaign activities and long-term monitoring in the framework of the CORSiCA Atmospheric Observatory will be presented (more details can be found in other more specific presentations). Acknowledgements are addressed to campaigns main sponsors: HyMeX: Météo-France, INSU, KIT; pre-ChArMEx: INSU, ADEME, CEA; VESSAER: FP7/Infrastructures/EUFAR, COST, Météo-France. The development of the CORSiCA platform is supported by Collectivité Territoriale de Corse (CPER) and EU FEDER funds.

  19. Pluripotency Activity of Nanog Requires Biochemical Stabilization by Variant Histone Protein H2A.Z.

    PubMed

    Wang, Jiaxu; Qiao, Mengran; He, Qianqian; Shi, Ronghua; Loh, Sharon Jia Hui; Stanton, Lawrence W; Wu, Mian

    2015-07-01

    The variant histone protein H2A.Z plays a critical role in early development. Likewise, Nanog, a master regulator of embryonic stem cells (ESCs), is essential for proper development in early embryogenesis. In this study, we establish that these two factors work together to maintain pluripotency. It is shown that H2A.Z influences the protein level of Nanog through the ubiquitin-proteasome pathway. Knockdown of H2A.Z causes differentiation of mouse ESCs and disrupts the reprogramming of somatic cells, which can be partially rescued by overexpression of Nanog. We conclude that the H2A.Z-Nanog partnership is involved in ESC pluripotency and reprogramming of somatic cells. Stem Cells 2015;33:2126-2134. PMID:25809870

  20. Selective Phthalate Activation of Naturally Occurring Human Constitutive Androstane Receptor Splice Variants and the Pregnane X Receptor

    PubMed Central

    DeKeyser, Joshua G.; Laurenzana, Elizabeth M.; Peterson, Eric C.; Chen, Tao; Omiecinski, Curtis J.

    2011-01-01

    Phthalates and other endocrine-disruptive chemicals are manufactured in large quantities for use as plasticizers and other commercial applications, resulting in ubiquitous human exposure and thus, concern regarding their toxicity. Innate defense against small molecule exposures is controlled in large part by the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). The human CAR gene undergoes multiple alternative splicing events resulting in the CAR2 and CAR3 variant receptors. Recent studies from our laboratory show that CAR2 is potently and specifically activated by di(2-ethylhexyl) phthalate (DEHP). We hypothesized that alternative splicing is a mechanism for increasing CAR’s functional diversity, broadening the human receptors’ repertoire of response to environmental xenobiotics. In these studies, we examine the interaction of alternatively spliced CARs and PXR with a range of suspected endocrine disruptors, including phthalates, bisphenol A (BPA), and 4-N-nonylphenol (NP). Transactivation and two-hybrid studies in COS-1 cells revealed differential selectivity of endocrine-disrupting chemicals for the variant CAR and PXR. Ex vivo studies showed DEHP and di-isononyl phthalate potently induced CYP2B6 and CYP3A4 expression in human hepatocytes. Mutation analysis of CAR2, in silico modeling, and ligand docking studies suggested that the SPTV amino acid insertion of CAR2 creates a unique ligand-binding pocket. Alternative gene splicing results in variant CAR receptors that selectively recognize phthalates and BPA. The interaction of phthalates with CAR and PXR suggests a xenobiotic response that is complex and biologically redundant. PMID:21227907

  1. Selective phthalate activation of naturally occurring human constitutive androstane receptor splice variants and the pregnane X receptor.

    PubMed

    DeKeyser, Joshua G; Laurenzana, Elizabeth M; Peterson, Eric C; Chen, Tao; Omiecinski, Curtis J

    2011-04-01

    Phthalates and other endocrine-disruptive chemicals are manufactured in large quantities for use as plasticizers and other commercial applications, resulting in ubiquitous human exposure and thus, concern regarding their toxicity. Innate defense against small molecule exposures is controlled in large part by the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). The human CAR gene undergoes multiple alternative splicing events resulting in the CAR2 and CAR3 variant receptors. Recent studies from our laboratory show that CAR2 is potently and specifically activated by di(2-ethylhexyl) phthalate (DEHP). We hypothesized that alternative splicing is a mechanism for increasing CAR's functional diversity, broadening the human receptors' repertoire of response to environmental xenobiotics. In these studies, we examine the interaction of alternatively spliced CARs and PXR with a range of suspected endocrine disruptors, including phthalates, bisphenol A (BPA), and 4-N-nonylphenol (NP). Transactivation and two-hybrid studies in COS-1 cells revealed differential selectivity of endocrine-disrupting chemicals for the variant CAR and PXR. Ex vivo studies showed DEHP and di-isononyl phthalate potently induced CYP2B6 and CYP3A4 expression in human hepatocytes. Mutation analysis of CAR2, in silico modeling, and ligand docking studies suggested that the SPTV amino acid insertion of CAR2 creates a unique ligand-binding pocket. Alternative gene splicing results in variant CAR receptors that selectively recognize phthalates and BPA. The interaction of phthalates with CAR and PXR suggests a xenobiotic response that is complex and biologically redundant. PMID:21227907

  2. Determining K/Ar age of fault activity through analysis of clay mineralogy: A case study of "El Doctor Fault", México

    NASA Astrophysics Data System (ADS)

    Garduño, D. E.; Pi, T.; Sole, J.; Martini, M.; Alcala, J. R.

    2013-05-01

    The upper continental crust of Mexico is cut by several major faults, some of which were interpreted as terrane boundaries. Although the age of such faults is key to reconstructing the tectonic evolution of Mexico, geochronologic studies focused on the absolute dating of a fault are scattered. The Doctor fault zone is a decakilometric NNW-SSE structure that produced the overriding of the Lower Cretaceus El Doctor carbonate platform onto foreland calcareous turbidites of Upper Cretaceous Soyatal Formation. In the fault zone, turbidites of the Soyatal Formation display a pervasive foliation at the submillimeter-scale. In calcareous layers, this foliation is defined by seams of opaque minerals concentrated along stilolitic surfaces, whereas in lutitic layers it is defined by iso-oriented fine-grained illite. We collected 17 samples from a traverse across the Doctor fault zone, in order to (1) defining and quantifying fault-related changes in clay mineralogy, (2) studying fabrics in clay-rich fault rocks and protolith, and (3) dating the fault activity by illite K/Ar with laser. Texture was studied with petrographic microscope on polished thin sections. Three size fractions (from 2 μm to 0.05 μm) were extracted using centrifugation. Clay mineralogy was determined using XRD in clay oriented samples and the illite crystallinity (IC) has been determined by the Kübler method (Kisch, 1990). The amount of 2M1 illite was quantified using XRD patterns from a randomly oriented sample, achieved using WILDFIRE (Reynolds, 1994, Haines and Van der Pluijm, 2008) and RIETVELD methods and the timing of fault main activity is determined using K/Ar dating. The mineralogy of the samples consists of quartz, calcite, plagioclase, hematite and clays. The clay mineralogy contain illite (zone 1, zone 2 and zone 3), smectite (zone 2), chlorite (zone 3), kaolinite (zone 1 and zone3), and vermiculite (zone 3). The range of IC (0.24 to 0.4) is attributed to heterogeneous origins of illite

  3. The ATP-sensitive K(+) channel ABCC8 S1369A type 2 diabetes risk variant increases MgATPase activity.

    PubMed

    Fatehi, Mohammad; Raja, Mobeen; Carter, Christian; Soliman, Daniel; Holt, Andrew; Light, Peter E

    2012-01-01

    Pancreatic β-cell ATP-sensitive K(+) (K(ATP)) channels are composed of Kir6.2 and SUR1 subunits encoded by the KCNJ11 and ABCC8 genes, respectively. Although rare monogenic activating mutations in these genes cause overt neonatal diabetes, the common variants E23K (KCNJ11) and S1369A (ABCC8) form a tightly heritable haplotype that is associated with an increased susceptibility to type 2 diabetes (T2D) risk. However, the molecular mechanism(s) underlying this risk remain to be elucidated. A homology model of the SUR1 nucleotide-binding domains (NBDs) indicates that residue 1369 is in close proximity to the major MgATPase site. Therefore, we investigated the intrinsic MgATPase activity of K(ATP) channels containing these variants. Electrophysiological and biochemical techniques were used to study the MgATPase activity of recombinant human K(ATP) channels or glutathione S-transferase and NBD2 fusion proteins containing the E23/S1369 (nonrisk) or K23/A1369 (risk) variant haplotypes. K(ATP) channels containing the K23/A1369 haplotype displayed a significantly increased stimulation by guanosine triphosphate compared with the E23/S1369 haplotype (3.2- vs. 1.8-fold). This effect was dependent on the presence of the A1369 variant and was lost in the absence of Mg(2+) ions or in the presence of the MgATPase inhibitor beryllium fluoride. Direct biochemical assays also confirmed an increase in MgATPase activity in NBD2 fusion proteins containing the A1369 variant. Our findings demonstrate that the A1369 variant increases K(ATP) channel MgATPase activity, providing a plausible molecular mechanism by which the K23/A1369 haplotype increases susceptibility to T2D in humans homozygous for these variants. PMID:22187380

  4. The ATP-Sensitive K+ Channel ABCC8 S1369A Type 2 Diabetes Risk Variant Increases MgATPase Activity

    PubMed Central

    Fatehi, Mohammad; Raja, Mobeen; Carter, Christian; Soliman, Daniel; Holt, Andrew; Light, Peter E.

    2012-01-01

    Pancreatic β-cell ATP-sensitive K+ (KATP) channels are composed of Kir6.2 and SUR1 subunits encoded by the KCNJ11 and ABCC8 genes, respectively. Although rare monogenic activating mutations in these genes cause overt neonatal diabetes, the common variants E23K (KCNJ11) and S1369A (ABCC8) form a tightly heritable haplotype that is associated with an increased susceptibility to type 2 diabetes (T2D) risk. However, the molecular mechanism(s) underlying this risk remain to be elucidated. A homology model of the SUR1 nucleotide-binding domains (NBDs) indicates that residue 1369 is in close proximity to the major MgATPase site. Therefore, we investigated the intrinsic MgATPase activity of KATP channels containing these variants. Electrophysiological and biochemical techniques were used to study the MgATPase activity of recombinant human KATP channels or glutathione S-transferase and NBD2 fusion proteins containing the E23/S1369 (nonrisk) or K23/A1369 (risk) variant haplotypes. KATP channels containing the K23/A1369 haplotype displayed a significantly increased stimulation by guanosine triphosphate compared with the E23/S1369 haplotype (3.2- vs. 1.8-fold). This effect was dependent on the presence of the A1369 variant and was lost in the absence of Mg2+ ions or in the presence of the MgATPase inhibitor beryllium fluoride. Direct biochemical assays also confirmed an increase in MgATPase activity in NBD2 fusion proteins containing the A1369 variant. Our findings demonstrate that the A1369 variant increases KATP channel MgATPase activity, providing a plausible molecular mechanism by which the K23/A1369 haplotype increases susceptibility to T2D in humans homozygous for these variants. PMID:22187380

  5. Serum paraoxonase activity is associated with variants in the PON gene cluster and risk of Alzheimer disease.

    PubMed

    Erlich, Porat M; Lunetta, Kathryn L; Cupples, L Adrienne; Abraham, Carmela R; Green, Robert C; Baldwin, Clinton T; Farrer, Lindsay A

    2012-05-01

    Previous studies have shown association of single nucleotide polymorphisms (SNPs) in 3 contiguous genes (PON1, PON2, and PON3) encoding paraoxonase with risk of Alzheimer disease (AD). We evaluated the association of serum paraoxonase activity measured by phenyl acetate (PA) and thiobutyl butyrolactone (TBBL) with risk of AD and with 26 SNPs spanning the PON gene cluster in 266 AD cases and 306 sibling controls from the MIRAGE study. The odds of AD (adjusted for age, gender, and ethnicity) increased 20% for each standard deviation decrease in PA or TBBL activity. There were association signals with activity in all 3 genes. Haplotypes including SNPs spanning the PON genes were generally more significant than haplotypes comprising SNPs from 1 gene. Significant interactions were observed between SNP pairs located across the PON cluster with either serum activity measure as the outcome, and between several PON SNPs and PA activity with AD status as the outcome. Our results suggest that low serum paraoxonase activity is a risk factor for AD. Furthermore, multiple variants in PON influence serum paraoxonase activity and their effects may be synergistic. PMID:20980077

  6. The ArsD As(III) metallochaperone

    PubMed Central

    Ajees, A. Abdul; Yang, Jianbo

    2013-01-01

    Arsenic, a toxic metalloid widely existing in the environment, causes a variety of health problems. The ars operon encoded by Escherichia coli plasmid R773 has arsD and arsA genes, where ArsA is an ATPase that is the catalytic subunit of the ArsAB As(III) extrusion pump, and ArsD is an arsenic chaperone for ArsA. ArsD transfers As(III) to ArsA and increases the affinity of ArsA for As(III), allowing resistance to environmental concentrations of arsenic. Cys12, Cys13 and Cys18 in ArsD form a three sulfur-coordinated As(III) binding site that is essential for metallochaperone activity. ATP hydrolysis by ArsA is required for transfer of As(III) from ArsD to ArsA, suggesting that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle. The 1.4 Å x-ray crystal structure of ArsD shows a core of four β-strands flanked by four α-helices in a thioredoxin fold. Docking of ArsD with ArsA was modeled in silico. Independently ArsD mutants exhibiting either weaker or stronger interaction with ArsA were selected. The locations of the mutations mapped on the surface of ArsD are consistent with the docking model. The results suggest that the interface with ArsA involves one surface of α1 helix and metalloid binding site of ArsD. PMID:21188475

  7. Intrinsically active variants of Erk oncogenically transform cells and disclose unexpected autophosphorylation capability that is independent of TEY phosphorylation

    PubMed Central

    Smorodinsky-Atias, Karina; Goshen-Lago, Tal; Goldberg-Carp, Anat; Melamed, Dganit; Shir, Alexei; Mooshayef, Navit; Beenstock, Jonah; Karamansha, Yael; Darlyuk-Saadon, Ilona; Livnah, Oded; Ahn, Natalie G.; Admon, Arie; Engelberg, David

    2016-01-01

    The receptor-tyrosine kinase (RTK)/Ras/Raf pathway is an essential cascade for mediating growth factor signaling. It is abnormally overactive in almost all human cancers. The downstream targets of the pathway are members of the extracellular regulated kinases (Erk1/2) family, suggesting that this family is a mediator of the oncogenic capability of the cascade. Although all oncogenic mutations in the pathway result in strong activation of Erks, activating mutations in Erks themselves were not reported in cancers. Here we used spontaneously active Erk variants to check whether Erk’s activity per se is sufficient for oncogenic transformation. We show that Erk1(R84S) is an oncoprotein, as NIH3T3 cells that express it form foci in tissue culture plates, colonies in soft agar, and tumors in nude mice. We further show that Erk1(R84S) and Erk2(R65S) are intrinsically active due to an unusual autophosphorylation activity they acquire. They autophosphorylate the activatory TEY motif and also other residues, including the critical residue Thr-207 (in Erk1)/Thr-188 (in Erk2). Strikingly, Erk2(R65S) efficiently autophosphorylates its Thr-188 even when dually mutated in the TEY motif. Thus this study shows that Erk1 can be considered a proto-oncogene and that Erk molecules possess unusual autoregulatory properties, some of them independent of TEY phosphorylation. PMID:26658610

  8. Effects of a genome-wide supported psychosis risk variant on neural activation during a theory-of-mind task.

    PubMed

    Walter, H; Schnell, K; Erk, S; Arnold, C; Kirsch, P; Esslinger, C; Mier, D; Schmitgen, M M; Rietschel, M; Witt, S H; Nöthen, M M; Cichon, S; Meyer-Lindenberg, A

    2011-04-01

    Schizophrenia is associated with marked deficits in theory of mind (ToM), a higher-order form of social cognition representing the thoughts, emotions and intentions of others. Altered brain activation in the medial prefrontal cortex and temporo-parietal cortex during ToM tasks has been found in patients with schizophrenia, but the relevance of these neuroimaging findings for the heritable risk for schizophrenia is unclear. We tested the hypothesis that activation of the ToM network is altered in healthy risk allele carriers of the single-nucleotide polymorphism rs1344706 in the gene ZNF804A, a recently discovered risk variant for psychosis with genome-wide support. In all, 109 healthy volunteers of both sexes in Hardy-Weinberg equilibrium for rs1344706 were investigated with functional magnetic resonance imaging during a ToM task. As hypothesised, risk carriers exhibited a significant (P<0.05 false discovery rate, corrected for multiple comparisons) risk allele dose effect on neural activity in the medial prefrontal cortex and left temporo-parietal cortex. Moreover, the same effect was found in the left inferior parietal cortex and left inferior frontal cortex, which are part of the human analogue of the mirror neuron system. In addition, in an exploratory analysis (P<0.001 uncorrected), we found evidence for aberrant functional connectivity between the frontal and temporo-parietal regions in risk allele carriers. To conclude, we show that a dysfunction of the ToM network is associated with a genome-wide supported genetic risk variant for schizophrenia and has promise as an intermediate phenotype that can be mined for the development of biological interventions targeted to social dysfunction in psychiatry. PMID:20231838

  9. Isolation of Intrinsically Active (MEK-independent) Variants of the ERK Family of Mitogen-activated Protein (MAP) Kinases*S⃞♦

    PubMed Central

    Levin-Salomon, Vered; Kogan, Konstantin; Ahn, Natalie G.; Livnah, Oded; Engelberg, David

    2008-01-01

    MAPKs are key components of cell signaling pathways with a unique activation mechanism: i.e. dual phosphorylation of neighboring threonine and tyrosine residues. The ERK enzymes form a subfamily of MAPKs involved in proliferation, differentiation, development, learning, and memory. The exact role of each Erk molecule in these processes is not clear. An efficient strategy for addressing this question is to activate individually each molecule, for example, by expressing intrinsically active variants of them. However, such molecules were not produced so far. Here, we report on the isolation, via a specifically designed genetic screen, of six variants (each carries a point mutation) of the yeast MAPK Mpk1/Erk that are active, independent of upstream phosphorylation. One of the activating mutations, R68S, occurred in a residue conserved in the mammalian Erk1 (Arg-84) and Erk2 (Arg-65) and in the Drosophila ERK Rolled (Arg-80). Replacing this conserved Arg with Ser rendered these MAPKs intrinsically active to very high levels when tested in vitro as recombinant proteins. Combination of the Arg to Ser mutation with the sevenmaker mutation (producing Erk2R65S+D319N and RolledR80S+D334N) resulted in even higher activity (45 and 70%, respectively, in reference to fully active dually phosphorylated Erk2 or Rolled). Erk2R65S and Erk2R65S+D319N were found to be spontaneously active also when expressed in human HEK293 cells. We further revealed the mechanism of action of the mutants and show that it involves acquisition of autophosphorylation activity. Thus, a first generation of Erk molecules that are spontaneously active in vitro and in vivo has been obtained. PMID:18829462

  10. Rare coding variants of the adenosine A3 receptor are increased in autism: on the trail of the serotonin transporter regulome

    PubMed Central

    2013-01-01

    Background Rare genetic variation is an important class of autism spectrum disorder (ASD) risk factors and can implicate biological networks for investigation. Altered serotonin (5-HT) signaling has been implicated in ASD, and we and others have discovered multiple, rare, ASD-associated variants in the 5-HT transporter (SERT) gene leading to elevated 5-HT re-uptake and perturbed regulation. We hypothesized that loci encoding SERT regulators harbor variants that impact SERT function and/or regulation and therefore could contribute to ASD risk. The adenosine A3 receptor (A3AR) regulates SERT via protein kinase G (PKG) and other signaling pathways leading to enhanced SERT surface expression and catalytic activity. Methods To test our hypothesis, we asked whether rare variants in the A3AR gene (ADORA3) were increased in ASD cases vs. controls. Discovery sequencing in a case-control sample and subsequent analysis of comparison exome sequence data were conducted. We evaluated the functional impact of two variants from the discovery sample on A3AR signaling and SERT activity. Results Sequencing discovery showed an increase of rare coding variants in cases vs. controls (P=0.013). While comparison exome sequence data did not show a significant enrichment (P=0.071), combined analysis strengthened evidence for association (P=0.0025). Two variants discovered in ASD cases (Leu90Val and Val171Ile) lie in or near the ligand-binding pocket, and Leu90Val was enriched individually in cases (P=0.040). In vitro analysis of cells expressing Val90-A3AR revealed elevated basal cGMP levels compared with the wildtype receptor. Additionally, a specific A3AR agonist increased cGMP levels across the full time course studied in Val90-A3AR cells, compared to wildtype receptor. In Val90-A3AR/SERT co-transfections, agonist stimulation elevated SERT activity over the wildtype receptor with delayed 5-HT uptake activity recovery. In contrast, Ile171-A3AR was unable to support agonist stimulation of

  11. Unique Thermal Histories from Whole-Rock 40Ar/39Ar Step-heating Data

    NASA Astrophysics Data System (ADS)

    Boehnke, P.; Harrison, M.; Heizler, M. T.; Lovera, O. M.; Warren, P. H.

    2014-12-01

    Step-heating 40Ar/39Ar analysis can reveal spatial distributions of 40Ar* at the micron scale imparted by post- crystallization heating events through complex, multi-diffusion domain models. These efforts have largely focused on single-phase, terrestrial samples with only scant attention paid to multi-phase or extra-terrestrial materials. Generalizing these models to incorporate the multiple activation energies (E) expected from bulk rock samples introduces significant interpretational ambiguity. This is because the thermal crossovers explicit in multi-E cases make the age spectrum a function of the lab heating schedule in thermally disturbed samples. A further difficulty is that unique interpretation of the associated Arrhenius plot is no longer possible and a range of E's can be fitted with equal goodness of fit. In order to address these challenges, we developed a new computational approach that simultaneously inverts the Arrhenius spectra and release pattern using a variant of the Adaptive Particle Swarm Optimization (APSO) algorithm for a square-pulse heating event. Our version uses a Levy Flight to break the swarm out of a local minima rather than randomly modifying a single dimension as in the original APSO. Further we explored issues of Pareto efficiency arising from fitting two fitness functions (i.e., the fit to the age spectra and to the Arrhenius plot) and found an adequate resolution to the classic inability to have a single best fit. By utilizing multiple-E samples, we are able to obtain unique thermal history solutions. Application of these methods to high resolution age spectra of the Jilin chondrite and Apollo 16 samples (North Ray Crater) and found fits of sufficiently high fidelity to constrain the absolute temperature of the thermal episode to better than ±10%.

  12. Campylobacter hyointestinalis Isolated from Pigs Produces Multiple Variants of Biologically Active Cytolethal Distending Toxin.

    PubMed

    Kamei, Kazumasa; Hatanaka, Noritoshi; Asakura, Masahiro; Somroop, Srinuan; Samosornsuk, Worada; Hinenoya, Atsushi; Misawa, Naoaki; Nakagawa, Shinsaku; Yamasaki, Shinji

    2015-11-01

    Campylobacter hyointestinalis isolated from swine with proliferative enteritis often is considered to be pathogenic. While the precise virulence mechanisms of this species remain unclear, we have recently identified a cytolethal distending toxin (cdt) gene cluster in C. hyointestinalis isolated from a patient with diarrhea (W. Samosornsuk et al., J Med Microbiol, 27 July 2015, http://dx.doi.org/10.1099/jmm.0.000145). However, the sequences of the cdt genes in C. hyointestinalis were found to be significantly different and the gene products are immunologically distinct from those of other Campylobacter species. In this study, we demonstrate the presence of a second variant of the cdt gene cluster in C. hyointestinalis, designated cdt-II, while the former is named cdt-I. Sequencing of the cdt-II gene cluster and deduced amino acid sequences revealed that homologies between the subunits CdtA, CdtB, and CdtC of ChCDT-I and ChCDT-II are 25.0, 56.0, and 24.8%, respectively. Furthermore, the CdtB subunit of ChCDT-II was found to be immunologically unrelated to that of ChCDT-I by Ouchterlony double gel diffusion test. Recombinant ChCDT-II also induced cell distention and death of HeLa cells by blocking the cell cycle at G2/M phase. Interestingly, the cdt-II genes were detected in all 23 animal isolates and in 1 human isolate of C. hyointestinalis, and 21 of these strains carried both cdt-I and cdt-II gene clusters. Altogether, our results indicate that ChCDT-II is an important virulence factor of C. hyointestinalis in animals. PMID:26283337

  13. Campylobacter hyointestinalis Isolated from Pigs Produces Multiple Variants of Biologically Active Cytolethal Distending Toxin

    PubMed Central

    Kamei, Kazumasa; Hatanaka, Noritoshi; Asakura, Masahiro; Somroop, Srinuan; Samosornsuk, Worada; Hinenoya, Atsushi; Misawa, Naoaki; Nakagawa, Shinsaku

    2015-01-01

    Campylobacter hyointestinalis isolated from swine with proliferative enteritis often is considered to be pathogenic. While the precise virulence mechanisms of this species remain unclear, we have recently identified a cytolethal distending toxin (cdt) gene cluster in C. hyointestinalis isolated from a patient with diarrhea (W. Samosornsuk et al., J Med Microbiol, 27 July 2015, http://dx.doi.org/10.1099/jmm.0.000145). However, the sequences of the cdt genes in C. hyointestinalis were found to be significantly different and the gene products are immunologically distinct from those of other Campylobacter species. In this study, we demonstrate the presence of a second variant of the cdt gene cluster in C. hyointestinalis, designated cdt-II, while the former is named cdt-I. Sequencing of the cdt-II gene cluster and deduced amino acid sequences revealed that homologies between the subunits CdtA, CdtB, and CdtC of ChCDT-I and ChCDT-II are 25.0, 56.0, and 24.8%, respectively. Furthermore, the CdtB subunit of ChCDT-II was found to be immunologically unrelated to that of ChCDT-I by Ouchterlony double gel diffusion test. Recombinant ChCDT-II also induced cell distention and death of HeLa cells by blocking the cell cycle at G2/M phase. Interestingly, the cdt-II genes were detected in all 23 animal isolates and in 1 human isolate of C. hyointestinalis, and 21 of these strains carried both cdt-I and cdt-II gene clusters. Altogether, our results indicate that ChCDT-II is an important virulence factor of C. hyointestinalis in animals. PMID:26283337

  14. Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo

    PubMed Central

    Kaiser, Constanze; Dobrikova, Elena Y.; Bradrick, Shelton S.; Shveygert, Mayya; Herbert, James T.; Gromeier, Matthias

    2008-01-01

    Protein synthesis is tightly controlled by assembly of an intricate ribonucleoprotein complex at the m7GTP-cap on eukaryotic mRNAs. Ensuing linear scanning of the 5′ untranslated region (UTR) is believed to transfer the preinitiation complex to the initiation codon. Eukaryotic mRNAs are characterized by significant 5′ UTR heterogeneity, raising the possibility of differential control of translation initiation rate at individual mRNAs. Curiously, many mRNAs with unconventional, highly structured 5′ UTRs encode proteins with central biological roles in growth control, metabolism, or stress response. The 5′ UTRs of such mRNAs may influence protein synthesis rate in multiple ways, but most significantly they have been implicated in mediating alternative means of translation initiation. Cap-independent initiation bypasses strict control over the formation of initiation intermediates at the m7GTP cap. However, the molecular mechanisms that favor alternative means of ribosome recruitment are not understood. Here we provide evidence that eukaryotic initiation factor (eIF) 4G controls cap-independent translation initiation at the c-myc and vascular endothelial growth factor (VEGF) 5′ UTRs in vivo. Cap-independent translation was investigated in tetracycline-inducible cell lines expressing either full-length eIF4G or a C-terminal fragment (Ct) lacking interaction with eIF4E and poly(A) binding protein. Expression of Ct, but not intact eIF4G, potently stimulated cap-independent initiation at the c-myc/VEGF 5′ UTRs. In vitro RNA-binding assays suggest that stimulation of cap-independent translation initiation by Ct is due to direct association with the c-myc/VEGF 5′ UTR, enabling 43S preinitiation complex recruitment. Our work demonstrates that variant translation initiation factors enable unconventional translation initiation at mRNA subsets with distinct structural features. PMID:18755839

  15. Differential activation of human constitutive androstane receptor and its SV23 and SV24 splice variants by rilpivirine and etravirine

    PubMed Central

    Sharma, Devinder; Lau, Aik Jiang; Sherman, Matthew A; Chang, Thomas K H

    2015-01-01

    Background and Purpose Rilpivirine and etravirine are second-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs) indicated for the treatment of HIV/AIDS. The constitutive androstane receptor (CAR) regulates the expression of genes involved in various biological processes, including the transport and biotransformation of drugs. We investigated the effect of rilpivirine and etravirine on the activity of the wild-type human CAR (hCAR-WT) and its hCAR-SV23 and hCAR-SV24 splice variants, and compared it with first-generation NNRTIs (efavirenz, nevirapine, and delavirdine). Experimental Approach Receptor activation, ligand-binding domain (LBD) transactivation, and co-activator recruitment were investigated in transiently transfected, NNRTI-treated HepG2 cells. Nuclear translocation of green fluorescent protein-tagged hCAR-WT and CYP2B6 gene expression were assessed in NNRTI-treated human hepatocytes. Key Results Rilpivirine and etravirine activated hCAR-WT, but not hCAR-SV23 or hCAR-SV24, and without transactivating the LBD or recruiting steroid receptor coactivators SRC-1, SRC-2, or SRC-3. Among the first-generation NNRTIs investigated, only efavirenz activated hCAR-WT, hCAR-SV23, and hCAR-SV24, but none of them transactivated the LBD of these receptors or substantively recruited SRC-1, SRC-2, or SRC-3. Rilpivirine, etravirine, and efavirenz triggered nuclear translocation of hCAR-WT and increased hCAR target gene (CYP2B6) expression. Conclusion and Implications NNRTIs activate hCAR-WT, hCAR-SV23, and hCAR-SV24 in a drug-specific and isoform-selective manner. The activation occurs by a mechanism that does not appear to involve binding to the LBD or recruitment of SRC-1, SRC-2, or SRC-3. PMID:25363652

  16. Three-dimensional magnetic restructuring in two homologous solar flares in the seismically active NOAA AR 11283

    SciTech Connect

    Liu, Chang; Deng, Na; Lee, Jeongwoo; Wang, Haimin; Wiegelmann, Thomas; Jiang, Chaowei; Dennis, Brian R.; Su, Yang; Donea, Alina

    2014-11-10

    We carry out a comprehensive investigation comparing the three-dimensional magnetic field restructuring, flare energy release, and the helioseismic response of two homologous flares, the 2011 September 6 X2.1 (FL1) and September 7 X1.8 (FL2) flares in NOAA AR 11283. In our analysis, (1) a twisted flux rope (FR) collapses onto the surface at a speed of 1.5 km s{sup –1} after a partial eruption in FL1. The FR then gradually grows to reach a higher altitude and collapses again at 3 km s{sup –1} after a fuller eruption in FL2. Also, FL2 shows a larger decrease of the flux-weighted centroid separation of opposite magnetic polarities and a greater change of the horizontal field on the surface. These imply a more violent coronal implosion with corresponding more intense surface signatures in FL2. (2) The FR is inclined northward and together with the ambient fields, it undergoes a southward turning after both events. This agrees with the asymmetric decay of the penumbra observed in the peripheral regions. (3) The amounts of free magnetic energy and nonthermal electron energy released during FL1 are comparable to those of FL2 within the uncertainties of the measurements. (4) No sunquake was detected in FL1; in contrast, FL2 produced two seismic emission sources S1 and S2 both lying in the penumbral regions. Interestingly, S1 and S2 are connected by magnetic loops, and the stronger source S2 has a weaker vertical magnetic field. We discuss these results in relation to the implosion process in the low corona and the sunquake generation.

  17. Three-dimensional Magnetic Restructuring in Two Homologous Solar Flares in the Seismically Active NOAA AR 11283

    NASA Astrophysics Data System (ADS)

    Liu, Chang; Deng, Na; Lee, Jeongwoo; Wiegelmann, Thomas; Jiang, Chaowei; Dennis, Brian R.; Su, Yang; Donea, Alina; Wang, Haimin

    2014-11-01

    We carry out a comprehensive investigation comparing the three-dimensional magnetic field restructuring, flare energy release, and the helioseismic response of two homologous flares, the 2011 September 6 X2.1 (FL1) and September 7 X1.8 (FL2) flares in NOAA AR 11283. In our analysis, (1) a twisted flux rope (FR) collapses onto the surface at a speed of 1.5 km s-1 after a partial eruption in FL1. The FR then gradually grows to reach a higher altitude and collapses again at 3 km s-1 after a fuller eruption in FL2. Also, FL2 shows a larger decrease of the flux-weighted centroid separation of opposite magnetic polarities and a greater change of the horizontal field on the surface. These imply a more violent coronal implosion with corresponding more intense surface signatures in FL2. (2) The FR is inclined northward and together with the ambient fields, it undergoes a southward turning after both events. This agrees with the asymmetric decay of the penumbra observed in the peripheral regions. (3) The amounts of free magnetic energy and nonthermal electron energy released during FL1 are comparable to those of FL2 within the uncertainties of the measurements. (4) No sunquake was detected in FL1; in contrast, FL2 produced two seismic emission sources S1 and S2 both lying in the penumbral regions. Interestingly, S1 and S2 are connected by magnetic loops, and the stronger source S2 has a weaker vertical magnetic field. We discuss these results in relation to the implosion process in the low corona and the sunquake generation.

  18. Three-Dimensional Magnetic Restructuring in Two Homologous Solar Flares in the Seismically Active NOAA AR 11283

    NASA Technical Reports Server (NTRS)

    Liu, Chang; Deng, Na; Lee, Jeongwoo; Wiegelmann, Thomas; JIang, Chaowei; Dennis, Brian R.; Su, Yang; Donea, Alina; Wang, Haimin

    2014-01-01

    We carry out a comprehensive investigation comparing the three-dimensional magnetic field restructuring, flare energy release, and the helioseismic response of two homologous flares, the 2011 September 6 X2.1 (FL1) and September 7 X1.8 (FL2) flares in NOAA AR 11283. In our analysis, (1) a twisted flux rope (FR) collapses onto the surface at a speed of 1.5 km s(exp-1) after a partial eruption in FL1. The FR then gradually grows to reach a higher altitude and collapses again at 3 km s(exp-1) after a fuller eruption in FL2. Also, FL2 shows a larger decrease of the flux-weighted centroid separation of opposite magnetic polarities and a greater change of the horizontal field on the surface. These imply a more violent coronal implosion with corresponding more intense surface signatures in FL2. (2) The FR is inclined northward and together with the ambient fields, it undergoes a southward turning after both events. This agrees with the asymmetric decay of the penumbra observed in the peripheral regions. (3) The amounts of free magnetic energy and nonthermal electron energy released during FL1 are comparable to those of FL2 within the uncertainties of the measurements. (4) No sunquake was detected in FL1; in contrast, FL2 produced two seismic emission sources S1 and S2 both lying in the penumbral regions. Interestingly, S1 and S2 are connected by magnetic loops, and the stronger source S2 has a weaker vertical magnetic field. We discuss these results in relation to the implosion process in the low corona and the sunquake generation.

  19. The Strength and Time Course of Lexical Activation of Pronunciation Variants

    ERIC Educational Resources Information Center

    Pitt, Mark A.

    2009-01-01

    Spoken words undergo frequent and often predictable variation in pronunciation. One form of variation is medial /t/ deletion, in which words like "center" and "cantaloupe" are pronounced without acoustic cues indicative of syllable-initial /t/. Three experiments examined the consequences of this missing phonetic information on lexical activation.…

  20. Epidermal growth factor receptor variant III mediates head and neck cancer cell invasion via STAT3 activation

    PubMed Central

    Suzuki, Shinsuke; Morgan, Sarah E.; Thomas, Sufi M.; Sen, Malabika; Leeman-Neill, Rebecca J.; Kuan, Chien-Tsun; Bigner, Darrell; Gooding, William E.; Lai, Stephen Y.; Grandis, Jennifer R.

    2009-01-01

    Epidermal Growth Factor Receptor (EGFR) is frequently over-expressed in head and neck squamous cell carcinoma (HNSCC) where aberrant signaling downstream of this receptor contributes to tumor growth. EGFR variant III (EGFRvIII) is the most commonly altered form of EGFR and contains a truncated ligand-binding domain. We previously reported that EGFRvIII is expressed in up to 40% of HNSCC tumors where it is associated with increased proliferation, tumor growth and chemoresistance to anti-tumor drugs including the EGFR targeting monoclonal antibody cetuximab. Cetuximab was FDA-approved in 2006 for HNSCC but has not been shown to prevent invasion or metastasis. The present study was undertaken to evaluate the mechanisms of EGFRvIII-mediated cell motility and invasion in HNSCC. We found that EGFRvIII induced HNSCC cell migration and invasion in conjunction with increased STAT3 activation, which was not abrogated by cetuximab treatment. Further investigation demonstrated that EGF-induced expression of the STAT3 target gene HIF1-α, was abolished by cetuximab in HNSCC cells expressing wild-type EGFR under hypoxic conditions, but not in EGFRvIII-expressing HNSCC cells. These results suggest that EGFRvIII mediates HNSCC cell migration and invasion via increased STAT3 activation and induction of HIF1-α, which contribute to cetuximab resistance in EGFRvIII-expressing HNSCC tumors. PMID:20622897

  1. Mechanistic Variants in Gas-Phase Metal-Oxide Mediated Activation of Methane at Ambient Conditions.

    PubMed

    Li, Jilai; Zhou, Shaodong; Zhang, Jun; Schlangen, Maria; Usharani, Dandamudi; Shaik, Sason; Schwarz, Helmut

    2016-09-01

    The C-H bond activation of methane mediated by a prototypical heteronuclear metal-oxide cluster, [Al2Mg2O5](•+), was investigated by using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) in conjunction with high-level quantum mechanical calculations. Experimentally, hydrogen-atom abstraction from methane by the cluster ion [Al2Mg2O5](•+) takes place at ambient conditions. As to the mechanism, according to our computational findings, both the proton-coupled electron transfer (PCET) and the conventional hydrogen-atom transfer (HAT) are feasible and compete with each other. This is in distinct contrast to the [XYO2](+) (X, Y = Mg, Al, Si) cluster oxide ions which activate methane exclusively via the PCET route (Li, J.; Zhou, S.; Zhang, J.; Schlangen, M.; Weiske, T.; Usharani, D.; Shaik, S.; Schwarz, H. J. Am. Chem. Soc. 2016, 138, 7973-7981). The electronic origins of the mechanistically rather complex reactivity scenarios of the [Al2Mg2O5](•+)/CH4 couple were elucidated. For the PCET mechanism, in which the Lewis acid-base pair [Al(+)-O(-)] of the cluster acts as the active site, a clear correlation has been established between the nature of the transition state, the corresponding barrier height, the Lewis acidity-basicity of the [M(+)-O(-)] unit, as well as the bond order of the M(+)-O(-) bond. Also addressed is the role of the spin and charge distributions of a terminal oxygen radical site in the direct HAT route. The knowledge of the factors that control the reactivity of PCET and HAT pathways not only deepens our mechanistic understanding of metal-oxide mediated C-H bond activation but may also provide guidance for the rational design of catalysts. PMID:27518766

  2. The drosomycin multigene family: three-disulfide variants from Drosophila takahashii possess antibacterial activity

    PubMed Central

    Gao, Bin; Zhu, Shunyi

    2016-01-01

    Drosomycin (DRS) is a strictly antifungal peptide in Drosophila melanogaster, which contains four disulfide bridges (DBs) with three buried in molecular interior and one exposed on molecular surface to tie the amino- and carboxyl-termini of the molecule together (called wrapper disulfide bridge, WDB). Based on computational analysis of genomes of Drosophila species belonging to the Oriental lineage, we identified a new multigene family of DRS in Drosphila takahashii that includes a total of 11 DRS-encoding genes (termed DtDRS-1 to DtDRS-11) and a pseudogene. Phylogenetic tree and synteny analyses reveal orthologous relationship between DtDRSs and DRSs, indicating that orthologous genes of DRS-1, DRS-2, DRS-3 and DRS-6 have undergone duplication in D. takahashii and three amplifications (DtDRS-9 to DtDRS-11) of DRS-3 have lost WDB. Among the 11 genes, five are transcriptionally active in adult fruitflies. The ortholog of DRS (DtDRS-1) shows high structural and functional similarity to DRS while two WDB-deficient members display antibacterial activity accompanying complete loss or remarkable reduction of antifungal activity. To the best of our knowledge, this is the first report on the presence of three-disulfide antibacterial DRSs in a specific Drosophila species, suggesting a potential role of DB loss in neofunctionalization of a protein via structural adjustment. PMID:27562645

  3. The drosomycin multigene family: three-disulfide variants from Drosophila takahashii possess antibacterial activity.

    PubMed

    Gao, Bin; Zhu, Shunyi

    2016-01-01

    Drosomycin (DRS) is a strictly antifungal peptide in Drosophila melanogaster, which contains four disulfide bridges (DBs) with three buried in molecular interior and one exposed on molecular surface to tie the amino- and carboxyl-termini of the molecule together (called wrapper disulfide bridge, WDB). Based on computational analysis of genomes of Drosophila species belonging to the Oriental lineage, we identified a new multigene family of DRS in Drosphila takahashii that includes a total of 11 DRS-encoding genes (termed DtDRS-1 to DtDRS-11) and a pseudogene. Phylogenetic tree and synteny analyses reveal orthologous relationship between DtDRSs and DRSs, indicating that orthologous genes of DRS-1, DRS-2, DRS-3 and DRS-6 have undergone duplication in D. takahashii and three amplifications (DtDRS-9 to DtDRS-11) of DRS-3 have lost WDB. Among the 11 genes, five are transcriptionally active in adult fruitflies. The ortholog of DRS (DtDRS-1) shows high structural and functional similarity to DRS while two WDB-deficient members display antibacterial activity accompanying complete loss or remarkable reduction of antifungal activity. To the best of our knowledge, this is the first report on the presence of three-disulfide antibacterial DRSs in a specific Drosophila species, suggesting a potential role of DB loss in neofunctionalization of a protein via structural adjustment. PMID:27562645

  4. High Basal Activity of the PTPN22 Gain-of-Function Variant Blunts Leukocyte Responsiveness Negatively Affecting IL-10 Production in ANCA Vasculitis

    PubMed Central

    Cao, Yali; Yang, Jiajin; Colby, Kerry; Hogan, Susan L.; Hu, Yichun; Jennette, Caroline E.; Berg, Elisabeth A.; Zhang, Youkang; Jennette, J. Charles; Falk, Ronald J.; Preston, Gloria A.

    2012-01-01

    Consequences of expression of the protein tyrosine phosphatase nonreceptor 22 (PTPN22) gain-of-function variant were evaluated in leukocytes from patients with anti-neutrophil cytoplasmic autoantibody (ANCA) disease. The frequency of the gain-of-function allele within the Caucasian patient cohort was 22% (OR 1.45), compared to general American Caucasian population (16.5%, p = 0.03). Examination of the basal phosphatase activity of PTPN22 gain-of-function protein indicated persistently elevated activity in un-stimulated peripheral leukocytes, while basal activity was undetectable in leukocytes from patients without the gain-of-function variant. To examine consequences of persistently high PTPN22 activity, the activation status of ERK and p38 MAPK were analyzed. While moderate levels of activated ERK were observed in controls, it was undetectable in leukocytes expressing PTPN22 gain-of-function protein and instead p38MAPK was up-regulated. IL-10 transcription, reliant on the ERK pathway, was negatively affected. Over the course of disease, patients expressing variant PTPN22 did not show a spike in IL-10 transcription as they entered remission in contrast to controls, implying that environmentally triggered signals were blunted. Sustained activity of PTPN22, due to the gain-of-function mutation, acts as a dominant negative regulator of ERK activity leading to blunted cellular responsiveness to environmental stimuli and expression of protective cytokines. PMID:22880107

  5. The Histone Variant H3.3 Is Enriched at Drosophila Amplicon Origins but Does Not Mark Them for Activation

    PubMed Central

    Paranjape, Neha P.; Calvi, Brian R.

    2016-01-01

    Eukaryotic DNA replication begins from multiple origins. The origin recognition complex (ORC) binds origin DNA and scaffolds assembly of a prereplicative complex (pre-RC), which is subsequently activated to initiate DNA replication. In multicellular eukaryotes, origins do not share a strict DNA consensus sequence, and their activity changes in concert with chromatin status during development, but mechanisms are ill-defined. Previous genome-wide analyses in Drosophila and other organisms have revealed a correlation between ORC binding sites and the histone variant H3.3. This correlation suggests that H3.3 may designate origin sites, but this idea has remained untested. To address this question, we examined the enrichment and function of H3.3 at the origins responsible for developmental gene amplification in the somatic follicle cells of the Drosophila ovary. We found that H3.3 is abundant at these amplicon origins. H3.3 levels remained high when replication initiation was blocked, indicating that H3.3 is abundant at the origins before activation of the pre-RC. H3.3 was also enriched at the origins during early oogenesis, raising the possibility that H3.3 bookmarks sites for later amplification. However, flies null mutant for both of the H3.3 genes in Drosophila did not have overt defects in developmental gene amplification or genomic replication, suggesting that H3.3 is not essential for the assembly or activation of the pre-RC at origins. Instead, our results imply that the correlation between H3.3 and ORC sites reflects other chromatin attributes that are important for origin function. PMID:27172191

  6. The Histone Variant H3.3 Is Enriched at Drosophila Amplicon Origins but Does Not Mark Them for Activation.

    PubMed

    Paranjape, Neha P; Calvi, Brian R

    2016-01-01

    Eukaryotic DNA replication begins from multiple origins. The origin recognition complex (ORC) binds origin DNA and scaffolds assembly of a prereplicative complex (pre-RC), which is subsequently activated to initiate DNA replication. In multicellular eukaryotes, origins do not share a strict DNA consensus sequence, and their activity changes in concert with chromatin status during development, but mechanisms are ill-defined. Previous genome-wide analyses in Drosophila and other organisms have revealed a correlation between ORC binding sites and the histone variant H3.3. This correlation suggests that H3.3 may designate origin sites, but this idea has remained untested. To address this question, we examined the enrichment and function of H3.3 at the origins responsible for developmental gene amplification in the somatic follicle cells of the Drosophila ovary. We found that H3.3 is abundant at these amplicon origins. H3.3 levels remained high when replication initiation was blocked, indicating that H3.3 is abundant at the origins before activation of the pre-RC. H3.3 was also enriched at the origins during early oogenesis, raising the possibility that H3.3 bookmarks sites for later amplification. However, flies null mutant for both of the H3.3 genes in Drosophila did not have overt defects in developmental gene amplification or genomic replication, suggesting that H3.3 is not essential for the assembly or activation of the pre-RC at origins. Instead, our results imply that the correlation between H3.3 and ORC sites reflects other chromatin attributes that are important for origin function. PMID:27172191

  7. Characterization of antimicrobial activity against Listeria and cytotoxicity of native melittin and its mutant variants.

    PubMed

    Wu, Xi; Singh, Atul K; Wu, Xiaoyu; Lyu, Yuan; Bhunia, Arun K; Narsimhan, Ganesan

    2016-07-01

    Antimicrobial peptides (AMPs) are relatively short peptides that have the ability to penetrate the cell membrane, form pores leading to cell death. This study compares both antimicrobial activity and cytotoxicity of native melittin and its two mutants, namely, melittin I17K (GIGAVLKVLTTGLPALKSWIKRKRQQ) with a higher charge and lower hydrophobicity and mutant G1I (IIGAVLKVLTTGLPALISWIKRKRQQ) of higher hydrophobicity. The antimicrobial activity against different strains of Listeria was investigated by bioassay, viability studies, fluorescence and transmission electron microscopy. Cytotoxicity was examined by lactate dehydrogenase (LDH) assay on mammalian Caco-2 cells. The minimum inhibitory concentration of native, mutant I17K, mutant G1I against Listeria monocytogenes F4244 was 0.315±0.008, 0.814±0.006 and 0.494±0.037μg/ml respectively, whereas the minimum bactericidal concentration values were 3.263±0.0034, 7.412±0.017 and 5.366±0.019μg/ml respectively. Lag time for inactivation of L. monocytogenes F4244 was observed at concentrations below 0.20 and 0.78μg/ml for native and mutant melittin I17K respectively. The antimicrobial activity against L. monocytogenes F4244 was in the order native>G1I>I17K. Native melittin was cytotoxic to mammalian Caco-2 cells above concentration of 2μg/ml, whereas the two mutants exhibited negligible cytotoxicity up to a concentration of 8μg/ml. Pore formation in cell wall/membrane was observed by transmission electron microscopy. Molecular dynamics (MD) simulation of native and its mutants indicated that (i) surface native melittin and G1I exhibited higher tendency to penetrate a mimic of bacterial cell membrane and (ii) transmembrane native and I17K formed water channel in mimics of bacterial and mammalian cell membranes. PMID:27011349

  8. The disease-protective complement factor H allotypic variant Ile62 shows increased binding affinity for C3b and enhanced cofactor activity

    PubMed Central

    Tortajada, Agustín; Montes, Tamara; Martinez-Barricarte, Ruben; Morgan, B. Paul; Harris, Claire L.; de Córdoba, Santiago Rodríguez

    2011-01-01

    Summary Mutations and polymorphisms in the gene encoding factor H (CFH) have been associated with atypical haemolytic uraemic syndrome, dense deposit disease and age-related macular degeneration. The disease-predisposing CFH variants show a differential association with pathology that has been very useful to unravel critical events in the pathogenesis of one or other disease. In contrast, the fH-Ile62 polymorphism confers strong protection to all three diseases. Using ELISA-based methods and surface plasmon resonance analyses we show here that the protective fH-Ile62 variant binds more efficiently to C3b than fH-Val62 and competes better with factor B in proconvertase formation. Functional analyses demonstrate an increased cofactor activity for fH-Ile62 in the factor I-mediated cleavage of fluid phase and surface-bound C3b; however, the two fH variants show no differences in decay accelerating activity. From these data we conclude that the protective effect of the fH-Ile62 variant is due to its better capacity to bind C3b, inhibit proconvertase formation and catalyse inactivation of fluid-phase and surface-bound C3b. This demonstration of the functional consequences of the fH-Ile62 polymorphism provides relevant insights into the complement regulatory activities of fH that will be useful in disease prediction and future development of effective therapeutics for disorders caused by complement dysregulation. PMID:19549636

  9. Ar-Ar ages and trapped Ar components in Martian shergottites RBT 04262 and LAR 06319

    NASA Astrophysics Data System (ADS)

    Park, Jisun; Bogard, Donald D.; Nyquist, Laurence E.; Garrison, Daniel H.; Mikouchi, Takashi

    2013-11-01

    We made 39Ar-40Ar (Ar-Ar) analyses of whole rock (WR) and mineral samples of two Martian shergottites, RBT 04262 (RBT) and LAR 06319 (LAR), in order to determine their Ar-Ar ages and the 40Ar/36Ar ratios of the trapped Martian Ar they contain. All samples released trapped (excess) 40Ar and 36Ar and suggested Ar-Ar ages older than their formation ages. Because trapped Ar components having different 40Ar/36Ar were released at different extraction temperatures, we utilized only a portion of the data to derive preferred Ar-Ar ages. We obtain Ar-Ar ages of 171 ± 8 Ma for RBT plagioclase and 163 ± 13 Ma for LAR whole rock. We identify two trapped Ar components. At low temperatures, particularly for plagioclase, Trapped-A with 40Ar/36Ar 285 ± 3 was released, and we believe this is most likely absorbed terrestrial air. At high extraction temperatures, particularly for pyroxene, Trapped-B with 40Ar/36Ar 1813 ± 127 was released. The poikilitic/non-poikilitic texture of RBT and the presence of large pyroxene oikocrysts allowed a clear definition of Trapped-B. This Ar component is Martian, and its isotopic similarity to the Martian atmospheric composition suggests that it may represent Martian atmospheric Ar incorporated into the shergottite melt via crustal rocks. Trapped-B partitioned into pyroxene at a constant molar ratio of K/36ArTr = 33.2 ± 9.5 × 106 for RBT 04262, and 80 ± 21 × 106 for LAR 06319. Trapped-A mixed in different proportions with Trapped-B could give apparently intermediate trapped 40Ar/36Ar compositions commonly observed in shergottites.

  10. Alpha1a-Adrenoceptor Genetic Variant Triggers Vascular Smooth Muscle Cell Hyperproliferation and Agonist Induced Hypertrophy via EGFR Transactivation Pathway

    PubMed Central

    Schwinn, Debra A.; Oganesian, Anush

    2015-01-01

    α1a Adrenergic receptors (α1aARs) are the predominant AR subtype in human vascular smooth muscle cells (SMCs). α1aARs in resistance vessels are crucial in the control of blood pressure, yet the impact of naturally occurring human α1aAR genetic variants in cardiovascular disorders remains poorly understood. To this end, we present novel findings demonstrating that 3D cultures of vascular SMCs expressing human α1aAR-247R (247R) genetic variant demonstrate significantly increased SMC contractility compared with cells expressing the α1aAR-WT (WT) receptor. Stable expression of 247R genetic variant also triggers MMP/EGFR-transactivation dependent serum- and agonist-independent (constitutive) hyperproliferation and agonist-dependent hypertrophy of SMCs. Agonist stimulation reduces contractility Using pathway-specific inhibitors we determined that the observed hyperproliferation of 247R-expressing cells is triggered via β-arrestin1/Src/MMP-2/EGFR/ERK-dependent mechanism. MMP-2-specific siRNA inhibited 247R-triggered hyperproliferation indicating MMP-2 involvement in 247R-triggered hyperproliferation in SMCs. β-arrestin1-specific shRNA also inhibited 247R-triggered hyperproliferation but did not affect hypertrophy in 247R-expressing SMCs, indicating that agonist-dependent hypertrophy is independent of β-arrestin1. Our data reveal that in different cardiovascular cells the same human receptor genetic variant can activate alternative modulators of the same signaling pathway. Thus, our findings in SMCs demonstrate that depending on the type of cells expressing the same receptor (or receptor variant), different target-specific inhibitors could be used to modulate aberrant hyperproliferative or hypertrophic pathways in order to restore normal phenotype. PMID:26571308

  11. Butyrylcholinesterase: K variant, plasma activity, molecular forms and rivastigmine treatment in Alzheimer's disease in a Southern Brazilian population.

    PubMed

    Bono, G F; Simão-Silva, D P; Batistela, M S; Josviak, N D; Dias, P F R; Nascimento, G A; Souza, R L R; Piovezan, M R; Souza, R K M; Furtado-Alle, L

    2015-02-01

    Alzheimer's disease (AD) is a neurodegenerative disorder in which there is a decline of cholinergic function. The symptomatic AD treatment involves the use of ChEIs (cholinesterase inhibitors) as rivastigimine, a dual inhibitor. The human butyrylcholinesterase (BChE) is an enzyme that has specific roles in cholinergic neurotransmission and it has been associated with AD. In the serum, BChE is found in four main molecular forms: G1 (monomer); G1-ALB (monomer linked to albumin); G2 (dimer); and G4 (tetramer). The interaction between the products of BCHE gene and CHE2 locus results in CHE2 C5+ and CHE2 C5- phenotypes. CHE2 C5+ phenotype and BChE-K are factors that influence on BChE activity. This work aimed to verify the proportions of BChE molecular forms, total and relative activity in 139 AD patients and 139 elderly controls, taking into account K variant, CHE2 locus, rivastigmine treatment and clinical dementia rating (CDR) of AD patients. Phenotypic frequencies of CHE2 C5+ and frequency of the carriers of the K allele were similar between groups. Total BChE activity in plasma was significantly lower in AD patients than in elderly controls. Furthermore, we found that reduction on plasma BChE activity is associated directly with AD progression in AD patients and that rivastigmine treatment has a stronger effect on BChE activity within the CDR2 group. The reduction in BChE activity did not occur proportionally in all molecular forms. Multiple regression analysis results confirmed that AD acts as the main factor in plasma BChE activity reduction and that severe stages are related with an even greater reduction. These findings suggest that the reduction of total plasma BChE and relative BChE molecular forms activity in AD patients is probably associated with a feedback mechanism and provides a future perspective of using this enzyme as a possible plasmatic secondary marker for AD. PMID:25624079

  12. Effector Activity of Peanut Allergens: A critical role for Ara h 2, Ara h 6, and their variants

    PubMed Central

    Porterfield, HS; Murray, KS; Schlichting, DG; Chen, X.; Hansen, KC; Duncan, MW; Dreskin, SC

    2009-01-01

    Rationale An important property of allergens is their ability to cross-link IgE and activate mast cells and basophils. The effector activity of peanut allergens has not been well characterized. Methods Crude extracts of fresh peanut flour were fractionated by gel filtration. Effector function was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from individual sera and from pools of sera of peanut allergic donors. Results Following gel filtration, 75±7% of the applied protein and 76±16% (n=3) of the applied activity (assayed with a pool of 11 sera) were recovered in the resultant fractions. The majority (85±2%; n=3) of the recovered activity resided in a fraction with a theoretical average molecular weight of ~20 kD and a range of 13–25 kD. When all the individual fractions were recombined, the measured activity was similar to that of the original extract (140±43% when measured with a pool of serum (n=2) and 66±7% when measured with individual sera (n=4)); when all individual fractions excluding the 20 kD fraction were recombined, the measured activity was only 8±2% (n=2) of the original extract when assayed with the serum pool and 10±4% (n=3) when assayed with the individual sera. Two dimensional gel electrophoresis of this biologically active fraction by revealed >60 protein spots . Analysis of 50 of the most prominent spots by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and of the full mixture by automated tandem mass spectrometry coupled to online capillary liquid chromatography revealed that greater than 97% of the protein mass consisted of Ara h 2.0101, Ara h 2.0201, Ara h 6 isoforms, and variants of these proteins. Conclusions Ara h 2 and Ara h 6 account for the majority of the effector activity found in a crude peanut extract. PMID:19438581

  13. Characterization of streptococcal platelet-activating factor acetylhydrolase variants that are involved in innate immune evasion.

    PubMed

    Liu, Guanghui; Liu, Mengyao; Xie, Gang; Lei, Benfang

    2013-09-01

    Human pathogen group A streptococcus (GAS) has developed mechanisms to subvert innate immunity. We recently reported that the secreted esterase produced by serotype M1 GAS (SsE(M1)) reduces neutrophil recruitment by targeting platelet-activating factor (PAF). SsE(M1) and SsE produced by serotype M28 GAS (SsE(M28)) have a 37% sequence difference. This study aims at determining whether SsE(M28) is also a PAF acetylhydrolase and participates in innate immune evasion. We also examined whether SsE evolved to target PAF by characterizing the PAF acetylhydrolase (PAF-AH) activity and substrate specificity of SsE(M1), SsE(M28), SeE, the SsE homologue in Streptococcus equi, and human plasma PAF-AH (hpPAF-AH). PAF incubated with SsE(M28) or SeE was converted into lyso-PAF. SsE(M1) and SsE(M28) had kcat values of 373 s(-1) and 467 s(-1), respectively, that were ≥ 30-fold greater than that of hpPAF-AH (12 s(-1)). The comparison of SsE(M1), SsE(M28), and hpPAF-AH in kcat and Km in hydrolyzing triglycerides, acetyl esters, and PAF indicates that the SsE proteins are more potent hydrolases against PAF and have high affinity for PAF. SsE(M28) possesses much lower esterase activities against triglycerides and other esters than SsE(M1) but have similar potency with SsE(M1) in PAF hydrolysis. Deletion of sse(M28) in a covS deletion mutant of GAS increased neutrophil recruitment and reduced skin infection, whereas in trans expression of SsE(M28) in GAS reduced neutrophil infiltration and increased skin invasion in subcutaneous infection of mice. These results suggest that the SsE proteins evolved to target PAF for enhancing innate immune evasion and skin invasion. PMID:23774595

  14. Characterization of Streptococcal Platelet-Activating Factor Acetylhydrolase Variants That Are Involved in Innate Immune Evasion

    PubMed Central

    Liu, Guanghui; Liu, Mengyao; Xie, Gang

    2013-01-01

    Human pathogen group A streptococcus (GAS) has developed mechanisms to subvert innate immunity. We recently reported that the secreted esterase produced by serotype M1 GAS (SsEM1) reduces neutrophil recruitment by targeting platelet-activating factor (PAF). SsEM1 and SsE produced by serotype M28 GAS (SsEM28) have a 37% sequence difference. This study aims at determining whether SsEM28 is also a PAF acetylhydrolase and participates in innate immune evasion. We also examined whether SsE evolved to target PAF by characterizing the PAF acetylhydrolase (PAF-AH) activity and substrate specificity of SsEM1, SsEM28, SeE, the SsE homologue in Streptococcus equi, and human plasma PAF-AH (hpPAF-AH). PAF incubated with SsEM28 or SeE was converted into lyso-PAF. SsEM1 and SsEM28 had kcat values of 373 s−1 and 467 s−1, respectively, that were ≥30-fold greater than that of hpPAF-AH (12 s−1). The comparison of SsEM1, SsEM28, and hpPAF-AH in kcat and Km in hydrolyzing triglycerides, acetyl esters, and PAF indicates that the SsE proteins are more potent hydrolases against PAF and have high affinity for PAF. SsEM28 possesses much lower esterase activities against triglycerides and other esters than SsEM1 but have similar potency with SsEM1 in PAF hydrolysis. Deletion of sseM28 in a covS deletion mutant of GAS increased neutrophil recruitment and reduced skin infection, whereas in trans expression of SsEM28 in GAS reduced neutrophil infiltration and increased skin invasion in subcutaneous infection of mice. These results suggest that the SsE proteins evolved to target PAF for enhancing innate immune evasion and skin invasion. PMID:23774595

  15. Overexpression of the active diacylglycerol acyltransferase variant transforms Saccharomyces cerevisiae into an oleaginous yeast.

    PubMed

    Kamisaka, Yasushi; Kimura, Kazuyoshi; Uemura, Hiroshi; Yamaoka, Masakazu

    2013-08-01

    Lipid production by Saccharomyces cerevisiae was improved by overexpression of the yeast diacylglycerol acyltransferase Dga1p lacking the N-terminal 29 amino acids (Dga1∆Np), which was previously found to be an active form in the ∆snf2 mutant. Overexpression of Dga1∆Np in the ∆snf2 mutant, however, did not increase lipid content as expected, which prompted us to search for a more suitable strain in which to study the role of Dga1∆Np in lipid accumulation. We found that the overexpression of Dga1∆Np in the ∆dga1 mutant effectively increased the lipid content up to about 45 % in the medium containing 10 % glucose. The high lipid content of the transformant was dependent on glucose concentration, nitrogen limitation, and active leucine biosynthesis. To better understand the effect of dga1 disruption on the ability of Dga1∆Np to stimulate lipid accumulation, the ∆dga1-1 mutant, in which the 3'-terminal 36 bp of the dga1 open reading frame (ORF) remained, and the ∆dga1-2 mutant, in which the 3'-terminal 36 bp were also deleted, were prepared with URA3 disruption cassettes. Surprisingly, the overexpression of Dga1∆Np in the ∆dga1-1 mutant had a lower lipid content than the original ∆dga1 mutant, whereas overexpression in the ∆dga1-2 mutant led to a high lipid content of about 45 %. These results indicated that deletion of the 3' terminal region of the dga1 ORF, rather than abrogation of genomic Dga1p expression, was crucial for the effect of Dga1∆Np on lipid accumulation. To investigate whether dga1 disruption affected gene expression adjacent to DGA1, we found that the overexpression of Esa1p together with Dga1∆Np in the ∆dga1 mutant reverted the lipid content to the level of the wild-type strain overexpressing Dga1∆Np. In addition, RT-qPCR analysis revealed that ESA1 mRNA expression in the ∆dga1 mutant was decreased compared to the wild-type strain at the early stages of culture, suggesting that lowered Esa1p expression is

  16. Excitation Energies with Cost-Reduced Variant of the Active-Space EOMCCSDT Method: The EOMCCSDt-3̅ Approach.

    PubMed

    Hu, Han-Shi; Kowalski, Karol

    2013-11-12

    In this paper, we discuss the performance of several simplified variants of equation-of-motion coupled cluster method (EOMCC) with iterative inclusion of singles, doubles, and active-space triples (EOMCCSDt). In particular, we explore simplified EOMCCSDt approaches that enable one to generate the triply excited amplitudes in an on-the-fly manner. The original EOMCCSDt formulation has already demonstrated great success in encapsulating the most important excited-state correlation effects due to triples. In analogy to the original EOMCCSDT-3 formulation, the proposed approach can bypass the typical bottlenecks associated with the need for storing triply excited amplitudes. In this paper, we illustrate the performance of several approximate EOMCCSDt methods, named EOMCCSDt-3̅ and EOMCCSDt-3̅, on typical benchmark systems including C2, N2, ozone, ethene, and E-butadiene molecules. These new methods yield excitation energies close to the EOMCCSDt ones. The extrapolation of excitation energies for basis sets ranging from cc-pVDZ to cc-pV6Z for N2 and C2 shows very good convergence to the experimental results for states dominated by single excitations. The performance of the EOMCCSDt-3̅x approach is also compared with the results obtained with popular CCSDR(3) and CC3 approaches. PMID:26583394

  17. Spectroscopic Studies of Single and Double Variants of M Ferritin: Lack of Conversion of a Biferrous Substrate Site into a Cofactor Site for O2 Activation

    PubMed Central

    2015-01-01

    Ferritin has a binuclear non-heme iron active site that functions to oxidize iron as a substrate for formation of an iron mineral core. Other enzymes of this class have tightly bound diiron cofactor sites that activate O2 to react with substrate. Ferritin has an active site ligand set with 1-His/4-carboxylate/1-Gln rather than the 2-His/4-carboxylate set of the cofactor site. This ligand variation has been thought to make a major contribution to this biferrous substrate rather than cofactor site reactivity. However, the Q137E/D140H double variant of M ferritin, has a ligand set that is equivalent to most of the diiron cofactor sites, yet did not rapidly react with O2 or generate the peroxy intermediate observed in the cofactor sites. Therefore, in this study, a combined spectroscopic methodology of circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD has been applied to evaluate the factors required for the rapid O2 activation observed in cofactor sites. This methodology defines the coordination environment of each iron and the bridging ligation of the biferrous active sites in the double and corresponding single variants of frog M ferritin. Based on spectral changes, the D140H single variant has the new His ligand binding, and the Q137E variant has the new carboxylate forming a μ-1,3 bridge. The spectra for the Q137E/D140H double variant, which has the cofactor ligand set, however, reflects a site that is more coordinately saturated than the cofactor sites in other enzymes including ribonucleotide reductase, indicating the presence of additional water ligation. Correlation of this double variant and the cofactor sites to their O2 reactivities indicates that electrostatic and steric changes in the active site and, in particular, the hydrophobic nature of a cofactor site associated with its second sphere protein environment, make important contributions to the activation of O2 by the binuclear non-heme iron enzymes. PMID

  18. Structural analysis, clay mineralogy and K-Ar dating of fault gouges from Centovalli Line (Central Alps) for reconstruction of their recent activity

    NASA Astrophysics Data System (ADS)

    Surace, Ivan R.; Clauer, Norbert; Thélin, Philippe; Pfeifer, Hans-Rudolf

    2011-09-01

    Between the cities of Domodossola and Locarno, the complex "Centovalli Line" tectonic zone of the Central Alps outlines deformation phases over a long period of time (probably starting ~ 30 Ma ago) and under variable P-T conditions. The last deformation phases developed gouge-bearing faults with a general E-W trend that crosscuts the roots of the Alpine Canavese zone and the Finero ultramafic body. Kinematic indicators show that the general motion was mainly dextral associated with back thrusting towards the S. The < 2 μm clay fractions of fault gouges from Centovalli Line consist mainly of illite, smectite and chlorite with varied illite-smectite, chlorite-smectite and chlorite-serpentine mixed-layers. Constrained with the illite crystallinity index, the thermal conditions induced by the tectonic activity show a gradual trend from anchizonal to diagenetic conditions. The < 2 and < 0.2 μm clay fractions, and hydrothermal K-feldspar separates all provide K-Ar ages between 14.2 ± 2.9 Ma and roughly 0 Ma, with major episodes at about 12, 8, 6 and close to 0 Ma. These ages set the recurrent tectonic activity and the associated fluid circulations between Upper Miocene and Recent. On the basis of the K-Ar ages and with a thermal gradient of 25-30 °C/km, the studied fault zones were located at a depth of 4-7 km. If they were active until now as observed in field, the exhumation was approximately 2.5-3.0 km for the last 12 Ma with a mean velocity of 0.4 mm/y. Comparison with available models on the recent Alpine evolution shows that the tectonic activity in the area relates to a continuum of the back-thrusting movements of the Canavese Line, and/or to several late-extensional phases of the Rhône-Simplon Line. The Centovalli-Val Vigezzo zone therefore represents a major tectonic zone of the Central-Western Alps resulting from different interacting tectonic events.

  19. Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface

    SciTech Connect

    Borgstahl, G.E.O.; Hickey, M.J.; Johnson, M.J.

    1996-04-09

    Human manganese superoxide dismutase (MnSOD) is a homotetrameric enzyme which protects mitochondria against oxygen-mediated free radical damage. Within each subunit, both the N-terminal helical hairpin and C-terminal {alpha}/{beta} domains contribute ligands to the catalytic manganese site. Two identical four-helix bundles,symmetrically assembled form the N-terminal helical hairpins, form a novel tetrameric interface that stabilizes the active sites. The 2.5 {angstrom} crystallographic structure of the naturally occurring polymorphic variant Ile58Thr MnSOD reveals that the helical hairpin mutation Thr58 causes two packing defects in each of the two four-helix bundles of the tetrameric interface. Similar mutations, expected to cause packing defects in the Cu,ZnSOD dimer interface, are associated with the degenerative disease amyotrophic lateral sclerosis. Ile58Thr MnSOD is primarily dimeric in solution and is significantly less thermostable than the normal enzyme, with decreases of 15{degrees}C in the main melting temperature and 20{degrees}C in the heat-inactivation temperature. Consequently, this mutant MnSOD is compromised at normal body temperatures: thermal inactivation, predicted from the decrease in thermal stability, occurs with a theoretical half-life of only 3.2h at 37{degrees}C (1.4 h at 41 {degrees}C), compared with 3.1 years for native MnSOD. This prediction is supported by direct measurements: incubation at 41.7{degrees}C for 3 h has no effect on the activity of native MnSOD but completely inactivates mutant MnSOD. Rapid inactivation of Ile58Thr MnSOD at the elevated temperatures associated with fever and inflammation could provide an early advantage by killing infected cells, but also would increase superoxide-mediated oxidative damage and perhaps contribute to late-onset diseases. 63 refs., 7 figs., 2 tabs.

  20. Requirement of a novel splicing variant of human histone deacetylase 6 for TGF-{beta}1-mediated gene activation

    SciTech Connect

    Zhuang, Yan; Nguyen, Hong T.; Lasky, Joseph A.; Cao, Subing; Li, Cui; Hu, Jiyao; Guo, Xinyue; Burow, Matthew E.; Shan, Bin

    2010-02-19

    Histone deacetylase 6 (HDAC6) belongs to the family of class IIb HDACs and predominantly deacetylates non-histone proteins in the cytoplasm via the C-terminal deacetylase domain of its two tandem deacetylase domains. HDAC6 modulates fundamental cellular processes via deacetylation of {alpha}-tubulin, cortactin, molecular chaperones, and other peptides. Our previous study indicates that HDAC6 mediates TGF-{beta}1-induced epithelial-mesenchymal transition (EMT) in A549 cells. In the current study, we identify a novel splicing variant of human HDAC6, hHDAC6p114. The hHDAC6p114 mRNA arises from incomplete splicing and encodes a truncated isoform of the hHDAC6p114 protein of 114 kDa when compared to the major isoform hHDAC6p131. The hHDAC6p114 protein lacks the first 152 amino acids from N-terminus in the hHDAC6p131 protein, which harbors a nuclear export signal peptide and 76 amino acids of the N-terminal deacetylase domain. hHDAC6p114 is intact in its deacetylase activity against {alpha}-tubulin. The expression hHDAC6p114 is elevated in a MCF-7 derivative that exhibits an EMT-like phenotype. Moreover, hHDAC6p114 is required for TGF-{beta}1-activated gene expression associated with EMT in A549 cells. Taken together, our results implicate that expression and function of hHDAC6p114 is differentially regulated when compared to hHDAC6p131.

  1. Analysis of the Enzymatic Activity of an NS3 Helicase Genotype 3a Variant Sequence Obtained from a Relapse Patient

    PubMed Central

    Provazzi, Paola J. S.; Mukherjee, Sourav; Hanson, Alicia M.; Nogueira, Mauricio L.; Carneiro, Bruno M.; Frick, David N.; Rahal, Paula

    2015-01-01

    The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people worldwide, causing chronic inflammation, cirrhosis and hepatocellular carcinoma. HCV genotype 3a is common in Brazil, and it is associated with a relatively poor response to current direct-acting antiviral therapies. The HCV NS3 protein cleaves part of the HCV polyprotein, and cellular antiviral proteins. It is therefore the target of several HCV drugs. In addition to its protease activity, NS3 is also an RNA helicase. Previously, HCV present in a relapse patient was found to harbor a mutation known to be lethal to HCV genotype 1b. The point mutation encodes the amino acid substitution W501R in the helicase RNA binding site. To examine how the W501R substitution affects NS3 helicase activity in a genotype 3a background, wild type and W501R genotype 3a NS3 alleles were sub-cloned, expressed in E. coli, and the recombinant proteins were purified and characterized. The impact of the W501R allele on genotype 2a and 3a subgenomic replicons was also analyzed. Assays monitoring helicase-catalyzed DNA and RNA unwinding revealed that the catalytic efficiency of wild type genotype 3a NS3 helicase was more than 600 times greater than the W501R protein. Other assays revealed that the W501R protein bound DNA less than 2 times weaker than wild type, and both proteins hydrolyzed ATP at similar rates. In Huh7.5 cells, both genotype 2a and 3a subgenomic HCV replicons harboring the W501R allele showed a severe defect in replication. Since the W501R allele is carried as a minor variant, its replication would therefore need to be attributed to the trans-complementation by other wild type quasispecies. PMID:26658750

  2. NOX2β: A Novel Splice Variant of NOX2 That Regulates NADPH Oxidase Activity in Macrophages

    PubMed Central

    Guida, Elizabeth; King, Paul T.; Sobey, Christopher G.; Drummond, Grant R.

    2012-01-01

    Nox2 oxidase is one isoform in a family of seven NADPH oxidases that generate reactive oxygen species (ROS) and thereby contribute to physiological and pathological processes including host defense, redox signaling and oxidative tissue damage. While alternative mRNA splicing has been shown to influence the activity of several Nox-family proteins, functionally relevant splice variants of Nox2 have not previously been identified. We immunoscreened several mouse tissues and cells for the presence of truncated Nox2 proteins and identified a 30 kDa protein in lung, spleen and macrophages. RT-PCR analysis of mRNA from primary and immortalised (RAW264.7) mouse macrophages, and from human alveolar macrophages, identified a truncated Nox2 transcript which, upon sequence analysis, was found to be a product of the ‘exon skipping’ mode of alternative splicing, lacking exons 4–10 of the Nox2 gene. The predicted protein is comparable in size to that identified by immunoscreening and contains two transmembrane helices and an extended cytosolic C-terminus with binding sites for NADPH and the Nox organiser protein p47phox. Importantly, selective siRNA-mediated knockdown of the transcript reduced expression of the 30 kDa protein in macrophages, and suppressed phorbol ester-stimulated ROS production by 50%. We thus provide the first evidence that Nox2 undergoes alternative mRNA splicing to yield a 30 kDa protein – herein termed Nox2β – that regulates NADPH oxidase activity in macrophages from mice and humans. The discovery of Nox2β paves the way for future examination of its role in physiological and pathological processes. PMID:23118986

  3. CCAR1 promotes chromatin loading of androgen receptor (AR) transcription complex by stabilizing the association between AR and GATA2

    PubMed Central

    Seo, Woo-Young; Jeong, Byong Chang; Yu, Eun Ji; Kim, Hwa Jin; Kim, Seok-Hyung; Lim, Joung Eun; Kwon, Ghee Young; Lee, Hyun Moo; Kim, Jeong Hoon

    2013-01-01

    Androgen receptor (AR), a ligand-dependent transcription factor, plays a critical role in prostate cancer onset and progression, and its transcriptional function is mediated largely by distinct nuclear receptor co-regulators. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1) functions as an AR co-activator. CCAR1 interacted with and enhanced the transcriptional activity of AR. Depletion of CCAR1 caused reduction in androgen-dependent expression of a subset of AR target genes. We further showed that CCAR1 is required for recruitment of AR, MED1 and RNA polymerase II to the enhancers of AR target genes and for androgen-induced long-range prostate specific antigen enhancer–promoter interaction. The molecular mechanism underlying CCAR1 function in AR-mediated transcription involves CCAR1-mediated enhanced recruitment of GATA2, a pioneer factor for AR, to AR-binding sites. CCAR1 stabilized the interaction between AR and GATA2 by interacting directly with both proteins, thereby facilitating AR and GATA2 occupancy on the enhancers. Furthermore, CCAR1 depletion inhibited the growth, migration, invasion of prostate cancer cells and reduced the tumorigenicity of prostate cancer cells in vivo. Our results firmly established CCAR1 as an AR co-activator that plays a key role in AR transcription complex assembly and has an important physiological role in androgen signaling and prostate tumorigenesis. PMID:23887938

  4. Multiple splice variants of the pituitary adenylate cyclase-activating polypeptide type 1 receptor detected by RT-PCR in single rat pituitary cells.

    PubMed

    Bresson-Bépoldin, L; Jacquot, M C; Schlegel, W; Rawlings, S R

    1998-10-01

    Alternative splicing of the rat type 1 pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PVR1) produces variants that couple either to both adenylyl cyclase (AC) and phospholipase C (PLC) (PVR1 short, PVR1 hop, PVR1 hiphop), or to AC alone (PVR1 hip). We have previously shown that populations of clonal alphaT3-1 gonadotrophs express PVR1 hop and PVR1 short mRNAs, whereas clonal GH4C1 somatotrophs do not. Here we have used the single cell RT-PCR technique to investigate whether normal rat gonadotrophs and somatotrophs express PVR1 mRNA, whether a single cell co-expresses multiple splice variant forms, and whether differential PVR1 mRNA expression correlates with differences in PACAP-stimulated Ca2+ signalling. We found that individual rat gonadotrophs expressed mRNA either for PVR1 hop, for PVR1 short, or co-expressed the two forms. Although we found no differences between the splice variant(s) expressed and the characteristics of PACAP-stimulated Ca2+ responses, the expression of PVR1 mRNA is consistent with the known PACAP stimulation of the PLC system in gonadotrophs. Individual rat somatotrophs also expressed PVR1 hop or PVR1 short (but not PVR1 hip) mRNAs although these forms were never co-expressed. The expression of PVR1 mRNA in somatotrophs can explain in part the activation by PACAP of the AC system in such cells. In conclusion, the single cell RT-PCR technique was used to demonstrate expression of multiple PVR1 splice variants in single identified pituitary cells. These findings open up important questions on the role of alternative splicing in cell biology. PMID:9801454

  5. 40Ar/39Ar ages of the AD 79 eruption of Vesuvius, Italy

    USGS Publications Warehouse

    Lanphere, M.; Champion, D.; Melluso, L.; Morra, V.; Perrotta, A.; Scarpati, C.; Tedesco, D.; Calvert, A.

    2007-01-01

    The Italian volcano, Vesuvius, erupted explosively in AD 79. Sanidine from pumice collected at Casti Amanti in Pompeii and Villa Poppea in Oplontis yielded a weighted-mean 40Ar/39Ar age of 1925??66 years in 2004 (1?? uncertainty) from incremental-heating experiments of eight aliquants of sanidine. This is the calendar age of the eruption. Our results together with the work of Renne et al. (1997) and Renne and Min (1998) demonstrate the validity of the 40Ar/39Ar method to reconstruct the recent eruptive history of young, active volcanoes. ?? Springer-Verlag 2006.

  6. Constitutive Activity of the Androgen Receptor

    PubMed Central

    Chan, Siu Chiu; Dehm, Scott M.

    2014-01-01

    Prostate cancer (PCa) is the most frequently diagnosed cancer in the United States. The androgen receptor (AR) signaling axis is central to all stages of PCa pathophysiology and serves as the main target for endocrine-based therapy. The most advanced stage of the disease, castration resistant prostate cancer (CRPC), is presently incurable and accounts for most PCa mortality. In this review, we highlight the mechanisms by which the AR signaling axis can bypass endocrine-targeted therapies and drive progression of CRPC. These mechanisms include alterations in growth factor, cytokine, and inflammatory signaling pathways, altered expression or activity of transcriptional co-regulators, AR point mutations, and AR gene amplification leading to AR protein overexpression. Additionally, we will discuss the mechanisms underlying the synthesis of constitutively active AR splice variants (AR-Vs) lacking the COOH-terminal ligand binding domain, as well as the role and regulation of AR-Vs in supporting therapeutic resistance in CRPC. Finally, we summarize the ongoing development of inhibitors targeting discrete AR functional domains as well as the status of new biomarkers for monitoring the AR signaling axis in patients. PMID:24931201

  7. n-back task performance and corresponding brain-activation patterns in women with restrictive and bulimic eating-disorder variants: preliminary findings.

    PubMed

    Israel, Mimi; Klein, Michael; Pruessner, Jens; Thaler, Lea; Spilka, Michael; Efanov, Simona; Ouellette, Anne-Sophie; Berlim, Marcelo; Ali, Nida; Beaudry, Thomas; Van den Eynde, Frederique; Walker, Claire-Dominique; Steiger, Howard

    2015-04-30

    Eating disorder (ED) variants characterized by "binge-eating/purging" symptoms differ from "restricting-only" variants along diverse clinical dimensions, but few studies have compared people with these different eating-disorder phenotypes on measures of neurocognitive function and brain activation. We tested the performances of 19 women with "restricting-only" eating syndromes and 27 with "binge-eating/purging" variants on a modified n-back task, and used functional magnetic resonance imaging (fMRI) to examine task-induced brain activations in frontal regions of interest. When compared with "binge-eating/purging" participants, "restricting-only" participants showed superior performance. Furthermore, in an intermediate-demand condition, "binge-eating/purging" participants showed significantly less event-related activation than did "restricting-only" participants in a right posterior prefrontal region spanning Brodmann areas 6-8-a region that has been linked to planning of motor responses, working memory for sequential information, and management of uncertainty. Our findings suggest that working memory is poorer in eating-disordered individuals with binge-eating/purging behaviors than in those who solely restrict food intake, and that observed performance differences coincide with interpretable group-based activation differences in a frontal region thought to subserve planning and decision making. PMID:25707581

  8. Stability of the Aryl hydrocarbon Receptor and its Regulated Genes in the Low activity Variant of Hepa-1 cell line

    PubMed Central

    Humphrey-Johnson, Andria; Abukalam, Rawia; Eltom, Sakina E.

    2015-01-01

    We examined the expression kinetics of some of the aryl hydrocarbon receptor (AhR)-regulated genes in LA1 variant cells compared to wild type (WT) Hepa-1 mouse hepatoma cell lines, and we investigated the stability of AhR protein as a key step in the function of this receptor. Treatment of both cell types with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in increased CYP1A1 and CYP1B1 mRNA with a subsequent down regulation of AhR. We show here that co-treatment with transcription inhibitor actinomycin D (ActD) has reversed the TCDD-induced depletion of AhR protein in WT. However, the proteolytic degradation of AhR in absence of TCDD was significantly higher in LA1 cells than in WT, and ActD treatment reduced this loss. Induction of CYP1A1 and CYP1B1 mRNA by TCDD in WT cells each exhibited bursts of activity in the initial hour which were about 3-fold greater than in LAI cells. The induced mRNA levels in LA1 exhibited a slow and sustained increase approximating the WT levels by 20 h. The induction of two other AhR-regulated genes also showed comparable turnover differences between the two cell types. Thus, altered regulation of the AhR responsive genes in LA1 may result from a difference in AhR stability. PMID:25637755

  9. Stability of the aryl hydrocarbon receptor and its regulated genes in the low activity variant of Hepa-1 cell line.

    PubMed

    Humphrey-Johnson, Andria; Abukalam, Rawia; Eltom, Sakina E

    2015-03-01

    We examined the expression kinetics of some of the aryl hydrocarbon receptor (AhR)-regulated genes in LA1 variant cells compared to wild type (WT) Hepa-1 mouse hepatoma cell lines, and we investigated the stability of AhR protein as a key step in the function of this receptor. Treatment of both cell types with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in increased CYP1A1 and CYP1B1 mRNA with a subsequent down regulation of AhR. We show here that co-treatment with transcription inhibitor actinomycin D (ActD) has reversed the TCDD-induced depletion of AhR protein in WT. However, the proteolytic degradation of AhR in absence of TCDD was significantly higher in LA1 cells than in WT, and ActD treatment reduced this loss. Induction of CYP1A1 and CYP1B1 mRNA by TCDD in WT cells each exhibited bursts of activity in the initial hour which were about 3-fold greater than in LAI cells. The induced mRNA levels in LA1 exhibited a slow and sustained increase approximating the WT levels by 20h. The induction of two other AhR-regulated genes also showed comparable turnover differences between the two types of cell. Thus, altered regulation of the AhR responsive genes in LA1 may result from a difference in AhR stability. PMID:25637755

  10. Receptor activity-modifying protein-dependent impairment of calcitonin receptor splice variant Δ(1–47)hCT(a) function

    PubMed Central

    Qi, T; Dong, M; Watkins, HA; Wootten, D; Miller, LJ; Hay, DL

    2013-01-01

    Background and Purpose Alternative splicing expands proteome diversity to GPCRs. Distinct receptor variants have been identified for a secretin family GPCR, the calcitonin receptor (CTR). The possible functional contributions of these receptor variants are further altered by their potential interactions with receptor activity-modifying proteins (RAMPs). One variant of the human CTR lacks the first 47 residues at its N terminus [Δ(1–47)hCT(a)]. However, very little is known about the pharmacology of this variant or its ability to interact with RAMPs to form amylin receptors. Experimental Approach Δ(1–47)hCT(a) was characterized both with and without RAMPs in Cos7 and/or HEK293S cells. The receptor expression (ELISA assays) and function (cAMP and pERK1/2 assays) for up to six agonists and two antagonists were determined. Key Results Despite lacking 47 residues at the N terminus, Δ(1–47)hCT(a) was still able to express at the cell surface, but displayed a generalized reduction in peptide potency. Δ(1–47)hCT(a) retained its ability to interact with RAMP1 and formed a functional amylin receptor; this also appeared to be the case with RAMP3. On the other hand, its interaction with RAMP2 and resultant amylin receptor was reduced to a greater extent. Conclusions and Implications Δ(1–47)hCT(a) acts as a functional receptor at the cell surface. It exhibits altered receptor function, depending on whether it associates with a RAMP and which RAMP it interacts with. Therefore, the presence of this variant in tissues will potentially contribute to altered peptide binding and signalling, depending on the RAMP distribution in tissues. PMID:22946511

  11. Structures of Mycobacterium tuberculosis Anthranilate Phosphoribosyltransferase Variants Reveal the Conformational Changes That Facilitate Delivery of the Substrate to the Active Site.

    PubMed

    Cookson, Tammie V M; Evans, Genevieve L; Castell, Alina; Baker, Edward N; Lott, J Shaun; Parker, Emily J

    2015-10-01

    Anthranilate phosphoribosyltransferase (AnPRT) is essential for the biosynthesis of tryptophan in Mycobacterium tuberculosis (Mtb). This enzyme catalyzes the second committed step in tryptophan biosynthesis, the Mg²⁺-dependent reaction between 5'-phosphoribosyl-1'-pyrophosphate (PRPP) and anthranilate. The roles of residues predicted to be involved in anthranilate binding have been tested by the analysis of six Mtb-AnPRT variant proteins. Kinetic analysis showed that five of six variants were active and identified the conserved residue R193 as being crucial for both anthranilate binding and catalytic function. Crystal structures of these Mtb-AnPRT variants reveal the ability of anthranilate to bind in three sites along an extended anthranilate tunnel and expose the role of the mobile β2-α6 loop in facilitating the enzyme's sequential reaction mechanism. The β2-α6 loop moves sequentially between a "folded" conformation, partially occluding the anthranilate tunnel, via an "open" position to a "closed" conformation, which supports PRPP binding and allows anthranilate access via the tunnel to the active site. The return of the β2-α6 loop to the "folded" conformation completes the catalytic cycle, concordantly allowing the active site to eject the product PRA and rebind anthranilate at the opening of the anthranilate tunnel for subsequent reactions. Multiple anthranilate molecules blocking the anthranilate tunnel prevent the β2-α6 loop from undergoing the conformational changes required for catalysis, thus accounting for the unusual substrate inhibition of this enzyme. PMID:26356348

  12. Identification of excess 40Ar by the 40Ar 39Ar, age spectrum technique

    USGS Publications Warehouse

    Lanphere, M.A.; Brent, Dalrymple G.

    1976-01-01

    40Ar 39Ar incremental heating experiments on igneous plagioclase, biotite, and pyroxene that contain known amounts of excess 40Ar indicate that saddle-shaped age spectra are diagnostic of excess 40Ar in igneous minerals as well as in igneous rocks. The minima in the age spectra approach but do not reach the crystallization age. Neither the age spectrum diagram nor the 40Ar 36Ar versus 39Ar 36Ar isochron diagram reliably reveal the crystallization age in such samples. ?? 1976.

  13. Splice Variants of the Dual Specificity Tyrosine Phosphorylation-regulated Kinase 4 (DYRK4) Differ in Their Subcellular Localization and Catalytic Activity*

    PubMed Central

    Papadopoulos, Chrisovalantis; Arato, Krisztina; Lilienthal, Eva; Zerweck, Johannes; Schutkowski, Mike; Chatain, Nicolas; Müller-Newen, Gerhard; Becker, Walter; de la Luna, Susana

    2011-01-01

    Dual specificity tyrosine phosphorylation-regulated kinases, DYRKs, are a family of conserved protein kinases that play key roles in the regulation of cell differentiation, proliferation, and survival. Of the five mammalian DYRKs, DYRK4 is the least studied family member. Here, we show that several splice variants of DYRK4 are expressed in tissue-specific patterns and that these variants have distinct functional capacities. One of these variants contains a nuclear localization signal in its extended N terminus that mediates its interaction with importin α3 and α5 and that is capable of targeting a heterologous protein to the nucleus. Consequently, the nucleocytoplasmic mobility of this variant differs from that of a shorter isoform in live cell imaging experiments. Other splicing events affect the catalytic domain, including a three-amino acid deletion within subdomain XI that markedly reduces the enzymatic activity of DYRK4. We also show that autophosphorylation of a tyrosine residue within the activation loop is necessary for full DYRK4 kinase activity, a defining feature of the DYRK family. Finally, by comparing the phosphorylation of an array of 720 peptides, we show that DYRK1A, DYRK2, and DYRK4 differ in their target recognition sequence and that preference for an arginine residue at position P −3 is a feature of DYRK1A but not of DYRK2 and DYRK4. Therefore, we highlight the use of subcellular localization as an important regulatory mechanism for DYRK proteins, and we propose that substrate specificity could be a source of functional diversity among DYRKs. PMID:21127067

  14. IL-7 splicing variant IL-7{delta}5 induces human breast cancer cell proliferation via activation of PI3K/Akt pathway

    SciTech Connect

    Pan, Deshun; Liu, Bing; Jin, Xiaobao; Zhu, Jiayong

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer This study confirms the role of IL-7{delta}5 in breast cancer cell proliferation. Black-Right-Pointing-Pointer IL-7{delta}5 promotes breast cancer cell proliferation and cell cycle progression. Black-Right-Pointing-Pointer IL-7{delta}5 promotes cell proliferation via activation of PI3K/Akt pathway. -- Abstract: Various tumor cells express interleukin 7 (IL-7) and IL-7 variants. IL-7 has been confirmed to stimulate solid tumor cell proliferation. However, the effect of IL-7 variants on tumor cell proliferation remains unclear. In this study, we evaluated the role of IL-7{delta}5 (an IL-7 variant lacking exon 5) on proliferation and cell cycle progression of human MDA-MB-231 and MCF-7 breast cancer cells. The results showed that IL-7{delta}5 promoted cell proliferation and cell cycle progression from G1 phase to G2/M phase, associated with upregulation of cyclin D1 expression and the downregulation of p27{sup kip1} expression. Mechanistically, we found that IL-7{delta}5 induced the activation of Akt. Inhibition of PI3K/Akt pathway by LY294002 reversed the proliferation and cell cycle progression of MDA-MB-231 and MCF-7 cells induced by IL-7{delta}5. In conclusion, our findings demonstrate that IL-7{delta}5 variant induces human breast cancer cell proliferation and cell cycle progression via activation of PI3K/Akt pathway. Thus, IL-7{delta}5 may be a potential target for human breast cancer therapeutics intervention.

  15. Characterization of human paraoxonase 1 variants suggest that His residues at 115 and 134 positions are not always needed for the lactonase/arylesterase activities of the enzyme

    PubMed Central

    Bajaj, Priyanka; Tripathy, Rajan K; Aggarwal, Geetika; Pande, Abhay H

    2013-01-01

    Human paraoxonase 1 (h-PON1) hydrolyzes variety of substrates and the hydrolytic activities of enzyme can be broadly grouped into three categories; arylesterase, phosphotriesterase, and lactonase. Current models of the catalytic mechanism of h-PON1 suggest that catalytic residues H115 and H134 mediate the lactonase and arylesterase activities of the enzyme. H-PON1 is a strong candidate for the development of catalytic bioscavenger for organophosphate poisoning in humans. Recently, Gupta et al. (Nat. Chem. Biol. 2011. 7, 120) identified amino acid substitutions that significantly increased the activity of chimeric-PON1 variant (4E9) against some organophosphate nerve agents. In this study we have examined the effect of these (L69G/S111T/H115W/H134R/R192K/F222S/T332S) and other substitutions (H115W/H134R and H115W/H134R/R192K) on the hydrolytic activities of recombinant h-PON1 (rh-PON1) variants. Our results show that the substitutions resulted in a significant increase in the organophosphatase activity of all the three variants of rh-PON1 enzyme while had a variable effect on the lactonase/arylesterase activities. The results suggest that H residues at positions 115 and 134 are not always needed for the lactonase/arylesterase activities of h-PON1 and force a reconsideration of the current model(s) of the catalytic mechanism of h-PON1. PMID:24123308

  16. 40Ar/39Ar geochronological constraints on the formation of the Dayingezhuang gold deposit: New implications for timing and duration of hydrothermal activity in the Jiaodong gold province, China

    USGS Publications Warehouse

    Yang, Li-Qiang; Deng, J.; Goldfarb, Richard J.; Zhang, Jiahua; Gao, Bang-Fei; Wang, Zhong-Liang

    2014-01-01

    China's largest gold resource is located in the highly endowed northwestern part of the Jiaodong gold province. Most gold deposits in this area are associated with the NE- to NNE-trending shear zones on the margins of the 130–126 Ma Guojialing granite. These deposits collectively formed at ca. 120 ± 5 Ma during rapid uplift of the granite. The Dayingezhuang deposit is a large (> 120 t Au) orogenic gold deposit in the same area, but located along the eastern margin of the Late Jurassic Linglong Metamorphic Core Complex. New 40Ar/39Ar geochronology on hydrothermal sericite and muscovite from the Dayingezhuang deposit indicate the gold event is related to evolution of the core complex at 130 ± 4 Ma and is the earliest important gold event that is well-documented in the province. The Dayingezhuang deposit occurs along the Linglong detachment fault, which defines the eastern edge of the ca. 160–150 Ma Linglong granite–granodiorite massif. The anatectic rocks of the massif were rapidly uplifted, at rates of at least 1 km/m.y. from depths of 25–30 km, to form the metamorphic core complex. The detachment fault, with Precambrian metamorphic basement rocks in the hangingwall and the Linglong granitoids and migmatites in the footwall, is characterized by early mylonitization and a local brittle overprinting in the footwall. Gold is associated with quartz–sericite–pyrite–K-feldspar altered footwall cataclasites at the southernmost area of the brittle deformation along the detachment fault. Our results indicate that there were two successive, yet distinct gold-forming tectonic episodes in northwestern Jiaodong. One event first reactivated the detachment fault along the edge of the Linglong massif between 134 and 126 Ma, and then a second reactivated the shears along the margins of the Guojialing granite. Both events may relate to a component of northwest compression after a middle Early Cretaceous shift from regional NW–SE extension to a NE

  17. A Newly Emerged Swine-Origin Influenza A(H3N2) Variant Dampens Host Antiviral Immunity but Induces Potent Inflammasome Activation.

    PubMed

    Cao, Weiping; Mishina, Margarita; Ranjan, Priya; De La Cruz, Juan A; Kim, Jin Hyang; Garten, Rebecca; Kumar, Amrita; García-Sastre, Adolfo; Katz, Jacqueline M; Gangappa, Shivaprakash; Sambhara, Suryaprakash

    2015-12-15

    We compared the innate immune response to a newly emerged swine-origin influenza A(H3N2) variant containing the M gene from 2009 pandemic influenza A(H1N1), termed "A(H3N2)vpM," to the immune responses to the 2010 swine-origin influenza A(H3N2) variant and seasonal influenza A(H3N2). Our results demonstrated that A(H3N2)vpM-induced myeloid dendritic cells secreted significantly lower levels of type I interferon (IFN) but produced significantly higher levels of proinflammatory cytokines and induced potent inflammasome activation. The reduction in antiviral immunity with increased inflammatory responses upon A(H3N2)vpM infection suggest that these viruses have the potential for increased disease severity in susceptible hosts. PMID:26068782

  18. Friedreich's Ataxia Variants I154F and W155R Diminish Frataxin-Based Activation of the Iron-Sulfur Cluster Assembly Complex

    SciTech Connect

    Tsai, Chi-Lin; Bridwell-Rabb, Jennifer; Barondeau, David P

    2011-11-07

    Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease that has been linked to defects in the protein frataxin (Fxn). Most FRDA patients have a GAA expansion in the first intron of their Fxn gene that decreases protein expression. Some FRDA patients have a GAA expansion on one allele and a missense mutation on the other allele. Few functional details are known for the ~15 different missense mutations identified in FRDA patients. Here in vitro evidence is presented that indicates the FRDA I154F and W155R variants bind more weakly to the complex of Nfs1, Isd11, and Isu2 and thereby are defective in forming the four-component SDUF complex that constitutes the core of the Fe-S cluster assembly machine. The binding affinities follow the trend Fxn ~ I154F > W155F > W155A ~ W155R. The Fxn variants also have diminished ability to function as part of the SDUF complex to stimulate the cysteine desulfurase reaction and facilitate Fe-S cluster assembly. Four crystal structures, including the first for a FRDA variant, reveal specific rearrangements associated with the loss of function and lead to a model for Fxn-based activation of the Fe-S cluster assembly complex. Importantly, the weaker binding and lower activity for FRDA variants correlate with the severity of disease progression. Together, these results suggest that Fxn facilitates sulfur transfer from Nfs1 to Isu2 and that these in vitro assays are sensitive and appropriate for deciphering functional defects and mechanistic details for human Fe-S cluster biosynthesis.

  19. Superdeformation of Ar hypernuclei

    NASA Astrophysics Data System (ADS)

    Isaka, Masahiro; Kimura, Masaaki; Hiyama, Emiko; Sagawa, Hiroyuki

    2015-10-01

    We investigate the differences in the Λ separation energies (S_Λ ) of the ground and superdeformed (SD) states in {}^{37}_Λ Ar, ^{39}_Λ Ar, and ^{41}_Λ Ar within the framework of antisymmetrized molecular dynamics (AMD). In this study, we find that the calculated S_Λ values in the SD states are much smaller than those in the ground states, unlike the result using the relativistic mean-field (RMF) calculation [B.-N. Lu et al., Phys. Rev. C, 89, 044307 (2014)]. One of the reasons for this difference between the present work and the RMF calculation is the difference in the density profile of the SD states in the core nuclei. We also find that the property of the Λ N odd-parity interaction affects the S_Λ trend between the ground and SD states.

  20. Crosstalk between the HpArsRS two-component system and HpNikR is necessary for maximal activation of urease transcription.

    PubMed

    Carpenter, Beth M; West, Abby L; Gancz, Hanan; Servetas, Stephanie L; Pich, Oscar Q; Gilbreath, Jeremy J; Hallinger, Daniel R; Forsyth, Mark H; Merrell, D Scott; Michel, Sarah L J

    2015-01-01

    Helicobacter pylori NikR (HpNikR) is a nickel dependent transcription factor that directly regulates a number of genes in this important gastric pathogen. One key gene that is regulated by HpNikR is ureA, which encodes for the urease enzyme. In vitro DNA binding studies of HpNikR with the ureA promoter (PureA ) previously identified a recognition site that is required for high affinity protein/DNA binding. As a means to determine the in vivo significance of this recognition site and to identify the key DNA sequence determinants required for ureA transcription, herein, we have translated these in vitro results to analysis directly within H. pylori. Using a series of GFP reporter constructs in which the PureA DNA target was altered, in combination with mutant H. pylori strains deficient in key regulatory proteins, we confirmed the importance of the previously identified HpNikR recognition sequence for HpNikR-dependent ureA transcription. Moreover, we identified a second factor, the HpArsRS two-component system that was required for maximum transcription of ureA. While HpArsRS is known to regulate ureA in response to acid shock, it was previously thought to function independently of HpNikR and to have no role at neutral pH. However, our qPCR analysis of ureA expression in wildtype, ΔnikR and ΔarsS single mutants as well as a ΔarsS/nikR double mutant strain background showed reduced basal level expression of ureA when arsS was absent. Additionally, we determined that both HpNikR and HpArsRS were necessary for maximal expression of ureA under nickel, low pH and combined nickel and low pH stresses. In vitro studies of HpArsR-P with the PureA DNA target using florescence anisotropy confirmed a direct protein/DNA binding interaction. Together, these data support a model in which HpArsRS and HpNikR cooperatively interact to regulate ureA transcription under various environmental conditions. This is the first time that direct "cross-talk" between HpArsRS and HpNikR at

  1. Crosstalk between the HpArsRS two-component system and HpNikR is necessary for maximal activation of urease transcription

    PubMed Central

    Carpenter, Beth M.; West, Abby L.; Gancz, Hanan; Servetas, Stephanie L.; Pich, Oscar Q.; Gilbreath, Jeremy J.; Hallinger, Daniel R.; Forsyth, Mark H.; Merrell, D. Scott; Michel, Sarah L. J.

    2015-01-01

    Helicobacter pylori NikR (HpNikR) is a nickel dependent transcription factor that directly regulates a number of genes in this important gastric pathogen. One key gene that is regulated by HpNikR is ureA, which encodes for the urease enzyme. In vitro DNA binding studies of HpNikR with the ureA promoter (PureA) previously identified a recognition site that is required for high affinity protein/DNA binding. As a means to determine the in vivo significance of this recognition site and to identify the key DNA sequence determinants required for ureA transcription, herein, we have translated these in vitro results to analysis directly within H. pylori. Using a series of GFP reporter constructs in which the PureA DNA target was altered, in combination with mutant H. pylori strains deficient in key regulatory proteins, we confirmed the importance of the previously identified HpNikR recognition sequence for HpNikR-dependent ureA transcription. Moreover, we identified a second factor, the HpArsRS two-component system that was required for maximum transcription of ureA. While HpArsRS is known to regulate ureA in response to acid shock, it was previously thought to function independently of HpNikR and to have no role at neutral pH. However, our qPCR analysis of ureA expression in wildtype, ΔnikR and ΔarsS single mutants as well as a ΔarsS/nikR double mutant strain background showed reduced basal level expression of ureA when arsS was absent. Additionally, we determined that both HpNikR and HpArsRS were necessary for maximal expression of ureA under nickel, low pH and combined nickel and low pH stresses. In vitro studies of HpArsR-P with the PureA DNA target using florescence anisotropy confirmed a direct protein/DNA binding interaction. Together, these data support a model in which HpArsRS and HpNikR cooperatively interact to regulate ureA transcription under various environmental conditions. This is the first time that direct “cross-talk” between HpArsRS and HpNikR at

  2. Association of Common Genetic Variants in Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 with Type 2 Diabetes Mellitus in a Chinese Han Population

    PubMed Central

    Li, Ting-Ting; Qiao, Hong; Tong, Hui-Xin; Zhuang, Tian-Wei; Wang, Tong-Tong

    2016-01-01

    Background: A study has identified several novel susceptibility variants of the mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) gene for type 2 diabetes mellitus (T2DM) within the German population. Among the variants, five single nucleotide polymorphisms (SNPs) of MAP4K4 (rs1003376, rs11674694, rs2236935, rs2236936, and rs6543087) showed significant association with T2DM or diabetes-related quantitative traits. We aimed to evaluate whether common SNPs in the MAP4K4 gene were associated with T2DM in the Chinese population. Methods: Five candidate SNPs were genotyped in 996 patients newly diagnosed with T2DM and in 976 control subjects, using the SNPscan™ method. All subjects were recruited from the Second Affiliated Hospital, Harbin Medical University from October 2010 to September 2013. We evaluated the T2DM risk conferred by individual SNPs and haplotypes using logistic analysis, and the association between the five SNPs and metabolic traits in the subgroups. Results: Of the five variants, SNP rs2236935T/C was significantly associated with T2DM in this study population (odds ratio = 1.293; 95% confidence interval: 1.034–1.619, P = 0.025). In addition, among the controls, rs1003376 was significantly associated with an increased body mass index (P = 0.045) and homeostatic model assessment-insulin resistance (P = 0.037). Conclusions: MAP4K4 gene is associated with T2DM in a Chinese Han population, and MAP4K4 gene variants may contribute to the risk toward the development of T2DM. PMID:27174326

  3. MEIS1 functions as a potential AR negative regulator

    SciTech Connect

    Cui, Liang; Yang, Yutao; Hang, Xingyi; Cui, Jiajun; Gao, Jiangping

    2014-10-15

    The androgen receptor (AR) plays critical roles in human prostate carcinoma progression and transformation. However, the activation of AR is regulated by co-regulators. MEIS1 protein, the homeodomain transcription factor, exhibited a decreased level in poor-prognosis prostate tumors. In this study, we investigated a potential interaction between MEIS1 and AR. We found that overexpression of MEIS1 inhibited the AR transcriptional activity and reduced the expression of AR target gene. A potential protein–protein interaction between AR and MEIS1 was identified by the immunoprecipitation and GST pull-down assays. Furthermore, MEIS1 modulated AR cytoplasm/nucleus translocation and the recruitment to androgen response element in prostate specific antigen (PSA) gene promoter sequences. In addition, MEIS1 promoted the recruitment of NCoR and SMRT in the presence of R1881. Finally, MEIS1 inhibited the proliferation and anchor-independent growth of LNCaP cells. Taken together, our data suggests that MEIS1 functions as a novel AR co-repressor. - Highlights: • A potential interaction was identified between MEIS1 and AR signaling. • Overexpression of MEIS1 reduced the expression of AR target gene. • MEIS1 modulated AR cytoplasm/nucleus translocation. • MEIS1 inhibited the proliferation and anchor-independent growth of LNCaP cells.

  4. ARS Culture Collection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The internationally recognized Agricultural Research Service (ARS) Culture Collection will be described to include the microorganisms maintained by the collection, preservation methods and worldwide distribution of cultures. The impact of the germplasm will be described to include discovery of the f...

  5. Variants of beta-glucosidase

    SciTech Connect

    Fidantsef, Ana; Lamsa, Michael; Gorre-Clancy, Brian

    2015-07-14

    The present invention relates to variants of a parent beta-glucosidase, comprising a substitution at one or more positions corresponding to positions 142, 183, 266, and 703 of amino acids 1 to 842 of SEQ ID NO: 2 or corresponding to positions 142, 183, 266, and 705 of amino acids 1 to 844 of SEQ ID NO: 70, wherein the variant has beta-glucosidase activity. The present invention also relates to nucleotide sequences encoding the variant beta-glucosidases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  6. Variants of beta-glucosidases

    SciTech Connect

    Fidantsef, Ana; Lamsa, Michael; Gorre-Clancy, Brian

    2014-10-07

    The present invention relates to variants of a parent beta-glucosidase, comprising a substitution at one or more positions corresponding to positions 142, 183, 266, and 703 of amino acids 1 to 842 of SEQ ID NO: 2 or corresponding to positions 142, 183, 266, and 705 of amino acids 1 to 844 of SEQ ID NO: 70, wherein the variant has beta-glucosidase activity. The present invention also relates to nucleotide sequences encoding the variant beta-glucosidases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  7. Variants of beta-glucosidases

    DOEpatents

    Fidantsef, Ana; Lamsa, Michael; Clancy, Brian Gorre

    2008-08-19

    The present invention relates to variants of a parent beta-glucosidase, comprising a substitution at one or more positions corresponding to positions 142, 183, 266, and 703 of amino acids 1 to 842 of SEQ ID NO: 2 or corresponding to positions 142, 183, 266, and 705 of amino acids 1 to 844 of SEQ ID NO: 70, wherein the variant has beta-glucosidase activity. The present invention also relates to nucleotide sequences encoding the variant beta-glucosidases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  8. Variants of beta-glucosidase

    DOEpatents

    Fidantsef, Ana; Lamsa, Michael; Gorre-Clancy, Brian

    2009-12-29

    The present invention relates to variants of a parent beta-glucosidase, comprising a substitution at one or more positions corresponding to positions 142, 183, 266, and 703 of amino acids 1 to 842 of SEQ ID NO: 2 or corresponding to positions 142, 183, 266, and 705 of amino acids 1 to 844 of SEQ ID NO: 70, wherein the variant has beta-glucosidase activity. The present invention also relates to nucleotide sequences encoding the variant beta-glucosidases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  9. The Histone Variant MacroH2A1.2 is Necessary for the Activation of Muscle Enhancers and Recruitment of the Transcription Factor Pbx1

    PubMed Central

    Dell’Orso, Stefania; Wang, A. Hongjun; Shih, Han-Yu; Saso, Kayoko; Berghella, Libera; Gutierrez-Cruz, Gustavo; Ladurner, Andreas G.; O’Shea, John J.; Sartorelli, Vittorio; Zare, Hossein

    2016-01-01

    SUMMARY Histone variants complement and integrate histone post-translational modifications in regulating transcription. The histone variant macroH2A1 (mH2A1) is almost three times the size of its canonical H2A counterpart due to the presence of a ~25kDa evolutionarily conserved non-histone macro domain. Strikingly, mH2A1 can mediate both gene repression and activation. However, the molecular determinants conferring these alternative functions remain elusive. Here, we report that mH2A1.2 is required for the activation of the myogenic gene regulatory network and muscle cell differentiation. H3K27 acetylation at prospective enhancers is exquisitely sensitive to mH2A1.2, indicating a role of mH2A1.2 in imparting enhancer activation. Both H3K27 acetylation and recruitment of the transcription factor Pbx1 at prospective enhancers are regulated by mH2A1.2. Overall, our findings indicate a role of mH2A1.2 in marking regulatory regions for activation. PMID:26832413

  10. A liver X receptor (LXR)-{beta} alternative splicing variant (LXRBSV) acts as an RNA co-activator of LXR-{beta}

    SciTech Connect

    Hashimoto, Koshi; Ishida, Emi; Matsumoto, Shunichi; Shibusawa, Nobuyuki; Okada, Shuichi; Monden, Tsuyoshi; Satoh, Tetsurou; Yamada, Masanobu; Mori, Masatomo

    2009-12-25

    We report the isolation and functional characterization of a novel transcriptional co-activator, termed LXRBSV. LXRBSV is an alternative splicing variant of liver X receptor (LXR)-{beta} LXRBSV has an intronic sequence between exons 2 and 3 in the mouse LXR-{beta} gene. The LXRBSV gene is expressed in various tissues including the liver and brain. We sub-cloned LXRBSV into pSG5, a mammalian expression vector, and LXRBSV in pSG5 augmented human Sterol Response Element Binding Protein (SREBP)-1c promoter activity in HepG2 cells in a ligand (TO901317) dependent manner. The transactivation mediated by LXRBSV is selective for LXR-{beta}. The LXRBSV protein was deduced to be 64 amino acids in length; however, a GAL4-LXRBSV fusion protein was not able to induce transactivation. Serial deletion constructs of LXRBSV demonstrated that the intronic sequence inserted in LXRBSV is required for its transactivation activity. An ATG mutant of LXRBSV was able to induce transactivation as wild type. Furthermore, LXRBSV functions in the presence of cycloheximide. Taken together, we have concluded that LXRBSV acts as an RNA transcript not as a protein. In the current study, we have demonstrated for the first time that an alternative splicing variant of a nuclear receptor acts as an RNA co-activator.

  11. Activity and protein kinase C regulate synaptic accumulation of N-methyl-D-aspartate (NMDA) receptors independently of GluN1 splice variant.

    PubMed

    Ferreira, Joana S; Rooyakkers, Amanda; She, Kevin; Ribeiro, Luis; Carvalho, Ana Luísa; Craig, Ann Marie

    2011-08-12

    NMDA receptors are calcium-permeable ionotropic receptors that detect coincident glutamate binding and membrane depolarization and are essential for many forms of synaptic plasticity in the mammalian brain. The obligatory GluN1 subunit of NMDA receptors is alternatively spliced at multiple sites, generating forms that vary in N-terminal N1 and C-terminal C1, C2, and C2' cassettes. Based on expression of GluN1 constructs in heterologous cells and in wild type neurons, the prevalent view is that the C-terminal cassettes regulate synaptic accumulation and its modulation by homeostatic activity blockade and by protein kinase C (PKC). Here, we tested the role of GluN1 splicing in regulated synaptic accumulation of NMDA receptors by lentiviral expression of individual GluN1 splice variants in hippocampal neurons cultured from GluN1 (-/-) mice. High efficiency transduction of GluN1 at levels similar to endogenous was achieved. Under control conditions, the C2' cassette mediated enhanced synaptic accumulation relative to the alternate C2 cassette, whereas the presence or absence of N1 or C1 had no effect. Surprisingly all GluN1 splice variants showed >2-fold increased synaptic accumulation with chronic blockade of NMDA receptor activity. Furthermore, in this neuronal rescue system, all GluN1 splice variants were equally rapidly dispersed upon activation of PKC. These results indicate that the major mechanisms mediating homeostatic synaptic accumulation and PKC dispersal of NMDA receptors occur independently of GluN1 splice isoform. PMID:21676872

  12. Sugar beet activities of the USDA-ARS East Lansing conducted in cooperation with Saginaw Valley Bean and Beet Farm during 2010

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cercospora leaf spot resistance evaluation plots were planted at the Saginaw Valley Research & Extension Center in Frankenmuth, MI in 2010. All trials were planted, following normal fall and spring tillage operations, with a USDA-ARS modified John Deere/Almaco research plot planter utilizing global ...

  13. Sugar beet activities of the USDA-ARS East Lansing conducted in cooperation with Saginaw Valley Bean and Beet Farm during 2011 (including Project 905)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Evaluation and rating plots were planted at the Saginaw Valley Research & Extension Center in Frankenmuth, MI in 2011 that focused on Cercospora leaf spot performance, conducted in conjunction with Beet Sugar Development Foundation and including USDA-ARS cooperators. 263 breeding lines were tested i...

  14. Sugar Beet Activities of the USDA-ARS East Lansing Conducted in Cooperation with Saginaw Valley Bean and Beet Farm During 2009

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two evaluation plots were planted at the Saginaw Valley Research & Extension Center in Frankenmuth, MI in 2009; one agronomic trial and one combined Cercospora evaluation trial. All trials were planted, following normal fall and spring tillage operations, with a USDA-ARS modified John Deere/Almaco ...

  15. A Novel New Delhi Metallo-β-Lactamase Variant, NDM-14, Isolated in a Chinese Hospital Possesses Increased Enzymatic Activity against Carbapenems

    PubMed Central

    Zou, Dayang; Huang, Yong; Zhao, Xiangna; Liu, Wei; Dong, Derong; Li, Huan; Wang, Xuesong; Huang, Simo; Wei, Xiao; Yan, Xiabei; Yang, Zhan; Tong, Yigang

    2015-01-01

    A novel New Delhi metallo-β-lactamase (NDM) variant, NDM-14, was identified in clinical isolate Acinetobacter lwoffii JN49-1, which was recovered from an intensive care unit patient at a local hospital in China. NDM-14, which differs from other existing enzymes by an amino acid substitution at position 130 (Asp130Gly), possesses enzymatic activity toward carbapenems that is greater than that of NDM-1. Kinetic data indicate that NDM-14 has a higher affinity for imipenem and meropenem. PMID:25645836

  16. AR3 messenger ribonucleic acid expression and its functional implication in human primary testicular failure.

    PubMed

    Tian, F; Wu, Y-S; Zhao, J; Li, W

    2014-10-01

    AR3, a major one of androgen receptor (AR) splice variants, has been shown to play a pivotal role in concert with AR signalling in prostate cancer. The present study was undertaken to characterise the expression pattern of AR3 in normal and impaired spermatogenesis. Expression of AR3 mRNA showed significantly lower level in testicular tissues with impaired spermatogenesis when compared to normal tissues. This aberrant expression profile of AR3 in human pathological testes was further confirmed by immunoblotting analysis. Moreover, in situ hybridisation studies revealed that the transcripts of the gene were dominantly localised in the pachytene spermatocytes and round spermatids, suggesting a potential involvement of this transcriptional regulator in the auto-/paracrine regulation of meiotic and post-meiotic differentiation. This hypothesis was strengthened by the observation that AR3 mRNA expression was positively correlated to average seminiferous tubule score and was negatively correlated to serum FSH level. To the best of our knowledge, such a distinct expression profile of AR3 has not been reported previously in human testis. Overall, our data are suggestive of a novel site of action of AR3 during human spermatogenesis and should shed light on the complicated circuit composed of AR and its splice variants. PMID:24124902

  17. Indirect activation of the SV23 and SV24 splice variants of human constitutive androstane receptor: analysis with 3-hydroxyflavone and its analogues

    PubMed Central

    Lau, Aik Jiang; Chang, Thomas K H

    2013-01-01

    BACKGROUND AND PURPOSE Naturally occurring splice variants of human CAR (hCAR), including hCAR-SV23 (insertion of amino acids SPTV) and hCAR-SV24 (APYLT), have been shown to be expressed in liver. However, little is known regarding how hCAR-SV23 and hCAR-SV24 are activated. Therefore, we investigated the mode of activation of these hCAR splice variants. EXPERIMENTAL APPROACH Cell-based reporter gene assays, including ligand-binding domain transactivation assays and coactivator recruitment assays, were conducted on cultured HepG2 cells transfected with various constructs and treated with 3-hydroxyflavone or a hydroxylated (galangin, datiscetin, kaempferol, morin, quercetin or myricetin) or methylated (isorhamnetin, tamarixetin, or syringetin) analogue. KEY RESULTS Among the flavonols investigated, only 3-hydroxyflavone increased hCAR-SV23 and hCAR-SV24 activities. 3-Hydroxyflavone did not transactivate the ligand-binding domain of these isoforms or recruit steroid receptor coactivators (SRC-1, SRC-2, or SRC-3). By comparison, 3-hydroxyflavone, galangin, datiscetin, kaempferol, quercetin, isorhamnetin and tamarixetin activated hCAR-WT, whereas none of the flavonols activated hCAR-SV25 (both SPTV and APYLT insertions). The flavonols 3-Hydroxyflavone, galangin, quercetin and tamarixetin transactivated the ligand-binding domain of hCAR-WT, but only 3-hydroxyflavone recruited SRC-1, SRC-2 and SRC-3 to the receptor. CONCLUSION AND IMPLICATIONS hCAR-SV23 and hCAR-SV24 can be activated by a mechanism that does not involve the ligand-binding domain of the receptor or recruitment of SRC-1, SRC-2, or SRC-3. 3-Hydroxyflavone and its structural analogues activated hCAR in an isoform-selective and chemical-specific manner. Overall, our study provides insight into a novel mode of ligand activation of hCAR-SV23 and hCAR-SV24. PMID:23809009

  18. Ar-Ar ages and thermal histories of enstatite meteorites

    NASA Astrophysics Data System (ADS)

    Bogard, Donald D.; Dixon, Eleanor T.; Garrison, Daniel H.

    2010-05-01

    Compared with ordinary chondrites, there is a relative paucity of chronological and other data to define the early thermal histories of enstatite parent bodies. In this study, we report 39Ar-40Ar dating results for five EL chondrites: Khairpur, Pillistfer, Hvittis, Blithfield, and Forrest; five EH chondrites: Parsa, Saint Marks, Indarch, Bethune, and Reckling Peak 80259; three igneous-textured enstatite meteorites that represent impact melts on enstatite chondrite parent bodies: Zaklodzie, Queen Alexandra Range 97348, and Queen Alexandra Range 97289; and three aubrites, Norton County, Bishopville, and Cumberland Falls Several Ar-Ar age spectra show unusual 39Ar recoil effects, possibly the result of some of the K residing in unusual sulfide minerals, such as djerfisherite and rodderite, and other age spectra show 40Ar diffusion loss. Few additional Ar-Ar ages for enstatite meteorites are available in the literature. When all available Ar-Ar data on enstatite meteorites are considered, preferred ages of nine chondrites and one aubrite show a range of 4.50-4.54Ga, whereas five other meteorites show only lower age limits over 4.35-4.46Ga. Ar-Ar ages of several enstatite chondrites are as old or older as the oldest Ar-Ar ages of ordinary chondrites, which suggests that enstatite chondrites may have derived from somewhat smaller parent bodies, or were metamorphosed to lower temperatures compared to other chondrite types. Many enstatite meteorites are brecciated and/or shocked, and some of the younger Ar-Ar ages may record these impact events. Although impact heating of ordinary chondrites within the last 1Ga is relatively common for ordinary chondrites, only Bethune gives any significant evidence for such a young event.

  19. Redox-Active Sites in Auricularia auricula-judae Dye-Decolorizing Peroxidase and Several Directed Variants: A Multifrequency EPR Study.

    PubMed

    Baratto, Maria Camilla; Sinicropi, Adalgisa; Linde, Dolores; Sáez-Jiménez, Verónica; Sorace, Lorenzo; Ruiz-Duenas, Francisco J; Martinez, Angel T; Basosi, Riccardo; Pogni, Rebecca

    2015-10-29

    Peroxide-activated Auricularia auricula-judae dye-decolorizing peroxidase (DyP) forms a mixed Trp377 and Tyr337 radical, the former being responsible for oxidation of the typical DyP substrates (Linde et al. Biochem. J., 2015, 466, 253-262); however, a pure tryptophanyl radical EPR signal is detected at pH 7 (where the enzyme is inactive), in contrast with the mixed signal observed at pH for optimum activity, pH 3. On the contrary, the presence of a second tyrosine radical (at Tyr147) is deduced by a multifrequency EPR study of a variety of simple and double-directed variants (including substitution of the above and other tryptophan and tyrosine residues) at different freezing times after their activation by H2O2 (at pH 3). This points out that subsidiary long-range electron-transfer pathways enter into operation when the main pathway(s) is removed by directed mutagenesis, with catalytic efficiencies progressively decreasing. Finally, self-reduction of the Trp377 neutral radical is observed when reaction time (before freezing) is increased in the absence of reducing substrates (from 10 to 60 s). Interestingly, the tryptophanyl radical is stable in the Y147S/Y337S variant, indicating that these two tyrosine residues are involved in the self-reduction reaction. PMID:26120933

  20. Prevalence of Human-Active and Variant 1 Strains of the Tick-Borne Pathogen Anaplasma phagocytophilum in Hosts and Forests of Eastern North America

    PubMed Central

    Keesing, Felicia; McHenry, Diana J.; Hersh, Michelle; Tibbetts, Michael; Brunner, Jesse L.; Killilea, Mary; LoGiudice, Kathleen; Schmidt, Kenneth A.; Ostfeld, Richard S.

    2014-01-01

    Anaplasmosis is an emerging infectious disease caused by infection with the bacterium Anaplasma phagocytophilum. In the eastern United States, A. phagocytophilum is transmitted to hosts through the bite of the blacklegged tick, Ixodes scapularis. We determined the realized reservoir competence of 14 species of common vertebrate hosts for ticks by establishing the probability that each species transmits two important strains of A. phagocytophilum (A. phagocytophilum human-active, which causes human cases, and A. phagocytophilum variant 1, which does not) to feeding larval ticks. We also sampled questing nymphal ticks from ∼150 sites in a single county over 2 years and sampled over 6 years at one location. White-footed mice (Peromyscus leucopus) and Eastern chipmunks (Tamias striatus) were the most competent reservoirs for infection with the A. phagocytophilum human-active strain. Across the county, prevalence in ticks for both strains together was 8.3%; ticks were more than two times as likely to be infected with A. phagocytophilum human-active as A. phagocytophilum variant 1. PMID:24865688

  1. Crystal structures of two monomeric triosephosphate isomerase variants identified via a directed-evolution protocol selecting for L-arabinose isomerase activity.

    PubMed

    Krause, Mirja; Kiema, Tiila Riikka; Neubauer, Peter; Wierenga, Rik K

    2016-06-01

    The crystal structures are described of two variants of A-TIM: Ma18 (2.7 Å resolution) and Ma21 (1.55 Å resolution). A-TIM is a monomeric loop-deletion variant of triosephosphate isomerase (TIM) which has lost the TIM catalytic properties. Ma18 and Ma21 were identified after extensive directed-evolution selection experiments using an Escherichia coli L-arabinose isomerase knockout strain expressing a randomly mutated A-TIM gene. These variants facilitate better growth of the Escherichia coli selection strain in medium supplemented with 40 mM L-arabinose. Ma18 and Ma21 differ from A-TIM by four and one point mutations, respectively. Ma18 and Ma21 are more stable proteins than A-TIM, as judged from CD melting experiments. Like A-TIM, both proteins are monomeric in solution. In the Ma18 crystal structure loop 6 is open and in the Ma21 crystal structure loop 6 is closed, being stabilized by a bound glycolate molecule. The crystal structures show only small differences in the active site compared with A-TIM. In the case of Ma21 it is observed that the point mutation (Q65L) contributes to small structural rearrangements near Asn11 of loop 1, which correlate with different ligand-binding properties such as a loss of citrate binding in the active site. The Ma21 structure also shows that its Leu65 side chain is involved in van der Waals interactions with neighbouring hydrophobic side-chain moieties, correlating with its increased stability. The experimental data suggest that the increased stability and solubility properties of Ma21 and Ma18 compared with A-TIM cause better growth of the selection strain when coexpressing Ma21 and Ma18 instead of A-TIM. PMID:27303904

  2. Dioxygen activation at non-heme diiron centers: oxidation of a proximal residue in the I100W variant of toluene/o-xylene monooxygenase hydroxylase.

    PubMed

    Murray, Leslie J; García-Serres, Ricardo; McCormick, Michael S; Davydov, Roman; Naik, Sunil G; Kim, Sun-Hee; Hoffman, Brian M; Huynh, Boi Hanh; Lippard, Stephen J

    2007-12-25

    At its carboxylate-bridged diiron active site, the hydroxylase component of toluene/o-xylene monooxygenase activates dioxygen for subsequent arene hydroxylation. In an I100W variant of this enzyme, we characterized the formation and decay of two species formed by addition of dioxygen to the reduced, diiron(II) state by rapid-freeze quench (RFQ) EPR, Mössbauer, and ENDOR spectroscopy. The dependence of the formation and decay rates of this mixed-valent transient on pH and the presence of phenol, propylene, or acetylene was investigated by double-mixing stopped-flow optical spectroscopy. Modification of the alpha-subunit of the hydroxylase after reaction of the reduced protein with dioxygen-saturated buffer was investigated by tryptic digestion coupled mass spectrometry. From these investigations, we conclude that (i) a diiron(III,IV)-W* transient, kinetically linked to a preceding diiron(III) intermediate, arises from the one-electron oxidation of W100, (ii) the tryptophan radical is deprotonated, (iii) rapid exchange of either a terminal water or hydroxide ion with water occurs at the ferric ion in the diiron(III,IV) cluster, and (iv) the diiron(III,IV) core and W* decay to the diiron(III) product by a common mechanism. No transient radical was observed by stopped-flow optical spectroscopy for reactions of the reduced hydroxylase variants I100Y, L208F, and F205W with dioxygen. The absence of such species, and the deprotonated state of the tryptophanyl radical in the diiron(III,IV)-W* transient, allow for a conservative estimate of the reduction potential of the diiron(III) intermediate as lying between 1.1 and 1.3 V. We also describe the X-ray crystal structure of the I100W variant of ToMOH. PMID:18044971

  3. Dioxygen Activation at Non-Heme Diiron Centers: Oxidation of a Proximal Residue in the I100W Variant of Toluene/o-Xylene Monooxygenase Hydroxylase†

    PubMed Central

    Murray, Leslie J.; García-Serres, Ricardo; McCormick, Michael S.; Davydov, Roman; Naik, Sunil; Kim, Sun-Hee; Hoffman, Brian M.; Huynh, Boi Hanh; Lippard, Stephen J.

    2008-01-01

    At its carboxylate-bridged diiron active site, the hydroxylase component of toluene/o-xylene monooxygenase activates dioxygen for subsequent arene hydroxylation. In an I100W variant of this enzyme, we characterized the formation and decay of two species formed by addition of dioxygen to the diiron(II) form by rapid-freeze quench (RFQ) EPR, Mössbauer, and ENDOR spectroscopy. The dependence of the formation and decay rates of this mixed-valent transient on pH and the presence of phenol, propylene, or acetylene, was investigated by double-mixing stopped-flow optical spectroscopy. Modification of the α-subunit of the hydroxylase after reaction of the reduced protein with dioxygen-saturated buffer was investigated by tryptic digestion coupled mass spectrometry. From these investigations, we conclude that (i) a diiron(III,IV)–W• transient, kinetically-linked to a preceding diiron(III) intermediate, arises from the one-electron oxidation of W100; (ii) the tryptophan radical is deprotonated; (iii) rapid exchange of either a terminal water or hydroxide ion with water occurs at the ferric ion in the diiron(III,IV) cluster; and (iv) the diiron(III,IV) core and W• decay to the diiron(III) product by a common mechanism. No transient radical was observed by stopped-flow optical spectroscopy for reactions of the reduced hydroxylase variants I100Y, L208F, and F205W with dioxygen. The absence of such species, and the deprotonated state of the tryptophanyl radical in the diiron(III,IV)–W• transient, allow for a conservative estimate of the reduction potential of the diiron(III) intermediate as lying between 1.1 and 1.3 V. We also describe the X-ray crystal structure of the I100W variant of ToMOH. PMID:18044971

  4. Previously Associated Type 2 Diabetes Variants May Interact With Physical Activity to Modify the Risk of Impaired Glucose Regulation and Type 2 Diabetes

    PubMed Central

    Brito, Ema C.; Lyssenko, Valeriya; Renström, Frida; Berglund, Göran; Nilsson, Peter M.; Groop, Leif; Franks, Paul W.

    2009-01-01

    OBJECTIVE Recent advances in type 2 diabetes genetics have culminated in the discovery and confirmation of multiple risk variants. Two important and largely unanswered questions are whether this information can be used to identify individuals most susceptible to the adverse consequences of sedentary behavior and to predict their response to lifestyle intervention; such evidence would be mechanistically informative and provide a rationale for targeting genetically susceptible subgroups of the population. RESEARCH DESIGN AND METHODS Gene × physical activity interactions were assessed for 17 polymorphisms in a prospective population-based cohort of initially nondiabetic middle-aged adults. Outcomes were 1) impaired glucose regulation (IGR) versus normal glucose regulation determined with either fasting or 2-h plasma glucose concentrations (n = 16,003), 2) glucose intolerance (in mmol/l, n = 8,860), or 3) incident type 2 diabetes (n = 2,063 events). RESULTS Tests of gene × physical activity interactions on IGR risk for 3 of the 17 polymorphisms were nominally statistically significant:CDKN2A/B rs10811661 (Pinteraction = 0.015), HNF1B rs4430796 (Pinteraction = 0.026), and PPARG rs1801282 (Pinteraction = 0.04). Consistent interactions were observed for the CDKN2A/B (Pinteraction = 0.013) and HNF1B (Pinteraction = 0.0009) variants on 2-h glucose concentrations. Where type 2 diabetes was the outcome, only one statistically significant interaction effect was observed, and this was for the HNF1B rs4430796 variant (Pinteraction = 0.0004). The interaction effects for HNF1B on IGR risk and incident diabetes remained significant after correction for multiple testing (Pinteraction = 0.015 and 0.0068, respectively). CONCLUSIONS Our observations suggest that the genetic predisposition to hyperglycemia is partially dependent on a person's lifestyle. PMID:19324937

  5. Variants of glycoside hydrolases

    SciTech Connect

    Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

    2013-02-26

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  6. Variants of glycoside hydrolases

    DOEpatents

    Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

    2011-04-26

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  7. Selective induction of CTL ‘helper’ rather than killer activity by natural epitope variants promotes DC-mediated HIV-1 dissemination

    PubMed Central

    Mailliard, Robbie B.; Smith, Kellie N.; Fecek, Ronald J.; Rappocciolo, Giovanna; Nascimento, Eduardo J. M.; Marques, Ernesto T.; Watkins, Simon C.; Mullins, James I.; Rinaldo, Charles R.

    2013-01-01

    The ability of HIV-1 to rapidly accumulate mutations provides the virus with an effective means of escaping CD8+ cytotoxic T lymphocyte (CTL) responses. Here we describe how subtle alterations in CTL epitopes expressed by naturally occurring HIV-1 variants can result in an incomplete escape from CTL recognition, providing the virus with a selective advantage. Rather than paralyzing the CTL response, these epitope modifications selectively induce the CTL to produce pro-inflammatory cytokines in the absence of target killing. Importantly, instead of dampening the immune response through CTL elimination of variant antigen-expressing immature dendritic cells (iDC), a positive CTL-to-DC immune feedback loop dominates whereby the iDC differentiate into mature pro-inflammatory DC. Moreover, these CTL-programmed DC exhibit a superior capacity to mediate HIV-1 trans-infection of T cells. This discordant induction of CTL helper activity in the absence of killing likely contributes to the chronic immune activation associated with HIV-1 infection, and can be utilized by HIV-1 to promote viral dissemination and persistence. Our findings highlight the need to address the detrimental potential of eliciting dysfunctional cross-reactive memory CTL responses when designing and implementing anti-HIV-1 immunotherapies. PMID:23913962

  8. MK-5172, a Selective Inhibitor of Hepatitis C Virus NS3/4a Protease with Broad Activity across Genotypes and Resistant Variants

    PubMed Central

    Summa, Vincenzo; McCauley, John A.; Fandozzi, Christine; Burlein, Christine; Claudio, Giuliano; Coleman, Paul J.; DiMuzio, Jillian M.; Ferrara, Marco; Di Filippo, Marcello; Gates, Adam T.; Graham, Donald J.; Harper, Steven; Hazuda, Daria J.; McHale, Carolyn; Monteagudo, Edith; Pucci, Vincenzo; Rowley, Michael; Rudd, Michael T.; Soriano, Aileen; Stahlhut, Mark W.; Vacca, Joseph P.; Olsen, David B.; Liverton, Nigel J.; Carroll, Steven S.

    2012-01-01

    HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens. PMID:22615282

  9. 40Ar/36Ar analyses of historic lava flows

    USGS Publications Warehouse

    Dalrymple, G.B.

    1969-01-01

    The ratio 40Ar/36Ar was measured for 26 subaerial historic lava flows. Approximately one-third of the samples had 40Ar/36Ar ratios either higher or lower than the atmospheric value of 295.5 at the 95% confidence level. Excess radiogenic 40Ar in five flows ranged from about 1 ?? 10-13 to 1.5 ?? 10-12 mol/g. Possible excess 36Ar in three flows was on the order of 10-16 to 10-15 mol/g. Upper 95% confidence limits for excess 40Ar in samples with normal 40Ar/36Ar ratios are generally less than 3 ?? 10-13 mol/g. The origin of the excess 36Ar is unknown but it may be due either to the incorporation of primitive argon that has been stored in the mantle in very low potassium environments or to enrichment in 36Ar as atmospheric argon diffuses into the rocks after they cool. ?? 1969.

  10. Community proteogenomics highlights microbial strain-variant protein expression within activated sludge performing enhanced biological phosphorus removal.

    SciTech Connect

    Wilmes, P; Andersson, Anders F.; Lefsrud, Mark G; Wexler, Margaret; Shah, Manesh B; Zhang, B; Hettich, Robert {Bob} L; Bond, P. L.; Verberkmoes, Nathan C; Banfield, Jillian F.

    2008-01-01

    Enhanced biological phosphorus removal (EBPR) selects for polyphosphate accumulating organisms to achieve phosphate removal from wastewater. We used highresolution community proteomics to identify key metabolic pathways in "Candidatus Accumulibacter phosphatis"-mediated EBPR and to evaluate the contributions of co- 5 existing strains within the dominant population. Results highlight the importance of denitrification, fatty acid cycling and the glyoxylate bypass in EBPR. Despite overall strong similarity in protein profiles under anaerobic and aerobic conditions, fatty acid degradation proteins were more abundant during the anaerobic phase. By comprehensive genome-wide alignment of orthologous proteins, we uncovered strong 10 functional partitioning for enzyme variants involved in both core-metabolism and EBPR-specific pathways among the dominant strains. These findings emphasize the importance of genetic diversity in maintaining the stable performance of EBPR systems and demonstrate the power of integrated cultivation-independent genomics and proteomics for analysis of complex biotechnological systems.

  11. Functional analysis of differences in transcriptional activity conferred by genetic variants in the 5' flanking region of the IL12RB2 gene.

    PubMed

    Kato-Kogoe, Nahoko; Ohyama, Hideki; Okano, Soichiro; Yamanegi, Koji; Yamada, Naoko; Hata, Masaki; Nishiura, Hiroshi; Abiko, Yoshimitsu; Terada, Nobuyuki; Nakasho, Keiji

    2016-01-01

    Interleukin 12 receptor β chain (IL12RB2) is a crucial regulatory factor involved in cell-mediated immune responses, and genetic variants of the gene encoding IL12RB2 are associated with susceptibility to various immune-related diseases. We previously demonstrated that haplotypes with single nucleotide polymorphisms (SNPs) in the 5' flanking region of IL12RB2, including -1035A>G (rs3762315) and -1023A>G (rs3762316), affect the expression of IL12RB2, thereby altering susceptibility to leprosy and periodontal diseases. In the present study, we identified transcription factors associated with the haplotype-specific transcriptional activity of IL12RB2 in T cells and NK cells. The -1023G polymorphism was found to create a consensus binding site for the transcription factor activating protein (AP)-1, and enzyme-linked immunosorbent assay (ELISA)-based binding assays showed that these SNPs enhanced AP-1 binding to this region. In reporter assays, suppression of JunB expression using siRNA eliminated differences in the -1035G/-1023G and -1035A/-1023A regions containing IL12RB2 promoter activity in Jurkat T cells and NK3.3 cells. These results suggested that the -1035/-1023 polymorphisms created differential binding affinities for JunB that could lead to differential IL12RB2 expression. Moreover, the -1035G and -1035A alleles formed binding sites for GATA-3 and myocyte enhancer factor-2 (MEF-2), respectively. Our data indicated that in addition to JunB, the SNP at -1035/-1023 influenced GATA-3 and MEF-2 binding affinity, potentially altering IL12RB2 transcriptional activity. These findings confirm the effects of rs3762315 and rs3762316 on IL12RB2 transcription. These genetic variants may alter cellular activation of T cells and NK cells and modify cell-mediated immune responses. PMID:26552659

  12. Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells

    PubMed Central

    Bolton, Eric C.

    2015-01-01

    The androgen receptor (AR) mediates the developmental, physiologic, and pathologic effects of androgens including 5α-dihydrotestosterone (DHT). However, the mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells are not well understood, though they are central to prostate development, homeostasis, and neoplasia. Here, we identify androgen-responsive genes that restrain cell cycle progression and proliferation of human prostate epithelial cell lines (HPr-1AR and PC3-Lenti-AR), and we investigate the mechanisms through which AR regulates their expression. DHT inhibited proliferation of HPr-1AR and PC3-Lenti-AR, and cell cycle analysis revealed a prolonged G1 interval. In the cell cycle, the G1/S-phase transition is initiated by the activity of cyclin D and cyclin-dependent kinase (CDK) complexes, which relieve growth suppression. In HPr-1AR, cyclin D1/2 and CDK4/6 mRNAs were androgen-repressed, whereas CDK inhibitor, CDKN1A, mRNA was androgen-induced. The regulation of these transcripts was AR-dependent, and involved multiple mechanisms. Similar AR-mediated down-regulation of CDK4/6 mRNAs and up-regulation of CDKN1A mRNA occurred in PC3-Lenti-AR. Further, CDK4/6 overexpression suppressed DHT-inhibited cell cycle progression and proliferation of HPr-1AR and PC3-Lenti-AR, whereas CDKN1A overexpression induced cell cycle arrest. We therefore propose that AR-mediated growth suppression of HPr-1AR involves cyclin D1 mRNA decay, transcriptional repression of cyclin D2 and CDK4/6, and transcriptional activation of CDKN1A, which serve to decrease CDK4/6 activity. AR-mediated inhibition of PC3-Lenti-AR proliferation occurs through a similar mechanism, albeit without down-regulation of cyclin D. Our findings provide insight into AR-mediated regulation of prostate epithelial cell proliferation. PMID:26372468

  13. Mitochondrial Fusion and ERK Activity Regulate Steroidogenic Acute Regulatory Protein Localization in Mitochondria

    PubMed Central

    Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J.; Poderoso, Cecilia

    2014-01-01

    The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR. PMID:24945345

  14. Activation of minority-variant Plasmodium vivax hypnozoites following artesunate + amodiaquine treatment in a 23-year old man with relapsing malaria in Antananarivo, Madagascar

    PubMed Central

    2013-01-01

    In endemic areas, Plasmodium vivax relapses are difficult to distinguish from new infections. Genotyping of patients who experience relapse after returning to a malaria-free area can be used to explore the nature of hypnozoite activation and relapse. This paper describes a person who developed P. vivax malaria for the first time after travelling to Boriziny in the malaria endemic coastal area of Madagascar, then suffered two P. vivax relapses 11 weeks and 21 weeks later despite remaining in Antananarivo in the malaria-free central highlands area. He was treated with the combination artesunate + amodiaquine according to the national malaria policy in Madagascar. Genotyping by PCR-RFLP at pvmsp-3α as well as pvmsp1 heteroduplex tracking assay (HTA) showed the same dominant genotype at each relapse. Multiple recurring minority variants were also detected at each relapse, highlighting the propensity for multiple hypnozoite clones to activate simultaneously to cause relapse. PMID:23721298

  15. Fractalkine (CX3CL1) enhances hippocampal N-methyl-d-aspartate receptor (NMDAR) function via d-serine and adenosine receptor type A2 (A2AR) activity

    PubMed Central

    2013-01-01

    Background N-Methyl-d-aspartate receptors (NMDARs) play fundamental roles in basic brain functions such as excitatory neurotransmission and learning and memory processes. Their function is largely regulated by factors released by glial cells, including the coagonist d-serine. We investigated whether the activation of microglial CX3CR1 induces the release of factors that modulate NMDAR functions. Methods We recorded the NMDAR component of the field excitatory postsynaptic potentials (NMDA-fEPSPs) elicited in the CA1 stratum radiatum of mouse hippocampal slices by Shaffer collateral stimulation and evaluated d-serine content in the extracellular medium of glial primary cultures by mass spectrometry analysis. Results We demonstrated that CX3CL1 increases NMDA-fEPSPs by a mechanism involving the activity of the adenosine receptor type A2 (A2AR) and the release of the NMDAR coagonist d-serine. Specifically (1) the selective A2AR blocker 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261) and the genetic ablation of A2AR prevent CX3CL1 action while the A2AR agonist 5-(6-amino-2-(phenethylthio)-9H-purin-9-yl)-N-ethyl-3,4-dihydroxytetrahydrofuran-2-carboxamide (VT7) mimics CX3CL1 effect, and (2) the selective blocking of the NMDAR glycine (and d-serine) site by 5,7-dicholorokynurenic acid (DCKA), the enzymatic degradation of d-serine by d-amino acid oxidase (DAAO) and the saturation of the coagonist site by d-serine, all block the CX3CL1 effect. In addition, mass spectrometry analysis demonstrates that stimulation of microglia and astrocytes with CX3CL1 or VT7 increases d-serine release in the extracellular medium. Conclusions CX3CL1 transiently potentiates NMDAR function though mechanisms involving A2AR activity and the release of d-serine. PMID:23981568

  16. A rare variant in human fibroblast activation protein associated with ER stress, loss of enzymatic function and loss of cell surface localisation.

    PubMed

    Osborne, Brenna; Yao, Tsun-Wen; Wang, Xin Maggie; Chen, Yiqian; Kotan, L Damla; Nadvi, Naveed A; Herdem, Mustafa; McCaughan, Geoffrey W; Allen, John D; Yu, Denise M T; Topaloglu, A Kemal; Gorrell, Mark D

    2014-07-01

    Fibroblast activation protein (FAP) is a focus of interest as a potential cancer therapy target. This membrane bound protease possesses the unique catalytic activity of hydrolysis of the post-proline bond two or more residues from the N-terminus of substrates. FAP is highly expressed in activated fibroblastic cells in tumours, arthritis and fibrosis. A rare, novel, human polymorphism, C1088T, encoding Ser363 to Leu, occurring in the sixth blade of the β propeller domain, was identified in a family. Both in primary human fibroblasts and in Ser363LeuFAP transfected cells, we showed that this single substitution ablates FAP dimerisation and causes loss of enzyme activity. Ser363LeuFAP was detectable only in endoplasmic reticulum (ER), in contrast to the distribution of wild-type FAP on the cell surface. The variant FAP showed decreased conformational antibody binding, consistent with an altered tertiary structure. Ser363LeuFAP expression was associated with upregulation of the ER chaperone BiP/GRP78, ER stress sensor ATF6, and the ER stress response target phospho-eIF2α, all indicators of ER stress. Proteasomal inhibition resulted in accumulation of Ser363LeuFAP, indicating the involvement of ER associated degradation (ERAD). Neither CHOP expression nor apoptosis was elevated, so ERAD is probably important for protecting Ser363LeuFAP expressing cells. These data on the first loss of function human FAP gene variant indicates that although the protein is vulnerable to an amino acid substitution in the β-propeller domain, inactive, unfolded FAP can be tolerated by cells. PMID:24717288

  17. Biochemical, thrombolytic and pharmacokinetic properties of rt-PA P47G, K49N, a substitution variant of human tissue-type plasminogen activator.

    PubMed

    Nelles, L; Li, X K; Vanlinthout, I; De Cock, F; Lijnen, H R; Collen, D

    1992-04-01

    rt-PA P47G, K49N, a substitution variant of recombinant human tissue-type plasminogen activator (rt-PA), in which proline at position 47 and lysine at position 49 were replaced by glycine and asparagine respectively, was previously described by Ahern et al. (J Biol Chem 1990; 265:5540-5) to have an extended in vivo half-life with unaltered in vitro fibrinolytic properties. Because this variant might possess an increased in vivo thrombolytic potency, we have constructed its cDNA, expressed it in Chinese hamster ovary cells and determined its biochemical, thrombolytic and pharmacokinetic properties relative to those of home-made rt-PA and of alteplase (Actilyse). The specific fibrinolytic activities on fibrin plates were 160,000 +/- 17,000, 210,000 +/- 88,000 and 460,000 +/- 72,000 IU/mg (mean +/- SEM) for rt-PA P47G, K49N, rt-PA and alteplase, respectively, while the catalytic efficiencies for plasminogen activation (k2/Km) in the absence of fibrin were comparable (1.1 to 1.7 x 10(-3) microM-1s-1). Fibrin enhanced the rate of plasminogen activation by rt-PA P47G, K49N 100-fold and by both wild-type molecules 390-fold. Binding of the variant rt-PA to fibrin was significantly reduced, but its affinity for lysine-Sepharose was unaltered. In an in vitro clot lysis system, consisting of a radiolabeled human plasma clot submersed in plasma, 50% clot lysis in 2 h required 0.67 +/- 0.14 micrograms/ml rt-PA P47G, K49N, 0.36 +/- 0.01 micrograms/ml rt-PA and 0.17 +/- 0.01 micrograms/ml alteplase, respectively (mean +/- SEM; n = 3 or 4). At these doses residual fibrinogen levels at 2 h were in excess of 80%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1631793

  18. Double substituted variant of Bacillus amyloliquefaciens esterase with enhanced enantioselectivity and high activity towards 1-(3',4'-methylenedioxyphenyl)ethyl acetate.

    PubMed

    Liu, Jia-Yan; Bian, Han-Ping; Tang, Yun; Bai, Yun-Peng; Xu, Jian-He

    2015-02-01

    Bacillus amyloliquefaciens esterase (BAE) was applied to produce (R)-1-(3',4'-methylenedioxyphenyl)ethanol, a chiral drug intermediate. In this study, we improved the enantioselectivity of BAE by protein engineering instead of process engineering as used in our previous work. Saturation mutagenesis was carried out on eight positions of BAE based on structure modeling and substrate docking. A double substituted variant V10 (K358D/A396C) showed an excellent enantioselectivity without decreasing the activity. The functions of these two mutations (K358D and A396C) were investigated, revealing a synergic effect on the BAE enantioselectivity. Using the variant V10, enantiopure (R)-1-(3',4'-methylenedioxyphenyl)ethanol could be readily prepared in >97 % ee, affording a high space-time yield (123 g L(-1) day(-1)) and a high ratio of substrate/catalyst (40 g g(-1)) in 1-L reaction. PMID:25104035

  19. A mix of S and ΔS variants of STAT3 enable survival of activated B-cell-like diffuse large B-cell lymphoma cells in culture.

    PubMed

    Zheng, M; Turton, K B; Zhu, F; Li, Y; Grindle, K M; Annis, D S; Lu, L; Drennan, A C; Tweardy, D J; Bharadwaj, U; Mosher, D F; Rui, L

    2016-01-01

    Activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL) is characterized by increased expression and activator of signal transducer and activator of transcription 3 (STAT3). ABC DLBCL cells require STAT3 for growth in culture. In ABC DLBCL cells, eosinophils and perhaps all cells, four variant STAT3 mRNAs (Sα, ΔSα, Sβ and ΔSβ) are present as a result of two alternative splicing events, one that results in the inclusion of a 55-residue C-terminal transactivation domain (α) or a truncated C-terminal domain with 7 unique residues (β) and a second that includes (S) or excludes (ΔS) the codon for Ser-701 in the linker between the SH2 and C-terminal domains. A substantial literature indicates that both α and β variants are required for optimal STAT3 function, but nothing is known about functions of ΔS variants. We used a knockdown/re-expression strategy to explore whether survival of ABC DLBCL cells requires that the four variants be in an appropriate ratio. No single variant rescued survival as well as STAT3Sα-C, Sα with activating mutations (A661C and N663C) in the SH2 domain. Better rescue was achieved when all four variants were re-expressed or Sα and ΔSα or Sβ and ΔSβ were re-expressed in pairs. Rescue correlated with expression of STAT3-sensitive genes NFKBIA and NFKBIZ. We consider a variety of explanations why a mix of S and ΔS variants of STAT3 should enable survival of ABC DLBCL cells. PMID:26727576

  20. One-electron pseudopotential investigation of the RbAr and FrAr van der Waals systems

    NASA Astrophysics Data System (ADS)

    Dhiflaoui, J.; Berriche, H.

    2012-12-01

    The potential energy curves of the ground state and many excited states of RbAr and FrAr van der Waals systems have been determined using a one-electron pseudopotential approach. The pseudopotential technique is used to replace the effect of the Rb+ and Fr+ cores and the electron-Ar interaction. In addition a core-core interaction is included. This has permitted to reduce the number of active electrons of the RbAr and FrAr systems to only one electron, the valence electron. This has led to use very large basis sets for Rb, Fr and Ar atoms. In this context, the potential energy curves of the ground and many excited states are performed at the SCF level. The core-core interactions for Rb+Ar and Fr+Ar are included using the CCSD(T) accurate potentials of Hickling et al. [H. Hickling, L. Viehland, D. Shepherd, P. Soldan, E. Lee and T. Wright, Phys. Chem. Chem. Phys. 6 (2004) 4233]. In addition, the spectroscopic constants of these states are derived and compared with the available theoretical works. Such comparison for RbAr has shown a very good agreement for the ground and the first excited states. However, the FrAr system was not studied previously and its spectroscopic constants are presented here for the first time.

  1. Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

    PubMed

    Stellmacher, Lena; Sandalova, Tatyana; Schneider, Sarah; Schneider, Gunter; Sprenger, Georg A; Samland, Anne K

    2016-04-01

    Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme. PMID:27050126

  2. Imaging Ca(2+) activity in mammalian cells and zebrafish with a novel red-emitting aequorin variant.

    PubMed

    Bakayan, Adil; Domingo, Beatriz; Miyawaki, Atsushi; Llopis, Juan

    2015-09-01

    Ca(2+) monitoring with aequorin is an established bioluminescence technique, whereby the photoprotein emits blue light when it binds to Ca(2+). However, aequorin's blue emission and low quantum yield limit its application for in vivo imaging because blue-green light is greatly attenuated in animal tissues. In earlier work, aequorin was molecularly fused with green, yellow, and red fluorescent proteins, producing an emission shift through bioluminescence resonance energy transfer (BRET). We have previously shown that the chimera tandem dimer Tomato-aequorin (tdTA) emits red light in mammalian cells and across the skin and other tissues of mice [1]. In this work, we varied the configuration of the linker in tdTA to maximize energy transfer. One variant, named Redquorin, improved BRET from aequorin to tdTomato to almost a maximum value, and the emission above 575 nm exceeded 73 % of total counts. By pairing Redquorin with appropriate synthetic coelenterazines, agonist-induced and spontaneous Ca(2+) oscillations in single HEK-293 cells were imaged. In addition, we also imaged Ca(2+) transients associated with twitching behavior in developing zebrafish embryos expressing Redquorin during the segmentation period. Furthermore, the emission profile of Redquorin resulted in significant luminescence crossing a blood sample, a highly absorbing tissue. This new tool will facilitate in vivo imaging of Ca(2+) from deep tissues of animals. PMID:25355614

  3. Induction of Prostatic Intraepithelial Neoplasia and Modulation of Androgen Receptor by ETS Variant 1/ETS-Related Protein 81

    PubMed Central

    Shin, Sook; Kim, Tae-Dong; Jin, Fang; van Deursen, Jan M.; Dehm, Scott M.; Tindall, Donald J.; Grande, Joseph P.; Munz, Jan-Marie; Vasmatzis, George; Janknecht, Ralf

    2014-01-01

    ETS variant 1 (ETV1), also known as ETS-related protein 81, is overexpressed in prostate tumors, but whether and how this transcription factor affects tumorigenesis has remained elusive. Here, we show that ETV1 is primarily overexpressed in the most aggressive human prostate tumors. Transgenic ETV1 mice developed prostatic intraepithelial neoplasia as well as hyperplasia/neoplasia in seminal vesicles. Moreover, ETV1 cooperated with the androgen receptor (AR) to bind to the prostate-specific antigen enhancer and stimulate gene transcription. Consistent with its ability to physically interact with AR, ETV1 rendered an ETV1 binding site–driven reporter androgen inducible, and, on the other hand, ETV1 superinduced transcription from an AR binding site on androgen stimulation. In conclusion, our study substantiates that ETV1 overexpression is an underlying cause in the development of prostate and possibly also seminal vesicle cancer. Its interaction with and activation of AR provides a molecular mechanism on how ETV1 exerts its deleterious function. Thus, inhibiting ETV1 or blocking its interaction with AR may represent novel strategies in prostate cancer therapy. PMID:19789348

  4. Development of AR-V7 as a putative treatment selection marker for metastatic castration-resistant prostate cancer

    PubMed Central

    Luo, Jun

    2016-01-01

    Prostate cancer cells demonstrate a remarkable “addiction” to androgen receptor (AR) signaling in all stages of disease progression. As such, suppression of AR signaling remains the therapeutic goal in systemic treatment of prostate cancer. A number of molecular alterations arise in patients treated with AR-directed therapies. These molecular alterations may indicate the emergence of treatment resistance and may be targeted for the development of novel agents for prostate cancer. The presence of functional androgen receptor splice variants may represent a potential explanation for resistance to abiraterone and enzalutamide, newer AR-directed agents developed to treat metastatic castration-resistant prostate cancer (mCRPC). In the last 8 years, many androgen receptor splice variants have been identified and characterized. Among these, androgen receptor splice variant-7 (AR-V7) has been investigated extensively. In AR-V7, the entire COOH-terminal ligand-binding domain of the canonical AR is truncated and replaced with a variant-specific peptide of 16 amino acids. Functionally, AR-V7 is capable of mediating constitutive nuclear localization and androgen receptor signaling in the absence of androgens, or in the presence of enzalutamide. In this review, we will focus on clinical translational studies involving detection/measurement of AR-V7. Methods have been developed to detect AR-V7 in clinical mCRPC specimens. AR-V7 can be reliably measured in both tissue and circulating tumor cells derived from mCRPC patients, making it possible to conduct both cross-sectional and longitudinal clinical correlative studies. Current evidence derived from studies focusing on detection of AR-V7 in mCRPC support its potential clinical utility as a treatment selection marker. PMID:27174161

  5. USDA-ARS RESEARCH ON BIOLOGICAL CONTROL OF ARTHROPODS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During 1999-2001, ARS scientists published over 100 papers on biocontrol of 30 insect pests. These papers address issues crucial to the three strategies of biological control: conservation, augmentation, and introduction. ARS scientists have been very active in determining the effects of pesticides...

  6. The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion

    PubMed Central

    Nalvarte, Ivan; Damdimopoulos, Anastasios E.; Rüegg, Joëlle; Spyrou, Giannis

    2015-01-01

    The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis. It transfers electrons from NADPH to a large variety of substrates, particularly to those containing redox-active cysteines. Previously, we reported that the classical form of cytosolic TrxR1 (TXNRD1_v1), when overexpressed in human embryonic kidney cells (HEK-293), prompted the cells to undergo differentiation [Nalvarte et al. (2004) J. Biol. Chem. 279, 54510–54517]. In the present study, we show that several genes associated with differentiation and adhesion are differentially expressed in HEK-293 cells stably overexpressing TXNRD1_v1 compared with cells expressing its splice variant TXNRD1_v2. Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity. Furthermore, differentiation of the neuroblastoma cell line SH-SY5Y induced by all-trans retinoic acid (ATRA) increased both TXNRD1_v1 and TXNRD1_v2 expressions along with several of the identified genes associated with differentiation and adhesion. Selenium supplementation in the SH-SY5Y cells also induced a differentiated morphology and changed expression of the adhesion protein fibronectin 1 and the differentiation marker cadherin 11, as well as different temporal expression of the studied TXNRD1 variants. These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1. PMID:26464515

  7. The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion.

    PubMed

    Nalvarte, Ivan; Damdimopoulos, Anastasios E; Rüegg, Joëlle; Spyrou, Giannis

    2015-01-01

    The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis. It transfers electrons from NADPH to a large variety of substrates, particularly to those containing redox-active cysteines. Previously, we reported that the classical form of cytosolic TrxR1 (TXNRD1_v1), when overexpressed in human embryonic kidney cells (HEK-293), prompted the cells to undergo differentiation [Nalvarte et al. (2004) J. Biol. Chem. 279: , 54510-54517]. In the present study, we show that several genes associated with differentiation and adhesion are differentially expressed in HEK-293 cells stably overexpressing TXNRD1_v1 compared with cells expressing its splice variant TXNRD1_v2. Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity. Furthermore, differentiation of the neuroblastoma cell line SH-SY5Y induced by all-trans retinoic acid (ATRA) increased both TXNRD1_v1 and TXNRD1_v2 expressions along with several of the identified genes associated with differentiation and adhesion. Selenium supplementation in the SH-SY5Y cells also induced a differentiated morphology and changed expression of the adhesion protein fibronectin 1 and the differentiation marker cadherin 11, as well as different temporal expression of the studied TXNRD1 variants. These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1. PMID:26464515

  8. Differential unroofing within the central metasedimentary Belt of the Grenville Orogen: constraints from 40Ar/39Ar thermochronology

    USGS Publications Warehouse

    Cosca, M.A.; Essene, E.J.; Kunk, M.J.; Sutter, J.F.

    1992-01-01

    An 40Ar/39Ar thermochronological investigation of upper greenschist to granulite facies gneiss, amphibolite and marble was conducted in the Central Metasedimentary Belt (CMB), Ontario, to constrain its cooling history. Incremental 40Ar/39Ar release spectra indicate that substantial differential unroofing occurred in the CMB between ??? 1000 and ??? 600 Ma. A consistent pattern of significantly older hornblende and phlogopite 40Ar/3Ar cooling ages on the southeast sides of major northeast striking shear zones is interpreted to reflect late displacement due to extensional deformation. Variations in hornblende 40Ar/39Ar age plateaus exceeding 200 Ma occur over distances less than 50 km with major age discontinuities occurring across the Robertson Lake shear zone and the Sharbot Lake mylonite zone which separate the Sharbot Lake terrane from the Elzevir and Frontenac terranes. Extensional displacements of up to 14 km are inferred between the Frontenac and Elzevir terranes of the CMB. No evidence for significant post argon-closure vertical displacement is indicated in the vicinity of the Perth Road mylonite within the Frontenac terrane. Variations of nearly 100 Ma in phlogopite 40Ar/39Ar plateau ages occur in undeformed marble on either side of the Bancroft Shear Zone. Phlogopites from sheared and mylonitized marble within the shear zone yield 40Ar/39Ar diffusional loss profiles, but have older geologically meaningless ages thought to reflect incorporation of excess argon. By ??? 900 Ma, southeast directed extension was occurring throughout the CMB, possibly initiated along previous zones of compressional shearing. An easterly migration of active zones of extension is inferred, possibly related to an earlier, overall easterly migration of active zones of regional thrusting and easterly migration of an ancient subduction zone. The duration of extensional shearing is not well constrained, but must have ceased before ??? 600 Ma as required by the deposition of overlying

  9. Production of 37Ar in The University of Texas TRIGA reactor facility

    SciTech Connect

    Egnatuk, Christine M.; Lowrey, Justin; Biegalski, S.; Bowyer, Ted W.; Haas, Derek A.; Orrell, John L.; Woods, Vincent T.; Keillor, Martin E.

    2011-06-19

    The detection of {sup 37}Ar is important for on-site inspections for the Comprehensive Nuclear-Test-Ban Treaty monitoring. In an underground nuclear explosion this radionuclide is produced by {sup 40}Ca(n,{alpha}){sup 37}Ar reaction in surrounding soil and rock. With a half-life of 35 days, {sup 37}Ar provides a signal useful for confirming the location of an underground nuclear event. An ultra-low-background proportional counter developed by Pacific Northwest National Laboratory is used to detect {sup 37}Ar, which decays via electron capture. The irradiation of Ar gas at natural enrichment in the 3L facility within the Mark II TRIGA reactor facility at The University of Texas at Austin provides a source of {sup 37}Ar for the calibration of the detector. The {sup 41}Ar activity is measured by the gamma activity using an HPGe detector after the sample is removed from the core. Using the {sup 41}Ar/{sup 37}Ar production ratio and the {sup 41}Ar activity, the amount of {sup 37}Ar created is calculated. The {sup 41}Ar decays quickly (half-life of 109.34 minutes) leaving a radioactive sample of high purity {sup 37}Ar and only trace levels of {sup 39}Ar.

  10. Laser {sup 40}Ar/{sup 39}Ar microprobe analyses of fine-grained illite

    SciTech Connect

    Onstott, T.C.; Mueller, C.; Vrolijk, P.J.; Pevear, D.R.

    1997-09-01

    Fine-grained (<0.02 {mu}m) to coarse-grained (2.0-0.2 {mu}m) illite separates and finely powdered muscovite standards were analyzed with a microencapsulation technique and an {sup 40}Ar/{sup 39}Ar laser microprobe. The integrated ages of the illite agreed within error with conventional K/Ar analyses, even though the sample sizes, 1-100 micrograms, were at least a 10,000-fold less. Incremental laser heating of an artificial mixture of illite and muscovite of two different ages yielded a stair step profile, where the youngest and oldest incremental ages approximately coincided with their K/Ar ages. The thermally activated argon release rate from illite was distinct from that of the muscovite and may result from differences in grain thickness, lower K concentration, and the presence of cis vs. trans-sited vacancies. Incremental heating, therefore, may prove capable of delineating detrital from authigenic components in illite extracted from shale and sandstone. Microencapsulation and laser {sup 40}Ar/{sup 39}Ar analyses, when combined with sophisticated techniques for separating clays, will permit dating of samples where clay is a minor constituent, such as sandstones and meteorites, and will enhance identification of endmember ages in naturally occurring clay. 45 refs., 9 figs., 2 tabs.

  11. AR-39-AR-40 "Age" of Basaltic Shergottite NWA-3171

    NASA Technical Reports Server (NTRS)

    Bogard, Donald D.; Park, Jisun

    2007-01-01

    North-West-Africa 3171 is a 506 g, relatively fresh appearing, basaltic shergottite with similarities to Zagami and Shergotty, but not obviously paired with any of the other known African basaltic shergottites. Its exposure age has the range of 2.5-3.1 Myr , similar to those of Zagami and Shergotty. We made AR-39-AR-40 analyses of a "plagioclase" (now shock-converted to maskelynite) separate and of a glass hand-picked from a vein connected to shock melt pockets.. Plagioclase was separated using its low magnetic susceptibility and then heavy liquid with density of <2.85 g/cm(exp 3). The AR-39-AR-40 age spectrum of NWA-317 1 plag displays a rise in age over 20-100% of the 39Ar release, from 0.24 Gyr to 0.27 Gy.

  12. Characterization of Estrogen and Androgen Activity of Food Contact Materials by Different In Vitro Bioassays (YES, YAS, ERα and AR CALUX) and Chromatographic Analysis (GC-MS, HPLC-MS)

    PubMed Central

    Osorio, Veronica; Grininger, Angelika; Richter, Alexander; Bergmair, Johannes; Pyerin, Michael; Washüttl, Michael; Tacker, Manfred

    2014-01-01

    Endocrine active substances (EAS) show structural similarities to natural hormones and are suspected to affect the human endocrine system by inducing hormone dependent effects. Recent studies with in vitro tests suggest that EAS can leach from packaging into food and may therefore pose a risk to human health. Sample migrates from food contact materials were tested for estrogen and androgen agonists and antagonists with different commonly used in vitro tests. Additionally, chemical trace analysis by GC-MS and HPLC-MS was used to identify potential hormone active substances in sample migrates. A GC-MS method to screen migrates for 29 known or potential endocrine active substances was established and validated. Samples were migrated according to EC 10/2011, concentrated by solid phase extraction and tested with estrogen and androgen responsive reporter gene assays based on yeast cells (YES and YAS) or human osteoblast cells (ERα and AR CALUX). A high level of agreement between the different bioassays could be observed by screening for estrogen agonists. Four out of 18 samples tested showed an estrogen activity in a similar range in both, YES and ERα CALUX. Two more samples tested positive in ERα CALUX due to the lower limits of detection in this assay. Androgen agonists could not be detected in any of the tested samples, neither with YAS nor with AR CALUX. When testing for antagonists, significant differences between yeast and human cell-based bioassays were noticed. Using YES and YAS many samples showed a strong antagonistic activity which was not observed using human cell-based CALUX assays. By GC-MS, some known or supposed EAS were identified in sample migrates that showed a biological activity in the in vitro tests. However, no firm conclusions about the sources of the observed hormone activity could be obtained from the chemical results. PMID:25000404

  13. Effects of time-variant extremely low-frequency (ELF) electromagnetic fields (EMF) on cholinesterase activity in Dictyostelium discoideum (Protista).

    PubMed

    Amaroli, Andrea; Trielli, Francesca; Bianco, Bruno; Giordano, Stefano; Moggia, Elsa; Corrado, Maria U Delmonte

    2005-12-15

    Recently, we detected propionylcholinesterase (PrChE) activity in single-cell amoebae of Dictyostelium discoideum using cytochemical, electrophoretic, and spectrophotometric methods. The involvement of this enzyme activity in cell-cell and cell-environment interactions was suggested. In this work, we found that exposure of single-cell amoebae to an extremely low-frequency electromagnetic fields (ELF-EMF) of 300 microT, 50 Hz, from 1 h up to 48 h at 21 +/- 1 degrees C affected PrChE activity. PMID:16425446

  14. Impact of cysteine variants on the structure, activity, and stability of recombinant human α-galactosidase A.

    PubMed

    Qiu, Huawei; Honey, Denise M; Kingsbury, Jonathan S; Park, Anna; Boudanova, Ekaterina; Wei, Ronnie R; Pan, Clark Q; Edmunds, Tim

    2015-09-01

    Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity. PMID:26044846

  15. Structure–activity correlations of variant forms of the B pentamer of Escherichia coli type II heat-labile enterotoxin LT-IIb with Toll-like receptor 2 binding

    SciTech Connect

    Cody, Vivian; Pace, Jim; Nawar, Hesham F.; King-Lyons, Natalie; Liang, Shuang; Connell, Terry D.; Hajishengallis, George

    2012-12-01

    Structural data for the S74D variant of the pentameric B subunit of type II heat-labile enterotoxin of Escherichia coli reveal a smaller pore opening that may explain its reduced Toll-like receptor binding affinity compared to that of the wild type enterotoxin. The explanation for the enhanced Toll-like receptor binding affinity of the S74A variant is more complex than simply being attributed to the pore opening. The pentameric B subunit of the type II heat-labile enterotoxin of Escherichia coli (LT-IIb-B{sub 5}) is a potent signaling molecule capable of modulating innate immune responses. It has previously been shown that LT-IIb-B{sub 5}, but not the LT-IIb-B{sub 5} Ser74Asp variant [LT-IIb-B{sub 5}(S74D)], activates Toll-like receptor (TLR2) signaling in macrophages. Consistent with this, the LT-IIb-B{sub 5}(S74D) variant failed to bind TLR2, in contrast to LT-IIb-B{sub 5} and the LT-IIb-B{sub 5} Thr13Ile [LT-IIb-B{sub 5}(T13I)] and LT-IIb-B{sub 5} Ser74Ala [LT-IIb-B{sub 5}(S74A)] variants, which displayed the highest binding activity to TLR2. Crystal structures of the Ser74Asp, Ser74Ala and Thr13Ile variants of LT-IIb-B{sub 5} have been determined to 1.90, 1.40 and 1.90 Å resolution, respectively. The structural data for the Ser74Asp variant reveal that the carboxylate side chain points into the pore, thereby reducing the pore size compared with that of the wild-type or the Ser74Ala variant B pentamer. On the basis of these crystallographic data, the reduced TLR2-binding affinity of the LT-IIb-B{sub 5}(S74D) variant may be the result of the pore of the pentamer being closed. On the other hand, the explanation for the enhanced TLR2-binding activity of the LT-IIb-B{sub 5}(S74A) variant is more complex as its activity is greater than that of the wild-type B pentamer, which also has an open pore as the Ser74 side chain points away from the pore opening. Data for the LT-IIb-B{sub 5}(T13I) variant show that four of the five variant side chains point to the outside

  16. Differences between the internal energy depositions induced by collisional activation and by electron transfer of W(CO){sub 6}{sup 2+} ions on collision with Ar and K targets

    SciTech Connect

    Hayakawa, Shigeo; Kitaguchi, Akihiro; Kameoka, Satoko; Toyoda, Michisato; Ichihara, Toshio

    2006-06-14

    Doubly charged tungsten hexacarbonyl W(CO){sub 6}{sup 2+} ions were made to collide with Ar and K targets to give singly and doubly charged positive ions by collision-induced dissociation (CID). The resulting ions were analyzed and detected by using a spherical electrostatic analyzer. Whereas the doubly charged fragment ions resulting from collisional activation (CA) were dominant with the Ar target, singly charged fragment ions resulting from electron transfer were dominant with the K target. The internal energy deposition in collisionally activated dissociation (CAD) evaluated with the Ar target was broad and decreased with increasing internal energy. The predominant peaks observed with the K target were associated with singly charged W(CO){sub 2}{sup +} and W(CO){sub 3}{sup +} ions: these ions were not the result of CA, but arose from dissociation induced by electron transfer (DIET). The internal energy deposition resulting from the electron transfer was very narrow and centered at a particular energy, 7.8 eV below the energy level of the W(CO){sub 6}{sup 2+} ion. This narrow internal energy distribution was explained in terms of electron transfer by Landau-Zener [Z. Phys. Soviet 2, 46 (1932); Proc. R. Soc. London, Ser. A 137, 646 (1952)] potential crossing at a separation of 5.9x10{sup -8} cm between a W(CO){sub 6}{sup 2+} ion and a K atom, and the coulombic repulsion between singly charged ions in the exit channel. A large cross section of 1.1x10{sup -14} cm{sup 2} was estimated for electron capture of the doubly charged W(CO){sub 6}{sup 2+} ion from the alkali metal target, whose ionization energy is very low. The term ''collision-induced dissociation,'' taken literally, includes all dissociation processes induced by collision, and therefore encompasses both CAD and DIET processes in the present work. Although the terms CID and CAD have been defined similarly, we would like to propose that they should not be used interchangeably, on the basis that there are

  17. Rapid degradation of an active formylglycine generating enzyme variant leads to a late infantile severe form of multiple sulfatase deficiency

    PubMed Central

    Schlotawa, Lars; Radhakrishnan, Karthikeyan; Baumgartner, Matthias; Schmid, Regula; Schmidt, Bernhard; Dierks, Thomas; Gärtner, Jutta

    2013-01-01

    Multiple sulfatase deficiency (MSD) is a rare inborn error of metabolism affecting posttranslational activation of sulfatases by the formylglycine generating enzyme (FGE). Due to mutations in the encoding SUMF1 gene, FGE's catalytic capacity is impaired resulting in reduced cellular sulfatase activities. Both, FGE protein stability and residual activity determine disease severity and have previously been correlated with the clinical MSD phenotype. Here, we report a patient with a late infantile severe course of disease. The patient is compound heterozygous for two so far undescribed SUMF1 mutations, c.156delC (p.C52fsX57) and c.390A>T (p.E130D). In patient fibroblasts, mRNA of the frameshift allele is undetectable. In contrast, the allele encoding FGE-E130D is expressed. FGE-E130D correctly localizes to the endoplasmic reticulum and has a very high residual molecular activity in vitro (55% of wildtype FGE); however, it is rapidly degraded. Thus, despite substantial residual enzyme activity, protein instability determines disease severity, which highlights that potential MSD treatment approaches should target protein folding and stabilization mechanisms. PMID:23321616

  18. Rapid degradation of an active formylglycine generating enzyme variant leads to a late infantile severe form of multiple sulfatase deficiency.

    PubMed

    Schlotawa, Lars; Radhakrishnan, Karthikeyan; Baumgartner, Matthias; Schmid, Regula; Schmidt, Bernhard; Dierks, Thomas; Gärtner, Jutta

    2013-09-01

    Multiple sulfatase deficiency (MSD) is a rare inborn error of metabolism affecting posttranslational activation of sulfatases by the formylglycine generating enzyme (FGE). Due to mutations in the encoding SUMF1 gene, FGE's catalytic capacity is impaired resulting in reduced cellular sulfatase activities. Both, FGE protein stability and residual activity determine disease severity and have previously been correlated with the clinical MSD phenotype. Here, we report a patient with a late infantile severe course of disease. The patient is compound heterozygous for two so far undescribed SUMF1 mutations, c.156delC (p.C52fsX57) and c.390A>T (p.E130D). In patient fibroblasts, mRNA of the frameshift allele is undetectable. In contrast, the allele encoding FGE-E130D is expressed. FGE-E130D correctly localizes to the endoplasmic reticulum and has a very high residual molecular activity in vitro (55% of wildtype FGE); however, it is rapidly degraded. Thus, despite substantial residual enzyme activity, protein instability determines disease severity, which highlights that potential MSD treatment approaches should target protein folding and stabilization mechanisms. PMID:23321616

  19. Role of C{sub 2}F{sub 4}, CF{sub 2}, and ions in C{sub 4}F{sub 8}/Ar plasma discharges under active oxide etch conditions in an inductively coupled GEC cell reactor

    SciTech Connect

    Barela, Marcos J.; Anderson, Harold M.; Oehrlein, Gottlieb S.

    2005-05-01

    Utilizing infrared diode-laser absorption spectroscopy (IRDLAS) and UV-Visible absorption spectroscopy (UV-Vis), we show that it is possible to make a near complete mass balance of etch reactants and products in a GEC inductively coupled fluorocarbon discharge while actively etching SiO{sub 2} substrates. Langmuir probe measurements were performed to measure the total ion current density. C{sub 2}F{sub 4} and CF{sub 2} are shown to be the main dissociation products in a C{sub 4}F{sub 8} plasma discharge. The C{sub 2}F{sub 4} concentration decreases as the SiO{sub 2} etching rate increases, along with CF{sub 2} and CF radicals, suggesting a role in the SiO{sub 2} etching process. The addition of Ar to the C{sub 4}F{sub 8} discharge increased the ion flux at the wafer surface, and the consumption rate of C{sub 2}F{sub 4} relative to CF{sub 2}. The increased ion flux enhanced the SiO{sub 2} etching rate, until at a very high degree of Ar dilution of C{sub 4}F{sub 8}/Ar the etching rate became neutral limited. We also monitored SiF{sub 2} using UV-Vis absorption and CO by IRDLAS. In our work we found SiF{sub 2} and CO to be the prevalent Si and C gas phase etch products for the SiO{sub 2} etching process.

  20. Molecular Characterization of an Ice Nucleation Protein Variant (InaQ) from Pseudomonas syringae and the Analysis of Its Transmembrane Transport Activity in Escherichia coli

    PubMed Central

    Li, Qianqian; Yan, Qi; Chen, Jinsi; He, Yan; Wang, Jing; Zhang, Hongxing; Yu, Ziniu; Li, Lin

    2012-01-01

    The ice nucleation protein (INP) of Pseudomonas syringae has gained scientific interest not only because of its pathogenicity of foliar necroses but also for its wide range of potential applications, such as in snow making, frozen food preparation, and surface-display system development. However, studies on the transport activity of INP remain lacking. In the present study, a newly identified INP-gene variant, inaQ, from a P. syringae MB03 strain was cloned. Its structural domains, signal sequences, and the hydrophilicity or hydrophobicity of each domain, were then characterized. The deduced amino acid sequence of InaQ shares similar protein domains with three P. syringae INPs, namely, InaK, InaZ, and InaV, which were identified as an N-terminal domain, a central repeating domain, and a C-terminal domain. The expression of the full-length InaQ and of various truncated variants was induced in Escherichia coli to analyze their transmembrane transport and surface-binding activities, while using the green fluorescence protein (GFP) as the fusion partner. With two transmembrane segments and a weak secretion signal, the N-terminal domain (InaQ-N) alone was found to be responsible for the transport process as well as for the binding to the outer membrane, whereas the C-terminal region was nonfunctional in protein transport. Increased membrane transport and surface-binding capacities were induced by a low isopropyl-β-D-thiogalactoside concentration (0.1 mmol/l) but not by culture temperatures (15 ºC to 37 ºC). Furthermore, by constructing the GFP-fused proteins with a single InaQ-N, as well as two and three tandemly aligned InaQ-N molecules, the transport and membrane-binding activities of these proteins were compared using Western blot analysis, immmunofluorescence microscopy, and assays of the GFP specific fluorescence intensity of subcellular fractions and flow cytometry, which showed that the increase of InaQ-N repeats resulted in a coordinated increase of the

  1. Comparison of conventional K-Ar and 40Ar/39Ar dating of young mafic volcanic rocks

    USGS Publications Warehouse

    Lanphere, M.A.

    2000-01-01

    K-Ar and 40Ar/39Ar ages have been measured on nine mafic volcanic rocks younger than 1 myr from the Snake River Plain (Idaho), Mount Adams (Washington), and Crater Lake (Oregon). The K-Ar ages were calculated from Ar measurements made by isotope dilution and K2O measurements by flame photometry. The 40Ar/39Ar ages are incremental-heating experiments using a low-blank resistance-heated furnace. The results indicate that high-quality ages can be measured on young, mafic volcanic rocks using either the K-Ar or the 40Ar/39Ar technique. The precision of an 40Ar/39Ar plateau age generally is better than the precision of a K-Ar age because the plateau age is calculated by pooling the ages of several gas increments. The precision of a plateau age generally is better than the precision of an isotope correlation (isochron) age for the same sample. For one sample the intercept of the isochron yielded an 40Ar/36Ar value significantly different from the atmospheric value of 295.5. Recalculation of increment ages using the isochron intercept for the composition of nonradiogenic Ar in the sample resulted in much better agreement of ages for this sample. The results of this study also indicate that, given suitable material and modern equipment, precise K-Ar and 40Ar/39Ar ages can be measured on volcanic rocks as young as the latest Pleistocene, and perhaps even the Holocene.

  2. Impaired coactivator activity of the Gly{sub 482} variant of peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) on mitochondrial transcription factor A (Tfam) promoter

    SciTech Connect

    Choi, Yon-Sik; Hong, Jung-Man; Lim, Sunny; Ko, Kyung Soo; Pak, Youngmi Kim . E-mail: ymkimpak@amc.seoul.kr

    2006-06-09

    Mitochondrial dysfunction may cause diabetes or insulin resistance. Peroxisome proliferation-activated receptor-{gamma} (PPAR-{gamma}) coactivator-1 {alpha} (PGC-1{alpha}) increases mitochondrial transcription factor A (Tfam) resulting in mitochondrial DNA content increase. An association between a single nucleotide polymorphism (SNP), G1444A(Gly482Ser), of PGC-1{alpha} coding region and insulin resistance has been reported in some ethnic groups. In this study, we investigated whether a change of glycine to serine at codon 482 of PGC-1{alpha} affected the Tfam promoter activity. The cDNA of PGC-1{alpha} variant bearing either glycine or serine at 482 codon was transfected into Chang human hepatocyte cells. The PGC-1{alpha} protein bearing glycine had impaired coactivator activity on Tfam promoter-mediated luciferase. We analyzed the PGC-1{alpha} genotype G1444A and mitochondrial DNA (mtDNA) copy number from 229 Korean leukocyte genomic DNAs. Subjects with Gly/Gly had a 20% lower amount of peripheral blood mtDNA than did subjects with Gly/Ser and Ser/Ser (p < 0.05). No correlation was observed between diabetic parameters and PGC-1{alpha} genotypes in Koreans. These results suggest that PGC-1{alpha} variants with Gly/Gly at 482nd amino acid may impair the Tfam transcription, a regulatory function of mitochondrial biogenesis, resulting in dysfunctional mtDNA replication.

  3. Evaluation of a multifunctional staphylokinase variant with thrombin inhibition and antiplatelet aggregation activities produced from salt-inducible E. coli GJ1158.

    PubMed

    Kumar, Anmol; Pulicherla, Krishna Kanth; Mayuren, Candasamy; Kotra, Seetharam; Rao, Krothapalli Rajasurya Sambasiva

    2013-10-01

    Reocclusion is one of the major root causes for secondary complications that arise during thrombolytic therapy. A multifunctional staphylokinase variant SRH (staphylokinase (SAK) linked with tripeptide RGD and didecapeptide Hirulog) with antiplatelet and antithrombin activities in addition to clot specific thrombolytic function, was developed to address the reocclusion problem. We preferred to use Escherichia coli GJ1158 as the host in this study for economic production of SRH by osmotic (0.3 mol/L sodium chloride) induction, to overcome the problems associated with the yeast expression system. The therapeutic potential of SRH was evaluated in the murine model of vascular thrombosis. The SAK protein (1 mg/kg body mass) and SRH protein (1 mg/kg and 2 mg/kg) were administered intravenously to the different treatment groups. The results have shown a dose-dependent antithrombotic effect in carrageenan-induced mouse tail thrombosis. The thrombin time, activated partial thromboplastin time, and prothrombin time were significantly prolonged (p < 0.05) in the SRH-infused groups. Moreover, SRH inhibited platelet aggregation in a dose-dependent manner (p < 0.05), while the bleeding time was significantly (p < 0.05) prolonged. All of these results inferred that the osmotically produced multifunctional fusion protein SRH (SAK-RGD-Hirulog) is a promising thrombolytic agent, and one which sustained its multifunctionality in the animal models. PMID:24144055

  4. Structural characteristics of two wheat histone H2A genes encoding distinct types of variants and functional differences in their promoter activity.

    PubMed

    Huh, G H; Nakayama, T; Meshi, T; Iwabuchi, M

    1997-03-01

    To investigate the regulation of plant histone H2A gene expression, we isolated two H2A genes (TH254 and TH274) from wheat, which encode two variants of H2A. Both genes had an intron in the coding region. In the promoters, some characteristic sequences, such as Oct and Nona motifs, which are conserved among plant histone genes, were located in a short region (about 120 bp) upstream from the putative TATA box. Transient expression analyses of promoter activity with H2A-GUS fusion genes using tobacco protoplasts revealed novel types of positive cis-acting sequences in the TH254 promoter: a direct repeat of a 13 bp sequence (AGTTACATTATTG) and a stretch composed of an AT-rich sequence (ATATAGAAAATTAAAA) and a G-box (CACGTG). Quantitative S1 assay of the mRNA amounts from the TH254/GUS and TH274/GUS chimeric genes in stably transformed and cell cycle-synchronized tobacco cell lines showed that the promoters of both genes contained at least one cis-acting element responsible for S phase-specific expression. Histochemical analysis of transgenic tobacco plants carrying the chimeric genes showed that the promoters of the two H2A genes were active in developing seedlings and flower organs but were regulated in a different manner. PMID:9106503

  5. A 3' untranslated region variant in FMR1 eliminates neuronal activity-dependent translation of FMRP by disrupting binding of the RNA-binding protein HuR.

    PubMed

    Suhl, Joshua A; Muddashetty, Ravi S; Anderson, Bart R; Ifrim, Marius F; Visootsak, Jeannie; Bassell, Gary J; Warren, Stephen T

    2015-11-24

    Fragile X syndrome is a common cause of intellectual disability and autism spectrum disorder. The gene underlying the disorder, fragile X mental retardation 1 (FMR1), is silenced in most cases by a CGG-repeat expansion mutation in the 5' untranslated region (UTR). Recently, we identified a variant located in the 3'UTR of FMR1 enriched among developmentally delayed males with normal repeat lengths. A patient-derived cell line revealed reduced levels of endogenous fragile X mental retardation protein (FMRP), and a reporter containing a patient 3'UTR caused a decrease in expression. A control reporter expressed in cultured mouse cortical neurons showed an expected increase following synaptic stimulation that was absent when expressing the patient reporter, suggesting an impaired response to neuronal activity. Mobility-shift assays using a control RNA detected an RNA-protein interaction that is lost with the patient RNA, and HuR was subsequently identified as an associated protein. Cross-linking immunoprecipitation experiments identified the locus as an in vivo target of HuR, supporting our in vitro findings. These data suggest that the disrupted interaction of HuR impairs activity-dependent translation of FMRP, which may hinder synaptic plasticity in a clinically significant fashion. PMID:26554012

  6. Expression of a constitutively active nitrate reductase variant in tobacco reduces tobacco-specific nitrosamine accumulation in cured leaves and cigarette smoke.

    PubMed

    Lu, Jianli; Zhang, Leichen; Lewis, Ramsey S; Bovet, Lucien; Goepfert, Simon; Jack, Anne M; Crutchfield, James D; Ji, Huihua; Dewey, Ralph E

    2016-07-01

    Burley tobaccos (Nicotiana tabacum) display a nitrogen-use-deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco-specific nitrosamines (TSNAs). Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen-assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase (NR) dramatically decreased free nitrate levels in the leaves. Field-grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well-documented animal carcinogens found in tobacco products. PMID:26800860

  7. Multi-Scale Soil Moisture Monitoring and Modeling at ARS Watersheds for NASA's Soil Moisture Active Passive (SMAP) Calibration/Validation Mission

    NASA Astrophysics Data System (ADS)

    Coopersmith, E. J.; Cosh, M. H.

    2014-12-01

    NASA's SMAP satellite, launched in November of 2014, produces estimates of average volumetric soil moisture at 3, 9, and 36-kilometer scales. The calibration and validation process of these estimates requires the generation of an identically-scaled soil moisture product from existing in-situ networks. This can be achieved via the integration of NLDAS precipitation data to perform calibration of models at each ­in-situ gauge. In turn, these models and the gauges' volumetric estimations are used to generate soil moisture estimates at a 500m scale throughout a given test watershed by leveraging, at each location, the gauge-calibrated models deemed most appropriate in terms of proximity, calibration efficacy, soil-textural similarity, and topography. Four ARS watersheds, located in Iowa, Oklahoma, Georgia, and Arizona are employed to demonstrate the utility of this approach. The South Fork watershed in Iowa represents the simplest case - the soil textures and topography are relative constants and the variability of soil moisture is simply tied to the spatial variability of precipitation. The Little Washita watershed in Oklahoma adds soil textural variability (but remains topographically simple), while the Little River watershed in Georgia incorporates topographic classification. Finally, the Walnut Gulch watershed in Arizona adds a dense precipitation network to be employed for even finer-scale modeling estimates. Results suggest RMSE values at or below the 4% volumetric standard adopted for the SMAP mission are attainable over the desired spatial scales via this integration of modeling efforts and existing in-situ networks.

  8. A Common Variant at the 14q32 Endometrial Cancer Risk Locus Activates AKT1 through YY1 Binding.

    PubMed

    Painter, Jodie N; Kaufmann, Susanne; O'Mara, Tracy A; Hillman, Kristine M; Sivakumaran, Haran; Darabi, Hatef; Cheng, Timothy H T; Pearson, John; Kazakoff, Stephen; Waddell, Nicola; Hoivik, Erling A; Goode, Ellen L; Scott, Rodney J; Tomlinson, Ian; Dunning, Alison M; Easton, Douglas F; French, Juliet D; Salvesen, Helga B; Pollock, Pamela M; Thompson, Deborah J; Spurdle, Amanda B; Edwards, Stacey L

    2016-06-01

    A recent meta-analysis of multiple genome-wide association and follow-up endometrial cancer case-control datasets identified a novel genetic risk locus for this disease at chromosome 14q32.33. To prioritize the functional SNP(s) and target gene(s) at this locus, we employed an in silico fine-mapping approach using genotyped and imputed SNP data for 6,608 endometrial cancer cases and 37,925 controls of European ancestry. Association and functional analyses provide evidence that the best candidate causal SNP is rs2494737. Multiple experimental analyses show that SNP rs2494737 maps to a silencer element located within AKT1, a member of the PI3K/AKT/MTOR intracellular signaling pathway activated in endometrial tumors. The rs2494737 risk A allele creates a YY1 transcription factor-binding site and abrogates the silencer activity in luciferase assays, an effect mimicked by transfection of YY1 siRNA. Our findings suggest YY1 is a positive regulator of AKT1, mediating the stimulatory effects of rs2494737 increasing endometrial cancer risk. Identification of an endometrial cancer risk allele within a member of the PI3K/AKT signaling pathway, more commonly activated in tumors by somatic alterations, raises the possibility that well tolerated inhibitors targeting this pathway could be candidates for evaluation as chemopreventive agents in individuals at high risk of developing endometrial cancer. PMID:27259051

  9. A novel human aquaporin-4 splice variant exhibits a dominant-negative activity: a new mechanism to regulate water permeability

    PubMed Central

    De Bellis, Manuela; Pisani, Francesco; Mola, Maria Grazia; Basco, Davide; Catalano, Francesco; Nicchia, Grazia Paola; Svelto, Maria; Frigeri, Antonio

    2014-01-01

    Two major isoforms of aquaporin-4 (AQP4) have been described in human tissue. Here we report the identification and functional analysis of an alternatively spliced transcript of human AQP4, AQP4-Δ4, that lacks exon 4. In transfected cells AQP4-Δ4 is mainly retained in the endoplasmic reticulum and shows no water transport properties. When AQP4-Δ4 is transfected into cells stably expressing functional AQP4, the surface expression of the full-length protein is reduced. Furthermore, the water transport activity of the cotransfectants is diminished in comparison to transfectants expressing only AQP4. The observed down-regulation of both the expression and water channel activity of AQP4 is likely to originate from a dominant-negative effect caused by heterodimerization between AQP4 and AQP4-Δ4, which was detected in coimmunoprecipitation studies. In skeletal muscles, AQP4-Δ4 mRNA expression inversely correlates with the level of AQP4 protein and is physiologically associated with different types of skeletal muscles. The expression of AQP4-Δ4 may represent a new regulatory mechanism through which the cell-surface expression and therefore the activity of AQP4 can be physiologically modulated. PMID:24356448

  10. Genetic Variants in the STMN1 Transcriptional Regulatory Region Affect Promoter Activity and Fear Behavior in English Springer Spaniels

    PubMed Central

    Zhang, Hanying; Xu, Yinxue

    2016-01-01

    Stathmin 1 (STMN1) is a neuronal growth-associated protein that is involved in microtubule dynamics and plays an important role in synaptic outgrowth and plasticity. Given that STMN1 affects fear behavior, we hypothesized that genetic variations in the STMN1 transcriptional regulatory region affect gene transcription activity and control fear behavior. In this study, two single nucleotide polymorphisms (SNPs), g. -327 A>G and g. -125 C>T, were identified in 317 English Springer Spaniels. A bioinformatics analysis revealed that both were loci located in the canine STMN1 putative promoter region and affected transcription factor binding. A statistical analysis revealed that the TT genotype at g.-125 C>T produced a significantly greater fear level than that of the CC genotype (P < 0.05). Furthermore, the H4H4 (GTGT) haplotype combination was significantly associated with canine fear behavior (P < 0.01). Using serially truncated constructs of the STMN1 promoters and the luciferase reporter, we found that a 395 bp (−312 nt to +83 nt) fragment constituted the core promoter region. The luciferase assay also revealed that the H4 (GT) haplotype promoter had higher activity than that of other haplotypes. Overall, our results suggest that the two SNPs in the canine STMN1 promoter region could affect canine fear behavior by altering STMN1 transcriptional activity. PMID:27390866

  11. Subtype-selective activation of K(v)7 channels by AaTXKβ₂₋₆₄, a novel toxin variant from the Androctonus australis scorpion venom.

    PubMed

    Landoulsi, Zied; Miceli, Francesco; Palmese, Angelo; Amoresano, Angela; Marino, Gennaro; El Ayeb, Mohamed; Taglialatela, Maurizio; Benkhalifa, Rym

    2013-11-01

    K(v)7.4 channel subunits are expressed in central auditory pathways and in inner ear sensory hair cells and skeletal and smooth muscle cells. Openers of K(v)7.4 channels have been suggested to improve hearing loss, systemic or pulmonary arterial hypertension, urinary incontinence, gastrointestinal and neuropsychiatric diseases, and skeletal muscle disorders. Scorpion venoms are a large source of peptides active on K⁺ channels. Therefore, we have optimized a combined purification/screening procedure to identify specific modulator(s) of K(v)7.4 channels from the venom of the North African scorpion Androctonus australis (Aa). We report the isolation and functional characterization of AaTXKβ₂₋₆₄, a novel variant of AaTXKβ₁₋₆₄, in a high-performance liquid chromatography fraction from Aa venom (named P8), which acts as the first peptide activator of K(v)7.4 channels. In particular, in both Xenopus oocytes and mammalian Chinese hamster ovary cells, AaTXKβ₂₋₆₄, but not AaTXKβ₁₋₆₄, hyperpolarized the threshold voltage of current activation and increased the maximal currents of heterologously expressed K(v)7.4 channels. AaTXKβ₂₋₆₄ also activated K(v)7.3, K(v)7.2/3, and K(v)7.5/3 channels, whereas homomeric K(v)1.1, K(v)7.1, and K(v)7.2 channels were unaffected. We anticipate that these results may prove useful in unraveling the novel biologic roles of AaTXKβ₂₋₆₄-sensitive K(v)7 channels and developing novel pharmacologic tools that allow subtype-selective targeting of K(v)7 channels. PMID:24019223

  12. Expression and phosphorylation of the AS160_v2 splice variant supports GLUT4 activation and the Warburg effect in multiple myeloma

    PubMed Central

    2013-01-01

    Background Multiple myeloma (MM) is a fatal plasma cell malignancy exhibiting enhanced glucose consumption associated with an aerobic glycolytic phenotype (i.e., the Warburg effect). We have previously demonstrated that myeloma cells exhibit constitutive plasma membrane (PM) localization of GLUT4, consistent with the dependence of MM cells on this transporter for maintenance of glucose consumption rates, proliferative capacity, and viability. The purpose of this study was to investigate the molecular basis of constitutive GLUT4 plasma membrane localization in MM cells. Findings We have elucidated a novel mechanism through which myeloma cells achieve constitutive GLUT4 activation involving elevated expression of the Rab-GTPase activating protein AS160_v2 splice variant to promote the Warburg effect. AS160_v2-positive MM cell lines display constitutive Thr642 phosphorylation, known to be required for inactivation of AS160 Rab-GAP activity. Importantly, we show that enforced expression of AS160_v2 is required for GLUT4 PM translocation and activation in these select MM lines. Furthermore, we demonstrate that ectopic expression of a full-length, phospho-deficient AS160 mutant is sufficient to impair constitutive GLUT4 cell surface residence, which is characteristic of MM cells. Conclusions This is the first study to tie AS160 de-regulation to increased glucose consumption rates and the Warburg effect in cancer. Future studies investigating connections between the insulin/IGF-1/AS160_v2/GLUT4 axis and FDG-PET positivity in myeloma patients are warranted and could provide rationale for therapeutically targeting this pathway in MM patients with advanced disease. PMID:24280290

  13. No Interactions Between Previously Associated 2-Hour Glucose Gene Variants and Physical Activity or BMI on 2-Hour Glucose Levels

    PubMed Central

    Scott, Robert A.; Chu, Audrey Y.; Grarup, Niels; Manning, Alisa K.; Hivert, Marie-France; Shungin, Dmitry; Tönjes, Anke; Yesupriya, Ajay; Barnes, Daniel; Bouatia-Naji, Nabila; Glazer, Nicole L.; Jackson, Anne U.; Kutalik, Zoltán; Lagou, Vasiliki; Marek, Diana; Rasmussen-Torvik, Laura J.; Stringham, Heather M.; Tanaka, Toshiko; Aadahl, Mette; Arking, Dan E.; Bergmann, Sven; Boerwinkle, Eric; Bonnycastle, Lori L.; Bornstein, Stefan R.; Brunner, Eric; Bumpstead, Suzannah J.; Brage, Soren; Carlson, Olga D.; Chen, Han; Chen, Yii-Der Ida; Chines, Peter S.; Collins, Francis S.; Couper, David J.; Dennison, Elaine M.; Dowling, Nicole F.; Egan, Josephine S.; Ekelund, Ulf; Erdos, Michael R.; Forouhi, Nita G.; Fox, Caroline S.; Goodarzi, Mark O.; Grässler, Jürgen; Gustafsson, Stefan; Hallmans, Göran; Hansen, Torben; Hingorani, Aroon; Holloway, John W.; Hu, Frank B.; Isomaa, Bo; Jameson, Karen A.; Johansson, Ingegerd; Jonsson, Anna; Jørgensen, Torben; Kivimaki, Mika; Kovacs, Peter; Kumari, Meena; Kuusisto, Johanna; Laakso, Markku; Lecoeur, Cécile; Lévy-Marchal, Claire; Li, Guo; Loos, Ruth J.F.; Lyssenko, Valeri; Marmot, Michael; Marques-Vidal, Pedro; Morken, Mario A.; Müller, Gabriele; North, Kari E.; Pankow, James S.; Payne, Felicity; Prokopenko, Inga; Psaty, Bruce M.; Renström, Frida; Rice, Ken; Rotter, Jerome I.; Rybin, Denis; Sandholt, Camilla H.; Sayer, Avan A.; Shrader, Peter; Schwarz, Peter E.H.; Siscovick, David S.; Stančáková, Alena; Stumvoll, Michael; Teslovich, Tanya M.; Waeber, Gérard; Williams, Gordon H.; Witte, Daniel R.; Wood, Andrew R.; Xie, Weijia; Boehnke, Michael; Cooper, Cyrus; Ferrucci, Luigi; Froguel, Philippe; Groop, Leif; Kao, W.H. Linda; Vollenweider, Peter; Walker, Mark; Watanabe, Richard M.; Pedersen, Oluf; Meigs, James B.; Ingelsson, Erik; Barroso, Inês; Florez, Jose C.; Franks, Paul W.; Dupuis, Josée; Wareham, Nicholas J.; Langenberg, Claudia

    2012-01-01

    Gene–lifestyle interactions have been suggested to contribute to the development of type 2 diabetes. Glucose levels 2 h after a standard 75-g glucose challenge are used to diagnose diabetes and are associated with both genetic and lifestyle factors. However, whether these factors interact to determine 2-h glucose levels is unknown. We meta-analyzed single nucleotide polymorphism (SNP) × BMI and SNP × physical activity (PA) interaction regression models for five SNPs previously associated with 2-h glucose levels from up to 22 studies comprising 54,884 individuals without diabetes. PA levels were dichotomized, with individuals below the first quintile classified as inactive (20%) and the remainder as active (80%). BMI was considered a continuous trait. Inactive individuals had higher 2-h glucose levels than active individuals (β = 0.22 mmol/L [95% CI 0.13–0.31], P = 1.63 × 10−6). All SNPs were associated with 2-h glucose (β = 0.06–0.12 mmol/allele, P ≤ 1.53 × 10−7), but no significant interactions were found with PA (P > 0.18) or BMI (P ≥ 0.04). In this large study of gene–lifestyle interaction, we observed no interactions between genetic and lifestyle factors, both of which were associated with 2-h glucose. It is perhaps unlikely that top loci from genome-wide association studies will exhibit strong subgroup-specific effects, and may not, therefore, make the best candidates for the study of interactions. PMID:22415877

  14. New insights into the mechanism of nickel insertion into carbon monoxide dehydrogenase: analysis of Rhodospirillum rubrum carbon monoxide dehydrogenase variants with substituted ligands to the [Fe3S4] portion of the active-site C-cluster.

    PubMed

    Jeon, Won Bae; Singer, Steven W; Ludden, Paul W; Rubio, Luis M

    2005-12-01

    Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum catalyzes the oxidation of CO to CO2. A unique [NiFe4S4] cluster, known as the C-cluster, constitutes the active site of the enzyme. When grown in Ni-deficient medium R. rubrum accumulates a Ni-deficient apo form of CODH that is readily activated by Ni. It has been previously shown that activation of apo-CODH by Ni is a two-step process involving the rapid formation of an inactive apo-CODH*Ni complex prior to conversion to the active holo-CODH. We have generated CODH variants with substitutions in cysteine residues involved in the coordination of the [Fe3S4] portion of the C-cluster. Analysis of the variants suggests that the cysteine residues at positions 338, 451, and 481 are important for CO oxidation activity catalyzed by CODH but not for Ni binding to the C-cluster. C451S CODH is the only new variant that retains residual CO oxidation activity. Comparison of the kinetics and pH dependence of Ni activation of the apo forms of wild-type, C451S, and C531A CODH allowed us to develop a model for Ni insertion into the C-cluster of CODH in which Ni reversibly binds to the C-cluster and subsequently coordinates Cys531 in the rate-determining step. PMID:16283394

  15. Histone variants and epigenetics.

    PubMed

    Henikoff, Steven; Smith, M Mitchell

    2015-01-01

    Histones package and compact DNA by assembling into nucleosome core particles. Most histones are synthesized at S phase for rapid deposition behind replication forks. In addition, the replacement of histones deposited during S phase by variants that can be deposited independently of replication provide the most fundamental level of chromatin differentiation. Alternative mechanisms for depositing different variants can potentially establish and maintain epigenetic states. Variants have also evolved crucial roles in chromosome segregation, transcriptional regulation, DNA repair, and other processes. Investigations into the evolution, structure, and metabolism of histone variants provide a foundation for understanding the participation of chromatin in important cellular processes and in epigenetic memory. PMID:25561719

  16. Variants of glutathione s-transferase pi 1 exhibit differential enzymatic activity and inhibition by heavy metals

    PubMed Central

    Goodrich, Jaclyn M.; Basu, Niladri

    2012-01-01

    Nonsynonymous single nucleotide polymorphisms in glutathione s-transferase pi 1 (GSTP1; Ile/Val 105, Ala/Val 114) have been associated with altered toxicant metabolism in epidemiological cohorts. We explored the impact of GSTP1 genotype on enzyme kinetics and heavy metal inhibition in vitro. Four GSTP1 allozymes (105/114: Ile/Ala, Val/Ala, Ile/Val, Val/Val) were expressed in and purified from E. coli. Enzyme activity assays quantifying the rate of glutathione conjugation with 1-chloro-2,4-dinitrobenzene (CDNB) revealed significant differences in kinetic parameters depending on genotype (p<0.01). Allozymes with Ile105 had better catalytic efficiency and greater affinity for CDNB (mean ±SEM: Ile105 Ala114 Km= 0.33±0.07 mM vs. Val105 Ala114 Km=1.15±0.07 mM). Inhibition of GSTP1 activity by heavy metals was assessed following treatment with mercury (inorganic- HgCl2, methylmercury- MeHg), selenium, cadmium, lead, arsenic, and manganese. All allozymes were inhibited by HgCl2 (IC50 range: 24.1–172 μM), MeHg (93.9–480 μM), and selenium (43.7–62.8 μM). Genotype significantly influenced the potency of mercury with GSTP1 Ile105 Val114 the least sensitive and Val105 Ala114 the most sensitive to inhibition by HgCl2 and MeHg. Overall, genotype of two nonsynonymous polymorphisms in GSTP1 influenced enzyme kinetics pertaining to an electrophilic substrate and inhibition by two mercury species. PMID:22401947

  17. Neutron-hole states in 45Ar from 1H(46Ar, d) 45Ar reactions

    NASA Astrophysics Data System (ADS)

    Lu, F.; Lee, Jenny; Tsang, M. B.; Bazin, D.; Coupland, D.; Henzl, V.; Henzlova, D.; Kilburn, M.; Lynch, W. G.; Rogers, A. M.; Sanetullaev, A.; Sun, Z. Y.; Youngs, M.; Charity, R. J.; Sobotka, L. G.; Famiano, M.; Hudan, S.; Horoi, M.; Ye, Y. L.

    2013-07-01

    To improve the effective interactions in the pf shell, it is important to measure the single-particle and single-hole states near the N = 28 shell gap. In this paper, the neutron spectroscopic factors of hole states from the unstable neutron-rich 45Ar (Z = 18,N = 27) nucleus have been studied using the 1H(46Ar,d) 45Ar transfer reaction in inverse kinematics. Comparison of our results with the particle states of 45Ar produced in 2H(44Ar, p) 45Ar reaction shows that the two reactions populate states with different angular momenta. Using the angular distributions, we are able to confirm the spin assignments of four low-lying states of 45Ar. These are the ground state (f7/2), the first-excited state (p3/2), and the s1/2 and d3/2 states. While large basis shell-model predictions describe spectroscopic properties of the ground and p3/2 states very well, they fail to describe the s1/2 and d3/2 hole states.

  18. Pulsed discharge production Ar* metastables

    NASA Astrophysics Data System (ADS)

    Han, Jiande; Heaven, Michael C.; Emmons, Daniel; Perram, Glen P.; Weeks, David E.; Bailey, William F.

    2016-03-01

    The production of relatively high densities of Ar* metastables (>1012 cm-3) in Ar/He mixtures, at total pressures close to 1 atm, is essential for the efficient operation of an optically pumped Ar* laser. We have used emission spectroscopy and diode laser absorption spectroscopy measurements to observe the production and decay of Ar* in a parallel plate pulsed discharge. With discharge pulses of 1 μs duration we find that metastable production is dominated by processes occurring within the first 100 ns of the gas break-down. Application of multiple, closely spaced discharge pulses yields insights concerning conditions that favor metastable production. This information has been combined with time-resolved measurements of voltage and current. The experimental results and preliminary modeling of the discharge kinetics are presented.

  19. A Δ38 Deletion Variant of Human Transketolase as a Model of Transketolase-Like Protein 1 Exhibits No Enzymatic Activity

    PubMed Central

    Schneider, Stefan; Lüdtke, Stefan; Schröder-Tittmann, Kathrin; Wechsler, Cindy; Meyer, Danilo; Tittmann, Kai

    2012-01-01

    Besides transketolase (TKT), a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1) has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a “pseudo-TKTL1” Δ38 deletion variant of human TKT (TKTΔ38) as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism. PMID:23118983

  20. Variants in Complement Factor H and Complement Factor H-Related Protein Genes, CFHR3 and CFHR1, Affect Complement Activation in IgA Nephropathy.

    PubMed

    Zhu, Li; Zhai, Ya-Ling; Wang, Feng-Mei; Hou, Ping; Lv, Ji-Cheng; Xu, Da-Min; Shi, Su-Fang; Liu, Li-Jun; Yu, Feng; Zhao, Ming-Hui; Novak, Jan; Gharavi, Ali G; Zhang, Hong

    2015-05-01

    Complement activation is common in patients with IgA nephropathy (IgAN) and associated with disease severity. Our recent genome-wide association study of IgAN identified susceptibility loci on 1q32 containing the complement regulatory protein-encoding genes CFH and CFHR1-5, with rs6677604 in CFH as the top single-nucleotide polymorphism and CFHR3-1 deletion (CFHR3-1∆) as the top signal for copy number variation. In this study, to explore the clinical effects of variation in CFH, CFHR3, and CFHR1 on IgAN susceptibility and progression, we enrolled two populations. Group 1 included 1178 subjects with IgAN and available genome-wide association study data. Group 2 included 365 subjects with IgAN and available clinical follow-up data. In group 1, rs6677604 was associated with mesangial C3 deposition by genotype-phenotype correlation analysis. In group 2, we detected a linkage between the rs6677604-A allele and CFHR3-1∆ and found that the rs6677604-A allele was associated with higher serum levels of CFH and lower levels of the complement activation split product C3a. Furthermore, CFH levels were positively associated with circulating C3 levels and negatively associated with mesangial C3 deposition. Moreover, serum levels of the pathogenic galactose-deficient glycoform of IgA1 were also associated with the degree of mesangial C3 deposition in patients with IgAN. Our findings suggest that genetic variants in CFH, CFHR3, and CFHR1 affect complement activation and thereby, predispose patients to develop IgAN. PMID:25205734

  1. Excitation energies with cost-reduced variant of the active-space EOMCCSDT method: the EOMCCSDt-(3) over-bar approach

    SciTech Connect

    Hu, Hanshi; Kowalski, Karol

    2013-11-12

    In this paper we discuss the performance of the several simplified variants of equation-of-motion coupled cluster method (EOMCC) with iterative inclusion of singles, doubles and active-space triples (EOMCCSDt). In particular, we explore simplified EOMCCSDt approaches which enable one to generate the triply excited amplitudes in on-the-fly manner. The original EOMCCSDt formulation has already demonstrated a great success in encapsulating the most important excited-state correlation effects due to triples. In analogy to the original EOMCCSDT formulation, the proposed approach can by-pass the typical bottlenecks associated with the need for storing triply-excited amplitudes. In this paper, we illustrate the performance of several approximate EOMCCSDt methods, named EOMCCSDt-3 and EOMCCSdt-3x, on typical benchmark systems including C2, N2, and the ozone molecules. These new methods yield excitation energies close to the EOMCCSDt ones. The extrapolation of excitation energies for basis sets ranging from cc-pVDZ to cc-pV6Z for N2 and C2 shows very good convergence to the experimental results for states dominated by single excitations.

  2. ASP2408 and ASP2409, novel CTLA4-Ig variants with CD86-selective ligand binding activity and improved immunosuppressive potency, created by directed evolution.

    PubMed

    Oshima, Shinsuke; Karrer, Erik E; Paidhungat, Madan M; Neighbors, Margaret; Chapin, Steven J; Fan, Rong A; Reed, Margaret A; Wu, Kuoting; Wong, Clifford; Chen, Yonghong; Whitlow, Marc; Anderson, Francisco A; Bam, Rujuta A; Zhang, Qian; Larsen, Brent R; Viswanathan, Sridhar; Devens, Bruce H; Bass, Steven H; Higashi, Yasuyuki

    2016-05-01

    The CTLA4-Ig therapeutics abatacept and belatacept inhibit CD28-mediated T cell activation by binding CD80 (B7-1) and CD86 (B7-2) co-stimulatory ligands. Both compounds preferentially bind CD80, yet CD86 has been implicated as the dominant co-stimulatory ligand. Using directed evolution methods, novel CTLA4-Ig variants were created with selective CD86 binding affinity, a property that confers increased immunosuppressive potency and potentially improved efficacy and safety profiles. Relative to abatacept (wild-type CTLA4-Ig), ASP2408 and ASP2409 have 83-fold and 220-fold enhanced binding affinity to CD86 while retaining 1.5-fold and 5.6-fold enhanced binding affinity to CD80, respectively. Improvements in CD86 binding affinity correlates with increased immunosuppressive potencyin vitroandin vivo Our results highlight the power of directed evolution methods to obtain non-intuitive protein engineering solutions and represent the first examples of CD86-selective CTLA4-Ig compounds that have entered clinical trials. PMID:26968452

  3. Effect of four classes of herbicides on growth and acetolactate-synthase activity in several variants of Arabidopsis thaliana.

    PubMed

    Mourad, G; King, J

    1992-11-01

    We have isolated a triazolopyrimidine-resistant mutant csrl-2, of Arabidopsis thaliana (L.) Heynh. Here, we compare csrl-2 with the previously isolated mutants csrl and csr1-1, and with wild-type Arabidopsis for responses to members of four classes of herbicides, namely, sulfonylureas, triazolopyrimidines, imidazolinones, and pyrimidyl-oxy-benzoates. Two separable herbicide binding sites have been identified previously on the protein of acetolactate synthase (ALS). Here, the mutation giving rise to csrl, originating in a coding sequence towards the 5' end of the ALS gene, and that in csrl-2, affected the inhibitory action on growth and ALS activity of sulfonylurea and triazolopyrimidine herbicides but not that of the imidazolinones or pyrimidyl-oxybenzoates. The other mutation, in csrl-1, originating in a coding sequence towards the 3' end of the ALS gene, affected the inhibitory action of imidazolinones and pyrimidyl-oxy-benzoates but not that of the sulfonylureas or triazolopyrimidines. Additional, stimulatory effects of some of these herbicides on growth of seedlings was unrelated to their effect on their primary target, ALS. The conclusion from these observations is that one of the two previously identified herbicide-binding sites may bind sulfonylureas and triazolopyrimidines while the other may bind imidazolinones and pyrimidyl-oxy-benzoates within a herbicide-binding domain on the ALS enzyme. Such a comparative study using near-isogenic mutants from the same species allows not only the further definition of the domain of herbicide binding on ALS but also could aid investigation of the relationship between herbicide-, substrate-, and allosteric-binding sites on this enzyme.This research was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to J.K. PMID:24178380

  4. Leptin and Leptin Receptor Genetic Variants Associate with Habitual Physical Activity and the Arm Body Composition Response to Resistance Training

    PubMed Central

    Walsh, S; Haddad, CJ; Kostek, MA; Angelopoulos, TJ; Clarkson, PM; Gordon, PM; Moyna, NM; Visich, PS; Zoeller, RF; Seip, RL; Bilbie, S; Thompson, PD; Devaney, J; Gordish-Dressman, H; Hoffman, EP; Price, Thomas B.; Pescatello, LS

    2012-01-01

    PURPOSE We investigated the influence of Leptin (LEP) and leptin receptor (LEPR) SNPs on habitual physical activity (PA) and body composition response to a unilateral, upper body resistance training (RT) program. METHODS European-derived American volunteers (men=111, women=131, 23.4±5.4 yr, 24.4±4.6 kg·m−2) were genotyped for LEP 19 G>A (rs2167270), and LEPR 326 A>G (rs1137100), 668 A>G (rs1137101), 3057 G>A (rs1805096), and 1968 G>C (rs8179183). They completed the Paffenbarger PA Questionnaire. Arm muscle and subcutaneous fat volumes were measured before and after 12wk of supervised RT with MRI. Multivariate and repeated measures ANCOVA tested differences among phenotypes by genotype and gender with age and body mass index as covariates. RESULTS Adults with the LEP 19 GG genotype reported more kcal/wk in vigorous intensity PA (1273.3±176.8, p=0.017) and sports/recreation (1922.8±226.0, p<0.04) than A allele carriers (718.0±147.2, 1328.6±188.2, respectively). Those with the LEP 19 GG genotype spent more hr/wk in light intensity PA (39.7±1.6) than A allele carriers (35.0±1.4, p=0.03). In response to RT, adults with the LEPR 668 G allele gained greater arm muscle volume (67687.05±3186.7 vs. 52321.87±5125.05 mm3, p=0.01) and subcutaneous fat volume (10599.89±3683.57 vs. −5224.73±5923.98 mm3, p=0.02) than adults with the LEPR 668 AA genotype, respectively. CONCLUSION LEP19 G>A and LEPR 668 A>G associated with habitual PA and the body composition response to RT. These LEP and LEPR SNPs are located in coding exons likely influencing LEP and LEPR function. Further investigation is needed to confirm our findings and establish mechanisms for LEP and LEPR genotype and PA and body composition associations we observed. PMID:22975643

  5. Mechanistic Support for Combined MET and AR Blockade in Castration-Resistant Prostate Cancer12

    PubMed Central

    Qiao, Yuanyuan; Feng, Felix Y.; Wang, Yugang; Cao, Xuhong; Han, Sumin; Wilder-Romans, Kari; Navone, Nora M.; Logothetis, Christopher; Taichman, Russell S.; Keller, Evan T.; Palapattu, Ganesh S.; Alva, Ajjai S.; Smith, David C.; Tomlins, Scott A.; Chinnaiyan, Arul M.; Morgan, Todd M.

    2016-01-01

    A recent phase III trial of the MET kinase inhibitor cabozantinib in men with castration-resistant prostate cancer (CRPC) failed to meet its primary survival end point; however, most men with CRPC have intact androgen receptor (AR) signaling. As previous work supports negative regulation of MET by AR signaling, we hypothesized that intact AR signaling may have limited the efficacy of cabozantinib in some of these patients. To assess the role of AR signaling on MET inhibition, we first performed an in silico analysis of human CRPC tissue samples stratified by AR signaling status (+ or −), which identified MET expression as markedly increased in AR− samples. In vitro, AR signaling inhibition in AR+ CRPC models increased MET expression and resulted in susceptibility to ligand (HGF) activation. Likewise, MET inhibition was only effective in blocking cancer phenotypes in cells with MET overexpression. Using multiple AR+ CRPC in vitro and in vivo models, we showed that combined cabozantinib and enzalutamide (AR antagonist) treatment was more efficacious than either inhibitor alone. These data provide a compelling rationale to combine AR and MET inhibition in CRPC and may explain the negative results of the phase III cabozantinib study in CRPC. Similarly, the expression of MET in AR− disease, whether due to AR inhibition or loss of AR signaling, suggests potential utility for MET inhibition in select patients with AR therapy resistance and in AR− prostate cancer. PMID:26806347

  6. Ar-39-Ar-40 Ages of Two Nakhlites, MIL03346 and Y000593: A Detailed Analysis

    NASA Technical Reports Server (NTRS)

    Park, Jisun; Garrison, Daniel; Bogard, Donald

    2007-01-01

    Radiometric dating of martian nakhlites by several techniques have given similar ages of approx.1.2-1.4 Ga [e.g. 1, 2]. Unlike the case with shergottites, where the presence of martian atmosphere and inherited radiogenic Ar-40 produce apparent Ar-39-Ar-40 ages older than other radiometric ages, Ar-Ar ages of nakhlites are similar to ages derived by other techniques. However, even in some nakhlites the presence of trapped martian Ar produces some uncertainty in the Ar-Ar age. We present here an analysis of such Ar-Ar ages from the MIL03346 and Y000593 nakhlites.

  7. Testing of a prototype of calibration facility for noble gas monitoring using 41Ar.

    PubMed

    Saibathulham, Holnisar; Wurdiyanto, Gatot; Marsum, Pujadi

    2012-09-01

    A prototype of a calibration facility for noble gas monitoring using (41)Ar in the PTKMR-BATAN has been tested. The facility was designed in such a way that the standard source of gas can be reused. The radioactive (41)Ar source was obtained by thermal neutron reaction of (40)Ar(n, γ)(41)Ar using a thermal neutron flux of 4.8×10(13) neutrons per cm(2) per second in two minutes on the multipurpose G.A. Siwabessy Reactor (Batan, Serpong, Indonesia). Gamma spectrometry was used to measure the radioactivity and purity of (41)Ar. The spectrum of the (41)Ar observed yields an energy of 1294 keV because of the highest intensity (99.2%). The activity of (41)Ar was 2821 kBq and 4% of the expanded uncertainty. The time required for (41)Ar to reach homogeneity was 7 min, and the effectiveness of resuse was 53%. PMID:22516716

  8. Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity

    SciTech Connect

    Bao, Baolong; Wijeratne, Subhashinee S.K.; Rodriguez-Melendez, Rocio; Zempleni, Janos

    2011-08-19

    Highlights: {yields} Unambiguous evidence is provided that methionine-58 serves as an in-frame alternative translation site for holocarboxylase synthetase (HLCS58). {yields} Full-length HLCS and HLCS58 enter the nucleus, but HLCS58 is the predominant variant. {yields} HLCS58 has biological activity as biotin protein ligase. -- Abstract: Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to both carboxylases in extranuclear structures and histones in cell nuclei, thereby mediating important roles in intermediary metabolism, gene regulation, and genome stability. HLCS has three putative translational start sites (methionine-1, -7, and -58), but lacks a strong nuclear localization sequence that would explain its participation in epigenetic events in the cell nucleus. Recent evidence suggests that small quantities of HLCS with a start site in methionine-58 (HLCS58) might be able to enter the nuclear compartment. We generated the following novel insights into HLCS biology. First, we generated a novel HLCS fusion protein vector to demonstrate that methionine-58 is a functional translation start site in human cells. Second, we used confocal microscopy and western blots to demonstrate that HLCS58 enters the cell nucleus in meaningful quantities, and that full-length HLCS localizes predominantly in the cytoplasm but may also enter the nucleus. Third, we produced recombinant HLCS58 to demonstrate its biological activity toward catalyzing the biotinylation of both carboxylases and histones. Collectively, these observations are consistent with roles of HLCS58 and full-length HLCS in nuclear events. We conclude this report by proposing a novel role for HLCS in epigenetic events, mediated by physical interactions between HLCS and other chromatin proteins as part of a larger multiprotein complex that mediates gene repression.

  9. Protein kinase D1 (PKD1) influences androgen receptor (AR) function in prostate cancer cells

    SciTech Connect

    Mak, Paul; Jaggi, Meena; Chauhan, Subhash C.; Balaji, K.C.

    2008-09-05

    Protein kinase D1 (PKD1), founding member of PKD protein family, is down-regulated in advanced prostate cancer (PCa). We demonstrate that PKD1 and androgen receptor (AR) are present as a protein complex in PCa cells. PKD1 is associated with a transcriptional complex which contains AR and promoter sequence of the Prostate Specific Antigen (PSA) gene. Ectopic expression of wild type PKD1 and the kinase dead mutant PKD1 (K628W) attenuated the ligand-dependent transcriptional activation of AR in prostate cancer cells and yeast cells indicating that PKD1 can affect AR transcription activity, whereas knocking down PKD1 enhanced the ligand-dependent transcriptional activation of AR. Co-expression of kinase dead mutant with AR significantly inhibited androgen-mediated cell proliferation in both LNCaP and DU145 PC cells. Our data demonstrate for the first time that PKD1 can influence AR function in PCa cells.

  10. High CYP2A6 Enzyme Activity as Measured by a Caffeine Test and Unique Distribution of CYP2A6 Variant Alleles in Ethiopian Population

    PubMed Central

    Djordjevic, Natasa; Carrillo, Juan Antonio; Makonnen, Eyasu; Bertilsson, Leif; Ingelman-Sundberg, Magnus

    2014-01-01

    Abstract CYP2A6 metabolizes clinically relevant drugs, including antiretroviral and antimalarial drugs of major public health importance for the African populations. CYP2A6 genotype–phenotype relationship in African populations, and implications of geographic differences on enzyme activity, remain to be investigated. We evaluated the influence of CYP2A6 genotype, geographical differences, gender, and cigarette smoking on enzyme activity, using caffeine as a probe in 100 healthy unrelated Ethiopians living in Ethiopia, and 72 living in Sweden. CYP2A6 phenotype was estimated by urinary 1,7-dimethyluric acid (17U)/1,7-dimethylxanthine or paraxanthine (17X) ratio. The frequencies of CYP2A6*1B, *1D, *2, *4, *9, and *1x2 in Ethiopians were 31.3, 29.4, 0.6, 0.6, 2.8, and 0.3%, respectively. The overall mean±SD for log 17U/17X was 0.12±0.24 and coefficient of variation 199%. No significant difference in the mean log 17U/17X ratio between Ethiopians living in Sweden versus Ethiopia was observed. Analysis of variance revealed CYP2A6 genotype (p=0.04, F=2.01) but not geographical differences, sex, or cigarette smoking as predictors of CYP2A6 activity. Importantly, the median (interquartile range) of 17U/17X ratio in Ethiopians 1.35 (0.99 to 1.84) was 3- and 11-fold higher than the previously reported value in Swedes 0.52 (0.27 to 1.00) and Koreans 0.13 (0.0 to 0.35), respectively (Djordjevic et al., 2013). Taken together, we report here the relevance of CYP2A6 genotype for enzyme activity in this Ethiopian sample, as well as high CYP2A6 activity and unique distribution of the CYP2A6 variant alleles in Ethiopians as compared other populations described hitherto. Because Omics biomarker research is rapidly accelerating in Africa, CYP2A6 pharmacogenetics and clinical pharmacology observations reported herein for the Ethiopian populations have clinical and biological importance to plan for future rational therapeutics efforts in the African continent as well as therapeutics

  11. Ar-Ar Age of Shergottite Dhofar 378: Formation or Early Shock Event?

    NASA Technical Reports Server (NTRS)

    Park, J.; Bogard, Don D.

    2006-01-01

    The Ar-39-Ar40 data for 16 stepwise temperature extractions of mixed mesostasis plus plagioclase show the following major characteristics. Changes in the K/Ca ratio and in the differential rate of Ar-39 release with extraction temperature suggest three distinct, but overlapping Ar diffusion domains: <13%, 13-45%, and >45% cumulative Ar-39 release:. The youngest Ar-Ar age, approx.162-165 Myr is observed at approx.28-40% Ar-39 release, which we attribute primarily to the mesostasis. Extractions releasing >45% Ar-39, probably from plagioclase, suggest older Ar-Ar ages and indicate release of trapped martian Ar-40. An isochron plot for 8 extractions, releasing 3-45% of the Ar-39 and corrected for 36Arcos using directly measured 36Arcos, gives an Ar-Ar age of 143+/-4 Myr (where the +/- ignores the uncertainty in applying a correction for Ar-36cos). Applying a correction assuming only one-half of the measured Ar-36cos gives an age of 159+/-2 Myr. Correcting for cos-Ar-36 using the minimum measured Ar-36/Ar-37 ratio gives a minimum possible age of 138+/-5 Myr. All of these ages are within combined uncertainties of the Sm-Nd age of 157+/-24 Myr [4]. The trapped Ar-40/Ar-36 ratio obtained from the isochron is largely defined by the highest [K] data.

  12. Physical Activity Attenuates the Influence of FTO Variants on Obesity Risk: A Meta-Analysis of 218,166 Adults and 19,268 Children

    PubMed Central

    Kilpeläinen, Tuomas O.; Qi, Lu; Brage, Soren; Sharp, Stephen J.; Sonestedt, Emily; Demerath, Ellen; Ahmad, Tariq; Mora, Samia; Kaakinen, Marika; Sandholt, Camilla Helene; Holzapfel, Christina; Autenrieth, Christine S.; Hyppönen, Elina; Cauchi, Stéphane; He, Meian; Kutalik, Zoltan; Kumari, Meena; Stančáková, Alena; Meidtner, Karina; Balkau, Beverley; Tan, Jonathan T.; Mangino, Massimo; Timpson, Nicholas J.; Song, Yiqing; Zillikens, M. Carola; Jablonski, Kathleen A.; Garcia, Melissa E.; Johansson, Stefan; Bragg-Gresham, Jennifer L.; Wu, Ying; van Vliet-Ostaptchouk, Jana V.; Onland-Moret, N. Charlotte; Zimmermann, Esther; Rivera, Natalia V.; Tanaka, Toshiko; Stringham, Heather M.; Silbernagel, Günther; Kanoni, Stavroula; Feitosa, Mary F.; Snitker, Soren; Ruiz, Jonatan R.; Metter, Jeffery; Larrad, Maria Teresa Martinez; Atalay, Mustafa; Hakanen, Maarit; Amin, Najaf; Cavalcanti-Proença, Christine; Grøntved, Anders; Hallmans, Göran; Jansson, John-Olov; Kuusisto, Johanna; Kähönen, Mika; Lutsey, Pamela L.; Nolan, John J.; Palla, Luigi; Pedersen, Oluf; Pérusse, Louis; Renström, Frida; Scott, Robert A.; Shungin, Dmitry; Sovio, Ulla; Tammelin, Tuija H.; Rönnemaa, Tapani; Lakka, Timo A.; Uusitupa, Matti; Rios, Manuel Serrano; Ferrucci, Luigi; Bouchard, Claude; Meirhaeghe, Aline; Fu, Mao; Walker, Mark; Borecki, Ingrid B.; Dedoussis, George V.; Fritsche, Andreas; Ohlsson, Claes; Boehnke, Michael; Bandinelli, Stefania; van Duijn, Cornelia M.; Ebrahim, Shah; Lawlor, Debbie A.; Gudnason, Vilmundur; Harris, Tamara B.; Sørensen, Thorkild I. A.; Mohlke, Karen L.; Hofman, Albert; Uitterlinden, André G.; Tuomilehto, Jaakko; Lehtimäki, Terho; Raitakari, Olli; Isomaa, Bo; Njølstad, Pål R.; Florez, Jose C.; Liu, Simin; Ness, Andy; Spector, Timothy D.; Tai, E. Shyong; Froguel, Philippe; Boeing, Heiner; Laakso, Markku; Marmot, Michael; Bergmann, Sven; Power, Chris; Khaw, Kay-Tee; Chasman, Daniel; Ridker, Paul; Hansen, Torben; Monda, Keri L.; Illig, Thomas; Järvelin, Marjo-Riitta; Wareham, Nicholas J.; Hu, Frank B.; Groop, Leif C.; Orho-Melander, Marju; Ekelund, Ulf; Franks, Paul W.; Loos, Ruth J. F.

    2011-01-01

    Background The FTO gene harbors the strongest known susceptibility locus for obesity. While many individual studies have suggested that physical activity (PA) may attenuate the effect of FTO on obesity risk, other studies have not been able to confirm this interaction. To confirm or refute unambiguously whether PA attenuates the association of FTO with obesity risk, we meta-analyzed data from 45 studies of adults (n = 218,166) and nine studies of children and adolescents (n = 19,268). Methods and Findings All studies identified to have data on the FTO rs9939609 variant (or any proxy [r2>0.8]) and PA were invited to participate, regardless of ethnicity or age of the participants. PA was standardized by categorizing it into a dichotomous variable (physically inactive versus active) in each study. Overall, 25% of adults and 13% of children were categorized as inactive. Interaction analyses were performed within each study by including the FTO×PA interaction term in an additive model, adjusting for age and sex. Subsequently, random effects meta-analysis was used to pool the interaction terms. In adults, the minor (A−) allele of rs9939609 increased the odds of obesity by 1.23-fold/allele (95% CI 1.20–1.26), but PA attenuated this effect (pinteraction  = 0.001). More specifically, the minor allele of rs9939609 increased the odds of obesity less in the physically active group (odds ratio  = 1.22/allele, 95% CI 1.19–1.25) than in the inactive group (odds ratio  = 1.30/allele, 95% CI 1.24–1.36). No such interaction was found in children and adolescents. Conclusions The association of the FTO risk allele with the odds of obesity is attenuated by 27% in physically active adults, highlighting the importance of PA in particular in those genetically predisposed to obesity. Please see later in the article for the Editors' Summary PMID:22069379

  13. Novel C-4 heteroaryl 13-cis-retinamide Mnk/AR degrading agents inhibit cell proliferation and migration and induce apoptosis in human breast and prostate cancer cells and suppress growth of MDA-MB-231 human breast and CWR22Rv1 human prostate tumor xenografts in mice.

    PubMed

    Mbatia, Hannah W; Ramalingam, Senthilmurugan; Ramamurthy, Vidya P; Martin, Marlena S; Kwegyir-Afful, Andrew K; Njar, Vincent C O

    2015-02-26

    The synthesis and in vitro and in vivo antibreast and antiprostate cancers activities of novel C-4 heteroaryl 13-cis-retinamides that modulate Mnk-eIF4E and AR signaling are discussed. Modifications of the C-4 heteroaryl substituents reveal that the 1H-imidazole is essential for high anticancer activity. The most potent compounds against a variety of human breast and prostate cancer (BC/PC) cell lines were compounds 16 (VNHM-1-66), 20 (VNHM-1-81), and 22 (VNHM-1-73). In these cell lines, the compounds induce Mnk1/2 degradation to substantially suppress eIF4E phosphorylation. In PC cells, the compounds induce degradation of both full-length androgen receptor (fAR) and splice variant AR (AR-V7) to inhibit AR transcriptional activity. More importantly, VNHM-1-81 has strong in vivo antibreast and antiprostate cancer activities, while VNHM-1-73 exhibited strong in vivo antibreast cancer activity, with no apparent host toxicity. Clearly, these lead compounds are strong candidates for development for the treatments of human breast and prostate cancers. PMID:25634130

  14. NeAr Dating: New Dimensions for Ar-Ar Dating Using Nucleogenic Neon Isotopes

    NASA Astrophysics Data System (ADS)

    Hall, C. M.

    2007-12-01

    The neutron reactions that produce 37Ar from Ca, 38Ar from Cl and 39Ar from K form the very heart of the 40Ar/39Ar dating system. Not only can ages be derived, but much can be deduced from the effective mineral separation performed by step-heating analysis. However, the normal suite of elements detected using Ar isotopes cannot determine the presence of some minerals. Specifically, the absence of Na means that it is not possible in principle to uniquely determine the compositions of degassing feldpsars and the inability to measure Mg limits the discrimination of some mafic phases. Mineral and glass samples of known composition have been irradiated to determine the important nucleogenic Ne isotopes produced from F, Na and Mg. Mg produces two isotopes from the reactions 24Mg(n,α)21Ne and 25Mg(n,α)22Ne, with a production ratio for (22Ne/21Ne)Mg of about 0.25. For Na, the important reactions are 23Na(n,α)20F(β-)20Ne with a production ratio for (20Ne/22Ne)Na of about 5.3. The thermal neutron reaction for F is 19F(n,γ)20F(β-)20Ne with (20Ne)F/(39Ar)K equal to about 1.1. Because there are only 3 isotopes and 4 end member isotopic compositions, it is not possible to uniquely deconvolve the above nucleogenic sources along with atmospheric Ne. Fortunately, most unirradiated minerals analyzed have had extremely low levels of atmospheric Ne. A maximal correction for atmospheric Ne can be done assuming an atmospheric 20Ne/36Ar ratio. Measuring Ne isotopes along with Ar isotopes is challenging, requiring extra time and cryo-separation of the two species. In addition, there are unresolved issues dealing with the relative rates of Ne and Ar diffusion and Ne recoil effects. However, there is promise for the method for all whole-rock samples, amphiboles, feldspars and any mineral with expected complex exsolution textures. Examples of a variety of Ne-enhanced argon age spectra will be shown.

  15. REGISTRATION OF BIRDSFOOT TREFOIL GERMPLASM ARS-2622

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ARS-2622 broadleafed birdsfoot trefoil (Lotus corniculatus L.) germplasm was released by the USDA-ARS in cooperation with the Missouri Agricultural Experiment Station in August 2002. The merit of ARS-2622 is that it is a rhizome producing population with a broad genetic base. ARS-2622 was developed ...

  16. Cancer-Associated Splicing Variant of Tumor Suppressor AIMP2/p38: Pathological Implication in Tumorigenesis

    PubMed Central

    Choi, Jin Woo; Kim, Dae Gyu; Lee, Al-Eum; Kim, Hye Rim; Lee, Jin Young; Kwon, Nam Hoon; Shin, Young Kee; Hwang, Soon-Kyung; Chang, Seung-Hee; Cho, Myung-Haing; Choi, Yoon-La; Kim, Jhingook; Oh, Seung Hyun; Kim, Bora; Kim, Soo-Youl; Jeon, Hyo-Sung; Park, Jae Yong; Kang, Hyunseok Peter; Park, Bum Joon; Han, Jung Min; Kim, Sunghoon

    2011-01-01

    Although ARS-interacting multifunctional protein 2 (AIMP2, also named as MSC p38) was first found as a component for a macromolecular tRNA synthetase complex, it was recently discovered to dissociate from the complex and work as a potent tumor suppressor. Upon DNA damage, AIMP2 promotes apoptosis through the protective interaction with p53. However, it was not demonstrated whether AIMP2 was indeed pathologically linked to human cancer. In this work, we found that a splicing variant of AIMP2 lacking exon 2 (AIMP2-DX2) is highly expressed by alternative splicing in human lung cancer cells and patient's tissues. AIMP2-DX2 compromised pro-apoptotic activity of normal AIMP2 through the competitive binding to p53. The cells with higher level of AIMP2-DX2 showed higher propensity to form anchorage-independent colonies and increased resistance to cell death. Mice constitutively expressing this variant showed increased susceptibility to carcinogen-induced lung tumorigenesis. The expression ratio of AIMP2-DX2 to normal AIMP2 was increased according to lung cancer stage and showed a positive correlation with the survival of patients. Thus, this work identified an oncogenic splicing variant of a tumor suppressor, AIMP2/p38, and suggests its potential for anti-cancer target. PMID:21483803

  17. Overexpression of lysine-specific demethylase 1 promotes androgen-independent transition of human prostate cancer LNCaP cells through activation of the AR signaling pathway and suppression of the p53 signaling pathway.

    PubMed

    Li, Xuechao; Li, Tao; Chen, Dehong; Zhang, Peng; Song, Yarong; Zhu, Hongxue; Xiao, Yajun; Xing, Yifei

    2016-01-01

    Lysine-specific demethylase 1 (LSD1) is the first defined histone demethylase, and was found to be closely correlated with the development and progression of various types of cancers, including prostate cancer (PCa). Previous research suggests that LSD1 is closely related with cell proliferation, angiogenesis, migration and invasion in PCa. However, it remains to be elucidated whether LSD1 is correlated with androgen-independent (AI) transition of PCa under androgen-ablated conditions. The present study aimed to investigate the correlation of LSD1 expression with AI transition of human androgen-dependent PCa LNCaP cells. Our data showed that LSD1 was overexpressed in human PCa specimens and in AI PCa LNCaP-AI cells, which were established through a three-month continuous culture of LNCaP cells in androgen-deprived medium. Under androgen-deprived conditions, LNCaP-AI cells grew perfectly with less apoptosis and G0/G1 cell cycle arrest. Overexpression of LSD1 protected the LNCaP cells from androgen deprivation-induced apoptosis and G0/G1 arrest, while knockdown of LSD1 drove LNCaP-AI cells into a higher rate of apoptosis and G0/G1 arrest. Furthermore, LSD1 was found to regulate the androgen receptor (AR) and p53 signaling pathways via demethylation, subsequently influencing apoptosis and cell cycle progression. These findings revealed that overexpression of LSD1 promoted AI transition of PCa LNCaP cells under androgen-ablated conditions via activation of the AR signaling pathway and suppression of the p53 signaling pathway. PMID:26534764

  18. Ethnic variation in allele distribution of the androgen receptor (AR) (CAG)n repeat.

    PubMed

    Ackerman, Christine M; Lowe, Lynn P; Lee, Hoon; Hayes, M Geoffrey; Dyer, Alan R; Metzger, Boyd E; Lowe, William L; Urbanek, Margrit

    2012-01-01

    The androgen receptor (AR) is important in reproductive organ development, as well as tissue homeostasis of the pancreas, liver, and skeletal muscle in adulthood. The trinucleotide (CAG)(n) repeat polymorphism in exon 1 of the AR gene is thought to regulate AR activity, with longer alleles conferring reduced receptor activity. Therefore, the evaluation of the allelic distribution of the AR (CAG)(n) repeat in various ethnic groups is crucial in understanding the interindividual variability in AR activity. We evaluated ethnic variation of this AR polymorphism by genotyping individuals from the multiethnic Hyperglycemia and Adverse Pregnancy Outcome study cohort. We genotyped 4421 Caucasian mothers and 3365 offspring of European ancestry; 1494 Thai mothers and 1742 offspring; 1119 Afro-Caribbean mothers and 1142 offspring; and 780 Hispanic mothers and 770 offspring of Mexican ancestry from Bellflower, California. The distributions of (CAG)(n) alleles among all 4 ethnic groups are significantly different (P < .0001). Pairwise tests confirmed significant differences between each pair of ethnicities tested (P < 10(-28)). The relative AR (CAG)(n) repeat length in the different groups was as follows: Afro-Caribbean (shortest repeat lengths and greatest predicted AR activity) < Caucasian < Hispanic < Thai (longest repeat length and lowest predicted AR activity). Significant interethnic differences in the allele frequencies of the AR exon 1 (CAG)(n) polymorphism exist. Our results suggest that there may be potential ethnic differences in androgenic pathway activity and androgen sensitivity. PMID:21597087

  19. A genome-wide identified risk variant for PTSD is a methylation quantitative trait locus and confers decreased cortical activation to fearful faces.

    PubMed

    Almli, Lynn M; Stevens, Jennifer S; Smith, Alicia K; Kilaru, Varun; Meng, Qian; Flory, Janine; Abu-Amara, Duna; Hammamieh, Rasha; Yang, Ruoting; Mercer, Kristina B; Binder, Elizabeth B; Bradley, Bekh; Hamilton, Steven; Jett, Marti; Yehuda, Rachel; Marmar, Charles R; Ressler, Kerry J

    2015-07-01

    Genetic factors appear to be highly relevant to predicting differential risk for the development of post-traumatic stress disorder (PTSD). In a discovery sample, we conducted a genome-wide association study (GWAS) for PTSD using a small military cohort (Systems Biology PTSD Biomarkers Consortium; SBPBC, N = 147) that was designed as a case-controlled sample of highly exposed, recently returning veterans with and without combat-related PTSD. A genome-wide significant single nucleotide polymorphism (SNP), rs717947, at chromosome 4p15 (N = 147, β = 31.34, P = 1.28 × 10(-8) ) was found to associate with the gold-standard diagnostic measure for PTSD (the Clinician Administered PTSD Scale). We conducted replication and follow-up studies in an external sample, a larger urban community cohort (Grady Trauma Project, GTP, N = 2006), to determine the robustness and putative functionality of this risk variant. In the GTP replication sample, SNP rs717947 associated with PTSD diagnosis in females (N = 2006, P = 0.005), but not males. SNP rs717947 was also found to be a methylation quantitative trait locus (meQTL) in the GTP replication sample (N = 157, P = 0.002). Further, the risk allele of rs717947 was associated with decreased medial and dorsolateral cortical activation to fearful faces (N = 53, P < 0.05) in the GTP replication sample. These data identify a genome-wide significant polymorphism conferring risk for PTSD, which was associated with differential epigenetic regulation and with differential cortical responses to fear in a replication sample. These results may provide new insight into understanding genetic and epigenetic regulation of PTSD and intermediate phenotypes that contribute to this disorder. PMID:25988933

  20. Sensitivity of human cells expressing low-fidelity or weak-catalytic-activity variants of DNA polymerase ζ to genotoxic stresses.

    PubMed

    Suzuki, Tetsuya; Grúz, Petr; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

    2016-09-01

    Translesion DNA polymerases (TLS pols) play critical roles in defense mechanisms against genotoxic agents. The defects or mutations of TLS pols are predicted to result in hypersensitivity of cells to environmental mutagens. In this study, human cells expressing DNA polymerase ζ (Pol ζ) variants with low fidelity or weak catalytic activity have been established with Nalm-6-MSH+ cells and their sensitivity to mutagenicity and cytotoxicity of benzo[a]pyrene diol epoxide (BPDE) and ultraviolet-C light (UV-C) was examined. The low-fidelity mutants were engineered by knocking-in DNA sequences that direct changes of leucine 2618 to either phenylalanine (L2618F) or methionine (L2618M) of Pol ζ. The weak-catalytic-activity mutants were generated by knocking-in DNA sequences that direct changes of either tyrosine 2779 to phenylalanine (Y2779F) or aspartate 2781 to asparagine (D2781N). In addition, a +1 frameshift mutation, i.e., CCC to CCCC, was introduced in the coding region of the TK1 gene to measure the mutant frequencies. Doubling time and spontaneous TK mutant frequencies of the established cell lines were similar to those of the wild-type cells. The low-fidelity mutants displayed, however, higher sensitivity to the mutagenicity of BPDE and UV-C than the wild-type cells although their cytotoxic sensitivity was not changed. In contrast, the weak-catalytic-activity mutants were more sensitive to the cytotoxicity of BPDE and UV-C than the wild-type cells, and displayed much higher sensitivity to the clastogenicity of BPDE than the wild-type cells in an in vitro micronucleus assay. These results indicate that human Pol ζ is involved in TLS across DNA lesions induced by BPDE and UV-C and also that the TLS plays important roles in induction of mutations, clastogenicity and in cellular survival of the damaged human cells. Similarities and differences in in vivo roles of yeast and human Pol ζ in genome integrity are discussed. PMID:27338670

  1. The Asian-American variant of human papillomavirus type 16 exhibits higher activation of MAPK and PI3K/AKT signaling pathways, transformation, migration and invasion of primary human keratinocytes.

    PubMed

    Hochmann, Jimena; Sobrinho, João S; Villa, Luisa L; Sichero, Laura

    2016-05-01

    Asian-American (AA) HPV-16 variants are associated with higher risk of cancer. Abnormal activation of intracellular signaling play a critical role in cancer development and progression. Our aim was to elucidate mechanisms underlying the higher oncogenic potential attributed to AA variant. We evaluated activation of MAPK and PI3K/AKT pathways in primary human keratinocytes (PHKs) transduced with E6/E7 of three HPV-16 variants: E-P, AA, E-350G. Phenotypes examined included migration, anchorage independent growth and invasion. AA PHKs presented the highest levels of active proteins involved in all cascades analyzed: MAPK-ERK, MAPK-p38 and PI3K-AKT. AA PHKs were more efficient in promoting anchorage independent growth, and in stimulating cell migration and invasion. MEK1 inhibition decreased migration. The mesenchymal phenotype marker vimentin was increased in AA PHKs. Our results suggest that MEK1, ERK2, AKT2 hyperactivation influence cellular behavior by means of GSK-3b inactivation and EMT induction prompting AA immortalized PHKs to more efficiently surpass carcinogenesis steps. PMID:26945151

  2. Mucopolysaccharidosis: A New Variant?

    ERIC Educational Resources Information Center

    Primrose, D. A.

    1972-01-01

    Described is a possibly new variant of mucopolysaccharidosis characterized by progressive mental and motor deficiency, bone abnormalities, a generalized skin lesion, and abnormal mucopolysaccharides in the urine as seen in a 20-year-old female. (DB)

  3. Normal Variants in Echocardiography.

    PubMed

    Sanchez, Daniel R; Bryg, Robert J

    2016-11-01

    Echocardiography is a powerful and convenient tool used routinely in the cardiac evaluation of many patients. Improved resolution and visualization of cardiac anatomy has led to the discovery of many normal variant structures that have no known pathologic consequence. Importantly, these findings may masquerade as pathology prompting unnecessary further evaluation at the expense of anxiety, cost, or potential harm. This review provides an updated and comprehensive collection of normal anatomic variants on both transthoracic and transesophageal imaging. PMID:27612473

  4. Variants of the Ankyrin Repeat Domain 6 Gene (ANKRD6) and Muscle and Physical Activity Phenotypes Among European-Derived American Adults

    PubMed Central

    Van Deveire, Katherine N.; Scranton, Sarah K.; Kostek, Mathew A.; Angelopoulos, Theodore J.; Clarkson, Priscilla M.; Gordon, Paul M.; Moyna, Niall M.; Visich, Paul S.; Zoeller, Robert F.; Thompson, Paul D.; Devaney, Joseph M.; Gordish-Dressman, Heather; Hoffman, Eric P.; Maresh, Carl M.; Pescatello, Linda S.

    2014-01-01

    Ankyrin repeat domain 6 (ANKRD6) is a ubiquitous protein that associates with early development in mammals and is highly expressed in the brain, spinal cord, and heart of humans. We examined the role of 8 ANKRD6 single-nucleotide polymorphisms (SNPs) on muscle performance and habitual physical activity (PA). Single-nucleotide polymorphisms were 545 T>A (rs9362667), 485 M>L (rs61736690), 233 T>M (rs2273238), 128 I>L (rs3748085), 631 P>L (rs61739327), 122 Q>E (rs16881983), 197805 G>A (rs9344950), and 710 L>X (NOVEL). This study consisted of 922 healthy, untrained, European-derived American men (n = 376, 23.6 ± 0.3 years, 25.0 ± 0.2 kg·m−2) and women (n = 546, 23.2 ± 0.2 years, 24.0 ± 0.2 kg·m−2). Muscle strength (maximum voluntary contraction [MVC] and 1 repetition maximum [1RM]) and size (cross-sectional area [CSA]) were assessed before and after 12 weeks of unilateral resistance training (RT). A subsample (n = 536, 23.4 ± 0.2 years, 24.6 ± 0.2 kg·m−2) completed the Paffenbarger Physical Activity Questionnaire. Associations among ANKRD6 genotypes and muscle phenotypes were tested with repeated measure analysis of covariance (ANCOVA) and PA phenotypes with multivariate ANCOVA, with age and body mass index as covariates. ANKRD6 122 Q>E was associated with increased baseline biceps CSA. ANKRD6 545 A>T and ANKRD6 710 L>X were associated with increased 1RM and MVC in response to RT, respectively. ANKRD6 631 P>L was associated with increased biceps CSA response to RT and time spent in moderate-intensity PA among the total sample and women. ANKRD6 genetic variants were associated with the muscle size and strength response to RT and habitual PA levels. Further research is needed to validate our results and explore mechanisms for the associations we observed. PMID:22580979

  5. Contribution of APOBEC3G/F activity to the development of low-abundance drug-resistant human immunodeficiency virus type 1 variants.

    PubMed

    Noguera-Julian, M; Cozzi-Lepri, A; Di Giallonardo, F; Schuurman, R; Däumer, M; Aitken, S; Ceccherini-Silberstein, F; D'Arminio Monforte, A; Geretti, A M; Booth, C L; Kaiser, R; Michalik, C; Jansen, K; Masquelier, B; Bellecave, P; Kouyos, R D; Castro, E; Furrer, H; Schultze, A; Günthard, H F; Brun-Vezinet, F; Metzner, K J; Paredes, R

    2016-02-01

    Plasma drug-resistant minority human immunodeficiency virus type 1 variants (DRMVs) increase the risk of virological failure to first-line non-nucleoside reverse transcriptase inhibitor antiretroviral therapy (ART). The origin of DRMVs in ART-naive patients, however, remains unclear. In a large pan-European case-control study investigating the clinical relevance of pre-existing DRMVs using 454 pyrosequencing, the six most prevalent plasma DRMVs detected corresponded to G-to-A nucleotide mutations (V90I, V106I, V108I, E138K, M184I and M230I). Here, we evaluated if such DRMVs could have emerged from apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3G/F (APOBEC3G/F) activity. Out of 236 ART-naive subjects evaluated, APOBEC3G/F hypermutation signatures were detected in plasma viruses of 14 (5.9%) individuals. Samples with minority E138K, M184I, and M230I mutations, but not those with V90I, V106I or V108I, were significantly associated with APOBEC3G/F activity (Fisher's P < 0.005), defined as the presence of > 0.5% of sample sequences with an APOBEC3G/F signature. Mutations E138K, M184I and M230I co-occurred in the same sequence as APOBEC3G/F signatures in 3/9 (33%), 5/11 (45%) and 4/8 (50%) of samples, respectively; such linkage was not found for V90I, V106I or V108I. In-frame STOP codons were observed in 1.5% of all clonal sequences; 14.8% of them co-occurred with APOBEC3G/F signatures. APOBEC3G/F-associated E138K, M184I and M230I appeared within clonal sequences containing in-frame STOP codons in 2/3 (66%), 5/5 (100%) and 4/4 (100%) of the samples. In a re-analysis of the parent case control study, the presence of APOBEC3G/F signatures was not associated with virological failure. In conclusion, the contribution of APOBEC3G/F editing to the development of DRMVs is very limited and does not affect the efficacy of non-nucleoside reverse transcriptase inhibitor ART. PMID:26482266

  6. Argon diffusion in Apollo 16 impact glass spherules: Implications for 40Ar/39Ar dating of lunar impact events

    NASA Astrophysics Data System (ADS)

    Gombosi, David J.; Baldwin, Suzanne L.; Watson, E. Bruce; Swindle, Timothy D.; Delano, John W.; Roberge, Wayne G.

    2015-01-01

    The 40Ar/39Ar technique applied to impact glass has been used to date both terrestrial and lunar impact events. The ability to utilize the 40Ar/39Ar technique rests on the assumption that impact glasses are closed to the loss of daughter product, 40Ar∗, after formation. Diffusion experiments were performed on three Apollo 16 lunar impact glasses and yielded activation energies for 39Ar of ∼17 to 20 kcal mol-1 and log10(D0/a2) values of -5.2 to -6.0 s-1. The resulting diffusion coefficients are interpreted as minimum values and the Apollo 16 glass is probably some of the least retentive of lunar glasses, as the degree of non-bridging oxygen is at one end of the range in lunar glasses. At temperatures below the glass transition temperature (i.e., ∼660 °C), the data can be explained by volume diffusion from a single diffusion domain. Modeling shows that Apollo 16 composition glass could lose significant quantities of radiogenic argon (40Ar∗) (∼90-100% over 20-40 Myr assuming a diffusion domain size (a) of 75 μm) due to diurnal temperature variations on the lunar surface, although 40Ar∗ loss is highly sensitive to exposure duration and effective diffusion domain size. Modeling shows that loss from transient thermal events (e.g., heating to ∼200 °C for 102 yr duration) can also cause partial resetting of apparent 40Ar/39Ar ages. In small (a = 75 μm) glasses a maximum of 50-60% of 40Ar∗ is lost over 4 Ga when buried to depths corresponding to temperatures of -15 °C. Results indicate that caution should be exercised in interpreting lunar impact glass 40Ar/39Ar ages, as the assumption of closed system behavior may have been violated, particularly in glasses with low fractions of non-bridging oxygen.

  7. Starving the addiction: new opportunities for durable suppression of AR signaling in prostate cancer

    PubMed Central

    Knudsen, Karen E.; Scher, Howard I.

    2009-01-01

    Clinical data and models of human disease indicate that androgen receptor (AR) activity is essential for prostate cancer development, growth and progression. The dependence of prostatic adenocarcinoma on AR signaling at all stages of disease has thereby been exploited in the treatment of disseminated tumors, for which ablation of AR function is the goal of first line therapy. Although these strategies are initially effective, recurrent tumors arise with restored AR activity, and no durable treatment has yet been identified to combat this stage of disease. New insights into AR regulation and the mechanisms underlying resurgent AR activity have provided fertile ground for the development of novel strategies to more effectively inhibit receptor activity and prolong the transition to therapeutic failure. PMID:19638458

  8. 40Ar/(39)Ar dating of the Kapthurin Formation, Baringo, Kenya.

    PubMed

    Deino, Alan L; McBrearty, Sally

    2002-01-01

    The(40)Ar/(39)Ar radiometric dating technique has been applied to tuffs and lavas of the Kapthurin Formation in the Tugen Hills, Kenya Rift Valley. Two variants of the(40)Ar/(39)Ar technique, single-crystal total fusion (SCTF) and laser incremental heating (LIH) have been employed to date five marker horizons within the formation: near the base, the Kasurein Basalt at 0.61+/-0.04 Ma; the Pumice Tuff at 0.543+/-0.004 Ma; the Upper Kasurein Basalt at 0.552+/-0.015 Ma; the Grey Tuff at 0.509+/-0.009 Ma; and within the upper part of the formation, the Bedded Tuff at 0.284+/-0.012 Ma. The new, precise radiometric age determination for the Pumice Tuff also provides an age for the widespread Lake Baringo Trachyte, since the Pumice Tuff is the early pyroclastic phase of this voluminous trachyte eruption. These results establish the age of fossil hominids KNM-BK 63-67 and KNM-BK 8518 at approximately 0.510-0.512 Ma, a significant finding given that few Middle Pleistocene hominids are radiometrically dated. The Kapthurin hominids are thus the near contemporaries of those from Bodo, Ethiopia and Tanzania. A flake and core industry from lacustrine sediments in the lower part of the formation is constrained by new dates of 0.55-0.52 Ma, a period during which the Acheulian industry, characterized by handaxes, is known throughout East Africa. Points, typical of the Middle Stone Age (MSA), are found in Kapthurin Formation sediments now shown to date to between 0.509+/-0.009 Ma and 0.284+/-0.012 Ma. This date exceeds previous estimates for the age of the MSA elsewhere in East Africa by 49 ka, and establishes the age of Acheulian to MSA transition for the region. Evidence of the use of the Levallois technique for the manufacture of both small flakes and biface preforms, the systematic production of blades, and the use and processing of red ochre also occurs in this interval. The presence of blades and red ochre at this depth is important as blades signify a high degree of technical

  9. ARS Grape Quality Research Update

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In ARS much of the research on grape quality is overseen by National Program 306, entitled Quality Preservation, Characterization and Enhancement and New Processes, New Uses and Value-Added Foods. The mission of the Processed Foods Research Unit at the Western Regional Research Center in Albany, CA...

  10. USDA-ARS Quartlerly News

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This quarterly article is an update of research going on at The USDA-ARS Thad Cochran Southern Horticultural Laboratory in Poplarville, MS to be published in the Louisiana Nursery and Landscape Associations (LANLA) quarterly newsletter. This is one of three publications that I am sending out to the ...

  11. 40Ar/39Ar dating of tourmaline as a tool for high-temperature metamorphism thermochronology

    NASA Astrophysics Data System (ADS)

    Jourdan, Fred; Thern, Eric

    2014-05-01

    Tourmaline is an ubiquitous mineral, with properties making it ideal for studying metamorphic processes as well as a useful tool for a wide range of applications (e.g, magmatism, metasomatism, ore deposits [1]), mostly because it is not sensitive to chemical or mechanical alteration and is stable over a wide range of pressure-temperature conditions (up to 6 GPa and 850° C [2]). Typical metamorphic tourmaline types include dravite and shorl which, along with elbaite, belong to the alkali group [1]. The alkali group is notable because tourmalines from this group tend to incorporate trace amounts of K2O and therefore, can be dated using the 40Ar/39Ar technique. In order to understand the maximum temperature below which the K/Ar chronometer stays closed to argon loss by thermally activated diffusion, we carried out temperature controlled furnace diffusion experiments on well-behaved 40Ar/39Ar plateau-forming Archean tourmaline of 2935 ± 9 Ma [3]. Each experiment yielded an Arrhenius profile (Do vs. 1/temperature) that shows that the 39Ar data form two linear arrays with two distinct slopes. The first array only includes a few % of the total gas, has a shallow slope and shows very fast diffusivity at low temperature. We interpret these data as indicating very fast release of argon by cracks and defects. The second array of data points includes most of the gas of each experiment and forms a much steeper slope. These data yielded Ea (activation energy) values ranging from 120 to 157 Kcal/mol and D0 (pre-exponential diffusion factor) values ranging from 1.9x106 to 2.5x109 cm2/s for crystals with an average radius of 100 ± 25 μm. Three additional experiments using a laser (resulting in poor temperature control) suggest similar values although the latter experiments are considered semi-quantitative. The furnace experiments suggest that tourmaline has a weighted mean closure temperature of 804 ± 90 ° C (1σ) for a cooling rate of 10° C/Ma. Monte Carlo simulations using

  12. Histone H3 Variants in Trichomonas vaginalis.

    PubMed

    Zubácová, Zuzana; Hostomská, Jitka; Tachezy, Jan

    2012-05-01

    The parabasalid protist Trichomonas vaginalis is a widespread parasite that affects humans, frequently causing vaginitis in infected women. Trichomonad mitosis is marked by the persistence of the nuclear membrane and the presence of an asymmetric extranuclear spindle with no obvious direct connection to the chromosomes. No centromeric markers have been described in T. vaginalis, which has prevented a detailed analysis of mitotic events in this organism. In other eukaryotes, nucleosomes of centromeric chromatin contain the histone H3 variant CenH3. The principal aim of this work was to identify a CenH3 homolog in T. vaginalis. We performed a screen of the T. vaginalis genome to retrieve sequences of canonical and variant H3 histones. Three variant histone H3 proteins were identified, and the subcellular localization of their epitope-tagged variants was determined. The localization of the variant TVAG_185390 could not be distinguished from that of the canonical H3 histone. The sequence of the variant TVAG_087830 closely resembled that of histone H3. The tagged protein colocalized with sites of active transcription, indicating that the variant TVAG_087830 represented H3.3 in T. vaginalis. The third H3 variant (TVAG_224460) was localized to 6 or 12 distinct spots at the periphery of the nucleus, corresponding to the number of chromosomes in G(1) phase and G(2) phase, respectively. We propose that this variant represents the centromeric marker CenH3 and thus can be employed as a tool to study mitosis in T. vaginalis. Furthermore, we suggest that the peripheral distribution of CenH3 within the nucleus results from the association of centromeres with the nuclear envelope throughout the cell cycle. PMID:22408228

  13. Antibacterial activity of class I and IIa bacteriocins combined with polymyxin E against resistant variants of Listeria monocytogenes and Escherichia coli.

    PubMed

    Naghmouchi, Karim; Belguesmia, Yanath; Baah, John; Teather, Ron; Drider, Djamel

    2011-01-01

    Development of resistance could render antimicrobial peptides ineffective as bio-preservatives in food. Variants resistant to nisin A or pediocin PA-1 were developed from Listeria monocytogenes and a variant resistant to polymyxin E was developed from Escherichia coli RR1. Inhibition of these organisms by these agents alone or in combination was assessed using critical dilution microassay and optical density measurement. The combination of pediocin or nisin with polymyxin E was synergistic against all five strains. The polymyxin/nisin combination at 9.3/32, 4.7/62.5 and 0.6/15.6 μg/ml inhibited growth of nisin-resistant L. monocytogenes, pediocin-resistant L. monocytogenes and polymyxin-resistant E. coli, respectively, by factors of 94%, 97% and 74% compared to controls. The pediocin/nisin combination was effective against L. monocytogenes and its variants, but not E. coli or its variant. Polymyxin (0.21 μg/ml) and polymyxin/nisin (0.3/7.8 μg/ml) reduced E. coli growth measured in the log phase by, respectively, 31.25% and 93.54%. L. monocytogenes growth in the logarithmic and stationary phases was reduced, respectively, by 90.46% and 77.52% by polymyxin/pediocin at 4.7/25 μg/ml. Our results suggest that the effective concentration of bacteriocin for control of resistant L. monocytogenes and E. coli variants could be lowered considerably by combination with polymyxin E. This suggests searching for polymyxin-like compounds to increase the efficiency of bacteriocins and slow the emergence of resistant mutants. This could be an important step towards the expanded use of bacteriocins in the medical arena. PMID:20868743

  14. SUMER-IRIS Observations of AR11875

    NASA Astrophysics Data System (ADS)

    Schmit, Donald; Innes, Davina

    2014-05-01

    We present results of the first joint observing campaign of IRIS and SOHO/SUMER. While the IRIS datasets provide information on the chromosphere and transition region, SUMER provides complementary diagnostics on the corona. On 2013-10-24, we observed an active region, AR11875, and the surrounding plage for approximately 4 hours using rapid-cadence observing programs. These datasets include spectra from a small C -class flare which occurs in conjunction with an Ellerman-bomb type event. Our analysis focusses on how the high spatial resolution and slit jaw imaging capabilities of IRIS shed light on the unresolved structure of transient events in the SUMER catalog.

  15. Atlas of Computed Tomography Variants

    SciTech Connect

    Kuhns, L.R.; Seeger, J.

    1983-01-01

    Atlas of Computed Tomography Variants is unique in that, while others of its kind may include plain film, roentgen variants, it concentrates solely on CT images of variants which may simulate disease. Organized into four regions, it presents dicussions covering CT variants of the skull, neck and spine; thorax; abdomen; and extremities-featuring a section on the head.

  16. A study of the trace 39Ar content in argon from deep underground sources

    NASA Astrophysics Data System (ADS)

    Xu, J.; Calaprice, F.; Galbiati, C.; Goretti, A.; Guray, G.; Hohman, T.; Holtz, D.; Ianni, An.; Laubenstein, M.; Loer, B.; Love, C.; Martoff, C. J.; Montanari, D.; Mukhopadhyay, S.; Nelson, A.; Rountree, S. D.; Vogelaar, R. B.; Wright, A.

    2015-06-01

    The discovery of argon from deep underground sources with significantly less 39Ar than atmospheric argon was an important step in the development of direct dark matter detection experiments using argon as the active target. We report on the design and operation of a low-background single-phase liquid argon detector that was built to study the 39Ar content of this underground argon. Underground argon from the Kinder Morgan CO2 plant in Cortez, Colorado was determined to have less than 0.65% of the 39Ar activity in atmospheric argon, or 6.6 mBq/kg specific 39Ar activity.

  17. [Genetic variants of platelet ADP receptor P2Y12 associated with changed platelet functional activity and development of cardiovascular diseases].

    PubMed

    Sirotkina, O V; Zabotina, A M; Berkovich, O A; Bazhenova, E A; Vavilova, T V; Shvartsman, A L

    2009-02-01

    The key role in platelet aggregation is played by the platelet ADP receptor P2Y12, which is the target for antiaggregant drugs, clopidogrel and ticlopidine. At present, only sporadic data on genetic variants of platelet ADP receptor P2Y12 are available from literature, and their association with thromboembolic and cardiovascular diseases still remains obscure. Analysis of the group of subjects with high platelet reactivity resulted in identification of two nucleotide substitutions, C18T and G36T, in the coding region of the P2Y12 gene. The frequency of the P2Y12 T1 8 allele was higher in control group than in the group of patients survived from myocardial infarction at the age under 45 years (39% versus 28%, respectively, P = 0.04). Moreover, in the T18 carriers, platelet aggregation activity was lower than in the carriers of the wild-type genotype (0.84 +/- 0.05% versus 1.01 +/- 0.08%, respectively, P = 0.03). In the group of patients with early myocardial infarctions, a tendency towards the increased frequency of 16T allele in comparison with control group (20 and 12%, respectively, P = 0.07) was observed. The rate of ADP-induced platelet aggregation in the carriers of 16T allele from the control group was somewhat higher than in the subjects with the GG36 genotype (1.31 +/- 0.16% versus 1.12 +/- 0.06%, respectively, P = 0.07). The nucleotide substitutions identified were in absolute disequilibrium, i.e., allele T18 conformed to allele G36. On the contrary, allele C18 conformed to allele T36. Haplotype T18G36 was found to be responsible for the decreased risk of myocardial infarction and decreased platelet reactivity. It is suggested that polymorphisms of the P2Y12 gene identified can be used for determination of the risk group for myocardial infarction in the young males. PMID:19334620

  18. Oldest human footprints dated by Ar/Ar

    NASA Astrophysics Data System (ADS)

    Scaillet, Stéphane; Vita-Scaillet, Grazia; Guillou, Hervé

    2008-11-01

    Fossilized human trackways are extremely rare in the geologic record. These bear indirect but invaluable testimony of human/hominid locomotion in open air settings and can provide critical information on biomechanical changes relating to bipedalism evolution throughout the primitive human lineage. Among these, the "Devil's footsteps" represent one of the best preserved human footprints suite recovered so far in a Pleistocene volcanic ash of the Roccamonfina volcano (southern Italy). Until recently, the age of these footprints remained speculative and indirectly correlated with a loosely dated caldera-forming eruption that produced the Brown Leucitic Tuff. Despite extensive hydrothermal alteration of the pyroclastic deposit and variable contamination with excess 40Ar, detailed and selective 40Ar/ 39Ar laser probe analysis of single leucite crystals recovered from the ash deposit shows that the pyroclastic layer and the footprints are 345 ± 6 kyr old (1 σ), confirming for the first time that these are the oldest human trackways ever dated, and that they were presumably left by the modern human predecessor, Homo heidelbergensis, close to Climatic Termination IV.

  19. ID4 promotes AR expression and blocks tumorigenicity of PC3 prostate cancer cells.

    PubMed

    Komaragiri, Shravan Kumar; Bostanthirige, Dhanushka H; Morton, Derrick J; Patel, Divya; Joshi, Jugal; Upadhyay, Sunil; Chaudhary, Jaideep

    2016-09-01

    Deregulation of tumor suppressor genes is associated with tumorigenesis and the development of cancer. In prostate cancer, ID4 is epigenetically silenced and acts as a tumor suppressor. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR. Previous study from our lab showed that ectopic ID4 expression in DU145 attenuates proliferation and promotes AR expression suggesting that ID4 dependent AR activity is tumor suppressive. In this study, we examined the effect of ectopic expression of ID4 on highly malignant prostate cancer cell, PC3. Here we show that stable overexpression of ID4 in PC3 cells leads to increased apoptosis and decreased cell proliferation and migration. In addition, in vivo studies showed a decrease in tumor size and volume of ID4 overexpressing PC3 cells, in nude mice. At the molecular level, these changes were associated with increased androgen receptor (AR), p21, and AR dependent FKBP51 expression. At the mechanistic level, ID4 may regulate the expression or function of AR through specific but yet unknown AR co-regulators that may determine the final outcome of AR function. PMID:27462022

  20. NF-κB and Androgen Receptor Variant 7 Induce Expression of SRD5A Isoforms and Confer 5ARI Resistance

    PubMed Central

    Austin, David C.; Strand, Douglas W.; Love, Harold L.; Franco, Omar E.; Grabowska, Magdalena M.; Miller, Nicole L.; Hameed, Omar; Clark, Peter E.; Matusik, Robert J.; Jin, Ren J.; Hayward, Simon W.

    2016-01-01

    BACKGROUND Benign prostatic hyperplasia (BPH) is treated with 5α-reductase inhibitors (5ARI). These drugs inhibit the conversion of testosterone to dihydrotestosterone resulting in apoptosis and prostate shrinkage. Most patients initially respond to 5ARIs; however, failure is common especially in inflamed prostates, and often results in surgery. This communication examines a link between activation of NF-κB and increased expression of SRD5A2 as a potential mechanism by which patients fail 5ARI therapy. METHODS Tissue was collected from “Surgical” patients, treated specifically for lower urinary tract symptoms secondary to advanced BPH; and, cancer free transition zone from “Incidental” patients treated for low grade, localized peripheral zone prostate cancer. Clinical, molecular and histopathological profiles were analyzed. Human prostatic stromal and epithelial cell lines were genetically modified to regulate NF-κB activity, androgen receptor (AR) full length (AR-FL), and AR variant 7 (AR-V7) expression. RESULTS SRD5A2 is upregulated in advanced BPH. SRD5A2 was significantly associated with prostate volume determined by Transrectal Ultrasound (TRUS), and with more severe lower urinary tract symptoms (LUTS) determined by American Urological Association Symptom Score (AUASS). Synthesis of androgens was seen in cells in which NF-κB was activated. AR-FL and AR-V7 expression increased SRD5A2 expression while forced activation of NF-κB increased all three SRD5A isoforms. Knockdown of SRD5A2 in the epithelial cells resulted in significant reduction in proliferation, AR target gene expression, and response to testosterone (T). In tissue recombinants, canonical NF-κB activation in prostatic epithelium elevated all three SRD5A isoforms and resulted in in vivo growth under castrated conditions. CONCLUSION Increased BPH severity in patients correlates with SRD5A2 expression. We demonstrate that NF-κB and AR-V7 upregulate SRD5A expression providing a mechanism

  1. Data on the evolutionary history of the V(D)J recombination-activating protein 1 - RAG1 coupled with sequence and variant analyses.

    PubMed

    Kumar, Abhishek; Bhandari, Anita; Sarde, Sandeep J; Muppavarapu, Sekhar; Tandon, Ravi

    2016-09-01

    RAG1 protein is one of the key component of RAG complex regulating the V(D)J recombination. There are only few studies for RAG1 concerning evolutionary history, detailed sequence and mutational hotspots. Herein, we present out datasets used for the recent comprehensive study of RAG1 based on sequence, phylogenetic and genetic variant analyses (Kumar et al., 2015) [1]. Protein sequence alignment helped in characterizing the conserved domains and regions of RAG1. It also aided in unraveling ancestral RAG1 in the sea urchin. Human genetic variant analyses revealed 751 mutational hotspots, located both in the coding and the non-coding regions. For further analysis and discussion, see (Kumar et al., 2015) [1]. PMID:27284568

  2. Resolvable miscalibration of the 40Ar/39Ar geochronometer

    NASA Astrophysics Data System (ADS)

    Mundil, R.; Renne, P. R.; Min, K. K.; Ludwig, K. R.

    2006-12-01

    U/Pb and 40Ar/39Ar isotopic dating techniques are the most widely applied geochronometers, both capable of 0.1% internal precision. A robust intercalibration between the two isotopic systems is fundamental for reconstructing short term processes and events in geologic time. However, whereas the U decay constants are known precisely (to ca 0.1%), the currently used 40K decay constant (5.543×10^{-10}/yr, (1)) is associated with an unstated uncertainty that is about an order of magnitude larger than the former, making high-resolution comparisons of ages from the two isotopic systems impossible. We present an indirect calibration by comparing radio-isotopic ages derived from both isotopic systems of rapidly cooled volcanic rocks in order to minimize effects from protracted cooling history. Eleven data pairs of 206Pb/238U and conventional 40Ar/39Ar ages exhibit a bias between the two isotopic systems ranging from >-1.5% for young rocks to ca -0.5% for rocks as old as 2 Ga (possibly even smaller for rocks >2 Ga), with the 40Ar/39Ar ages being consistently younger. All Mesozoic and Paleozoic samples display a bias of about -1%. Most of this bias is probably the result of miscalibration of the electron capture decay constant of 404→ 40Ar (λ40Kec) by ca -1%, in combination with a miscalibration of smaller magnitude and opposite sense of the β- decay constant (λ40Kβ-) of 40K→ 40Ca. Bias greater than 1% for younger Cenozoic samples probably reflects pre-eruptive zircon saturation (magma residence time) whose effects become proportionately negligible beyond ca. 200 Ma. Whereas the currently used decay constant for 40K (see above) is based on an arguably arbitrary selection from counting experiments associated with large and sometimes incomprehensible uncertainties (mostly from experiments conducted in the 1940s to 1960s) two recent recalibrations of λ40Ktotal using liquid scintillation counting techniques suggest precise and mutually consistent values of 5.553 ± 0

  3. CEAP ARS watershed assessment study - overview

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The symposium “Conservation Effects Assessment Project: Accomplishments from USDA-ARS Benchmark Watersheds” will illustrate the opportunities for ARS research accomplishments to support conservation policy. Specifically, their long-term databases provide scientific bases for regional assessment outc...

  4. C3 variants in Japanese.

    PubMed

    Nishimukai, H; Kitamura, H; Sano, Y; Tamaki, Y

    1985-01-01

    By high-voltage agarose gel electrophoresis, seven phenotypes of C3 were found in Japanese. The allele frequencies for C3*S, C3*S025, C3*S02, C3*F, C3*F06, C3*F065, and C3*F08 were 0.9943, 0.0003, 0.0003, 0.0006, 0.0003, 0.0021, and 0.0021, respectively. CH50, C3/C3c protein concentrations, and C3 hemolytic activities in fresh sera with variant C3 phenotypes were within the normal ranges. PMID:3988301

  5. Mycosis Fungoides Variants.

    PubMed

    Martínez-Escala, M Estela; González, Belén Rubio; Guitart, Joan

    2014-06-01

    Mycosis fungoides (MF) is a cutaneous T-cell lymphoma that usually manifests as patches and plaques with a propensity for nonphotoexposed areas. MF is a common mimicker of inflammatory and infectious skin diseases, because it can be manifested with a wide variety of clinical and pathologic presentations. These atypical presentations of MF may be difficult to diagnose, requiring a high level of suspicion and careful clinicopathologic correlation. Within this array of clinical presentations, the World Health Organization classification recognizes 3 MF variants: folliculotropic MF, pagetoid reticulosis, and granulomatous slack skin. These 3 variants, as well as hypopigmented MF, are addressed in this article. PMID:26837197

  6. arXiv.org and Physics Education

    ERIC Educational Resources Information Center

    Ramlo, Susan

    2007-01-01

    The website arXiv.org (pronounced "archive") is a free online resource for full-text articles in the fields of physics, mathematics, computer science, nonlinear science, and quantitative biology that has existed for about 15 years. Available directly at http://www.arXiv.org, this e-print archive is searchable. As of Jan. 3, 2007, arXiv had open…

  7. Acetalated Dextran Encapsulated AR-12 as a Host-directed Therapy to Control Salmonella Infection

    PubMed Central

    Hoang, Ky V.; Borteh, Hassan M.; Rajaram, Murugesan V. S.; Peine, Kevin J.; Curry, Heather; Collier, Michael A.; Homsy, Michael L.; Bachelder, Eric M.; Gunn, John S.; Schlesinger, Larry S.; Ainslie, Kristy M.

    2014-01-01

    AR-12 has been evaluated in clinical trials as an anti-cancer agent but also has demonstrated host-directed, broad-spectrum clearance of bacteria. We have previously shown that AR-12 has activity in vitro against Salmonella enterica serovar Typhimurium and Francisella species by inducing autophagy and other host immune pathways. AR-12 treatment of S. Typhimurium-infected mice resulted in a 10-fold reduction in bacterial load in the liver and spleen and an increased survival time. However, AR-12 treatment did not protect mice from death, likely due poor formulation. In the current study, AR-12 was encapsulated in a microparticulate carrier formulated from the novel degradable biopolymer acetalated dextran (Ace-DEX) and subsequently evaluated for its activity in human monocyte-derived macrophages (hMDMs). Our results show that hMDMs efficiently internalized Ace-DEX microparticles (MPs), and that encapsulation significantly reduced host cell cytotoxicity compared to unencapsulated AR-12. Efficient macrophage internalization of AR-12 loaded MPs (AR-12/MPs) was further demonstrated by autophagosome formation that was comparable to free AR-12 and resulted in enhanced clearance of intracellular Salmonella. Taken together, these studies provide support that Ace-DEX encapsulated AR-12 may be a promising new therapeutic agent to control intracellular bacterial pathogens of macrophages by targeting delivery and reducing drug toxicity. PMID:25447826

  8. 40Ar-39Ar Analyses of Antarctic Dust Particles

    NASA Astrophysics Data System (ADS)

    Knott, S. F.; Turner, G.; Maurette, M.

    1993-07-01

    Eleven particles from the 100-400-micrometer-sized fraction of a sample of dust (vial G1-35) retrieved from Antarctica early in 1991 [1] have been analyzed using high-sensitivity noble gas mass spectrometry and, where possible, SEM/EDX techniques. The bulk sample was possibly heavily contaminated with terrestrial material but an attempt was made to preselect angular unmelted grains of extraterrestrial origin. Particles were examined optically and then split, where possible, into three parts to provide samples for 40Ar-39Ar, SEM, and He isotope analysis. Samples for 40Ar-39Ar studies were irradiated in the University of Michigan reactor, where they received a fast neutron fluence of approximately 10^18 cm^-2 (J = 0.0097, beta = 3.16). SEM analyses indicated that three particles (SK64, SK69, and SK72) have chondritic compositions, while a fourth (SK71) was thought to be extraterrestrial on the basis of its morphology. Two particles (SK65 and SK73) appeared to be terrestrial based on their location on an Mg-Fe-Si plot [2]. No SEM analyses are available for five of the samples (SK63, SK66, SK67, SK70, and SK71), and their origins are unknown. Gas was extracted from the samples for the argon analyses using a pulsed Nd laser. Step-heating was performed on each particle by defocusing the laser beam to reduce the heating effect. The laser delivered about 200 mJ per pulse; the initial heating was done with the beam covering approximately 150 micrometers. A broad overview of the data from nine particles analyzed in a seven-day sequence is shown in Fig. 1. Gas release, in units of 10^-12 ccSTP, is plotted as a function of run number with sample analyses interspersed with system blanks. Only two terrestrial particles, with well-defined ages of 200 Ma and 1000 Ma, released large amounts of gas and are omitted from the plot. The remaining particles analyzed so far released very little 40Ar and contrast sharply with the much larger amounts observed by Saxton et al. [3] in a suite

  9. Heterogeneity of adenovirus type 5 E1A proteins: multiple serine phosphorylations induce slow-migrating electrophoretic variants but do not affect E1A-induced transcriptional activation or transformation.

    PubMed Central

    Richter, J D; Slavicek, J M; Schneider, J F; Jones, N C

    1988-01-01

    The 289-amino-acid product encoded by the adenovirus E1A 13S mRNA has several pleiotropic activities, including transcriptional activation, transcriptional repression, and when acting in concert with certain oncogene products, cell transformation. In all cell types in which E1A has been introduced (except bacteria), E1A protein is extensively posttranslationally modified to yield several isoelectric and molecular weight variants. The most striking variant is one that has a retarded mobility, by about Mr = 2,000, in sodium dodecyl sulfate gels. We have investigated the nature of this modification and have assessed its importance for E1A activity. Phosphorylation is responsible for the altered mobility of E1A, since acid phosphatase treatment eliminates the higher apparent molecular weight products. By using several E1A deletion mutants, we show that at least two seryl residues, residing between residues 86 and 120 and 224 and 289, are the sites of phosphorylation and that each phosphorylation can independently induce the mobility shift. However, E1A mutants lacking these seryl residues transcriptionally activate the adenovirus E3 and E2A promoters and transform baby rat kidney cells to near wild-type levels. Images PMID:2835499

  10. ARS-NLT-SALT AND ARS-NLT-SALT/B SALINE TOLERANT NARROW LEAF TREFOIL GERMPLASM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ARS-NLT-SALT and ARS-NLT-SALT/B are narrow leaf trefoil germplasm that are tolerant of saline germination conditions that were developed from the broad based narrow leaf trefoil germplasm ARS-1207 using two cycles of saline condition selection during seed germination. ARS-NLT-SALT was developed usin...

  11. Functional analysis of the sea urchin-derived arylsulfatase (Ars)-element in mammalian cells.

    PubMed

    Watanabe, Satoshi; Watanabe, Sachiko; Sakamoto, Naoaki; Sato, Masahiro; Akasaka, Koji

    2006-09-01

    An insulator is a DNA sequence that has both enhancer-blocking activity, through its ability to modify the influence of neighboring cis-acting elements, and a barrier function that protects a transgene from being silenced by surrounding chromatin. Previously, we isolated and characterized a 582-bp-long element from the sea urchin arylsulfatase gene (Ars). This Ars-element was effective in sea urchin and Drosophila embryos and in plant cells. To investigate Ars-element activity in mammalian cells, we placed the element between the cytomegalovirus enhancer and a luciferase (luc) expression cassette. In contrast to controls lacking the Ars-element, NIH3T3 and 293T cells transfected with the element-containing construct displayed reduced luciferase activities. The Ars-element therefore acts as an enhancer-blocking element in mammalian cells. We assessed the barrier activity of the Ars-element using vectors in which a luc expression cassette was placed between two elements. Transfection experiments demonstrated that luc activity in these vectors was approximately ten-fold higher than in vectors lacking elements. Luc activities were well maintained even after 12 weeks in culture. Our observations demonstrate that the Ars-element has also a barrier activity. These results indicated that the Ars-element act as an insulator in mammalian cells. PMID:16923122

  12. [Variant of Bacillus anthracoides].

    PubMed

    Galanina, L A; Bekhtereva, M N; Kraĭnova, O A

    1979-01-01

    A comparative study of the Bacillus anthracoides culture and its variant has shown that the latter differs drastically from the parent culture in the shape and consistence of colonies, the size of spores and vegetative cells, the rate of spore germination in MPB, and the resistence to steam treatment and chloroactive disinfectants. PMID:423806

  13. Interaction of human GTP cyclohydrolase I with its splice variants

    PubMed Central

    Pandya, Maya J.; Golderer, Georg; Werner, Ernst R.; Werner-Felmayer, Gabriele

    2006-01-01

    Tetrahydrobiopterin is an essential cofactor for aromatic amino acid hydroxylases, ether lipid oxidase and nitric oxide synthases. Its biosynthesis in mammals is regulated by the activity of the homodecameric enzyme GCH (GTP cyclohydrolase I; EC 3.5.4.16). In previous work, catalytically inactive human GCH splice variants differing from the wild-type enzyme within the last 20 C-terminal amino acids were identified. In the present study, we searched for a possible role of these splice variants. Gel filtration profiles of purified recombinant proteins showed that variant GCHs form high-molecular-mass oligomers similar to the wild-type enzyme. Co-expression of splice variants together with wild-type GCH in mammalian cells revealed that GCH levels were reduced in the presence of splice variants. Commensurate with these findings, the GCH activity obtained for wild-type enzyme was reduced 2.5-fold through co-expression with GCH splice variants. Western blots of native gels suggest that splice variants form decamers despite C-terminal truncation. Therefore one possible explanation for the effect of GCH splice variants could be that inactive variants are incorporated into GCH heterodecamers, decreasing the enzyme stability and activity. PMID:16848765

  14. Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene.

    PubMed

    Känsäkoski, Johanna; Jääskeläinen, Jarmo; Jääskeläinen, Tiina; Tommiska, Johanna; Saarinen, Lilli; Lehtonen, Rainer; Hautaniemi, Sampsa; Frilander, Mikko J; Palvimo, Jorma J; Toppari, Jorma; Raivio, Taneli

    2016-01-01

    Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5' splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation. PMID:27609317

  15. Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene

    PubMed Central

    Känsäkoski, Johanna; Jääskeläinen, Jarmo; Jääskeläinen, Tiina; Tommiska, Johanna; Saarinen, Lilli; Lehtonen, Rainer; Hautaniemi, Sampsa; Frilander, Mikko J.; Palvimo, Jorma J.; Toppari, Jorma; Raivio, Taneli

    2016-01-01

    Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5′ splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation. PMID:27609317

  16. High-resolution 40Ar 39Ar chronology of Oligocene volcanic rocks, San Juan Mountains, Colorado

    USGS Publications Warehouse

    Lanphere, M.A.

    1988-01-01

    The central San Juan caldera complex consists of seven calderas from which eight major ash-flow tuffs were erupted during a period of intense volcanic activity that lasted for approximately 2 m.y. about 26-28 Ma. The analytical precision of conventional K-Ar dating in this time interval is not sufficient to unambiguously resolve this complex history. However, 40Ar 39Ar incremental-heating experiments provide data for a high-resolution chronology that is consistent with stratigraphie relations. Weighted-mean age-spectrum plateau ages of biotite and sanidine are the most precise with standard deviations ranging from 0.08 to 0.21 m.y. The pooled estimate of standard deviation for the plateau ages of 12 minerals is about 0.5 percent or about 125,000 to 135,000 years. Age measurements on coexisting minerals from one tuff and on two samples of each of two other tuffs indicate that a precision in the age of a tuff of better than 100,000 years can be achieved at 27 Ma. New data indicate that the San Luis caldera is the youngest caldera in the central complex, not the Creede caldera as previously thought. ?? 1988.

  17. Mechanistic and computational studies of the reductive half-reaction of tyrosine to phenylalanine active site variants of D-arginine dehydrogenase.

    PubMed

    Gannavaram, Swathi; Sirin, Sarah; Sherman, Woody; Gadda, Giovanni

    2014-10-21

    The flavin-mediated enzymatic oxidation of a CN bond in amino acids can occur through hydride transfer, carbanion, or polar nucleophilic mechanisms. Previous results with D-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) using multiple deuterium kinetic isotope effects (KIEs) and computational studies established preferred binding of the substrate protonated on the α-amino group, with cleavages of the NH and CH bonds occurring in asynchronous fashion, consistent with the three possible mechanisms. The hydroxyl groups of Y53 and Y249 are ≤4 Å from the imino and carboxylate groups of the reaction product iminoarginine, suggesting participation in binding and catalysis. In this study, we have investigated the reductive half-reactions of the Y53F and Y249F variants of PaDADH using substrate and solvent deuterium KIEs, solvent viscosity and pH effects, and quantum mechanical/molecular mechanical computational approaches to gain insights into the catalytic roles of the tyrosines and evaluate whether their mutations affect the transition state for substrate oxidation. Both Y53F and Y249F enzymes oxidized D-arginine with steady-state kinetic parameters similar to those of the wild-type enzyme. Rate constants for flavin reduction (k(red)) with D-leucine, a slow substrate amenable to rapid kinetics, were 3-fold smaller than the wild-type value with similar pKa values for an unprotonated group of ∼10.0. Similar pKa values were observed for (app)Kd in the variant and wild-type enzymes. However, cleavage of the substrate NH and CH bonds in the enzyme variants occurred in synchronous fashion, as suggested by multiple deuterium KIEs on k(red). These data can be reconciled with a hydride transfer mechanism, but not with carbanion and polar nucleophilic mechanisms. PMID:25243743

  18. Chandra's First Decade Observing AR Lac

    NASA Astrophysics Data System (ADS)

    Ratzlaff, Peter; Drake, Jeremy J.; Durham, R. Nicholas; Kashyap, Vinay; Posson-Brown, Jennifer; Wargelin, Bradford J.

    2009-09-01

    X-ray observations of the eclipsing RS CVn-type binary AR Lacertae have been obtained every year from 1999 to 2008 with the Chandra High Resolution Camera imaging and spectroscopic detectors (HRC-I, HRC-S) as part of their gain and point spread function calibration. These represent the best quality data yet obtained on the long term variability of the X-ray emission of an RS CVn star, and are rendered especially valuable for the multi-epoch coverage of the AR Lac eclipses. The data are characterised by stochastic variability by factors of ˜2 on timescales of one to several ks, and by minor flaring events in which count rates are observed to be elevated by slightly larger factors. During primary eclipse, the X-ray count rate is generally observed at approximately 60% of its value outside of eclipse and during periods of relative quiescence. Little evidence for secondary eclipses is present in the data, reminiscent of earlier X-ray and EUV observations. The X-ray count rate modulation through the eclipses allow us to place an upper limit on the extent of a spherically symmetric coronae of about two stellar radii, the exact limit depending on the details of the coronal models and partition of emission between the component stars. We compare the observed Chandra count rates to earlier EUVE, EINSTEIN, EXOSAT and ROSAT observations and comment on the apparent lack of cyclic coronal activity on RS CVn-type binaries.

  19. Preclinical evaluation of the AR inhibitor enzalutamide in triple-negative breast cancer cells.

    PubMed

    Caiazza, Francesco; Murray, Alyson; Madden, Stephen F; Synnott, Naoise C; Ryan, Elizabeth J; O'Donovan, Norma; Crown, John; Duffy, Michael J

    2016-04-01

    The androgen receptor (AR) is present in approximately 80% of invasive breast cancer patients and in up to 30% of patients with triple-negative breast cancer (TNBC). Therefore, our aim was to investigate the targeting of AR as a possible hormonal approach to the treatment of TNBC. Analysis of 2091 patients revealed an association between AR expression and poor overall survival, selectively in patients with the basal subtype of breast cancer, the vast majority of which are TNBC. IC50 values for the second-generation anti-androgen enzalutamide across 11 breast cancer cell lines varied from 4 µM to >50 µM. The activity of enzalutamide was similar in TN and non-TN cell lines but was dependent on the presence of AR. Enzalutamide reduced clonogenic potential and cell growth in a 3D matrix in AR-positive cells. In addition, enzalutamide also inhibited cell migration and invasion in an AR-dependent manner. Enzalutamide appeared to mediate these processes through down-regulation of the transcription factors AP-1 and SP-1. The first-generation anti-androgen flutamide similarly blocked cell growth, migration and invasion. AR-positive TNBC cells clustered separately from AR-negative cells based on an androgen-related gene expression signature, independently of TNBC subtype. We conclude that targeting of the AR with drugs such as enzalutamide may provide an alternative treatment strategy for patients with AR-positive TNBC. PMID:26932782

  20. Challenge of human Jurkat T-cells with the adenylate cyclase activator forskolin elicits major changes in cAMP phosphodiesterase (PDE) expression by up-regulating PDE3 and inducing PDE4D1 and PDE4D2 splice variants as well as down-regulating a novel PDE4A splice variant.

    PubMed Central

    Erdogan, S; Houslay, M D

    1997-01-01

    The cAMP phosphodiesterase (PDE) 3 and PDE4 isoforms provide the major cAMP-hydrolysing PDE activities in Jurkat T-cells, with additional contributions from the PDE1 and PDE2 isoforms. Challenge of cells with the adenylate cyclase activator forskolin led to a rapid, albeit transient, increase in PDE3 activity occurring over the first 45 min, followed by a sustained increase in PDE3 activity which began after approximately 3 h and continued for at least 24 h. Only this second phase of increase in PDE3 activity was blocked by the transcriptional inhibitor actinomycin D. After approximately 3 h of exposure to forskolin, PDE4 activity had increased, via a process that could be inhibited by actinomycin D, and it remained elevated for at least a 24 h period. Such actions of forskolin were mimicked by cholera toxin and 8-bromo-cAMP. Forskolin increased intracellular cAMP concentrations in a time-dependent fashion and its action was enhanced when PDE induction was blocked with actinomycin D. Reverse transcription (RT)-PCR analysis, using generic primers designed to detect transcripts representing enzymically active products of the four PDE4 genes, identified transcripts for PDE4A and PDE4D but not for PDE4B or PDE4C in untreated Jurkat T-cells. Forskolin treatment did not induce transcripts for either PDE4B or PDE4C; however, it reduced the RT-PCR signal for PDE4A transcripts and markedly enhanced that for PDE4D transcripts. Using RT-PCR primers for PDE4 splice variants, a weak signal for PDE4D1 was evident in control cells whereas, in forskolin-treated cells, clear signals for both PDE4D1 and PDE4D2 were detected. RT-PCR analysis of the PDE4A species indicated that it was not the PDE4A isoform PDE-46 (PDE4A4B). Immunoblotting of control cells for PDE4 forms identified a single PDE4A species of approximately 118 kDa, which migrated distinctly from the PDE4A4B isoform PDE-46, with immunoprecipitation analyses showing that it provided all of the PDE4 activity in control

  1. Spatial control of the βAR system in heart failure: the transverse tubule and beyond

    PubMed Central

    Gorelik, Julia; Wright, Peter T.; Lyon, Alexander R.; Harding, Sian E.

    2013-01-01

    The beta1-adrenoceptors (β1AR) and beta-2 (β2AR) adrenoceptors represent the predominant pathway for sympathetic control of myocardial function. Diverse mechanisms have evolved to translate signalling via these two molecules into differential effects on physiology. In this review, we discuss how the functions of the βAR are organized from the level of secondary messengers to the whole heart to achieve this. Using novel microscopy and bio-imaging methods researchers have uncovered subtle organization of the control of cyclic adenosine monophosphate (cAMP), the predominant positively inotropic pathway for the βAR. The β2AR in particular is demonstrated to give rise to highly compartmentalized, spatially confined cAMP signals. Organization of β2AR within the T-tubule and caveolae of cardiomyocytes concentrates this receptor with molecules which buffer and shape its cAMP signal to give fine control. This situation is undermined in various forms of heart failure. Human and animal models of heart failure demonstrate disruption of cellular micro-architecture which contributes to the change in response to cardiac βARs. Loss of cellular structure has proved key to the observed loss of confined β2AR signalling. Some pharmacological and genetic treatments have been successful in returning failing cells to a more structured phenotype. Within these cells it has been possible to observe the partial restoration of normal β2AR signalling. At the level of the organ, the expression of the two βAR subtypes varies between regions with the β2AR forming a greater proportion of the βAR population at the apex. This distribution may contribute to regional wall motion abnormalities in Takotsubo cardiomyopathy, a syndrome of high sympathetic activity, where the phosphorylated β2AR can signal via Gi protein to produce negatively inotropic effects. PMID:23345264

  2. Cellobiohydrolase I gene and improved variants

    DOEpatents

    Adney, William S.; Decker, Stephen R.; Mc Carter, Suzanne; Baker, John O.; Nieves, Raphael; Himmel, Michael E.; Vinzant, Todd B.

    2008-05-20

    The disclosure provides a method for preparing an active exoglucanase in a heterologous host of eukaryotic origin. The method includes mutagenesis to reduce glycosylation of the exoglucanase when expressed in a heterologous host. It is further disclosed a method to produce variant cellobiohydrolase that is stable at high temperature through mutagenesis.

  3. Age measurements of potassium-bearing sulfide minerals by the 40Ar/39Ar technique

    USGS Publications Warehouse

    Czamanske, G.K.; Lanphere, M.A.; Erd, Richard C.; Blake, M.C., Jr.

    1978-01-01

    K-Ar ages have been determined for sulfide minerals for the first time. The occurrence of adequate amounts of potassium-bearing sulfides with ideal compositions K3Fe10S14 (???10 wt.% K) and KFe2S3 (???16 wt.% K) in samples from a mafic alkalic diatreme at Coyote Peak, California, prompted an attempt to date these materials. K3Fe10S14, a massive mineral with conchoidal fracture, gives an age of 29.4 ?? 0.5 m.y. (40Ar/39Ar), indistinguishable from the 28.3 ?? 0.4 m.y. (40Ar/39Ar) and 30.2 ?? 1.0 m.y.8 (conventional K-Ar) ages obtained for associated phlogopite (8.7 wt.% K). KFe2S3, a bladed, fibrous sulfide, gives a younger age, 26.5 ?? 0.5 m.y. (40Ar/39Ar), presumably owing to Ar loss. ?? 1978.

  4. Fetal bovine serum and human constitutive androstane receptor: Evidence for activation of the SV23 splice variant by artemisinin, artemether, and arteether in a serum-free cell culture system

    SciTech Connect

    Lau, Aik Jiang; Chang, Thomas K.H.

    2014-06-01

    The naturally occurring SV23 splice variant of human constitutive androstane receptor (hCAR-SV23) is activated by di-(2-ethylhexyl)phthalate (DEHP), which is detected as a contaminant in fetal bovine serum (FBS). In our initial experiment, we compared the effect of dialyzed FBS, charcoal-stripped, dextran-treated FBS (CS-FBS), and regular FBS on the basal activity and ligand-activation of hCAR-SV23 in a cell-based reporter gene assay. In transfected HepG2 cells cultured in medium supplemented with 10% FBS, basal hCAR-SV23 activity varied with the type of FBS (regular > dialyzed > CS). DEHP increased hCAR-SV23 activity when 10% CS-FBS, but not regular FBS or dialyzed FBS, was used. With increasing concentrations (1–10%) of regular FBS or CS-FBS, hCAR-SV23 basal activity increased, whereas in DEHP-treated cells, hCAR-SV23 activity remained similar (regular FBS) or slightly increased (CS-FBS). Subsequent experiments identified a serum-free culture condition to detect DEHP activation of hCAR-SV23. Under this condition, artemisinin, artemether, and arteether increased hCAR-SV23 activity, whereas they decreased it in cells cultured in medium supplemented with 10% regular FBS. By comparison, FBS increased the basal activity of the wild-type isoform of hCAR (hCAR-WT), whereas it did not affect the basal activity of the SV24 splice variant (hCAR-SV24) or ligand activation of hCAR-SV24 and hCAR-WT by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). The use of serum-free culture condition was suitable for detecting CITCO activation of hCAR-WT and hCAR-SV24. In conclusion, FBS leads to erroneous classification of pharmacological ligands of hCAR-SV23 in cell-based assays, but investigations on functional ligands of hCAR isoforms can be conducted in serum-free culture condition. - Highlights: • FBS leads to erroneous pharmacological classification of hCAR-SV23 ligands. • Artemisinin, artemether, and arteether activate h

  5. Repression of Runx2 by Androgen Receptor (AR) in Osteoblasts and Prostate Cancer Cells: AR Binds Runx2 and Abrogates Its Recruitment to DNA

    PubMed Central

    Baniwal, Sanjeev K.; Khalid, Omar; Sir, Donna; Buchanan, Grant; Coetzee, Gerhard A.; Frenkel, Baruch

    2009-01-01

    Runx2 and androgen receptor (AR) are master transcription factors with pivotal roles in bone metabolism and prostate cancer (PCa). We dissected AR-mediated repression of Runx2 in dihydrotestosterone (DHT)-treated osteoblastic and PCa cells using reporter assays and endogenous Runx2 target genes. Repression required DHT, but not AR’s transactivation function, and was associated with nuclear colocalization of the two proteins. Runx2 and AR coimmunoprecipitated and interacted directly in glutathione-S-transferase pull-down assays. Interaction was ionic in nature. Intact AR DNA-binding domain (DBD) was necessary and sufficient for both interaction with Runx2 and its repression. Runx2 sequences required for interaction were the C-terminal 132 amino acid residues together with the Runt DBD. Runx2 DNA binding was abrogated by endogenous AR in chromatin immunoprecipitation assays and by recombinant AR-DBD in gel shift assays. Furthermore, AR caused increased nuclear mobility of Runx2 as indicated by faster fluorescence recovery after photobleaching. Thus, AR binds Runx2 and abrogates its binding to DNA and possibly to other nuclear components. Clinical relevance of our results was suggested by an inverse correlation between expression of AR-responsive prostate-specific antigen and osteocalcin genes in PCa biopsies. Given the tumor suppressor properties of Runx2, its repression by AR may constitute a mechanism of hormone carcinogenesis. Attenuation of Runx2 by AR in osteoblasts may play a role in skeletal metabolism: the bone-sparing effect of androgens is attributable, in part, to keeping Runx2 activity in check and preventing high-turnover bone disease such as seen after castration and in transgenic mice overexpressing Runx2 in osteoblasts. PMID:19389811

  6. StAR enhances transcription of genes encoding the mitochondrial proteases involved in its own degradation.

    PubMed

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Lauria, Ines; Langer, Thomas; Orly, Joseph

    2014-02-01

    Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR. PMID:24422629

  7. Etching characteristics and mechanisms of Mo thin films in Cl2/Ar and CF4/Ar inductively coupled plasmas

    NASA Astrophysics Data System (ADS)

    Lim, Nomin; Efremov, Alexander; Yeom, Geun Young; Choi, Bok-Gil; Kwon, Kwang-Ho

    2014-11-01

    The etching characteristics and mechanism of Mo thin films in Cl2/Ar and CF4/Ar inductively coupled plasmas under the same operating conditions (pressure, 6 mTorr; input power, 700 W; bias power, 200 W) were investigated. For both gas mixtures, an increase in the Ar fraction or gas pressure at a fixed gas mixing ratio was found to cause a non-monotonic change in the Mo etching rates. The X-ray photoelectron spectroscopy (XPS) diagnostics indicated contamination of the etched surfaces by reaction products. The Cl2/Ar and CF4/Ar plasma parameters were also investigated using a combination of a zero-dimensional plasma model and plasma diagnostics using Langmuir probes. An analysis of the etching kinetics with the model-predicted fluxes of the plasma active species suggests that: 1) the Mo etching process occurs in the transitional regime of the ion-assisted chemical reaction, and 2) the non-monotonic Mo etching rate is probably associated with opposing changes in the fluxes of the reactive neutral species and ion energy.

  8. Artificial neutrino source based on the {sup 37}Ar isotope

    SciTech Connect

    Barsanov, V. I.; Dzhanelidze, A. A.; Zlokazov, S. B.; Kotelnikov, N. A.; Markov, S. Yu.; Selin, V. V.; Shakirov, Z. N.; Abdurashitov, D. N.; Veretenkin, E. P.; Gavrin, V. N.; Gorbachev, V. V.; Ibragimova, T. V.; Kalikhov, A. V.; Mirmov, I. N. Shikhin, A. A.; Yants, V. E.; Khomyakov, Yu. S.; Cleveland, B. T.

    2007-02-15

    In April 2004, a neutrino source was produced by irradiating a 330-kg piece of pressed calcium oxide at the fast-neutron reactor BN-600 (Zarechny, Russia) for six months. The {sup 37}Ar isotope was obtained via the (n, {alpha}) reaction on {sup 40}Ca, and {sup 37}Ar was extracted from an aqueous solution of nitric acid in which the solid target was dissolved. After that, {sup 37}Ar was purified and sealed into a capsule. This source was used to measure the neutrino-capture rate in metalic gallium for neutrinos from {sup 37}Ar decay, which have an energy close to that of the main line of solar {sup 7}Be neutrinos (863 keV). The target of the SAGE Gallium-Germanium Neutrino Telescope was irradiated by using this source at the Baksan Neutrino Observatory (Institute for Nuclear Research, Russian Academy of Sciences). The source activity was measured by several methods during its production, in the course of irradiation, and after its completion. The weighted mean of the activity for six measurements was 409 {+-} 2 kCi at the beginning of irradiation of the gallium target (04:00 Moscow time, 30.04.2004). The scatter in the activity values obtained by different methods does not exceed 5%.

  9. Isoproterenol acts as a biased agonist of the alpha-1A-adrenoceptor that selectively activates the MAPK/ERK pathway.

    PubMed

    Copik, Alicja J; Baldys, Aleksander; Nguyen, Khanh; Sahdeo, Sunil; Ho, Hoangdung; Kosaka, Alan; Dietrich, Paul J; Fitch, Bill; Raymond, John R; Ford, Anthony P D W; Button, Donald; Milla, Marcos E

    2015-01-01

    The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e., not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and β2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α1A-AR. α1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α1A-AR. Iso induced signaling at α1A-AR was further interrogated by probing steps along the Gαq /PLC, Gαs and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α1A-AR, and CHO_α1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by α1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical Gαq- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α1A-AR partial agonist with signaling bias toward MAPK

  10. Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

    PubMed Central

    Copik, Alicja. J.; Baldys, Aleksander; Nguyen, Khanh; Sahdeo, Sunil; Ho, Hoangdung; Kosaka, Alan; Dietrich, Paul J.; Fitch, Bill; Raymond, John R.; Ford, Anthony P. D. W.; Button, Donald; Milla, Marcos E.

    2015-01-01

    The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and β2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α1A-AR. α1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α1A-AR. Iso induced signaling at α1A-AR was further interrogated by probing steps along the Gαq /PLC, Gαs and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α1A-AR, and CHO_α1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by α1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical Gαq- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α1A-AR partial agonist with signaling bias toward MAPK

  11. New 40Ar/39Ar Ages From Southwest Bolivia Refine the Timing of APVC Volcanism

    NASA Astrophysics Data System (ADS)

    Salisbury, M.; de Silva, S. L.; Jicha, B.; Singer, B.; Jiménez, N.; Ort, M.

    2008-12-01

    The Altiplano-Puna Volcanic Complex (APVC) of the Central Andes has produced prodigious silicic volcanism (at least 11,000 km3 of magma) over the last 10 Ma including some of the largest known ignimbrites on Earth. Despite excellent exposure, little previous work had been conducted on the timing and distribution of ignimbrite volcanism in the Lípez region of southwestern Bolivia, the heart of the APVC. To address this deficiency we have performed ~612 single crystal laser-fusion 40Ar/39Ar analyses from 39 pumice and bulk matrix samples collected from the main ignimbrite units within the Lípez region. Geochemistry of pumice and mineral samples, and paleomagnetic data are also being used to correlate individual ignimbrite units. Our new 40Ar/39Ar results establish new or refined eruption ages (with 2σ error) from the Vilama caldera at 8.41±0.02 Ma, Pastos Grandes caldera at 5.45±0.02 and 2.94±0.01 Ma, and Guacha caldera at 5.65±0.01, and 3.57±0.02 Ma. New ages were also determined for eruptions from the Panizos ignimbrite shield (6.86±0.03 Ma), Juvina ignimbrite shield (5.23±0.01 Ma), and the Laguna Colorado ignimbrite shield (2.21±0.05 and 1.95±0.03 Ma). The oldest ignimbrite we have found in the area is 10.33±0.64 Ma, a local unit beneath the Vilama ignimbrite. The youngest units have been identified west of the Guacha caldera with eruption ages of 1.70±0.6 Ma and 0.70±0.01 Ma. These results demonstrate that ignimbrite-producing eruptions in the Lípez region span the age of APVC volcanism previously established, with the largest eruptions occurring from long-lived, cyclic supervolcano caldera systems like Guacha and Pastos Grandes. The aggregate data from the APVC support the hypothesis that the APVC developed predominantly during distinct pulses of massive ignimbrite eruptions at ~8, 6, and 4 Ma and attest to episodic behavior of the magmatic system. Ignimbrites of <1 Ma, the cyclical nature of activity, and the continued geothermal presence and

  12. Joint determination of 40K decay constants and 40Ar∗/ 40K for the Fish Canyon sanidine standard, and improved accuracy for 40Ar/ 39Ar geochronology

    NASA Astrophysics Data System (ADS)

    Renne, Paul R.; Mundil, Roland; Balco, Greg; Min, Kyoungwon; Ludwig, Kenneth R.

    2010-09-01

    40Ar/ 39Ar and K-Ar geochronology have long suffered from large systematic errors arising from imprecise K and Ar isotopic data for standards and imprecisely determined decay constants for the branched decay of 40K by electron capture and β - emission. This study presents a statistical optimization approach allowing constraints from 40K activity data, K-Ar isotopic data, and pairs of 238U- 206Pb and 40Ar/ 39Ar data for rigorously selected rocks to be used as inputs for estimating the partial decay constants ( λ ɛ and λ β) of 40K and the 40Ar∗/ 40K ratio ( κFCs) of the widely used Fish Canyon sanidine (FCs) standard. This yields values of κFCs = (1.6418 ± 0.0045) × 10 -3, λ ɛ = (0.5755 ± 0.0016) × 10 -10 a -1 and λ β = (4.9737 ± 0.0093) × 10 -10 a -1. These results improve uncertainties in the decay constants by a factor of >4 relative to values derived from activity data alone. Uncertainties in these variables determined by our approach are moderately to highly correlated (cov( κFCs, λ ɛ) = 7.1889 × 10 -19, cov( κFCs, λ β) = -7.1390 × 10 -19, cov( λ ɛ, λ β) = -3.4497 × 10 -26) and one must take account of the covariances in error propagation by either linear or Monte Carlo methods. 40Ar/ 39Ar age errors estimated from these results are significantly reduced relative to previous calibrations. Also, age errors are smaller for a comparable level of isotopic measurement precision than those produced by the 238U/ 206Pb system, because the 40Ar/ 39Ar system is now jointly calibrated by both the 40K and 238U decay constants, and because λ ɛ( 40K) < λ( 238U). Based on this new calibration, the age of the widely used Fish Canyon sanidine standard is 28.305 ± 0.036 Ma. The increased accuracy of 40Ar/ 39Ar ages is now adequate to provide meaningful validation of high-precision U/Pb or astronomical tuning ages in cases where closed system behavior of K and Ar can be established.

  13. Ar-Ar chronology of the Martian meteorite ALH84001: evidence for the timing of the early bombardment of Mars.

    PubMed

    Turner, G; Knott, S F; Ash, R D; Gilmour, J D

    1997-09-01

    ALH84001, a cataclastic cumulate orthopyroxenite meteorite from Mars, has been dated by Ar-Ar stepped heating and laser probe methods. Both methods give ages close to 3,900 Ma. The age calculated is dependent on assumptions made about 39Ar recoil effects and on whether significant quantities of 40Ar from the Martian atmosphere are trapped in the meteorite. If, as suggested by xenon and nitrogen isotope studies, Martian atmospheric argon is present, then it must reside predominantly in the K-rich phase maskelynite. Independently determined 129Xe abundances in the maskelynite can be used to place limits on the concentration of the atmospheric 40Ar. These indicate a reduction of around 80 Ma to ages calculated on the assumption that no Martian atmosphere is present. After this correction, the nominal ages obtained are: 3940 +/- 50, 3870 +/- 80, and 3970 +/- 100 Ma. by stepped heating, and 3900 +/- 90 Ma by laser probe (1 sigma statistical errors), giving a weighted mean value of 3,920 Ma. Ambiguities in the interpretation of 39Ar recoil effects and in the contribution of Martian atmospheric 40Ar lead to uncertainties in the Ar-Ar age which are difficult to quantify, but we suggest that the true value lies somewhere between 4,050 and 3,800 Ma. This age probably dates a period of annealing of the meteorite subsequent to the shock event which gave it its cataclastic texture. The experiments provide the first evidence of an event occurring on Mars coincident with the time of the late heavy bombardment of the Moon and may reflect a similar period of bombardment in the Southern Highlands of Mars. Whether the age determined bears any relationship to the time of carbonate deposition in ALH84001 is not known. Such a link depends on whether the temperature associated with the metasomatic activity was sufficient to cause argon loss from the maskelynite and/or whether the metasomatism and metamorphism were linked in time through a common heat source. PMID:11541217

  14. Combination of carmustine and selenite effectively inhibits tumor growth by targeting androgen receptor, androgen receptor-variants, and Akt in preclinical models: New hope for patients with castration resistant prostate cancer.

    PubMed

    Thamilselvan, Vijayalakshmi; Menon, Mani; Thamilselvan, Sivagnanam

    2016-10-01

    Despite established androgen receptor (AR) antagonists, AR/AR-variants signaling remain a major obstacle for the successful treatment of castration resistant prostate cancer (CRPC). In addition, CRPC cells adapt to survive via AR-independent pathways to escape next generation therapies. Therefore, there is an urgent need for drugs that can target these signaling pathways in CRPC. In this study, we sought to determine whether carmustine and selenite in combination could induce apoptosis and inhibit growth of CRPC in-vitro and in-vivo. CRPC (22Rv1, VCaP, and PC-3) cell lines in culture and xenograft mouse were used. Combination of carmustine and selenite treatment significantly increased reactive oxygen species, apoptosis and growth inhibition in CRPC cells with down regulation of anti-apoptotic (Bcl-2 and Mcl-1) and proliferative proteins (c-Myc and cyclin-D1). This effect was associated with complete reduction of AR/AR-variants, AR-V7, PSA and significant induction of p27Kip1. Combination treatment substantially abolished phospho-Akt, phospho-GSK-3β, and anchorage-independent growth in AR-positive and AR-negative cells. Consistent with in-vitro results, combination treatment effectively induced apoptosis and completely inhibited xenograft tumor growth and markedly reduced AR/AR-variants, AR-V7, PSA, and Bcl-2 in xenograft tumors without causing genotoxicity in host mice. Individual agent treatment showed only partial effect. The combination treatment showed a significant synergistic effect. The present study is the first to demonstrate that the combination of carmustine and selenite treatment completely suppressed CRPC tumor growth by reducing AR/AR-variants and Akt signaling. Our findings suggest that the combination of carmustine and selenite could constitute a promising next-generation therapy for successful treatment of patients with CRPC. PMID:27198552

  15. 40Ar/39Ar Interlaboratory Calibration into the Holocene.

    NASA Astrophysics Data System (ADS)

    Heizler, M. T.; Jicha, B.; Koppers, A. A. P.; Miggins, D. P.

    2015-12-01

    Advances in 40Ar/39Ar analytical precision for very young rocks requires collaborative efforts amongst argon geochronology labs to demonstrate age reproducibility commensurate with high precision. NM Tech (NMT), the University of Wisconsin (UW) and Oregon State University (OSU) have each dated Quaternary flux monitor standard AC-2 sanidine (~1.185 Ma), a blind sanidine described as being 50-100 ka (BS) and sanidine from the Qixiangshan (QIX) flow (~10 ka), Changbaishan volcano, China. The samples were irradiated in a single package with FC-2 sanidine (28.201 Ma) as the flux monitor and the irradiated material was distributed amongst the labs. Heizler was present during analysis at both OSU and UW and Jicha attended OSU during analysis. Physical presence was key towards gaining understanding of individual protocols and prompted valuable discussions. Analyses were carried out on single crystals using total fusion and/or step heating approaches. Age agreement was achieved within 2s uncertainty that ranged between (0.03-0.3%, 0.13-0.37% and 1.8-2.6%) for AC-2, BS and QIX, respectively. Each lab found AC-2 to vary somewhat beyond a normal distribution and to yield an age relative to FC-2 of ~1.185 Ma that is ~1.3% (~5-10 sigma) lower than some published estimates. A key cause of the variation between this study and previous results may be variable gas pressure equilibration times between extraction line and mass spectrometer coupled with variable choices to estimate time zero by other laboratories. The majority of our efforts concentrated on the QIX sanidine where prior data obtained by our labs revealed a factor of two spread in age (~11 and 23 ka) based on experiments carried out by total fusion and bulk incremental heating. By conducting single crystal age spectrum analysis we were able to mitigate effects of melt inclusion hosted excess argon and xenocrystic contamination towards obtaining analytical agreement with apparent ages near 10 ka. However, philosophical

  16. Uncertainty Quantification of Ar-37 Transport in Fractured Rock

    NASA Astrophysics Data System (ADS)

    Sun, Y.; Carrigan, C. R.; Chen, M.; Wagoner, J. L.

    2011-12-01

    Underground nuclear explosions produce radioactive noble gas isotopes, such as Ar-37 that may migrate through fractured rock and soil from the detonation site to the ground surface. For the on site inspection monitoring protocol of the Comprehensive Nuclear Test Ban Treaty, the detection of Ar-37 above its background level is therefore an indicator of a nuclear test. However, Ar-37 is also produced in the subsurface due to cosmic-neutron activation of calcium by the 40Ca(n,α)37Ar reaction. The cosmic-neutron induced production rate of Ar-37 in the subsurface depends on many uncertain parameters, including the calcium content, the depth below ground surface, the geological structure, and other rock/soil properties. It is therefore important to distinguish the cosmic-neutron induced and test relevant Ar-37 transport in fractured rock and soil. The physical model is conceptualized as a deep dual permeability bedrock layer consisting of overlapping fracture and porous matrix continua overlain by a shallow layer of interconnected clay and sand alluvium. In this study supporting a subsurface gas tracer migration experiment at the National Center for Nuclear Security, we use numerical simulation of non-isothermal multi-phase and multi-component transport to investigate gas-component production, release, and transport in this combined fractured rock and clay-sand alluvium system. In addition to the spatial and temporal domain, we extend the modeling to a high-dimensional space including parameters characterized by a range of uncertainties using the PSUADE code, developed at Lawrence Livermore National Laboratory. Using PSUADE, we consider the dependence of the detectability on these uncertain parameters with the goal of understanding how to optimize the detection of an underground nuclear test. (LLNL-ABS-491792). This work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.

  17. 39Ar-40Ar Dating of Thermal Events on Meteorite Parent Bodies

    NASA Astrophysics Data System (ADS)

    Bogard, D. D.; Garrison, D. H.

    1999-03-01

    A summary of 39Ar-40Ar ages reveals the impact and thermal history of several meteorite parent bodies, i.e., eucrites, chondrites, mesosiderites, acapulcoites/lodranites, winonaites, enstatites, and IAB and IIE irons.

  18. The Chelyabinsk Meteorite: Variable Shock Effects Recorded by the 40Ar-39Ar System

    NASA Astrophysics Data System (ADS)

    Korochantseva, E. V.; Buikin, A. I.; Hopp, J.; Lorenz, C. A.; Trieloff, M.

    2015-07-01

    Shocked lithologies of the Chelyabinsk LL chondrite have higher apparent 40Ar-39Ar ages than the very young light lithology. We interpret previous impact events made shocked lithologies more retentive and resistant against thermal reset.

  19. First-principles calibration of 40Ar/39Ar mineral standards and complete extraction of 40Ar* from sanidine

    NASA Astrophysics Data System (ADS)

    Morgan, L. E.; Kuiper, K.; Mark, D.; Postma, O.; Villa, I. M.; Wijbrans, J. R.

    2010-12-01

    40Ar/39Ar geochronology relies on comparing argon isotopic data for unknowns to those for knowns. Mineral standards used as neutron fluence monitors must be dated by the K-Ar method (or at least referenced to a mineral of known K-Ar age). The commonly used age of 28.02 ± 0.28 Ma for the Fish Canyon sanidine (FCs) (Renne et al., 1998) is based upon measurements of radiogenic 40Ar in GA1550 biotite (McDougall and Roksandic, 1974), but underlying full data were not published (these measurements were never intended for use as an international standard), so uncertainties are difficult to assess. Recent developments by Kuiper et al. (2008) and Renne et al. (2010) are limited by their reliance on the accuracy of other systems. Modern technology should allow for more precise and accurate calibration of primary K-Ar and 40Ar/39Ar standards. From the ideal gas law, the number of moles of 40Ar in a system can be calculated from measurements of pressure, volume, and temperature. Thus we have designed and are proceeding to build a pipette system to introduce well-determined amounts of 40Ar into noble gas extraction lines and mass spectrometers. This system relies on components with calibrations traceable to SI unit prototypes, including a diaphragm pressure gauge (MKS Instruments), thermocouples, and a “slug” of an accurately determined volume to be inserted into the reservoir for volume determinations of the reservoir and pipette. The system will be renewable, with a lifetime of ca. 1 month for gas in the reservoir, and portable, to permit interlaboratory calibrations. The quantitative extraction of 40Ar* from the mineral standard is of highest importance; for sanidine standards this is complicated by high melt viscosity during heating. Experiments adding basaltic “zero age glass” (ZAG) to decrease melt viscosity are underway. This has previously been explored by McDowell (1983) with a resistance furnace, but has not been quantitatively addressed with laser heating

  20. 40Ar/39Ar chronology and paleomagnetism of Quaternary basaltic lavas from the Perşani Mountains (East Carpathians)

    NASA Astrophysics Data System (ADS)

    Panaiotu, C. G.; Jicha, B. R.; Singer, B. S.; Ţugui, A.; Seghedi, I.; Panaiotu, A. G.; Necula, C.

    2013-08-01

    Quaternary volcanism in the Perşani Mountains forms an Na-alkali basaltic province inside the bend area of the Carpathians in the southeastern part of Europe. Previous K-Ar ages and paleomagnetic data reveal several transitional virtual geomagnetic poles, which were tentatively associated with the Cobb Mountain subchron and a Brunhes chron excursion. We report a new paleomagnetic and rock-magnetic study coupled with 40Ar/39Ar geochronology to better constrain the age of geomagnetic reversals or excursions that might be recorded and the timing of volcanism. Of the paleomagnetic directions obtained from sampled lava flows 4 are reversed polarity, 19 are normal polarity and 16 have transitional polarity. 40Ar/39Ar plateau ages determined from incremental heating experiments on groundmass indicate that two of the reversely magnetized lavas erupted at 1142 ± 41 and 800 ± 25 ka, four of the normally magnetized lavas erupted at 1060 ± 10, 1062 ± 24, 684 ± 21, and 683 ± 28 ka, and two transitionally magnetized lavas formed at 1221 ± 11 and 799 ± 21 ka. Both the new 40Ar/39Ar ages and the paleomagnetic data suggest at least five episodes of volcanic activity with the most active periods during the Jaramillo and Brunhes chrons. This results shows that the last phases of alkalic and calc-alkaline magmatism in the South-East Carpathians were contemporaneous. The age of the older transitionally magnetized lava flow is within error of recent unspiked K-Ar and astrochronologic ages for the reversal that defines the onset of the Cobb Mountain normal polarity subchron. The age of the younger transitional lava is similar to that of an excursion that preceded the Matuyama-Brunhes polarity reversal and which has come to be known as the Matuyama-Brunhes precursor. Omitting the excursion data, the dispersion of the virtual geomagnetic poles (around 19°) is larger than the expected value around 45°N from the global compilation, but closer to the value obtained only from the

  1. Ion kinetics in Ar/H2 cold plasmas: the relevance of ArH+

    PubMed Central

    Jiménez-Redondo, Miguel; Cueto, Maite; Doménech, José Luis; Tanarro, Isabel; Herrero, Víctor J.

    2015-01-01

    The recent discovery of ArH+ in the interstellar medium has awakened the interest in the chemistry of this ion. In this work, the ion-molecule kinetics of cold plasmas of Ar/H2 is investigated in glow discharges spanning the whole range of [H2]/([H2]+[Ar]) proportions for two pressures, 1.5 and 8 Pa. Ion concentrations are determined by mass spectrometry, and electron temperatures and densities, with Langmuir probes. A kinetic model is used for the interpretation of the results. The selection of experimental conditions evinces relevant changes with plasma pressure in the ion distributions dependence with the H2 fraction, particularly for the major ions: Ar+, ArH+ and H3+. At 1.5 Pa, ArH+ prevails for a wide interval of H2 fractions: 0.3<[H2]/([H2]+[Ar])<0.7. Nevertheless, a pronounced displacement of the ArH+ maximum towards the lowest H2 fractions is observed at 8 Pa, in detriment of Ar+, which becomes restricted to very small [H2]/([H2]+[Ar]) ratios, whereas H3+ becomes dominant for all [H2]/([H2]+[Ar]) > 0.1. The analysis of the data with the kinetic model allows the identification of the sources and sinks of the major ions over the whole range of experimental conditions sampled. Two key factors turn out to be responsible for the different ion distributions observed: the electron temperature, which determines the rate of Ar+ formation and thus of ArH+, and the equilibrium ArH+ + H2 ⇄ H3+ + Ar, which can be strongly dependent of the degree of vibrational excitation of H3+. The results are discussed and compared with previously published data on other Ar/H2 plasmas. PMID:26702354

  2. Re-Evaluation of Ar-39 - Ar-40 Ages for Apollo Lunar Rocks 15415 and 60015

    NASA Technical Reports Server (NTRS)

    Park, J.; Nyquist, L. E.; Bogard, D. D.; Garrison, D. H.; Shih, C.-Y.

    2010-01-01

    We re-analyzed 39Ar-40Ar ages of Apollo lunar highland samples 15415 and 60015, two ferroan anorthosites analyzed previously in the 1970 s, with a more detailed approach and with revised decay constants. From these samples we carefully prepared 100-200 mesh mineral separates for analysis at the Noble Gas Laboratory at NASA-Johnson Space Center. The Ar-39-Ar-40 age spectra for 15415 yielded an age of 3851 +/- 38 Ma with 33-99% of Ar39 release, roughly in agreement with previously reported Ar-Ar ages. For 60015, we obtained an age of 3584 +/- 152 Ma in 23-98% of Ar39 release, also in agreement with previously reported Ar-Ar ages of approximately 3.5 Ga. Highland anorthosites like these are believed by many to be the original crust of the moon, formed by plagioclase floatation atop a magma ocean, however the Ar-Ar ages of 15415 and 60015 are considerably younger than lunar crust formation. By contrast, recently recovered lunar anorthosites such as Dhofar 489, Dhofar 908, and Yamato 86032 yield older Ar-Ar ages, up to 4.35 Ga, much closer to time of formation of the lunar crust. It follows that the Ar-Ar ages of the Apollo samples must have been reset by secondary heating, and that this heating affected highland anorthosites at both the Apollo 15 and Apollo 16 landing sites but did not affect lunar highland meteorites. One obvious consideration is that while the Apollo samples were collected from the near side of the moon, these lunar meteorites are thought to have originated from the lunar far side

  3. 40Ar/39Ar age spectra of some undisturbed terrestrial samples

    USGS Publications Warehouse

    Brent, Dalrymple G.; Lanphere, M.A.

    1974-01-01

    40Ar/39Ar age spectra and 40Ar/36Ar vs 39Ar/36Ar isochrons were determined by incremental heating for 11 terrestrial rocks and minerals whose geology indicates that they represent essentially undisturbed systems. The samples include muscovite, biotite, hornblende, sanidine, plagioclase, dacite, diabase and basalt and range in age from 40 to 1700 m.y. For each sample, the 40Ar/39Ar ratios, corrected for atmospheric and neutron-generated argon isotopes, are the same for most of the gas fractions released and the age spectra, which show pronounced plateaus, thus are consistent with models previously proposed for undisturbed samples. Plateau ages and isochron ages calculated using plateau age fractions are concordant and appear to be meaningful estimates of the crystallization and cooling ages of these samples. Seemingly anomalous age spectrum points can be attributed entirely to small amounts of previously unrecognized argon loss and to gas fractions that contain too small (less than 2 per cent) a proportion of the 39Ar released to be geologically significant. The use of a quantitative abscissa for age spectrum diagrams is recommended so that the size of each gas fraction is readily apparent. Increments containing less than about 4-5 per cent of the total 39Ar released should be interpreted cautiously. Both the age spectrum and isochron methods of data reduction for incremental heating experiments are worthwhile, as each gives slightly different but complementary information about the sample from the same basic data. Use of a least-squares fit that allows for correlated errors is recommended for 40Ar/36Ar vs 39Ar/36Ar isochrons. The results indicate that the 40Ar/39Ar incremental heating technique can be used to distinguish disturbed from undisturbed rock and mineral systems and will be a valuable geochronological tool in geologically complex terranes. ?? 1994.

  4. LRP5 variants may contribute to ADPKD.

    PubMed

    Cnossen, Wybrich R; te Morsche, René H M; Hoischen, Alexander; Gilissen, Christian; Venselaar, Hanka; Mehdi, Soufi; Bergmann, Carsten; Losekoot, Monique; Breuning, Martijn H; Peters, Dorien J M; Veltman, Joris A; Drenth, Joost P H

    2016-02-01

    Mutations in Polycystic Kidney Disease proteins (PKD1 or PKD2) are causative for autosomal dominant polycystic kidney disease (ADPKD). However, a small subset of ADPKD probands do not harbor a mutation in any of the known genes. Low density lipoprotein Receptor-related Protein 5 (LRP5) was recently associated with hepatic cystogenesis in isolated polycystic liver disease (PCLD). Here, we demonstrate that this gene may also have a role in unlinked and sporadic ADPKD patients. In a cohort of 79 unrelated patients with adult-onset ADPKD, we identified a total of four different LRP5 variants that were predicted to be pathogenic by in silico tools. One ADPKD patient has a positive family history for ADPKD and variant LRP5 c.1680G>T; p.(Trp560Cys) segregated with the disease. Although also two PKD1 variants probably affecting protein function were identified, luciferase activity assays presented for three LRP5 variants significant decreased signal activation of canonical Wnt signaling. This study contributes to the genetic spectrum of ADPKD. Introduction of the canonical Wnt signaling pathway provides new avenues for the study of the pathophysiology. PMID:25920554

  5. Ar-Ar Age of NWA-1460 and Evidence For Young Formation Ages of the Shergottites

    NASA Technical Reports Server (NTRS)

    Bogard, Donald D.; Park, Jisun

    2006-01-01

    Agreement of Ar-Ar, Sm-Nd, and Rb-Sr ages for NWA1460, and the inconsistency between a low shock-heating temperature for Zagami and the proposition that a 4.0 Gyr-old Zagami lost most of its Ar-40 are inconsistent with ancient formation ages for these shergottites, but are consistent with relatively young igneous formation ages.

  6. 40Ar/39Ar constraints on the temporal evolution of Graciosa Island, Azores (Portugal)

    NASA Astrophysics Data System (ADS)

    Larrea, Patricia; Wijbrans, Jan R.; Galé, Carlos; Ubide, Teresa; Lago, Marceliano; França, Zilda; Widom, Elisabeth

    2014-02-01

    Lava flows spanning the eruptive record of Graciosa Island (Azores archipelago) and a gabbro xenolith were dated by 40Ar/39Ar in order to constrain the Pleistocene and Holocene volcanic evolution of the island. The results range from 1.05 Ma to 3.9 ka, whereas prior published K-Ar and 14C ages range from 620 to 2 ka. The formation of the Serra das Fontes shield volcano started at minimum 1.05 Ma, and the magmatic system was active for ca. 600 ky, as suggested by the formation of the gabbro xenolith by magmatic differentiation. Evolved magmas making up the Serra das Fontes-Serra Branca composite volcano were generated at ca. 450 ka. After a period of ca. 110 ky of volcanic inactivity and erosion of volcanic edifices, volcanism was reactivated with the formation of the Vitória Unit NW platform. Later, the development of the Vulcão Central Unit started with the formation of monogenetic cones located to the south of the Serra das Fontes-Serra Branca-Vitória Unit. This volcanism became progressively more evolved and was concentrated in a main eruptive center, forming the Vulcão Central stratovolcano with an age older than 50 ka. The caldera related to this stratovolcano is older than 47 ka and was followed by effusion of basaltic magmas into the caldera, resulting in the formation of a lava lake, which ultimately spilled over the caldera rim at ca. 11 ka. The most recent eruptions on Graciosa formed two small pyroclastic cones within the caldera and the Pico do Timão cone within the Vitória Unit at ca 3.9 ka.

  7. Structural model of haptoglobin and its complex with the anticoagulant ecotin variants: structure-activity relationship study and analysis of interactions.

    PubMed

    Sathler, Plínio Cunha; Lourenço, André Luiz; Miceli, Leonardo Alves; Rodrigues, Carlos Rangel; Albuquerque, Magaly Girão; Cabral, Lúcio Mendes; Castro, Helena Carla

    2014-04-01

    Recently the literature described the binding of Haptoglobin (HP) with ecotin, a fold-specific serine-proteases inhibitor with an anticoagulant profile and produced by Escherichia coli. In this work, we used some in silico and in vitro techniques to evaluate HP 3D-fold and its interaction with wild-type ecotin and two variants. Our data showed HP models conserved trypsin fold, in agreement to the in vitro immunological recognition of HP by trypsin antibodies. The analysis of the three ecotin-HP complexes using the mutants RR and TSRR/R besides the wild type revealed several hydrogen bonds between HP and ecotin secondary site. These data are in agreement with the in vitro PAGE assays that showed the HP-RR complex in native gel conditions. Interestingly, the ternary complex interactions varied depending on the inhibitor structure and site-directed mutation. The interaction of HP with TSRR/R involved new residues compared to wild type, which infers a binding energy increase caused by the mutation. PMID:23477410

  8. Ubiquitous Argonium, ArH^+, in the Diffuse Interstellar Medium

    NASA Astrophysics Data System (ADS)

    Schilke, P.; Müller, Holger S. P.; Comito, C.; Sanchez-Monge, A.; Neufeld, D. A.; Indriolo, Nick; Bergin, Edwin; Lis, D. C.; Gerin, Maryvonne; Black, J. H.; Wolfire, M. G.; Pearson, John; Menten, Karl; Winkel, B.

    2014-06-01

    ArH^+ is isoelectronic with HCl. The J = 1 - 0 and 2 - 1 transitions of 36ArH^+ near 617.5 and 1234.6 GHz, respectively, have been identified very recently as emission lines in spectra obtained with Herschel toward the Crab Nebula supernova remnant. On Earth, 40Ar is by far the most abundant isotope, being almost exclusively formed by the radioactive decay of 40K. However, 36Ar is the dominant isotope in the Universe. In the course of unbiased line surveys of the massive and very luminous Galactic Center star-forming regions Sagittarius B2(M) and (N) with the high-resolution instrument HIFI on board of Herschel, we detected the J = 1 - 0 transition of 36ArH^+ as a moderately strong absorption line initially associated with an unidentified carrier. In both cases, the absorption feature is unique in its appearance at all velocity components associated with diffuse foreground molecular clouds, together with its conspicuous absence at velocities related to the denser sources themselves. Model calculations are able to reproduce the derived ArH^+ column densities and suggest that argonium resides in the largely atomic, diffuse interstellar medium with a molecular fraction of no more than ˜10-4. The 38ArH^+ isotopolog was also detected. Subsequent observations toward the continuum sources W51, W49, W31C, and G34.3+0.1 resulted in unequivocal detections of 36ArH^+ absorption. Hence, argonium is a good probe of the transition zone between atomic and molecular gas, in particular in combination with OH^+ and H_2O^+, whose abundances peak at a molecular fraction of ˜0.1. Moreover, argonium is a good indicator of an enhanced cosmic ray ionization rate. Therefore, it may be prominent toward, e.g., active galactic nuclei (AGNs) in addition to supernova remnants. M. J. Barlow et al., Science 342 (2013) 1343. H. S. P. Müller et al., Proceedings of the IAU Symposium 297, 2013, "The Diffuse Interstellar Bands", Eds. J. Cami & N. Cox.

  9. 244-AR Vault Interim Stabilization Project Plan

    SciTech Connect

    LANEY, T.

    2000-03-24

    The 244-AR Vault Facility, constructed between 1966 and 1968, was designed to provide lag storage and treatment for the Plutonium-Uranium Extraction Facility (PUREX) tank farm sludges. Tank farm personnel transferred the waste from the 244-AR Vault Facility to B Plant for recovery of cesium and strontium. B Plant personnel then transferred the treatment residuals back to the tank farms for storage of the sludge and liquids. The last process operations, which transferred waste supporting the cesium/strontium recovery mission, occurred in April 1978. After the final transfer in 1978, the 244-AR facility underwent a cleanout. However, 2,271 L (600 gal) of sludge were left in Tank 004AR from an earlier transfer from Tank 241-AX-104. When the cleanout was completed, the facility was placed in a standby status. The sludge had been transferred to Tank 004AR to support Pacific Northwest National Laboratory [PNNL] vitrification work. Documentation of waste transfers suggests that a portion of the sludge may have been moved from Tank 004AR to Tank 002AR in preparation for transfer back to the AX Tank Farm; however, quantities of the sludge that were moved to Tank 002AR from that transfer must be estimated.

  10. USDA/ARS Organic Production Research

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For much of its history, USDA/ARS had little to do with research on organic agriculture, however research in organic systems has made considerable gains at the agency over the past decade. In the 1980's and 1990's, as the organic food industry was taking off, ARS researchers who wanted to serve orga...

  11. Geology and 40Ar/39Ar geochronology of the medium- to high-K Tanaga volcanic cluster, western Aleutians

    USGS Publications Warehouse

    Jicha, Brian R.; Coombs, Michelle L.; Calvert, Andrew T.; Singer, Brad S.

    2012-01-01

    We used geologic mapping and geochemical data augmented by 40Ar/39Ar dating to establish an eruptive chronology for the Tanaga volcanic cluster in the western Aleutian arc. The Tanaga volcanic cluster is unique in comparison to other central and western Aleutian volcanoes in that it consists of three closely spaced, active, volumetrically significant edifices (Sajaka, Tanaga, and Takawangha), the eruptive products of which have unusually high K2O contents. Thirty-five new 40Ar/39Ar ages obtained in two different laboratories constrain the duration of Pleistocene–Holocene subaerial volcanism to younger than 295 ka. The eruptive activity has been mostly continuous for the last 150 k.y., unlike most other well-characterized arc volcanoes, which tend to grow in discrete pulses. More than half of the analyzed Tanaga volcanic cluster lavas are basalts that have erupted throughout the lifetime of the cluster, although a considerable amount of basaltic andesite and basaltic trachyandesite has also been produced since 200 ka. Major- and trace-element variations suggest that magmas from Sajaka and Tanaga volcanoes are likely to have crystallized pyroxene and/or amphibole at greater depths than the older Takawangha magmas, which experienced a larger percentage of plagioclase-dominated fractionation at shallower depths. Magma output from Takawangha has declined over the last 86 k.y. At ca. 19 ka, the focus of magma flux shifted to the west beneath Tanaga and Sajaka volcanoes, where hotter, more mafic magma erupted.

  12. The effect of SEM imaging on the Ar/Ar system in feldspars

    NASA Astrophysics Data System (ADS)

    Flude, S.; Sherlock, S.; Lee, M.; Kelley, S. P.

    2010-12-01

    Complex microtextures form in K-feldspar crystals as they cool and are affected by deuteric alteration. This complex structure is the cause of variable closure temperatures for Ar-Ar, a phenomenon which has been utilized in multi domain diffusion (MDD) modelling to recover thermal histories [1]. However, there has been substantial controversy regarding the precise interaction between feldspar microtextures and Ar-diffusion [2,3]. A number of studies have addressed this issue using coupled SEM imaging and Ar/Ar UV laser ablation microprobe (UV-LAMP) analysis on the same sample, to enable direct comparison of microtextures with Ar/Ar age data [4]. Here we have tested the idea that SEM work may affect Ar/Ar ages, leading to inaccurate results in subsequent Ar/Ar analyses. Three splits of alkali feldspar from the Dartmoor Granite in SW England were selected for Ar/Ar UV-LAMP analysis. Split 1 (“control”) was prepared as a polished thick section for Ar/Ar analysis. Split 2 (“SEM”) was prepared as a polished thick section, was chemically-mechanically polished with colloidal silica and underwent SEM imaging (uncoated) and focussed ion beam (FIB) milling (gold coated); electron beam damage in the SEM was maximised by leaving the sample at high magnification for eight minutes. Split 3 (“Etch”) is a cleavage fragment that was etched with HF vapour and underwent low to moderate magnification SEM imaging. The control split gave a range of laser-spot ages consistent with the expected cooling age of the granite and high yields of radiogenic 40Ar* (>90%). The area of the “SEM” split that experienced significant electron beam damage gave younger than expected ages and 40Ar* yields as low as 57%. These are interpreted as a combination of implantation of atmospheric Ar and local redistribution of K within the sample. The area of “SEM” that underwent FIB milling gave ages and 40Ar* yields comparable to the control split, suggesting that the Au-coat minimises FIB

  13. The Ensembl Variant Effect Predictor.

    PubMed

    McLaren, William; Gil, Laurent; Hunt, Sarah E; Riat, Harpreet Singh; Ritchie, Graham R S; Thormann, Anja; Flicek, Paul; Cunningham, Fiona

    2016-01-01

    The Ensembl Variant Effect Predictor is a powerful toolset for the analysis, annotation, and prioritization of genomic variants in coding and non-coding regions. It provides access to an extensive collection of genomic annotation, with a variety of interfaces to suit different requirements, and simple options for configuring and extending analysis. It is open source, free to use, and supports full reproducibility of results. The Ensembl Variant Effect Predictor can simplify and accelerate variant interpretation in a wide range of study designs. PMID:27268795

  14. 40Ar/39Ar geochronology, paleomagnetism, and evolution of the Boring volcanic field, Oregon and Washington, USA

    USGS Publications Warehouse

    Fleck, Robert J.; Hagstrum, Jonathan T.; Calvert, Andrew T.; Evarts, Russell C.; Conrey, Richard M.

    2014-01-01

    The 40Ar/39Ar investigations of a large suite of fine-grained basaltic rocks of the Boring volcanic field (BVF), Oregon and Washington (USA), yielded two primary results. (1) Using age control from paleomagnetic polarity, stratigraphy, and available plateau ages, 40Ar/39Ar recoil model ages are defined that provide reliable age results in the absence of an age plateau, even in cases of significant Ar redistribution. (2) Grouping of eruptive ages either by period of activity or by composition defines a broadly northward progression of BVF volcanism during latest Pliocene and Pleistocene time that reflects rates consistent with regional plate movements. Based on the frequency distribution of measured ages, periods of greatest volcanic activity within the BVF occurred 2.7–2.2 Ma, 1.7–0.5 Ma, and 350–50 ka. Grouped by eruptive episode, geographic distributions of samples define a series of northeast-southwest–trending strips whose centers migrate from south-southeast to north-northwest at an average rate of 9.3 ± 1.6 mm/yr. Volcanic activity in the western part of the BVF migrated more rapidly than that to the east, causing trends of eruptive episodes to progress in an irregular, clockwise sense. The K2O and CaO values of dated samples exhibit well-defined temporal trends, decreasing and increasing, respectively, with age of eruption. Divided into two groups by K2O, the centers of these two distributions define a northward migration rate similar to that determined from eruptive age groups. This age and compositional migration rate of Boring volcanism is similar to the clockwise rotation rate of the Oregon Coast Range with respect to North America, and might reflect localized extension on the trailing edge of that rotating crustal block.

  15. Revised error propagation of 40Ar/39Ar data, including covariances

    NASA Astrophysics Data System (ADS)

    Vermeesch, Pieter

    2015-12-01

    The main advantage of the 40Ar/39Ar method over conventional K-Ar dating is that it does not depend on any absolute abundance or concentration measurements, but only uses the relative ratios between five isotopes of the same element -argon- which can be measured with great precision on a noble gas mass spectrometer. The relative abundances of the argon isotopes are subject to a constant sum constraint, which imposes a covariant structure on the data: the relative amount of any of the five isotopes can always be obtained from that of the other four. Thus, the 40Ar/39Ar method is a classic example of a 'compositional data problem'. In addition to the constant sum constraint, covariances are introduced by a host of other processes, including data acquisition, blank correction, detector calibration, mass fractionation, decay correction, interference correction, atmospheric argon correction, interpolation of the irradiation parameter, and age calculation. The myriad of correlated errors arising during the data reduction are best handled by casting the 40Ar/39Ar data reduction protocol in a matrix form. The completely revised workflow presented in this paper is implemented in a new software platform, Ar-Ar_Redux, which takes raw mass spectrometer data as input and generates accurate 40Ar/39Ar ages and their (co-)variances as output. Ar-Ar_Redux accounts for all sources of analytical uncertainty, including those associated with decay constants and the air ratio. Knowing the covariance matrix of the ages removes the need to consider 'internal' and 'external' uncertainties separately when calculating (weighted) mean ages. Ar-Ar_Redux is built on the same principles as its sibling program in the U-Pb community (U-Pb_Redux), thus improving the intercomparability of the two methods with tangible benefits to the accuracy of the geologic time scale. The program can be downloaded free of charge from

  16. The B AB AR detector

    NASA Astrophysics Data System (ADS)

    Aubert, B.; Bazan, A.; Boucham, A.; Boutigny, D.; De Bonis, I.; Favier, J.; Gaillard, J.-M.; Jeremie, A.; Karyotakis, Y.; Le Flour, T.; Lees, J. P.; Lieunard, S.; Petitpas, P.; Robbe, P.; Tisserand, V.; Zachariadou, K.; Palano, A.; Chen, G. P.; Chen, J. C.; Qi, N. D.; Rong, G.; Wang, P.; Zhu, Y. S.; Eigen, G.; Reinertsen, P. L.; Stugu, B.; Abbott, B.; Abrams, G. S.; Amerman, L.; Borgland, A. W.; Breon, A. B.; Brown, D. N.; Button-Shafer, J.; Clark, A. R.; Dardin, S.; Day, C.; Dow, S. F.; Fan, Q.; Gaponenko, I.; Gill, M. S.; Goozen, F. R.; Gowdy, S. J.; Gritsan, A.; Groysman, Y.; Hernikl, C.; Jacobsen, R. G.; Jared, R. C.; Kadel, R. W.; Kadyk, J.; Karcher, A.; Kerth, L. T.; Kipnis, I.; Kluth, S.; Kral, J. F.; Lafever, R.; LeClerc, C.; Levi, M. E.; Lewis, S. A.; Lionberger, C.; Liu, T.; Long, M.; Luo, L.; Lynch, G.; Luft, P.; Mandelli, E.; Marino, M.; Marks, K.; Matuk, C.; Meyer, A. B.; Minor, R.; Mokhtarani, A.; Momayezi, M.; Nyman, M.; Oddone, P. J.; Ohnemus, J.; Oshatz, D.; Patton, S.; Pedrali-Noy, M.; Perazzo, A.; Peters, C.; Pope, W.; Pripstein, M.; Quarrie, D. R.; Rasson, J. E.; Roe, N. A.; Romosan, A.; Ronan, M. T.; Shelkov, V. G.; Stone, R.; Strother, P. D.; Telnov, A. V.; von der Lippe, H.; Weber, T. F.; Wenzel, W. A.; Zizka, G.; Bright-Thomas, P. G.; Hawkes, C. M.; Kirk, A.; Knowles, D. J.; O'Neale, S. W.; Watson, A. T.; Watson, N. K.; Deppermann, T.; Koch, H.; Krug, J.; Kunze, M.; Lewandowski, B.; Peters, K.; Schmuecker, H.; Steinke, M.; Andress, J. C.; Barlow, N. R.; Bhimji, W.; Chevalier, N.; Clark, P. J.; Cottingham, W. N.; De Groot, N.; Dyce, N.; Foster, B.; Mass, A.; McFall, J. D.; Wallom, D.; Wilson, F. F.; Abe, K.; Hearty, C.; McKenna, J. A.; Thiessen, D.; Camanzi, B.; Harrison, T. J.; McKemey, A. K.; Tinslay, J.; Antohin, E. I.; Blinov, V. E.; Bukin, A. D.; Bukin, D. A.; Buzykaev, A. R.; Dubrovin, M. S.; Golubev, V. B.; Ivanchenko, V. N.; Kolachev, G. M.; Korol, A. A.; Kravchenko, E. A.; Mikhailov, S. F.; Onuchin, A. P.; Salnikov, A. A.; Serednyakov, S. I.; Skovpen, Yu. I.; Telnov, V. I.; Yushkov, A. N.; Booth, J.; Lankford, A. J.; Mandelkern, M.; Pier, S.; Stoker, D. P.; Zioulas, G.; Ahsan, A.; Arisaka, K.; Buchanan, C.; Chun, S.; Faccini, R.; MacFarlane, D. B.; Prell, S. A.; Rahatlou, Sh.; Raven, G.; Sharma, V.; Burke, S.; Callahan, D.; Campagnari, C.; Dahmes, B.; Hale, D.; Hart, P. A.; Kuznetsova, N.; Kyre, S.; Levy, S. L.; Long, O.; Lu, A.; May, J.; Richman, J. D.; Verkerke, W.; Witherell, M.; Yellin, S.; Beringer, J.; DeWitt, J.; Dorfan, D. E.; Eisner, A. M.; Frey, A.; Grillo, A. A.; Grothe, M.; Heusch, C. A.; Johnson, R. P.; Kroeger, W.; Lockman, W. S.; Pulliam, T.; Rowe, W.; Sadrozinski, H.; Schalk, T.; Schmitz, R. E.; Schumm, B. A.; Seiden, A.; Spencer, E. N.; Turri, M.; Walkowiak, W.; Wilder, M.; Williams, D. C.; Chen, E.; Dubois-Felsmann, G. P.; Dvoretskii, A.; Hanson, J. E.; Hitlin, D. G.; Kolomensky, Yu. G.; Metzler, S.; Oyang, J.; Porter, F. C.; Ryd, A.; Samuel, A.; Weaver, M.; Yang, S.; Zhu, R. Y.; Devmal, S.; Geld, T. L.; Jayatilleke, S.; Jayatilleke, S. M.; Mancinelli, G.; Meadows, B. T.; Sokoloff, M. D.; Bloom, P.; Broomer, B.; Erdos, E.; Fahey, S.; Ford, W. T.; Gaede, F.; van Hoek, W. C.; Johnson, D. R.; Michael, A. K.; Nauenberg, U.; Olivas, A.; Park, H.; Rankin, P.; Roy, J.; Sen, S.; Smith, J. G.; Wagner, D. L.; Blouw, J.; Harton, J. L.; Krishnamurthy, M.; Soffer, A.; Toki, W. H.; Warner, D. W.; Wilson, R. J.; Zhang, J.; Brandt, T.; Brose, J.; Dahlinger, G.; Dickopp, M.; Dubitzky, R. S.; Eckstein, P.; Futterschneider, H.; Kocian, M. L.; Krause, R.; Müller-Pfefferkorn, R.; Schubert, K. R.; Schwierz, R.; Spaan, B.; Wilden, L.; Behr, L.; Bernard, D.; Bonneaud, G. R.; Brochard, F.; Cohen-Tanugi, J.; Ferrag, S.; Fouque, G.; Gastaldi, F.; Matricon, P.; Mora de Freitas, P.; Renard, C.; Roussot, E.; T'Jampens, S.; Thiebaux, C.; Vasileiadis, G.; Verderi, M.; Anjomshoaa, A.; Bernet, R.; Di Lodovico, F.; Muheim, F.; Playfer, S.; Swain, J. E.; Falbo, M.; Bozzi, C.; Dittongo, S.; Folegani, M.; Piemontese, L.; Ramusino, A. C.; Treadwell, E.; Anulli, F.; Baldini-Ferroli, R.; Calcaterra, A.; de Sangro, R.; Falciai, D.; Finocchiaro, G.; Patteri, P.; Peruzzi, I. M.; Piccolo, M.; Xie, Y.; Zallo, A.; Bagnasco, S.; Buzzo, A.; Contri, R.; Crosetti, G.; Fabbricatore, P.; Farinon, S.; Lo Vetere, M.; Macri, M.; Minutoli, S.; Monge, M. R.; Musenich, R.; Pallavicini, M.; Parodi, R.; Passaggio, S.; Pastore, F. C.; Patrignani, C.; Pia, M. G.; Priano, C.; Robutti, E.; Santroni, A.; Bartoldus, R.; Dignan, T.; Hamilton, R.; Mallik, U.; Cochran, J.; Crawley, H. B.; Fischer, P. A.; Lamsa, J.; McKay, R.; Meyer, W. T.; Rosenberg, E. I.; Albert, J. N.; Beigbeder, C.; Benkebil, M.; Breton, D.; Cizeron, R.; Du, S.; Grosdidier, G.; Hast, C.; Höcker, A.; Lacker, H. M.; LePeltier, V.; Lutz, A. M.

    2002-02-01

    B AB AR, the detector for the SLAC PEP-II asymmetric e +e - B Factory operating at the ϒ(4 S) resonance, was designed to allow comprehensive studies of CP-violation in B-meson decays. Charged particle tracks are measured in a multi-layer silicon vertex tracker surrounded by a cylindrical wire drift chamber. Electromagnetic showers from electrons and photons are detected in an array of CsI crystals located just inside the solenoidal coil of a superconducting magnet. Muons and neutral hadrons are identified by arrays of resistive plate chambers inserted into gaps in the steel flux return of the magnet. Charged hadrons are identified by d E/d x measurements in the tracking detectors and by a ring-imaging Cherenkov detector surrounding the drift chamber. The trigger, data acquisition and data-monitoring systems, VME- and network-based, are controlled by custom-designed online software. Details of the layout and performance of the detector components and their associated electronics and software are presented.

  17. 40Ar/39Ar and K-Ar data bearing on the metamorphic and tectonic history of western New England.

    USGS Publications Warehouse

    Sutter, J.F.; Ratcliffe, N.M.; Mukasa, S.B.

    1985-01-01

    40Ar/39Ar ages of coexisting biotite and hornblende from Proterozoic Y gneisses of the Berkshire and Green Mt massifs, as well as 40Ar/39Ar and K/Ar mineral and whole-rock ages from Palaeozoic metamorphic rocks, suggest that the thermal peaks for the dominant metamorphic recrystallization in western New England occurred 465 + or - 5 m.y. (Taconian). 40Ar/39Ar age data from a poorly-defined terrain along the eastern strip of the area suggests that the area has been retrograded during a metamorphism that peaked at least 376 + or - 5 m.y. (Acadian). Available age and petrological data from western New England indicate the presence of at least three separate metamorphic-structure domains of Taconic age: 1) a small area of relict high-P and low-T metamorphism, 2) a broad area of normal Barrovian metamorphism from chlorite to garnet grade characterized by a gentle metamorphic gradient and, 3) a rather narrow belt of steep-gradient, Barrovian series metamorphic rocks. Areas of maximum metamorphic intensity within the last domain coincide with areas of maximum crustal thickening in the later stage of Taconic orogeny. -L.di H

  18. Toward a high-resolution 40Ar/39Ar geochronology of the Tatun Volcano Group, Taiwan

    NASA Astrophysics Data System (ADS)

    Mesko, G. T.; Song, S.; Chang, S.; Hemming, S. R.; Turrin, B. D.

    2010-12-01

    The Tatun Volcano Group [TVG] consists of five volcanic subgroups of which ~30 edifices have been identified, all in close proximity to the densely populated Taipei Basin to its south (Song et al., 2000, Journal of the Geological Society of China, in Chinese). Evidence of eruptions is in the form of mostly lava flows, with pyroclastic flows, and ash deposition (Tsai et al., 2010, TAO), consistent with vulcanian and plinian eruptions that are only minimally preserved because of the region’s high weathering rate (Belousov et al., 2010, Journal of Volcanology and Geothermal Research). The TVG is made up of calc-alkaline andesite, with few interspersed basaltic lava flows that bear geochemical signatures consistent with subduction volcanism, yet due to tectonic location Teng (1996, Geology) describes it as Ryukyu back-arc basin volcanism, and still others attribute volcanism here to post-collisional collapse of the Taiwan orogen (Wang et al., 1999, Tectonophysics and 2004, Journal of Petrology). Various TVG samples were previously K-Ar dated by Juang and Chen (1989, Bulletin of Central Geological Survey, in Chinese), Tsao (1994, Bulletin of Central Geological Survey, in Chinese), and 40Ar/39Ar whole rock analyses by Lee (1996, masters thesis, National Taiwan University) to suggest volcanism from 2.8-2.5Ma and then from 1.5-.22Ma after which volcanic events ceased. In contrast, radiocarbon dates obtained from charcoal in related sediment by Chen et al. (2010, TAO) and Belousov et al. (2010, Journal of Volcanology Geothermal Research) suggest volcanic activity was present at 20ka and 6ka respectively. The andesite samples are microcrystalline; therefore hand picked aliquots of groundmass from the hand magnetic fraction were subjected to several iterations of sonic rinse in glycine-based soap, then 4N HNO3, then quartz-distilled water in a preparation modified from Nicolaysen et al. (2000, EPSL). Samples were co-irradiated at the USGS facility in Denver using Alder

  19. Ar-Ar investigations on Quarternary volcanic sequences of Monte Vulture (Southern Italy)

    NASA Astrophysics Data System (ADS)

    Buettner, A.; Principe, C.; Villa, I. M.

    2003-04-01

    Volcanic sanidines and phlogopites from feldspathoid- or sanidine-bearing volcanic sequences of Monte Vulture (Southern Italy) were analysed using Ar-Ar stepwise heating. In all samples phlogopite and sanidine are internally inhomogeneous as observed in a plot of Cl/K ratio vs step-age. At least two phases that are degassed during different temperature steps can be distinguished, resulting in internal discordance. Hence, all samples are affected by impurities and have to be treated as heterochemical mixtures. These effects are strongest in phlogopite. The high uncertainty of the calculated ages mirrors the statistically significant age discordance. The calculation of average ages was focused on isochemical steps. Isochron calculations necessarily give less precise ages because of the excessive scatter of datapoints. Moreover, most coarse grained phlogopites could contain excess 40Ar (as suggested by non-atmospheric intercepts in isochron plots) which could be of mantle origin. The analysed phlogopites from carbonatitic and melilite-foiditolite rocks of the upper stratigraphical sequence of Monte Vulture are Mg-rich, which indicates a mantle provenance (Stoppa and Woolley, 1997). All calculated phlogopite ages have, therefore, to be considered with caution. One sample, PG5, contains both sanidine and matrix phlogopite. This offers the possibility to directly compare the analytical results and further decipher possible inconsistencies. Ages were calculated as the average of isochemical steps, and errors are indicated at the 95% confidence level. Sanidine gives a weighted average age of 752±13 ka in agreement with the phlogopite age of 801±88 ka. In comparison to these average step ages, an isochron over five sanidine-steps yields an age of 737±35 ka (MSWD=1.5) and an atmospheric intercept of 295±49. The eruptive time-span covered by the analysed samples is 75±20 ka, confirming that the eruption history of Monte Vulture has been organised in clusters of activity

  20. Canatoxin, a toxic protein from jack beans (Canavalia ensiformis), is a variant form of urease (EC 3.5.1.5): biological effects of urease independent of its ureolytic activity.

    PubMed Central

    Follmer, C; Barcellos, G B; Zingali, R B; Machado, O L; Alves, E W; Barja-Fidalgo, C; Guimarães, J A; Carlini, C R

    2001-01-01

    Canatoxin is a toxic protein from Canavalia ensiformis seeds, lethal to mice (LD(50)=2 mg/kg) and insects. Further characterization of canatoxin showed that its main native form (184 kDa) is a non-covalently linked dimer of a 95 kDa polypeptide containing zinc and nickel. Partial sequencing of internal peptides indicated homology with urease (EC 3.5.1.5) from the same seed. Canatoxin has approx. 30% of urease's activity for urea, and K(m) of 2-7 mM. The proteins differ in their affinities for metal ions and were separated by affinity chromatography on a Zn(2+) matrix. Similar to canatoxin, urease activates blood platelets and interacts with glycoconjugates. In contrast with canatoxin, no lethality was seen in mice injected with urease (10 mg/kg). Pretreatment with p-hydroxymercuribenzoate irreversibly abolished the ureolytic activity of both proteins. On the other hand, p-hydroxymercuribenzoate-treated canatoxin was still lethal to mice, and both treated proteins were fully active in promoting platelet aggregation and binding to glycoconjugates. Taken together, our data indicate that canatoxin is a variant form of urease. Moreover, we show for the first time that these proteins display several biological effects that are unrelated to their enzymic activity for urea. PMID:11696010

  1. Late-stage volcano geomorphic evolution of the Pleistocene San Francisco Mountain, Arizona (USA), based on high-resolution DEM analysis and 40Ar/39Ar chronology

    NASA Astrophysics Data System (ADS)

    Karátson, Dávid; Telbisz, Tamás; Singer, Brad S.

    2010-09-01

    The cone-building volcanic activity and subsequent erosion of San Francisco Mountain, AZ, USA, were studied by using high-resolution digital elevation model (DEM) analysis and new 40Ar/39Ar dating. By defining remnants or planèzes of the volcano flanks in DEM-derived images, the original edifice can be reconstructed. We propose a two-cone model with adjacent summit vents which were active in different times. The reconstructed cones were 4,460 and 4,350 m high a.s.l., corresponding to ˜2,160 and 2,050 m relative height, respectively. New 40Ar/39Ar data allow us to decipher the chronological details of the cone-building activity. We dated the Older and Younger Andesites of the volcano that, according to previous mapping, built the stage 2 and stage 3 stratocones, respectively. The new 40Ar/39Ar plateau ages yielded 589-556 ka for the Older and 514-505 ka for the Younger Andesites, supporting their distinct nature with a possible dormant period between. The obtained ages imply an intense final (≤100 ka long) cone-building activity, terminating ˜100 ka earlier than indicated by previous K-Ar ages. Moreover, 40Ar/39Ar dating constrains the formation of the Inner Basin, an elliptical depression in the center of the volcano initially created by flank collapse. A 530 ka age (with a ±58.4 ka 2σ error) for a post-depression dacite suggests that the collapse event is geochronologically indistinguishable from the termination of the andesitic cone-building activity. According to our DEM analysis, the original cone of San Francisco Mountain had a volume of about 80 km3. Of this volume, ˜7.5 km3 was removed by the flank collapse and subsequent glacial erosion, creating the present-day enlarged Inner Basin, and ˜2 km3 was removed from the outer valleys by erosion. Based on volumetric analysis and previous and new radiometric ages, the average long-term eruption rate of San Francisco Mountain was ˜0.2 km3/ka, which is a medium rate for long-lived stratovolcanoes. However

  2. Drosophila alcohol dehydrogenase: developmental studies on cryptic variant lines.

    PubMed

    Miglani, G S; Ampy, F R

    1981-10-01

    Thirty-five cryptic variant lines were used to examine the mechanisms involved in genetic modulation of alcohol metabolism in Drosophila. Late third-instar larval, preemergence pupal, and adult stages cultured at 18 and 28 C were examined. Spectrophotometric analyses for native alcohol dehydrogenase (ADH) activity and residual ADH activity after treatment with guanidine hydrochloride and heat were performed. Differential response of cryptic variants to treatment with the denaturants during development suggested that this variation may have an adaptive significance. PMID:6800354

  3. Mutations in the control of virulence sensor gene from Streptococcus pyogenes after infection in mice lead to clonal bacterial variants with altered gene regulatory activity and virulence.

    PubMed

    Mayfield, Jeffrey A; Liang, Zhong; Agrahari, Garima; Lee, Shaun W; Donahue, Deborah L; Ploplis, Victoria A; Castellino, Francis J

    2014-01-01

    The cluster of virulence sensor (CovS)/responder (CovR) two-component operon (CovRS) regulates ∼15% of the genes of the Group A Streptococcal pyogenes (GAS) genome. Bacterial clones containing inactivating mutations in the covS gene have been isolated from patients with virulent invasive diseases. We report herein an assessment of the nature and types of covS mutations that can occur in both virulent and nonvirulent GAS strains, and assess whether a nonvirulent GAS can attain enhanced virulence through this mechanism. A group of mice were infected with a globally-disseminated clonal M1T1 GAS (isolate 5448), containing wild-type (WT) CovRS (5448/CovR+S+), or less virulent engineered GAS strains, AP53/CovR+S+ and Manfredo M5/CovR+S+. SpeB negative GAS clones from wound sites and/or from bacteria disseminated to the spleen were isolated and the covS gene was subjected to DNA sequence analysis. Numerous examples of inactivating mutations were found in CovS in all regions of the gene. The mutations found included frame-shift insertions and deletions, and in-frame small and large deletions in the gene. Many of the mutations found resulted in early translation termination of CovS. Thus, the covS gene is a genomic mutagenic target that gives GAS enhanced virulence. In cases wherein CovS- was discovered, these clonal variants exhibited high lethality, further suggesting that randomly mutated covS genes occur during the course of infection, and lead to the development of a more invasive infection. PMID:24968349

  4. Mutations in the Control of Virulence Sensor Gene from Streptococcus pyogenes after Infection in Mice Lead to Clonal Bacterial Variants with Altered Gene Regulatory Activity and Virulence

    PubMed Central

    Mayfield, Jeffrey A.; Liang, Zhong; Agrahari, Garima; Lee, Shaun W.; Donahue, Deborah L.; Ploplis, Victoria A.; Castellino, Francis J.

    2014-01-01

    The cluster of virulence sensor (CovS)/responder (CovR) two-component operon (CovRS) regulates ∼15% of the genes of the Group A Streptococcal pyogenes (GAS) genome. Bacterial clones containing inactivating mutations in the covS gene have been isolated from patients with virulent invasive diseases. We report herein an assessment of the nature and types of covS mutations that can occur in both virulent and nonvirulent GAS strains, and assess whether a nonvirulent GAS can attain enhanced virulence through this mechanism. A group of mice were infected with a globally-disseminated clonal M1T1 GAS (isolate 5448), containing wild-type (WT) CovRS (5448/CovR+S+), or less virulent engineered GAS strains, AP53/CovR+S+ and Manfredo M5/CovR+S+. SpeB negative GAS clones from wound sites and/or from bacteria disseminated to the spleen were isolated and the covS gene was subjected to DNA sequence analysis. Numerous examples of inactivating mutations were found in CovS in all regions of the gene. The mutations found included frame-shift insertions and deletions, and in-frame small and large deletions in the gene. Many of the mutations found resulted in early translation termination of CovS. Thus, the covS gene is a genomic mutagenic target that gives GAS enhanced virulence. In cases wherein CovS− was discovered, these clonal variants exhibited high lethality, further suggesting that randomly mutated covS genes occur during the course of infection, and lead to the development of a more invasive infection. PMID:24968349

  5. 40Ar/39Ar Ages of Carbonaceous Xenoliths in 2 HED Meteorites

    NASA Technical Reports Server (NTRS)

    Turrin, B.; Lindsay, F. N.; Park, J.; Herzog, G. F.; Delaney, J. S.; Swisher, C. C., III; Johnson, J.; Zolensky, M.

    2016-01-01

    The generally young K/Ar and 40Ar/39Ar ages of CM chondrites made us wonder whether carbonaceous xenoliths (CMX) entombed in Howardite–Eucrite–Diogenite (HED) meteorites might retain more radiogenic 40Ar than do ‘free-range’ CM-chondrites. To find out, we selected two HED breccias with carbonaceous inclusions in order to compare the 40Ar/39Ar release patterns and ages of the inclusions with those of nearby HED material. Carbonaceous inclusions (CMXs) in two HED meteorites lost a greater fraction of radiogenic 40Ar than did surrounding host material, but a smaller fraction of it than did free-range CM-chondrites such as Murchison or more heavily altered ones. Importantly, however, the siting of the CMXs in HED matrix did not prevent the 40Ar loss of about 40 percent of the radiogenic 40Ar, even from phases that degas at high laboratory temperatures. We infer that carbonaceous asteroids with perihelia of 1 astronomical unit probably experience losses of at least this size. The usefulness of 40Ar/39Ar dating for samples returned from C-type asteroids may hinge, therefore, on identifying and analyzing separately small quantities of the most retentive phases of carbonaceous chondrites.

  6. Geology and 40Ar/39Ar Geochronology of Akutan Volcano, Eastern Aleutian Islands

    NASA Astrophysics Data System (ADS)

    Coombs, M. L.; Jicha, B. R.

    2013-12-01

    40Ar/39Ar dating and new whole-rock geochemical analyses are used to establish an eruptive chronology for Akutan volcano, Akutan Island, in the eastern Aleutian island arc. Akutan Island (166° W, 54.1° N) is the site of long-lived volcanism and the entire island comprises volcanic rocks as old as 3.3 Ma (Richter et al., 1998, USGS Open-File 98-135). Our current focus is on the 225 km2 western half of the island, which is home to the Holocene active cone, Holocene to latest Pleistocene satellite vents, and underlying middle Pleistocene volcanic basement rocks. Eruptive products span the tholeiitic-calc-alkaline boundary, are medium-K, and range from basalt to dacite. Furnace incremental heating experiments on groundmass separates of 38 samples resulted in 29 40Ar/39Ar ages. The remainder did not yield radiogenic 40Ar contents and are likely Holocene in age. The oldest ages (1251×10 and 1385×12 ka) are from a wedge of flat-lying dissected lavas north of the Holocene cone; these likely represent the upper part of the volcanic basement that underlies the entire island. Above a major unconformity lie basaltic andesite to dacite lavas that range from 765× 4 to 522×8 ka. The eroded remnants of the source volcano for these flows appears to crop out as a series of variably hydrothermally altered breccias and domes 5 km east-northeast of the current summit. A 625 m-tall eroded basaltic center, Lava Peak, sits 6 km northwest of the summit; its deeply incised western flank exposes lava flows and a plug. Two flows are dated at 598×16 and 602×15 ka. A high ridge 1.5 km south of the summit is made of oxidized, mostly andesitic lavas 284-249 ka old; these are presumably the remnants of an eruptive center located near the current cone. Flat Top Peak, 3.5 km southwest of the summit, produced almost exclusively basalts and six dated lavas range from 155×8 to 98×18 ka. Lavas from Flat Top (1065 m asl) are deeply eroded suggesting extensive ice cover during marine isotope

  7. 40Ar/39Ar ages of lunar impact glasses: Relationships among Ar diffusivity, chemical composition, shape, and size

    NASA Astrophysics Data System (ADS)

    Zellner, N. E. B.; Delano, J. W.

    2015-07-01

    Lunar impact glasses, which are quenched melts produced during cratering events on the Moon, have the potential to provide not only compositional information about both the local and regional geology of the Moon but also information about the impact flux over time. We present in this paper the results of 73 new 40Ar/39Ar analyses of well-characterized, inclusion-free lunar impact glasses and demonstrate that size, shape, chemical composition, fraction of radiogenic 40Ar retained, and cosmic ray exposure (CRE) ages are important for 40Ar/39Ar investigations of these samples. Specifically, analyses of lunar impact glasses from the Apollo 14, 16, and 17 landing sites indicate that retention of radiogenic 40Ar is a strong function of post-formation thermal history in the lunar regolith, size, and chemical composition. This is because the Ar diffusion coefficient (at a constant temperature) is estimated to decrease by ∼3-4 orders of magnitude with an increasing fraction of non-bridging oxygens, X(NBO), over the compositional range of most lunar impact glasses with compositions from feldspathic to basaltic. Based on these relationships, lunar impact glasses with compositions and sizes sufficient to have retained ∼90% of their radiogenic Ar during 750 Ma of cosmic ray exposure at time-integrated temperatures of up to 290 K have been identified and are likely to have yielded reliable 40Ar/39Ar ages of formation. Additionally, ∼50% of the identified impact glass spheres have formation ages of ⩽500 Ma, while ∼75% of the identified lunar impact glass shards and spheres have ages of formation ⩽2000 Ma. Higher thermal stresses in lunar impact glasses quenched from hyperliquidus temperatures are considered the likely cause of poor survival of impact glass spheres, as well as the decreasing frequency of lunar impact glasses in general with increasing age. The observed age-frequency distribution of lunar impact glasses may reflect two processes: (i) diminished

  8. Ar-40/Ar-39 laser-probe dating of diamond inclusions from the Premier kimberlite

    NASA Technical Reports Server (NTRS)

    Phillips, D.; Onstott, T. C.; Harris, J. W.

    1989-01-01

    The results of Ar-40/Ar-39 laser-probe analyses of individual eclogitic clinopyroxene inclusions from Premier diamonds are reported which yield a mean age of 1198 + or - 14 Myr. This age agrees well with Sm-Nd and Ar-40/Ar-39 analyses on similar Premier inclusions and is indistinguishable from the inferred time of emplacement of the host kimberlite, which implies that diamond formation was essentially synchronous with kimberlite generation. The extrapolated nonradiogenic Ar-40/Ar-36 ratio of 334 + or - 102 is similar to the present-day atmospheric composition. This value is inconsistent with Sr and Nd isotopic signatures from Premier eclogite inclusions, which suggest a depleted mantle source. Preentrapment equilibration of the inclusions with an Ar-36-rich fluid is the most probable explanation for the low nonradiogenic composition.

  9. Group B streptococcal opacity variants.

    PubMed Central

    Pincus, S H; Cole, R L; Wessels, M R; Corwin, M D; Kamanga-Sollo, E; Hayes, S F; Cieplak, W; Swanson, J

    1992-01-01

    Colony opacity variants were detected for type III group B streptococci (GBS). Transparent colonies predominate in the parent GBS, with occasional colonies having opaque portions. Two stable opaque variants (1.1 and 1.5) were compared with three transparent clones (1.2, 1.3, and 1.4). All grew well on blood agar and on GC medium, but variant 1.1 failed to grow on Todd-Hewitt medium. Scanning and transmission electron microscopy demonstrated that colony opacity correlated with bacterial aggregation status, with opaque variants forming longer and more organized chains. Opaque-transparent switches were observed in both directions for most variants, with transparent to opaque noted most frequently, but 1.5 did not switch at all. Switching of the opacity phenotype was observed both in vitro and in neonatal mice. Relationships between colony opacity and several cell surface phenomena were explored. (i) Opaque variant 1.1 had two surface proteins (46 and 75 kDa) that were either unique or greatly overexpressed. (ii) Variant 1.1 was deficient in type III polysaccharide, while 1.5 lacked group B antigen. Diminished capsular polysaccharide of variant 1.1 was reflected in reduced negative electrophoretic mobility and in increased buoyant density. (iii) Transparent variant colonies growing closest to a penicillin disk were opaque, but colonial variants did not differ in their sensitivity to penicillin. These data indicate that GBS can exist in both opaque and transparent forms, with opaque appearance occurring by multiple routes. Opaque variants grow poorly on Todd-Hewitt medium generally used for isolation of GBS, so any possible relationships between opacity variation and pathogenesis of GBS infection are unknown. Images PMID:1592825

  10. Variants of windmill nystagmus.

    PubMed

    Choi, Kwang-Dong; Shin, Hae Kyung; Kim, Ji-Soo; Kim, Sung-Hee; Choi, Jae-Hwan; Kim, Hyo-Jung; Zee, David S

    2016-07-01

    Windmill nystagmus is characterized by a clock-like rotation of the beating direction of a jerk nystagmus suggesting separate horizontal and vertical oscillators, usually 90° out of phase. We report oculographic characteristics in three patients with variants of windmill nystagmus in whom the common denominator was profound visual loss due to retinal diseases. Two patients showed a clock-like pattern, while in the third, the nystagmus was largely diagonal (in phase or 180° out of phase) but also periodically changed direction by 180°. We hypothesize that windmill nystagmus is a unique manifestation of "eye movements of the blind." It emerges when the central structures, including the cerebellum, that normally keep eye movements calibrated and gaze steady can no longer perform their task, because they are deprived of the retinal image motion that signals a need for adaptive recalibration. PMID:27159990

  11. Phenylketonuria variants in Ontario.

    PubMed Central

    1976-01-01

    Since mass screening of the newborn population for phenylketonuria (PKU) by the Guthrie test was begun in Ontario in July 1965 many variants of PKU have been recognized in the 96 to 97% screened. Seventy-one cases of classic PKU were detected (four were missed). Of 48 cases of persistent hyperphenylalaninemia discovered, 18 were classified as atypical PKU and 30 as persistent benign hyperphenylalaninemia. Numerous infants with transient hyperphenylalaninemia (initial values over 10 mg/dl in 12), in many instances the result of transient neonatal tyrosinemia, were discovered. There was a slight predominance of males. Serum phenylalanine values of up to 15 mg/dl seemed to be harmless to the developing brain. A survey of 67 247 adults in the general population revealed 1 person with PKU and 1 with persistent benign hyperphenylalaninemia; both had normal intelligence quotients. Of 1548 mothers of retarded children tested, none had hyperphenylalaninemia. PMID:953933

  12. Progress in methods for rare variant association.

    PubMed

    Santorico, Stephanie A; Hendricks, Audrey E

    2016-01-01

    Empirical studies and evolutionary theory support a role for rare variants in the etiology of complex traits. Given this motivation and increasing affordability of whole-exome and whole-genome sequencing, methods for rare variant association have been an active area of research for the past decade. Here, we provide a survey of the current literature and developments from the Genetics Analysis Workshop 19 (GAW19) Collapsing Rare Variants working group. In particular, we present the generalized linear regression framework and associated score statistic for the 2 major types of methods: burden and variance components methods. We further show that by simply modifying weights within these frameworks we arrive at many of the popular existing methods, for example, the cohort allelic sums test and sequence kernel association test. Meta-analysis techniques are also described. Next, we describe the 6 contributions from the GAW19 Collapsing Rare Variants working group. These included development of new methods, such as a retrospective likelihood for family data, a method using genomic structure to compare cases and controls, a haplotype-based meta-analysis, and a permutation-based method for combining different statistical tests. In addition, one contribution compared a mega-analysis of family-based and population-based data to meta-analysis. Finally, the power of existing family-based methods for binary traits was compared. We conclude with suggestions for open research questions. PMID:26866487

  13. Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis.

    PubMed

    Gu, Hongmei; Fisher, Amanda J; Mickler, Elizabeth A; Duerson, Frank; Cummings, Oscar W; Peters-Golden, Marc; Twigg, Homer L; Woodruff, Trent M; Wilkes, David S; Vittal, Ragini

    2016-06-01

    Complement activation, an integral arm of innate immunity, may be the critical link to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Whereas we have previously reported elevated anaphylatoxins-complement component 3a (C3a) and complement component 5a (C5a)-in IPF, which interact with TGF-β and augment epithelial injury in vitro, their role in IPF pathogenesis remains unclear. The objective of the current study is to determine the mechanistic role of the binding of C3a/C5a to their respective receptors (C3aR and C5aR) in the progression of lung fibrosis. In normal primary human fetal lung fibroblasts, C3a and C5a induces mesenchymal activation, matrix synthesis, and the expression of their respective receptors. We investigated the role of C3aR and C5aR in lung fibrosis by using bleomycin-injured mice with fibrotic lungs, elevated local C3a and C5a, and overexpression of their receptors via pharmacologic and RNA interference interventions. Histopathologic examination revealed an arrest in disease progression and attenuated lung collagen deposition (Masson's trichrome, hydroxyproline, collagen type I α 1 chain, and collagen type I α 2 chain). Pharmacologic or RNA interference-specific interventions suppressed complement activation (C3a and C5a) and soluble terminal complement complex formation (C5b-9) locally and active TGF-β1 systemically. C3aR/C5aR antagonists suppressed local mRNA expressions of tgfb2, tgfbr1/2, ltbp1/2, serpine1, tsp1, bmp1/4, pdgfbb, igf1, but restored the proteoglycan, dcn Clinically, compared with pathologically normal human subjects, patients with IPF presented local induction of C5aR, local and systemic induction of soluble C5b-9, and amplified expression of C3aR/C5aR in lesions. The blockade of C3aR and C5aR arrested the progression of fibrosis by attenuating local complement activation and TGF-β/bone morphologic protein signaling as well as restoring decorin, which suggests a promising therapeutic strategy for patients with

  14. arXiv.org and Physics Education

    NASA Astrophysics Data System (ADS)

    Ramlo, Susan

    2007-09-01

    The website arXiv.org (pronounced archive) is a free online resource for full-text articles in the fields of physics, mathematics, computer science, nonlinear science, and quantitative biology that has existed for about 15 years. Available directly at http://www.arXiv.org, this e-print archive is searchable. As of Jan. 3, 2007, arXiv had open access to 401,226 e-prints in the topic areas. Those who sign up for an ID and password can also sign up for daily submission abstract emails for specific subject classes of arXiv, including physics education, physics and society, and history of physics. Founded and developed by Paul Ginsparg when he was at Los Alamos National Laboratory, arXiv's original name was the LANL preprint archive or xxx.lanl.gov. The location and name changed after Ginsparg moved to the physics department at Cornell University. Today, arXiv is hosted and operated by Cornell University library. Mirror sites for arXiv exist worldwide.2

  15. Identification and functional characterization of NifA variants that are independent of GlnB activation in the photosynthetic bacterium Rhodospirillum rubrum.

    PubMed

    Zou, Xiaoxiao; Zhu, Yu; Pohlmann, Edward L; Li, Jilun; Zhang, Yaoping; Roberts, Gary P

    2008-09-01

    The activity of NifA, the transcriptional activator of the nitrogen fixation (nif) gene, is tightly regulated in response to ammonium and oxygen. However, the mechanisms for the regulation of NifA activity are quite different among various nitrogen-fixing bacteria. Unlike the well-studied NifL-NifA regulatory systems in Klebsiella pneumoniae and Azotobacter vinelandii, in Rhodospirillum rubrum NifA is activated by a direct protein-protein interaction with the uridylylated form of GlnB, which in turn causes a conformational change in NifA. We report the identification of several substitutions in the N-terminal GAF domain of R. rubrum NifA that allow NifA to be activated in the absence of GlnB. Presumably these substitutions cause conformational changes in NifA necessary for activation, without interaction with GlnB. We also found that wild-type NifA can be activated in a GlnB-independent manner under certain growth conditions, suggesting that some other effector(s) can also activate NifA. An attempt to use Tn5 mutagenesis to obtain mutants that altered the pool of these presumptive effector(s) failed, though much rarer spontaneous mutations in nifA were detected. This suggests that the necessary alteration of the pool of effector(s) for NifA activation cannot be obtained by knockout mutations. PMID:18757802

  16. Rutaecarpine and evodiamine selected as β1-AR inhibitor candidates using β1-AR/CMC-offline-UPLC/MS prevent cardiac ischemia-reperfusion injury via energy modulation.

    PubMed

    Xue, Hui; Cheng, Yongjie; Wang, Xin; Yue, Yuan; Zhang, Weifang; Li, Xiaoni

    2015-11-10

    In the present study, an offline analytical method combining β1-adrenergic receptor/cell membrane chromatography (β1-AR/CMC) with ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) was used for direct recognition, separation, and identification of β1-AR inhibitors from Evodia rutaecarpa (Juss) Benth, by which rutaecarpine and evodiamine were screened and identified as potential β1-AR antagonists and the β1-AR inhibition activity of them was confirmed by downregulation of cAMP and PKA in vitro test. In addition, the results of in vivo pharmacological trials revealed that rutaecarpine (1.1mg/ml) and evodiamine (1.1mg/ml) attenuated myocardial infarct size injured by myocardial ischemia/reperfusion, improved metabolism disorders between fatty acid and glucose, increased the content of ATP, Ca(2+)-ATPase activity and reduced the content of peroxisome proliferator-activated receptor α (PPARα) protein level. Thus, the β1-AR/CMC-offline-UPLC/MS method developed in this study could be used as an effective alternative for screening β1-AR binding bioactive components in traditional Chinese medicines and the bioactive components could be used to remedy cardiac diseases via energy modulation. PMID:26263059

  17. K-Ar dating and delta O-18-delta D characterization of nanometric illite from Ordovician K-bentonites of the Appalachians: illitization and the Acadian-Alleghenian tectonic activity

    USGS Publications Warehouse

    Clauer, Norbert; Fallick, Anthony E.; Eberl, Dennis D.; Honty, Miroslav; Huff, Warren D.; Auberti, Amelie

    2013-01-01

    Nanometric (2 diagram that illitization occurred in all fractions by simultaneous nucleation and crystal growth, except for one sample. In that sample, a period of growth without nucleation was detected on top of the nucleation and growth episode. The K-Ar ages organize into two isochrons, the first at 319.9 ± 2.0 Ma with an initial 40Ar/36Ar ratio of 271 ± 66 Ma, and the second at 284.9 ± 1.2 Ma with an initial 40Ar/36Ar ratio of 310 ± 44. One data point above the older isochron and three between the two isochrons suggest a detrital contamination for the former separate and a possible further generation of nanoparticles for the three others. The samples with the older crystallization age consist of illite and illite-rich mixed-layers, and those with the younger age contain smectite-rich mixed-layers without illite, or illite-enriched illite-smectite mixed-layers. The K-Ar ages fit the age trends published previously for similar K-bentonites with regional age patterns between 240 and 270 Ma in the southwestern region, between 270 and 300 Ma in the central zone and the southern Appalachians, and between 315 and 370 Ma in the northernmost. Each of the two generations of illite crystals yields very consistent δ18O (V-SMOW) values at 17 ± 1‰ for the older and at 21 ± 1‰ for the younger. If crystallization temperatures of the nanometric illite were between 100 and 200 °C, as suggested by microthermometric determinations, the hydrothermal fluids had δ18O values of 4 ± 1‰ in the Dalton district and of 8 ± 1‰ in the Lafayette, Trenton, and Dirtseller districts at 100 °C, and of 11 ± 1 and 15 ± 1‰ in the same locations at 200 °C, probably because the water-rock isotope exchanges at elevated temperature occurred in rock-dominated systems. The δ18O of the fluids remained unchanged during local crystal growth, but varied depending on the geographic location of the samples and timing of illitization. The δD (V-SMOW) values of the different size

  18. Exome sequencing of case-unaffected-parents trios reveals recessive and de novo genetic variants in sporadic ALS

    PubMed Central

    Steinberg, Karyn Meltz; Yu, Bing; Koboldt, Daniel C.; Mardis, Elaine R.; Pamphlett, Roger

    2015-01-01

    The contribution of genetic variants to sporadic amyotrophic lateral sclerosis (ALS) remains largely unknown. Either recessive or de novo variants could result in an apparently sporadic occurrence of ALS. In an attempt to find such variants we sequenced the exomes of 44 ALS-unaffected-parents trios. Rare and potentially damaging compound heterozygous variants were found in 27% of ALS patients, homozygous recessive variants in 14% and coding de novo variants in 27%. In 20% of patients more than one of the above variants was present. Genes with recessive variants were enriched in nucleotide binding capacity, ATPase activity, and the dynein heavy chain. Genes with de novo variants were enriched in transcription regulation and cell cycle processes. This trio study indicates that rare private recessive variants could be a mechanism underlying some case of sporadic ALS, and that de novo mutations are also likely to play a part in the disease. PMID:25773295

  19. Volcanic history and 40Ar/39Ar and 14C geochronology of Terceira Island, Azores, Portugal

    USGS Publications Warehouse

    Calvert, A.T.; Moore, R.B.; McGeehin, J.P.; Rodrigues da Silva, A.M.

    2006-01-01

    Seven new 40Ar/39Ar and 23 new radiocarbon ages of eruptive units, in support of new geologic mapping, improve the known chronology of Middle to Late Pleistocene and Holocene volcanic activity on the island of Terceira, Azores and define an east-to-west progression in stratovolcano growth. The argon ages indicate that Cinco Picos Volcano, the oldest on Terceira, completed its main subaerial cone building activity by about 370-380??ka. Collapse of the upper part of the stratovolcanic edifice to form a 7 ?? 9??km caldera occurred some time after 370??ka. Postcaldera eruptions of basalt from cinder cones on and near the caldera floor and trachytic pyroclastic flow and pumice fall deposits from younger volcanoes west of Cinco Picos have refilled much of the caldera. The southern portion of Guilherme Moniz Volcano, in the central part of the island, began erupting prior to 270??ka and produced trachyte domes, flows, and minor pyroclastic deposits until at least 111??ka. The northern part of Guilherme Moniz Caldera is less well exposed than the southern part, but reflects a similar age range. The northwest portion of the caldera was formed sometime after 44??ka. Several well-studied ignimbrites that blanket much of the island likely erupted from Guilherme Moniz Volcano. The Pico Alto Volcanic Center, a tightly spaced cluster of trachyte domes and short flows, is a younger part of Guilherme Moniz Volcano. Stratigraphic studies and our new radiocarbon ages suggest that most of the Pico Alto eruptions occurred during the period from about 9000 to 1000??years BP. Santa Barbara Volcano is the youngest stratovolcano on Terceira, began erupting prior to 29??ka, and has been active historically. ?? 2006.

  20. Selective β2-AR Blockage Suppresses Colorectal Cancer Growth Through Regulation of EGFR-Akt/ERK1/2 Signaling, G1-Phase Arrest, and Apoptosis.

    PubMed

    Chin, Chih-Chien; Li, Jhy-Ming; Lee, Kam-Fai; Huang, Yun-Ching; Wang, Kuan-Chieh; Lai, Hsiao-Ching; Cheng, Chih-Chung; Kuo, Yi-Hung; Shi, Chung-Sheng

    2016-02-01

    The stress-upregulated catecholamines-activated β1- and β2-adrenergic receptors (β1/2-ARs) have been shown to accelerate the progression of cancers such as colorectal cancer (CRC). We investigated the underlying mechanism of the inhibition of β1/2-ARs signaling for the treatment of CRC and elucidated the significance of β2-AR expression in CRC in vitro and in clinical samples. The impacts of β1/2-AR antagonists in CRC in vitro and CRC-xenograft in vivo were examined. We found that repression of β2-AR but not β1-AR signaling selectively suppressed cell viability, induced G1-phase cell cycle arrest, caused both intrinsic and extrinsic pathways-mediated apoptosis of specific CRC cells and inhibited CRC-xenograft growth in vivo. Moreover, the expression of β2-AR was not consistent with the progression of CRC in vitro or in clinical samples. Our data evidence that the expression profiles, signaling, and blockage of β2-AR have a unique pattern in CRC comparing to other cancers. β2-AR antagonism selectively suppresses the growth of CRC accompanying active β2-AR signaling, which potentially carries wild-type KRAS, in vitro and in vivo via the inhibition of β2-AR transactivated EFGR-Akt/ERK1/2 signaling pathway. Thus, β2-AR blockage might be a potential therapeutic strategy for combating the progressions of β2-AR-dependent CRC. PMID:26189563

  1. Isolation of myeloma variants with predefined variant surface immunoglobulin by cell sorting.

    PubMed Central

    Liesegang, B; Radbruch, A; Rajewsky, K

    1978-01-01

    We describe a procedure for the isolation of somatic cell variants in which gene products are expressed on the cell surface that are not expressed in the wild type. Cloned cells of the myeloma line MPC 11, which expresses an IgG2b protein, were incubated with an antiserum specific for IgGI and IgG2a. Cells reacting with this antiserum were stained with a fluorescent anti-antiserum and enriched in three cycles of sorting in the fluorescence-activated cell sorter and subsequent growth in vitro. From the enriched population two variants were isolated by cloning in soft agar. One of them expressed a variant immunoglobulin that types serologically as an IgG2a but whose variable portion was idiotypically related to that of the MPC 11 wild-type protein. Images PMID:308657

  2. The 40Ar/39Ar dating technique applied to planetary sciences

    NASA Astrophysics Data System (ADS)

    Jourdan, F.

    2012-12-01

    The 40Ar/39Ar technique is a powerful geochronological method that can help to unravel the evolution of the solar system. The 40Ar/39Ar system can not only record the timing of volcanic and metamorphic processes on asteroids and planets, it finds domain of predilection in dating impact events throughout the solar system. However, the 40Ar/39Ar method is a robust analytical technique if, and only if, the events to be dated are well understood and data are not over interpreted. Yet, too many 'ages' reported in the literature are still based on over-interpretation of perturbed age spectra which tends to blur the big picture. This presentation is centred on the most recent applications of the 40Ar/39Ar technique applied to planetary material and through several examples, will attempt to demonstrate the benefit of focusing on statistically robust data. For example, 40Ar/39Ar dating of volcanic events on the Moon suggests that volcanism was mostly concentrated between ca. 3.8 and 3.1 Ga but statistical filtering of the data allow identifying a few well-defined eruptive events. The study of lunar volcanism would also benefit from dating of volcanic spherules. Rigorous filtering of the 40Ar/39Ar age database of lunar melt breccias yielded concordant and ages with high precision for two major basins (i.e. Imbrium & Serenitatis) of the Moon. 40Ar/39Ar dating of lunar impact spherules recovered from four different sites and with high- and low-K compositions shows an increase of ages younger than 400 Ma suggesting a recent increase in the impact flux. The impact history of the LL parent body (bodies?) has yet to be well constrained but may mimic the LHB observed on the Moon, which would indicate that the LL parent body was quite large. 40Ar/39Ar dating (in progress) of grains from the asteroid Itokawa recovered by the japanese Hayabusa mission have the potential to constrain the formation history and exposure age of Itokawa and will allow us to compare the results with the

  3. Instrumentation development for planetary in situ 40Ar/39Ar geochronology

    NASA Astrophysics Data System (ADS)

    Davidheiser-Kroll, B.; Morgan, L. E.; Munk, M.; Warner, N. H.; Gupta, S.; Slaybaugh, R.; Harkness, P.; Mark, D. F.

    2015-12-01

    The chronology of the Solar System, particularly the timing of formation of extraterrestrial bodies and their features, is a major outstanding problem in planetary science. Although various chronological methods for in situ geochronology have been proposed (e.g. Rb-Sr, K-Ar), and even applied (K-Ar, Farley et al., 2014), the reliability, accuracy, and applicability of the 40Ar/39Ar method makes it by far the most desirable chronometer for dating extraterrestrial bodies. The method however relies on the neutron irradiation of samples, and thus a neutron source. We will discuss the challenges and feasibility of deploying a passive neutron source to planetary surfaces for the in situ application of the 40Ar/39Ar chronometer. Requirements in generating and shielding neutrons, as well as analyzing samples are discussed, along with an exploration of limitations such as mass, power, and cost. Two potential solutions for the in situ extraterrestrial deployment of the 40Ar/39Ar method will be presented. Although this represents a challenging task, developing the technology to apply the 40Ar/39Ar method on planetary surfaces would represent a major advance towards constraining the timescale of solar system formation and evolution.

  4. Ar-40/Ar-39 Studies of Martian Meteorite RBT 04262 and Terrestrial Standards

    NASA Technical Reports Server (NTRS)

    Park, J.; Herzog, G. F.; Turrin, B.; Lindsay, F. N.; Delaney, J. S.; Swisher, C. C., III; Nagao, K.; Nyquist, L. E.

    2014-01-01

    Park et al. recently presented an Ar-40/Ar-39 dating study of maskelynite separated from the Martian meteorite RBT 04262. Here we report an additional study of Ar-40/Ar-39 patterns for smaller samples, each consisting of only a few maskelynite grains. Considered as a material for Ar-40/Ar-39 dating, the shock-produced glass maskelynite has both an important strength (relatively high K concentration compared to other mineral phases) and some potentially problematic weaknesses. At Rutgers, we have been analyzing small grains consisting of a single phase to explore local effects that might be averaged and remain hidden in larger samples. Thus, to assess the homogeneity of the RBT maskelynite and for comparison with the results of, we analyzed six approx. 30 microgram samples of the same maskelynite separate they studied. Furthermore, because most Ar-40/Ar-39 are calculated relative to the age of a standard, we present new Ar-40/Ar-39 age data for six standards. Among the most widely used standards are sanidine from Fish Canyon (FCs) and various hornblendes (hb3gr, MMhb-1, NL- 25), which are taken as primary standards because their ages have been determined by independent, direct measurements of K and A-40.

  5. GPR88 in A2AR Neurons Enhances Anxiety-Like Behaviors

    PubMed Central

    Meirsman, Aura Carole; Robé, Anne

    2016-01-01

    Abstract GPR88 is an orphan G-protein-coupled receptor highly expressed in striatal dopamine D1 (receptor) R- and D2R-expressing medium spiny neurons. This receptor is involved in activity and motor responses, and we previously showed that this receptor also regulates anxiety-like behaviors. To determine whether GPR88 in D2R-expressing neurons contributes to this emotional phenotype, we generated conditional Gpr88 knock-out mice using adenosine A2AR (A2AR)-Cre-driven recombination, and compared anxiety-related responses in both total and A2AR-Gpr88 KO mice. A2AR-Gpr88 KO mice showed a selective reduction of Gpr88 mRNA in D2R-expressing, but not D1R-expressing, neurons. These mutant mice showed increased locomotor activity and decreased anxiety-like behaviors in light/dark and elevated plus maze tests. These phenotypes were superimposable on those observed in total Gpr88 KO mice, demonstrating that the previously reported anxiogenic activity of GPR88 operates at the level of A2AR-expressing neurons. Further, A2AR-Gpr88 KO mice showed no change in novelty preference and novelty-suppressed feeding, while these responses were increased and decreased, respectively, in the total Gpr88 KO mice. Also, A2AR-Gpr88 KO mice showed intact fear conditioning, while the fear responses were decreased in total Gpr88 KO. We therefore also show for the first time that GPR88 activity regulates approach behaviors and conditional fear; however, these behaviors do not seem mediated by receptors in A2AR neurons. We conclude that Gpr88 expressed in A2AR neurons enhances ethological anxiety-like behaviors without affecting conflict anxiety and fear responses. PMID:27570825

  6. GPR88 in A2AR Neurons Enhances Anxiety-Like Behaviors.

    PubMed

    Meirsman, Aura Carole; Robé, Anne; de Kerchove d'Exaerde, Alban; Kieffer, Brigitte Lina

    2016-01-01

    GPR88 is an orphan G-protein-coupled receptor highly expressed in striatal dopamine D1 (receptor) R- and D2R-expressing medium spiny neurons. This receptor is involved in activity and motor responses, and we previously showed that this receptor also regulates anxiety-like behaviors. To determine whether GPR88 in D2R-expressing neurons contributes to this emotional phenotype, we generated conditional Gpr88 knock-out mice using adenosine A2AR (A2AR)-Cre-driven recombination, and compared anxiety-related responses in both total and A2AR-Gpr88 KO mice. A2AR-Gpr88 KO mice showed a selective reduction of Gpr88 mRNA in D2R-expressing, but not D1R-expressing, neurons. These mutant mice showed increased locomotor activity and decreased anxiety-like behaviors in light/dark and elevated plus maze tests. These phenotypes were superimposable on those observed in total Gpr88 KO mice, demonstrating that the previously reported anxiogenic activity of GPR88 operates at the level of A2AR-expressing neurons. Further, A2AR-Gpr88 KO mice showed no change in novelty preference and novelty-suppressed feeding, while these responses were increased and decreased, respectively, in the total Gpr88 KO mice. Also, A2AR-Gpr88 KO mice showed intact fear conditioning, while the fear responses were decreased in total Gpr88 KO. We therefore also show for the first time that GPR88 activity regulates approach behaviors and conditional fear; however, these behaviors do not seem mediated by receptors in A2AR neurons. We conclude that Gpr88 expressed in A2AR neurons enhances ethological anxiety-like behaviors without affecting conflict anxiety and fear responses. PMID:27570825

  7. Improvements Needed in the 40Ar/39Ar Study of Geomagnetic Excursion Chronology

    NASA Astrophysics Data System (ADS)

    Champion, D. E.; Turrin, B. D.

    2015-12-01

    Our knowledge of the existence and frequency of brief geomagnetic polarity. excursions only increases with time. Precise and accurate 40Ar/39Ar ages will be required to document this, because 25 or more excursions may have occurred within the Brunhes Epoch (780ky) separated in time by as little as 10ky. Excursions are and will dominantly be discovered in mafic, low K2O rocks. Improvements in the analytical protocol to 40Ar/39Ar date low K2O, "young", and thus low 40Arrad rocks are required. While conventional K/Ar dating "worked", the assumption of perfect atmospheric equilibration is flawed. In particular, using a measured isochron intercept (±2s) to embrace an atmospheric intercept assumption turns a 40Ar/39Ar diffusive extraction into a series of "K/Ar-lite" experiments. The near ubiquitous excess 40Ar exhibited in final steps of "matrix" or "groundmass" fractions from whole-rock experiments (no glass, crystals) suggests equilibration with the atmosphere is not achieved. Removing magnetic sample splits (glass?) thought subject to poor argon retention, and crystals subject to 40Ar inheritance are routinely done without documenting different isochrons. Short 15 to 20 minute irradiation times effectively eliminate recoil and dramatically minimize isotopic corrections, and the assumption of equivalence in Ar isotope recoil behavior. Assuming no pressure dependency and constancy of mass discrimination value ignores knowledge from other gas mass spectroscopy (O, H, He, Ne). Dynamic mass spectroscopy in stable isotopic analysis allows routine per mil and 0.1 per mil ratios to be measured. Maintaining more than daily bracketing air pipette measurements at differing pressures, and controlling the range of pressures from each diffusive step will approximate this dynamic precision. Experiments will be discussed that exhibit aspects of 40Ar/39Ar dating protocols with which precision and accuracy can be improved.

  8. Xrt And Shinx Joint Flare Study: Ar 11024

    NASA Astrophysics Data System (ADS)

    Engell, Alexander; Sylwester, J.; Siarkowski, M.

    2010-05-01

    From 12:00 UT on July 3 through July 7, 2009 SphinX (Solar Photometer IN X-rays) observes 130 flares with active region (AR) 11024 being the only AR on disk. XRT (X-Ray Telescope) is able to observe 64 of these flare events. The combination of both instruments results in a flare study revealing (1) a relationship between flux emergence and flare rate, (2) that the presence of active region loops typically results in different flare morphologies (single and multiple loop flares) then when there is a lack of an active region loop environment where more cusp and point-like flares are observed, (3) cusp and point-like flares often originate from the same location, and (4) a distribution of flare temperatures corresponding to the different flare morphologies. The differences between the observed flare morphologies may occur as the result of the heated plasma through the flaring process being confined by the proximity of loop structures as for the single and multiple loop flares, while for cusp and point-like flares they occur in an early-phase environment that lack loop presence. The continuing flux emergence of AR 11024 likely provides different magnetic interactions and may be the source responsible for all of the flares.

  9. Estimate of the 42Ar content in the Earth's atmosphere

    NASA Astrophysics Data System (ADS)

    Barabash, A. S.; Kornoukhov, V. N.; Jants, V. E.

    1997-02-01

    42Ar is a potential source of background in large volume argon-based detectors. The production of the 42Ar isotope both by cosmic rays and by neutrons produced by testing of nuclear weapons is discussed. We demonstrate that main channel of the 42Ar production is from atmospheric testing of nuclear bombs from 1945 to 1962 and the 42Ar content must be less than 1.3 × 10 -23 parts of 42Ar per part of natAr.

  10. Cellobiohydrolase variants and polynucleotides encoding same

    SciTech Connect

    Wogulis, Mark

    2014-10-14

    The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

  11. Cellobiohydrolase variants and polynucleotides encoding same

    DOEpatents

    Wogulis, Mark

    2013-09-24

    The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

  12. Cellobiohydrolase variants and polynucleotides encoding the same

    DOEpatents

    Wogulis, Mark

    2014-09-09

    The present invention relates to variants of a parent cellobiohydrolase. The present invention also relates to polynucleotides encoding the cellobiohydrolase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the cellobiohydrolase variants.

  13. Measurements of the 40Ar(n, γ)41Ar radiative-capture cross section between 0.4 and 14.8 MeV

    NASA Astrophysics Data System (ADS)

    Bhike, Megha; Fallin, B.; Tornow, W.

    2014-09-01

    The 40Ar(n, γ)41Ar neutron capture cross section has been measured between 0.4 and 14.8 MeV neutron energy using the activation technique. The data are important for estimating backgrounds in argon-based neutrino and dark-matter detectors and in the neutrino-less double-beta decay search GERDA, which uses liquid argon as cooling and shielding medium. For the first time the 40Ar(n, γ)41Ar cross section has been measured for neutron energies above 1 MeV. Our results are compared to the evaluation ENDF/B-VII.1 and the calculated prediction TENDL-2013. The latter agrees very well with the present results.

  14. Geochemical and 40Ar/39Ar constraints on the evolution of volcanism in the Woodlark Rift, Papua New Guinea

    NASA Astrophysics Data System (ADS)

    Catalano, Joseph P.

    The tectonic mechanisms producing Pliocene to active volcanism in eastern Papua New Guinea (PNG) have been debated for decades. In order to assess mechanisms that produce volcanism in the Woodlark Rift, we evaluate the evolution of volcanism in eastern PNG using 40Ar/39Ar thermochronology and whole rock geochemistry. Active volcanism in southeastern Papua New Guinea occurs on the Papuan Peninsula (Mt. Lamington, Mt. Victory and Waiwa), in the Woodlark Rift (Dobu Island, SE Goodenough Island, and Western Fergusson Island), and in the Woodlark Basin. In the Woodlark Basin, seafloor spreading is active and decompression melting of the upper mantle is producing basaltic magmatism. However, the cause of Pliocene and younger volcanism in the Woodlark Rift is controversial. Two hypotheses for the tectonic setting have been proposed to explain Pliocene and younger volcanism in the Woodlark Rift: (1) southward subduction of Solomon Sea lithosphere beneath eastern PNG at the Trobriand Tough and (2) decompression melting of mantle, previously modified by subduction, as the lithosphere undergoes extension associated with the opening of the Woodlark Basin. A comparison of 40Ar/39Ar ages with high field strength element (HFSE) concentrations in primary magmas indicates that HFSE concentrations correlate with age in the Woodlark rift. These data support the hypothesis that Pliocene to active volcanism in the Woodlark Rise and D'Entrecasteaux Islands results from decompression melting of a relict mantle wedge. The subduction zone geochemical signatures (negative HFSE anomalies) in Woodlark Rift lavas younger than 4 m.y. are a relict from older subduction beneath eastern Papua, likely in the middle Miocene. As the lithosphere is extended ahead of the tip of the westward propagating seafloor spreading center in the Woodlark Basin, the composition of volcanism is inherited from prior arc magmatism (via flux melting) and through time evolves toward magmatism associated with a rifting

  15. Search for cosmogenic Ar-42 in meteorites

    NASA Astrophysics Data System (ADS)

    Cini Castagnoli, G.; Cane, D.; Taricco, C.; Bhandari, N.

    2003-04-01

    We have evidence for decreasing galactic cosmic ray (GCR) flux in the past 3 centuries by a factor about two [1]. The measurements of the activity of cosmogenic 44Ti (T1/2 = 59.2 y) produced by GCR in stony meteorites that fell during the last two centuries are in good agreement with the calculated values and validate the decreasing trend of GCR flux. The measurements were obtained by an hyperpure (372 cm3) Ge-NaI(Tl) spectrometer operating in the Monte dei Cappuccini laboratory in Torino[2]. To further improve upon statistical precision of 44Ti data and also to be able to measure the 42Ar (T1/2 = 33 y) which is produced in even smaller amounts by GCR in meteorites, we have set up in the same laboratory a larger (581 cm3) hyperpure Ge detector operating in coincidence with a 100 kg NaI(Tl) crystal assembly. This should enable us to confirm the above results about GCR variations. We wish to acknowledge our deep gratitude to professor Bonino, deceased on September 29, 2002, to whom the assemblage of the new equipment is due. [1] G. Bonino, G. Cini Castagnoli, D. Cane, C. Taricco and N. Bhandari,Proc. XXVII Intern. Cosmic Ray Conf. (Hamburg, 2001) 3769-3772. [2] Bonino G., Cini Castagnoli G., Cane D., Taricco C., Bhandari N, textit {34th COSPAR Sci. Ass. Houston 2002 (Adv. Space Res.)}, in press

  16. The ΔC splice-variant of TRPM2 is the hypertonicity-induced cation channel in HeLa cells, and the ecto-enzyme CD38 mediates its activation

    PubMed Central

    Numata, Tomohiro; Sato, Kaori; Christmann, Jens; Marx, Romy; Mori, Yasuo; Okada, Yasunobu; Wehner, Frank

    2012-01-01

    Hypertonicity-induced cation channels (HICCs) are key-players in proliferation and apoptosis but their molecular correlate remains obscure. Furthermore, the activation profile of HICCs is not well defined yet. We report here that, in HeLa cells, intracellular adenosine diphosphate ribose (ADPr) and cyclic ADPr (cADPr), as supposed activators of TRPM2, elicited cation currents that were virtually identical to the osmotic activation of HICCs. Silencing of the expression of TRPM2 and of the ecto-enzyme CD38 (as a likely source of ADPr and cADPr) inhibited HICC as well as nucleotide-induced currents and, in parallel, the hypertonic volume response of cells (the regulatory volume increase, RVI) was attenuated. Quantification of intracellular cADPr levels and the systematic application of extra- vs. intracellular nucleotides indicate that the outwardly directed gradient rather than the cellular activity of ADPr and cADPr triggers TRPM2 activation, probably along with a simultaneous biotransformation of nucleotides. Cloning of TRPM2 identified the ΔC-splice variant as the molecular correlate of the HICC, which could be strongly supported by a direct comparison of the respective Ca2+ selectivity. Finally, immunoprecipitation and high-resolution FRET/FLIM imaging revealed the interaction of TRPM2 and CD38 in the native as well as in a heterologous (HEK293T) expression system. We propose transport-related nucleotide export via CD38 as a novel mechanism of TRPM2/HICC activation. With the biotransformation of nucleotides running in parallel, continuous zero trans-conditions are achieved which will render the system infinitely sensitive. PMID:22219339

  17. Geology and preliminary [sup 40]Ar/[sup 39]Ar geochronology of the Sliderock Mountain volcano, south-central Montana

    SciTech Connect

    Du Bray, E.A.; Harlan, S.S. )

    1993-04-01

    The Sliderock Mountain Volcano is a deeply eroded, Upper Cretaceous basaltic andesite stratovolcano complex located along the northeastern margin of the Laramide Beartooth uplift of south-central Montana. Historically, these rocks have been included in the Livingston Group and correlated with Upper Cretaceous, dominantly epiclastic sedimentary rocks of the Livingston Group in the Crazy Mountains Basin. Recent geologic mapping has identified several map units including: basaltic andesite of Derby Ridge (lava flows, and minor interbedded pyroclastic flows including welded tuff, block and ash flows, and lahars); volcaniclastic sedimentary rocks; lahar deposits; dioritic plutons and sills (including the diorite of Sliderock Mountain); basaltic andesite lavas; and basaltic trachyandesite dikes. Stratigraphic relations indicate that initial volcanic activity was dominated by eruption of the basaltic andesite of Derby Ridge. Cross-cutting relations indicate that dioritic plutons and sills are younger than the basaltic andesite of Derby Ridge and the lahars but age relations with the second set of basaltic andesite lavas are indeterminate. The volcanic and dike rocks of the stratovolcano are cpx-plag rocks, characterized by limited compositional variation, whereas intrusive rocks are hbl-plag rocks whose compositions are principally that of diorite, but range to granite. [sup 40]Ar/[sup 39]Ar isotopic analysis of hornblende from the Lodgepole intrusion, a hypabyssal intrusion that may constitute part of the volcano's solidified magma chamber, gives an age of 76.2 [+-] 0.3 Ma (1[sigma]), significantly younger than a previously reported K-Ar biotite age of 82 Ma. [sup 40]Ar/[sup 39]Ar analyses of sericite from weakly mineralized Au-bearing quartz veins hosted by the diorite of Sliderock Mountain give slightly younger isochron ages of 73--74 Ma indicating that gold mineralization is probably associated with the late stages of cooling of the Sliderock Mountain magma system.

  18. 40Ar/39Ar impact ages and time-temperature argon diffusion history of the Bunburra Rockhole anomalous basaltic achondrite

    NASA Astrophysics Data System (ADS)

    Jourdan, Fred; Benedix, Gretchen; Eroglu, Ela.; Bland, Phil. A.; Bouvier, Audrey.

    2014-09-01

    The Bunburra Rockhole meteorite is a brecciated anomalous basaltic achondrite containing coarse-, medium- and fine-grained lithologies. Petrographic observations constrain the limited shock pressure to between ca. 10 GPa and 20 GPa. In this study, we carried out nine 40Ar/39Ar step-heating experiments on distinct single-grain fragments extracted from the coarse and fine lithologies. We obtained six plateau ages and three mini-plateau ages. These ages fall into two internally concordant populations with mean ages of 3640 ± 21 Ma (n = 7; P = 0.53) and 3544 ± 26 Ma (n = 2; P = 0.54), respectively. Based on these results, additional 40Ar/39Ar data of fusion crust fragments, argon diffusion modelling, and petrographic observations, we conclude that the principal components of the Bunburra Rockhole basaltic achondrite are from a melt rock formed at ∼3.64 Ga by a medium to large impact event. The data imply that this impact generated high enough energy to completely melt the basaltic target rock and reset the Ar systematics, but only partially reset the Pb-Pb age. We also conclude that a complete 40Ar∗ resetting of pyroxene and plagioclase at this time could not have been achieved at solid-state conditions. Comparison with a terrestrial analog (Lonar crater) shows that the time-temperature conditions required to melt basaltic target rocks upon impact are relatively easy to achieve. Ar data also suggest that a second medium-size impact event occurred on a neighbouring part of the same target rock at ∼3.54 Ga. Concordant low-temperature step ages of the nine aliquots suggest that, at ∼3.42 Ga, a third smaller impact excavated parts of the ∼3.64 Ga and ∼3.54 Ga melt rocks and brought the fragments together. The lack of significant impact activity after 3.5 Ga, as recorded by the Bunburra Rockhole suggests that (1) either the meteorite was ejected in a small secondary parent body where it resided untouched by large impacts, or (2) it was covered by a porous heat

  19. Molybdenite Re-Os and muscovite 40Ar/39Ar dating of the Xihuashan tungsten deposit, central Nanling district, South China

    NASA Astrophysics Data System (ADS)

    Hu, Rui-Zhong; Wei, Wen-Feng; Bi, Xian-Wu; Peng, Jian-Tang; Qi, You-Qiang; Wu, Li-Yan; Chen, You-Wei

    2012-10-01

    The Xihuashan tungsten deposit in the central Nanling region, South China, is an important vein-type ore deposit hosted in Cambrian strata and Mesozoic granitic intrusions. Wolframite and molybdenite are the principal ore minerals. The gangue minerals are mainly quartz and muscovite. Wolframite and molybdenite are products of the first stage hydrothermal activity, whereas muscovite formed dominantly at the second stage. Molybdenite Re-Os and muscovite 40Ar/39Ar dating have been carried out to investigate the age of mineralization. Re-Os isotopic dating for molybdenite associated with wolframite yield a precise, well-constrained isochron age of 157.8 ± 0.9 Ma (MSWD = 1.5). Ar-Ar isotopic analyses of muscovite yield a plateau age of 152.8 ± 1.6 Ma, in agreement with an inverse isochron age of 152.8 ± 1.6 Ma, which is ~ 5 mys younger than the Re-Os age. The molybdenite Re-Os age is interpreted as the age of tungsten mineralization. This age coincides well with the zircon U-Pb age of the host granitic intrusion reported previously. The ~ 5 mys difference between molybdenite Re-Os and muscovite 40Ar/39Ar ages probably represents the duration of hydrothermal activity. The results show that the Xihuashan tungsten deposit is one of many important tungsten-tin deposits formed during 150 to 160 Ma associated with large-scale lithospheric extension in South China.

  20. Chronostratigraphy of Monte Vulture volcano (southern Italy): secondary mineral microtextures and 39Ar-40Ar systematics

    NASA Astrophysics Data System (ADS)

    Villa, Igor M.; Buettner, Annett

    2009-12-01

    The eruptive history of Monte Vulture has been the subject of several geochronological investigations during the past decades, which reliably dated only a small number of eruptions. Understanding the causes of sub-optimum data yield in the past requires an interdisciplinary approach. We re-analyzed samples from previous works and present new data on samples from the main volcano-stratigraphic units of Monte Vulture, so as to provide an improved, consistent chronostratigraphic database. Imaging of minerals by cathodoluminescence and backscattered electrons reveals that heterochemical, high-temperature deuteric reaction textures are ubiquitous. Such observations are common in metamorphic rocks but had not frequently been reported from volcanic rocks. In view of the mineralogical complexity, we base our chronological interpretation on isochemical steps, defined as steps for which the Cl/K and/or the Ca/K ratios are constant. Isochemical steps carry the isotopic signature of chemically homogeneous mineral phases and therefore allow a well-constrained age interpretation. Comparison of old and new 39Ar-40Ar data proves the reproducibility of age spectra and their shapes. This quantifies the analytical reliability of the irradiation and mass-spectrometric analyses. Anomalous age spectra are a reproducible property of some specific samples and correlate with mineralogical anomalies. The present data allow us to fine-tune the age of the volcanostratigraphic units of Monte Vulture during the known interval of main volcanic activity from ca. 740 to 610 ka. After a very long stasis, the volcanic activity in the Monte Vulture area resumed with diatremic eruptions, one of which (Lago Piccolo di Monticchio, the site of a palynological-paleoclimatological drilling) was dated at ca. 140 ka.

  1. Rare Copy Number Variants

    PubMed Central

    Grozeva, Detelina; Kirov, George; Ivanov, Dobril; Jones, Ian R.; Jones, Lisa; Green, Elaine K.; St Clair, David M.; Young, Allan H.; Ferrier, Nicol; Farmer, Anne E.; McGuffin, Peter; Holmans, Peter A.; Owen, Michael J.; O’Donovan, Michael C.; Craddock, Nick

    2015-01-01

    Context Recent studies suggest that copy number variation in the human genome is extensive and may play an important role in susceptibility to disease, including neuropsychiatric disorders such as schizophrenia and autism. The possible involvement of copy number variants (CNVs) in bipolar disorder has received little attention to date. Objectives To determine whether large (>100 000 base pairs) and rare (found in <1% of the population) CNVs are associated with susceptibility to bipolar disorder and to compare with findings in schizophrenia. Design A genome-wide survey of large, rare CNVs in a case-control sample using a high-density microarray. Setting The Wellcome Trust Case Control Consortium. Participants There were 1697 cases of bipolar disorder and 2806 nonpsychiatric controls. All participants were white UK residents. Main Outcome Measures Overall load of CNVs and presence of rare CNVs. Results The burden of CNVs in bipolar disorder was not increased compared with controls and was significantly less than in schizophrenia cases. The CNVs previously implicated in the etiology of schizophrenia were not more common in cases with bipolar disorder. Conclusions Schizophrenia and bipolar disorder differ with respect to CNV burden in general and association with specific CNVs in particular. Our data are consistent with the possibility that possession of large, rare deletions may modify the phenotype in those at risk of psychosis: those possessing such events are more likely to be diagnosed as having schizophrenia, and those without them are more likely to be diagnosed as having bipolar disorder. PMID:20368508

  2. LASER MICROPROBE **4**0Ar/**3**9Ar DATING OF MINERAL GRAINS IN SITU.

    USGS Publications Warehouse

    Sutter, J.F.; Hartung, J.B.

    1984-01-01

    A laser-microprobe attached to a mass spectrometer for **4**0Ar/**3**9Ar age determination of single mineral grains in geological materials has been made operational at the US Geological Survey, Reston, VA. This microanalytical technique involves focusing a pulsed laser beam onto a sample contained in an ultra-high vacuum chamber attached to a rare-gas mass spectrometer. Argon in the neutron-irradiated sample is released by heating with the laser pulse and its isotopic composition is measured to yield an **4**0Ar/**3**9Ar age. Laser probe **4**0Ar/**3**9Ar ages of single mineral grains measured in situ can aid greatly in understanding the chronology of many geological situations where datable minerals are present but are not physically separable in quantities needed for conventional age dating.

  3. Ar-39-Ar-40 Ages of Euerites and the Thermal History of Asteroid 4-Vesta

    NASA Technical Reports Server (NTRS)

    Bogard, Donald D.; Garrison, Daniel H.

    2002-01-01

    Eucrite meteorites are igneous rocks that derive from a large asteroid, probably 4 Vesta. Prior studies have shown that after eucrites formed, most were subsequently metamorphosed to temperatures up to equal to or greater than 800 C, and much later many were brecciated and heated by large impacts into the parent body surface. The uncommon basaltic, unbrecciated eucrites also formed near the surface but presumably escaped later brecciation, whereas the cumulate eucrites formed at depth where metamorphism may have persisted for a considerable period. To further understand the complex HED parent body thermal history, we determined new Ar-39-Ar-40 ages for nine eucrites classified as basaltic but unbrecciated, six eucrites classified as cumulate, and several basaltic-brecciated eucrites. Relatively precise Ar-Ar ages of two cumulate eucrites (Moama and EET87520) and four unbrecciated eucrites give a tight cluster at 4.48 +/1 0.01 Gyr. Ar-Ar ages of six additional unbrecciated eucrites are consistent with this age, within their larger age uncertainties. In contrast, available literature data on Pb-Pb isochron ages of four cumulate eucrites and one unbrecciated eucrite vary over 4