Science.gov

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

    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

    2015-10-13

    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.

  2. 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

  3. AR intragenic deletions linked to androgen receptor splice variant expression and activity in models of prostate cancer progression.

    PubMed

    Li, Y; Hwang, T H; Oseth, L A; Hauge, A; Vessella, R L; Schmechel, S C; Hirsch, B; Beckman, K B; Silverstein, K A; Dehm, S M

    2012-11-08

    Reactivation of the androgen receptor (AR) during androgen depletion therapy (ADT) underlies castration-resistant prostate cancer (CRPCa). Alternative splicing of the AR gene and synthesis of constitutively active COOH-terminally truncated AR variants lacking the AR ligand-binding domain has emerged as an important mechanism of ADT resistance in CRPCa. In a previous study, we demonstrated that altered AR splicing in CRPCa 22Rv1 cells was linked to a 35-kb intragenic tandem duplication of AR exon 3 and flanking sequences. In this study, we demonstrate that complex patterns of AR gene copy number imbalances occur in PCa cell lines, xenografts and clinical specimens. To investigate whether these copy number imbalances reflect AR gene rearrangements that could be linked to splicing disruptions, we carried out a detailed analysis of AR gene structure in the LuCaP 86.2 and CWR-R1 models of CRPCa. By deletion-spanning PCR, we discovered a 8579-bp deletion of AR exons 5, 6 and 7 in the LuCaP 86.2 xenograft, which provides a rational explanation for synthesis of the truncated AR v567es AR variant in this model. Similarly, targeted resequencing of the AR gene in CWR-R1 cells led to the discovery of a 48-kb deletion in AR intron 1. This intragenic deletion marked a specific CWR-R1 cell population with enhanced expression of the truncated AR-V7/AR3 variant, a high level of androgen-independent AR transcriptional activity and rapid androgen independent growth. Together, these data demonstrate that structural alterations in the AR gene are linked to stable gain-of-function splicing alterations in CRPCa.

  4. AR intragenic deletions linked to androgen receptor splice variant expression and activity in models of prostate cancer progression

    PubMed Central

    Li, Yingming; Hwang, Tae Hyun; Oseth, LeAnn; Hauge, Adam; Vessella, Robert L.; Schmechel, Stephen C.; Hirsch, Betsy; Beckman, Kenneth B.; Silverstein, Kevin A.; Dehm, Scott M.

    2011-01-01

    Reactivation of the androgen receptor (AR) during androgen depletion therapy (ADT) underlies castration-resistant prostate cancer (CRPCa). Alternative splicing of the AR gene and synthesis of constitutively-active COOH-terminally truncated AR variants lacking the AR ligand binding domain has emerged as an important mechanism of ADT-resistance in CRPCa. In a previous study, we demonstrated that altered AR splicing in CRPCa 22Rv1 cells was linked to a 35 kb intragenic tandem duplication of AR exon 3 and flanking sequences. In this study, we demonstrate that complex patterns of AR gene copy number imbalances occur in PCa cell lines, xenografts, and clinical specimens. To investigate whether these copy number imbalances reflect AR gene rearrangements that could be linked to splicing disruptions, we carried out a detailed analysis of AR gene structure in the LuCaP 86.2 and CWR-R1 models of CRPCa. By deletion-spanning PCR, we discovered a 8,579 bp deletion of AR exons 5, 6, and 7 in the LuCaP 86.2 xenograft, which provides a rational explanation for synthesis of the truncated AR v567es AR variant in this model. Similarly, targeted re-sequencing of the AR gene in CWR-R1 cells led to the discovery of a 48 kb deletion in AR intron 1. This intragenic deletion marked a specific CWR-R1 cell population with enhanced expression of the truncated AR-V7/AR3 variant, a high level of androgen-independent AR transcriptional activity, and rapid androgen independent growth. Together, these data demonstrate that structural alterations in the AR gene are linked to stable gain-of-function splicing alterations in CRPCa. PMID:22266865

  5. Targeting chromatin binding regulation of constitutively active AR variants to overcome prostate cancer resistance to endocrine-based therapies

    PubMed Central

    Chan, Siu Chiu; Selth, Luke A.; Li, Yingming; Nyquist, Michael D.; Miao, Lu; Bradner, James E.; Raj, Ganesh V.; Tilley, Wayne D.; Dehm, Scott M.

    2015-01-01

    Androgen receptor (AR) variants (AR-Vs) expressed in prostate cancer (PCa) lack the AR ligand binding domain (LBD) and function as constitutively active transcription factors. AR-V expression in patient tissues or circulating tumor cells is associated with resistance to AR-targeting endocrine therapies and poor outcomes. Here, we investigated the mechanisms governing chromatin binding of AR-Vs with the goal of identifying therapeutic vulnerabilities. By chromatin immunoprecipitation and sequencing (ChIP-seq) and complementary biochemical experiments, we show that AR-Vs display a binding preference for the same canonical high-affinity androgen response elements (AREs) that are preferentially engaged by AR, albeit with lower affinity. Dimerization was an absolute requirement for constitutive AR-V DNA binding and transcriptional activation. Treatment with the bromodomain and extraterminal (BET) inhibitor JQ1 resulted in inhibition of AR-V chromatin binding and impaired AR-V driven PCa cell growth in vitro and in vivo. Importantly, this was associated with a novel JQ1 action of down-regulating AR-V transcript and protein expression. Overall, this study demonstrates that AR-Vs broadly restore AR chromatin binding events that are otherwise suppressed during endocrine therapy, and provides pre-clinical rationale for BET inhibition as a strategy for inhibiting expression and chromatin binding of AR-Vs in PCa. PMID:25908785

  6. 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.

  7. 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 Central

    Mancini, Michael A; Marcelli, Marco

    2016-01-01

    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

  8. Androgen receptor splice variants circumvent AR blockade by microtubule-targeting agents

    PubMed Central

    Zhang, Guanyi; Liu, Xichun; Li, Jianzhuo; Ledet, Elisa; Alvarez, Xavier; Qi, Yanfeng; Fu, Xueqi; Sartor, Oliver; Dong, Yan; Zhang, Haitao

    2015-01-01

    Docetaxel-based chemotherapy is established as a first-line treatment and standard of care for patients with metastatic castration-resistant prostate cancer. However, half of the patients do not respond to treatment and those do respond eventually become refractory. A better understanding of the resistance mechanisms to taxane chemotherapy is both urgent and clinical significant, as taxanes (docetaxel and cabazitaxel) are being used in various clinical settings. Sustained signaling through the androgen receptor (AR) has been established as a hallmark of CRPC. Recently, splicing variants of AR (AR-Vs) that lack the ligand-binding domain (LBD) have been identified. These variants are constitutively active and drive prostate cancer growth in a castration-resistant manner. In taxane-resistant cell lines, we found the expression of a major variant, AR-V7, was upregulated. Furthermore, ectopic expression of two clinically relevant AR-Vs (AR-V7 and ARV567es), but not the full-length AR (AR-FL), reduced the sensitivities to taxanes in LNCaP cells. Treatment with taxanes inhibited the transcriptional activity of AR-FL, but not those of AR-Vs. This could be explained, at least in part, due to the inability of taxanes to block the nuclear translocation of AR-Vs. Through a series of deletion constructs, the microtubule-binding activity was mapped to the LBD of AR. Finally, taxane-induced cytoplasm sequestration of AR-FL was alleviated when AR-Vs were present. These findings provide evidence that constitutively active AR-Vs maintain the AR signaling axis by evading the inhibitory effects of microtubule-targeting agents, suggesting that these AR-Vs play a role in resistance to taxane chemotherapy. PMID:26160840

  9. Targeting AR Variant-Coactivator Interactions to Exploit Prostate Cancer Vulnerabilities.

    PubMed

    Magani, Fiorella; Peacock, Stephanie O; Rice, Meghan A; Martinez, Maria J; Greene, Ann M; Magani, Pablo S; Lyles, Rolando; Weitz, Jonathan R; Burnstein, Kerry L

    2017-08-15

    Castration-resistant prostate cancer (CRPC) progresses rapidly and is incurable. Constitutively active androgen receptor splice variants (AR-Vs) represent a well-established mechanism of therapeutic resistance and disease progression. These variants lack the AR ligand-binding domain and, as such, are not inhibited by androgen deprivation therapy (ADT), which is the standard systemic approach for advanced prostate cancer. Signaling by AR-Vs, including the clinically relevant AR-V7, is augmented by Vav3, an established AR coactivator in CRPC. Using mutational and biochemical studies, we demonstrated that the Vav3 Diffuse B-cell lymphoma homology (DH) domain interacted with the N-terminal region of AR-V7 (and full length AR). Expression of the Vav3 DH domain disrupted Vav3 interaction with and enhancement of AR-V7 activity. The Vav3 DH domain also disrupted AR-V7 interaction with other AR coactivators: Src1 and Vav2, which are overexpressed in PC. This Vav3 domain was used in proof-of-concept studies to evaluate the effects of disrupting the interaction between AR-V7 and its coactivators on CRPC cells. This disruption decreased CRPC cell proliferation and anchorage-independent growth, caused increased apoptosis, decreased migration, and resulted in the acquisition of morphological changes associated with a less aggressive phenotype. While disrupting the interaction between FL-AR and its coactivators decreased N-C terminal interaction, disrupting the interaction of AR-V7 with its coactivators decreased AR-V7 nuclear levels.Implications: This study demonstrates the potential therapeutic utility of inhibiting constitutively active AR-V signaling by disrupting coactivator binding. Such an approach is significant, as AR-Vs are emerging as important drivers of CRPC that are particularly recalcitrant to current therapies. Mol Cancer Res; 1-12. ©2017 AACR. ©2017 American Association for Cancer Research.

  10. Androgen Receptor Variants and Prostate Cancer in Humanized AR Mice

    PubMed Central

    Albertelli, Megan A.; O’Mahony, Orla A.

    2008-01-01

    SUMMARY Androgen, acting via the androgen receptor (AR), is central to male development, differentiation and hormone-dependent diseases such as prostate cancer. AR is actively involved in the initiation of prostate cancer, the transition to androgen independence, and many mechanisms of resistance to therapy. To examine genetic variation of AR in cancer, we created mice by germ-line gene targeting in which human AR sequence replaces that of the mouse. Since shorter length of a polymorphic N-terminal glutamine (Q) tract has been linked to prostate cancer risk, we introduced alleles with 12, 21 or 48 Qs to test this association. The three “humanized” AR mouse strains (h/mAR) are normal physiologically, as well as by cellular and molecular criteria, although slight differences are detected in AR target gene expression, correlating inversely with Q tract length. However, distinct allele-dependent differences in tumorigenesis are evident when these mice are crossed to a transgenic prostate cancer model. Remarkably, Q tract variation also differentially impacts disease progression following androgen depletion. This finding emphasizes the importance of AR function in androgen-independent as well as –dependent disease. These mice provide a novel genetic paradigm in which to dissect opposing functions of AR in tumor suppression vs. oncogenesis. PMID:17936615

  11. Melatonin Inhibits Androgen Receptor Splice Variant-7 (AR-V7)-Induced Nuclear Factor-Kappa B (NF-κB) Activation and NF-κB Activator-Induced AR-V7 Expression in Prostate Cancer Cells: Potential Implications for the Use of Melatonin in Castration-Resistant Prostate Cancer (CRPC) Therapy

    PubMed Central

    Liu, Vincent Wing Sun; Yau, Wing Lung; Tam, Chun Wai; Yao, Kwok-Ming; Shiu, Stephen Yuen Wing

    2017-01-01

    A major current challenge in the treatment of advanced prostate cancer, which can be initially controlled by medical or surgical castration, is the development of effective, safe, and affordable therapies against progression of the disease to the stage of castration resistance. Here, we showed that in LNCaP and 22Rv1 prostate cancer cells transiently overexpressing androgen receptor splice variant-7 (AR-V7), nuclear factor-kappa B (NF-κB) was activated and could result in up-regulated interleukin (IL)-6 gene expression, indicating a positive interaction between AR-V7 expression and activated NF-κB/IL-6 signaling in castration-resistant prostate cancer (CRPC) pathogenesis. Importantly, both AR-V7-induced NF-κB activation and IL-6 gene transcription in LNCaP and 22Rv1 cells could be inhibited by melatonin. Furthermore, stimulation of AR-V7 mRNA expression in LNCaP cells by betulinic acid, a pharmacological NF-κB activator, was reduced by melatonin treatment. Our data support the presence of bi-directional positive interactions between AR-V7 expression and NF-κB activation in CRPC pathogenesis. Of note, melatonin, by inhibiting NF-κB activation via the previously-reported MT1 receptor-mediated antiproliferative pathway, can disrupt these bi-directional positive interactions between AR-V7 and NF-κB and thereby delay the development of castration resistance in advanced prostate cancer. Apparently, this therapeutic potential of melatonin in advanced prostate cancer/CRPC management is worth translation in the clinic via combined androgen depletion and melatonin repletion. PMID:28561752

  12. Melatonin Inhibits Androgen Receptor Splice Variant-7 (AR-V7)-Induced Nuclear Factor-Kappa B (NF-κB) Activation and NF-κB Activator-Induced AR-V7 Expression in Prostate Cancer Cells: Potential Implications for the Use of Melatonin in Castration-Resistant Prostate Cancer (CRPC) Therapy.

    PubMed

    Liu, Vincent Wing Sun; Yau, Wing Lung; Tam, Chun Wai; Yao, Kwok-Ming; Shiu, Stephen Yuen Wing

    2017-05-31

    A major current challenge in the treatment of advanced prostate cancer, which can be initially controlled by medical or surgical castration, is the development of effective, safe, and affordable therapies against progression of the disease to the stage of castration resistance. Here, we showed that in LNCaP and 22Rv1 prostate cancer cells transiently overexpressing androgen receptor splice variant-7 (AR-V7), nuclear factor-kappa B (NF-κB) was activated and could result in up-regulated interleukin (IL)-6 gene expression, indicating a positive interaction between AR-V7 expression and activated NF-κB/IL-6 signaling in castration-resistant prostate cancer (CRPC) pathogenesis. Importantly, both AR-V7-induced NF-κB activation and IL-6 gene transcription in LNCaP and 22Rv1 cells could be inhibited by melatonin. Furthermore, stimulation of AR-V7 mRNA expression in LNCaP cells by betulinic acid, a pharmacological NF-κB activator, was reduced by melatonin treatment. Our data support the presence of bi-directional positive interactions between AR-V7 expression and NF-κB activation in CRPC pathogenesis. Of note, melatonin, by inhibiting NF-κB activation via the previously-reported MT₁ receptor-mediated antiproliferative pathway, can disrupt these bi-directional positive interactions between AR-V7 and NF-κB and thereby delay the development of castration resistance in advanced prostate cancer. Apparently, this therapeutic potential of melatonin in advanced prostate cancer/CRPC management is worth translation in the clinic via combined androgen depletion and melatonin repletion.

  13. The RNA helicase DDX39B and its paralog DDX39A regulate androgen receptor splice variant AR-V7 generation.

    PubMed

    Nakata, Daisuke; Nakao, Shoichi; Nakayama, Kazuhide; Araki, Shinsuke; Nakayama, Yusuke; Aparicio, Samuel; Hara, Takahito; Nakanishi, Atsushi

    2017-01-29

    Mounting evidence suggests that constitutively active androgen receptor (AR) splice variants, typified by AR-V7, are associated with poor prognosis and resistance to androgen deprivation therapy in prostate cancer patients. However, mechanisms governing the generation of AR splice variants are not fully understood. In this study, we aimed to investigate the dynamics of AR splice variant generation using the JDCaP prostate cancer model that expresses AR splice variants under androgen depletion. Microarray analysis of JDCaP xenografts before and after expression of AR splice variants suggested that dysregulation of RNA processing pathways is likely involved in AR splice variant generation. To explore factors contributing to generation of AR-V7 mRNA, we conducted a focused RNA interference screen in AR-V7-positive JDCaP-hr cells using an shRNA library targeting spliceosome-related genes. This screen identified DDX39B as a regulator of AR-V7 mRNA expression. Simultaneous knockdown of DDX39B and its paralog DDX39A drastically and selectively downregulated AR-V7 mRNA expression in multiple AR-V7-positive prostate cancer cell lines. DDX39B was upregulated in relapsed JDCaP xenografts expressing AR splice variants, suggesting its role in expression of AR splice variants. Taken together, our findings offer insight into the mechanisms of AR splice variant generation and identify DDX39 as a potential drug target for the treatment of AR splice variant-positive prostate cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Lin28 Induces Resistance to Anti-Androgens Via Promotion of AR Splice Variant Generation

    PubMed Central

    Tummala, Ramakumar; Nadiminty, Nagalakshmi; Lou, Wei; Evans, Christopher P.; Gao, Allen C.

    2017-01-01

    BACKGROUND Prostate cancer (PCa) is androgen-dependent initially and progresses to a castration-resistant state after androgen deprivation therapy. Treatment options for castration-resistant PCa include the potent second-generation anti-androgen enzalutamide or CYP17A1 inhibitor abiraterone. Recent clinical observations point to the development of resistance to these therapies which may be mediated by constitutively active alternative splice variants of the androgen receptor (AR). METHODS Sensitivity of LNCaP cells overexpressing Lin28 (LN-Lin28) to enzalutamide, abiraterone, or bicalutamide was compared to that of control LN-neo cells using cell growth assays, proliferation assays using MTT, anchorage-dependent clonogenic ability assays and soft agar assays. Ability of LN-Lin28 cells to maintain AR activation after treatment with enzalutamide, abiraterone, or bicalutamide was tested using immunofluorescence, Western blotting, ChIP assays, and qRT-PCR. Importance of Lin28 in enzalutamide resistance was assessed by the downregulation of Lin28 expression in C4-2B and 22Rv1 cells chronically treated with enzalutamide. Requirement for sustained AR signaling in LN-Lin28 cells was examined by the downregulation of either full length AR or AR-V7 using siRNA. RESULTS We show that Lin28 promotes the development of resistance to currently used targeted therapeutics by enhancing the expression of AR splice variants such as AR-V7. PCa cells overexpressing Lin28 exhibit resistance to treatment with enzalutamide, abiraterone, or bicalutamide. Downregulation of Lin28 resensitizes enzalutamide-resistant PCa cells to enzalutamide treatment. We also show that the upregulation of splicing factors such as hnRNPA1 by Lin28 may mediate the enhanced generation of AR splice variants in Lin28-expressing cells. CONCLUSIONS Our findings suggest that Lin28 plays a key role in the acquisition of resistance to AR-targeted therapies by PCa cells and establish the importance of Lin28 in PCa

  15. Lin28 induces resistance to anti-androgens via promotion of AR splice variant generation.

    PubMed

    Tummala, Ramakumar; Nadiminty, Nagalakshmi; Lou, Wei; Evans, Christopher P; Gao, Allen C

    2016-04-01

    Prostate cancer (PCa) is androgen-dependent initially and progresses to a castration-resistant state after androgen deprivation therapy. Treatment options for castration-resistant PCa include the potent second-generation anti-androgen enzalutamide or CYP17A1 inhibitor abiraterone. Recent clinical observations point to the development of resistance to these therapies which may be mediated by constitutively active alternative splice variants of the androgen receptor (AR). Sensitivity of LNCaP cells overexpressing Lin28 (LN-Lin28) to enzalutamide, abiraterone, or bicalutamide was compared to that of control LN-neo cells using cell growth assays, proliferation assays using MTT, anchorage-dependent clonogenic ability assays and soft agar assays. Ability of LN-Lin28 cells to maintain AR activation after treatment with enzalutamide, abiraterone, or bicalutamide was tested using immunofluorescence, Western blotting, ChIP assays, and qRT-PCR. Importance of Lin28 in enzalutamide resistance was assessed by the downregulation of Lin28 expression in C4-2B and 22Rv1 cells chronically treated with enzalutamide. Requirement for sustained AR signaling in LN-Lin28 cells was examined by the downregulation of either full length AR or AR-V7 using siRNA. We show that Lin28 promotes the development of resistance to currently used targeted therapeutics by enhancing the expression of AR splice variants such as AR-V7. PCa cells overexpressing Lin28 exhibit resistance to treatment with enzalutamide, abiraterone, or bicalutamide. Downregulation of Lin28 resensitizes enzalutamide-resistant PCa cells to enzalutamide treatment. We also show that the upregulation of splicing factors such as hnRNPA1 by Lin28 may mediate the enhanced generation of AR splice variants in Lin28-expressing cells. Our findings suggest that Lin28 plays a key role in the acquisition of resistance to AR-targeted therapies by PCa cells and establish the importance of Lin28 in PCa progression. © 2015 Wiley Periodicals, Inc.

  16. Armenian Astronomical Society (ArAS) activities

    NASA Astrophysics Data System (ADS)

    Mickaelian, A. M.

    2016-09-01

    A review on the activities and achievements of Armenian Astronomical Society (ArAS) and Armenian astronomy in general during the last years is given. ArAS membership, ArAS electronic newsletters (ArASNews), ArAS webpage, Annual Meetings, Annual Prize for Young Astronomers (Yervant Terzian Prize) and other awards, international relations, presence in international organizations, local and international summer schools, science camps, astronomical Olympiads and other events, matters related to astronomical education, astronomical heritage, amateur astronomy, astronomy outreach and ArAS further projects are described and discussed.

  17. Androgen Receptor and its Splice Variant, AR-V7, Differentially Regulate FOXA1 Sensitive Genes in LNCaP Prostate Cancer Cells

    PubMed Central

    Krause, William C.; Shafi, Ayesha A.; Nakka, Manjula; Weigel, Nancy L.

    2014-01-01

    Prostate cancer (PCa) is an androgen-dependent disease, and tumors that are resistant to androgen ablation therapy often remain androgen receptor (AR) dependent. Among the contributors to castration-resistant PCa are AR splice variants that lack the ligand-binding domain (LBD). Instead, they have small amounts of unique sequence derived from cryptic exons or from out of frame translation. The AR-V7 (or AR3) variant is constitutively active and is expressed under conditions consistent with CRPC. AR-V7 is reported to regulate a transcriptional program that is similar but not identical to that of AR. However, it is unknown whether these differences are due to the unique sequence in AR-V7, or simply to loss of the LBD. To examine transcriptional regulation by AR-V7, we have used lentiviruses encoding AR-V7 (amino acids 1-627 of AR with the 16 amino acids unique to the variant) to prepare a derivative of the androgen-dependent LNCaP cells with inducible expression of AR-V7. An additional cell line was generated with regulated expression of AR-NTD (amino acids 1-660 of AR); this mutant lacks the LBD but does not have the AR-V7 specific sequence. We find that AR and AR-V7 have distinct activities on target genes that are co-regulated by FOXA1. Transcripts regulated by AR-V7 were similarly regulated by AR-NTD, indicating that loss of the LBD is sufficient for the observed differences. Differential regulation of target genes correlates with preferential recruitment of AR or AR-V7 to specific cis-regulatory DNA sequences providing an explanation for some of the observed differences in target gene regulation. PMID:25008967

  18. Kava components down-regulate expression of AR and AR splice variants and reduce growth in patient-derived prostate cancer xenografts in mice.

    PubMed

    Li, Xuesen; Liu, Zhongbo; Xu, Xia; Blair, Christopher A; Sun, Zheng; Xie, Jun; Lilly, Michael B; Zi, Xiaolin

    2012-01-01

    Men living in Fiji and drinking kava have low incidence of prostate cancer (PCa). However, the PCa incidence among Fijian men who had migrated to Australia, increased by 5.1-fold. We therefore examined the potential effects of kava root extracts and its active components (kavalactones and flavokawains) on PCa growth and androgen receptor (AR) expression. PCa cell lines (LNCaP, LAPC-4, 22Rv1, C4-2B, DU145 and PC-3) with different AR expression, and a transformed prostate myofibroblast cell line (WPMY-1), were treated with a commercial kava extract, kavalactones (kawain, 5'6'-dehydrokawain, yangonin, methysticin) and flavokawain B. Expression of AR and its target genes (PSA and TMPRSS2) was examined. Two novel patient-derived PCa xenograft models from high grade PCa specimens were established by implanting the specimens into nude mice and passing tumor pieces through subcutaneous injection in nude mice, and then treated with kava extract and flavokawain B to examine their effects on tumor growth, AR expression and serum PSA levels. The kava extract and flavokawain B effectively down-regulated the expression of both the full-length AR and AR splice variants. The kava extract and kavalactones accelerated AR protein degradation, while flavokawain B inhibited AR mRNA transcription via decreasing Sp1 expression and the binding of Sp1 to the AR promoter. The kava root extract and flavokawain B reduce tumor growth, AR expression in tumor tissues and levels of serum PSA in the patient-derived PCa xenograft models. These results suggest a potential usefulness of a safe kava product or its active components for prevention and treatment of advanced PCa by targeting AR.

  19. Kava Components Down-Regulate Expression of AR and AR Splice Variants and Reduce Growth in Patient-Derived Prostate Cancer Xenografts in Mice

    PubMed Central

    Li, Xuesen; Liu, Zhongbo; Xu, Xia; Blair, Christopher A.; Sun, Zheng; Xie, Jun; Lilly, Michael B.; Zi, Xiaolin

    2012-01-01

    Men living in Fiji and drinking kava have low incidence of prostate cancer (PCa). However, the PCa incidence among Fijian men who had migrated to Australia, increased by 5.1-fold. We therefore examined the potential effects of kava root extracts and its active components (kavalactones and flavokawains) on PCa growth and androgen receptor (AR) expression. PCa cell lines (LNCaP, LAPC-4, 22Rv1, C4-2B, DU145 and PC-3) with different AR expression, and a transformed prostate myofibroblast cell line (WPMY-1), were treated with a commercial kava extract, kavalactones (kawain, 5′6′-dehydrokawain, yangonin, methysticin) and flavokawain B. Expression of AR and its target genes (PSA and TMPRSS2) was examined. Two novel patient-derived PCa xenograft models from high grade PCa specimens were established by implanting the specimens into nude mice and passing tumor pieces through subcutaneous injection in nude mice, and then treated with kava extract and flavokawain B to examine their effects on tumor growth, AR expression and serum PSA levels. The kava extract and flavokawain B effectively down-regulated the expression of both the full-length AR and AR splice variants. The kava extract and kavalactones accelerated AR protein degradation, while flavokawain B inhibited AR mRNA transcription via decreasing Sp1 expression and the binding of Sp1 to the AR promoter. The kava root extract and flavokawain B reduce tumor growth, AR expression in tumor tissues and levels of serum PSA in the patient-derived PCa xenograft models. These results suggest a potential usefulness of a safe kava product or its active components for prevention and treatment of advanced PCa by targeting AR. PMID:22347450

  20. Androgen Receptor Variant AR-V9 Is Coexpressed with AR-V7 in Prostate Cancer Metastases and Predicts Abiraterone Resistance.

    PubMed

    Kohli, Manish; Ho, Yeung; Hillman, David W; Van Etten, Jamie L; Henzler, Christine; Yang, Rendong; Sperger, Jamie M; Li, Yingming; Tseng, Elizabeth; Hon, Ting; Clark, Tyson; Tan, Winston; Carlson, Rachel E; Wang, Liguo; Sicotte, Hugues; Thai, Ho; Jimenez, Rafael; Huang, Haojie; Vedell, Peter T; Eckloff, Bruce W; Quevedo, Jorge F; Pitot, Henry C; Costello, Brian A; Jen, Jin; Wieben, Eric D; Silverstein, Kevin A T; Lang, Joshua M; Wang, Liewei; Dehm, Scott M

    2017-08-15

    Purpose: Androgen receptor (AR) variant AR-V7 is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies. Accordingly, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The purpose of this study was to understand whether other AR variants may be coexpressed with AR-V7 and promote resistance to AR-targeted therapies.Experimental Design: We utilized complementary short- and long-read sequencing of intact AR mRNA isoforms to characterize AR expression in CRPC models. Coexpression of AR-V7 and AR-V9 mRNA in CRPC metastases and circulating tumor cells was assessed by RNA-seq and RT-PCR, respectively. Expression of AR-V9 protein in CRPC models was evaluated with polyclonal antisera. Multivariate analysis was performed to test whether AR variant mRNA expression in metastatic tissues was associated with a 12-week progression-free survival endpoint in a prospective clinical trial of 78 CRPC-stage patients initiating therapy with the androgen synthesis inhibitor, abiraterone acetate.Results: AR-V9 was frequently coexpressed with AR-V7. Both AR variant species were found to share a common 3' terminal cryptic exon, which rendered AR-V9 susceptible to experimental manipulations that were previously thought to target AR-V7 uniquely. AR-V9 promoted ligand-independent growth of prostate cancer cells. High AR-V9 mRNA expression in CRPC metastases was predictive of primary resistance to abiraterone acetate (HR = 4.0; 95% confidence interval, 1.31-12.2; P = 0.02).Conclusions: AR-V9 may be an important component of therapeutic resistance in CRPC. Clin Cancer Res; 23(16); 4704-15. ©2017 AACR. ©2017 American Association for Cancer Research.

  1. High levels of the AR-V7 Splice Variant and Co-Amplification of the Golgi Protein Coding YIPF6 in AR Amplified Prostate Cancer Bone Metastases.

    PubMed

    Djusberg, Erik; Jernberg, Emma; Thysell, Elin; Golovleva, Irina; Lundberg, Pia; Crnalic, Sead; Widmark, Anders; Bergh, Anders; Brattsand, Maria; Wikström, Pernilla

    2017-05-01

    The relation between androgen receptor (AR) gene amplification and other mechanisms behind castration-resistant prostate cancer (CRPC), such as expression of constitutively active AR variants and steroid-converting enzymes has been poorly examined. Specific aim was to examine AR amplification in PC bone metastases and to explore molecular and functional consequences of this, with the long-term goal of identifying novel molecular targets for treatment. Gene amplification was assessed by fluorescence in situ hybridization in cryo-sections of clinical PC bone metastases (n = 40) and by PCR-based copy number variation analysis. Whole genome mRNA expression was analyzed using H12 Illumina Beadchip arrays and specific transcript levels were quantified by qRT-PCR. Protein localization was analyzed using immunohistochemistry and confocal microscopy. The YIPF6 mRNA expression was transiently knocked down and stably overexpressed in the 22Rv1 cell line as representative for CRPC, and effects on cell proliferation, colony formation, migration, and invasion were determined in vitro. Extracellular vesicles (EVs) were isolated from cell cultures using size-exclusion chromatography and enumerated by nanoparticle tracking analysis. Protein content was identified by LC-MS/MS analysis. Blood coagulation was measured as activated partial thromboplastin time (APTT). Functional enrichment analysis was performed using the MetaCore software. AR amplification was detected in 16 (53%) of the bone metastases examined from CRPC patients (n = 30), and in none from the untreated patients (n = 10). Metastases with AR amplification showed high AR and AR-V7 mRNA levels, increased nuclear AR immunostaining, and co-amplification of genes such as YIPF6 in the AR proximity at Xq12. The YIPF6 protein was localized to the Golgi apparatus. YIPF6 overexpression in 22Rv1 cells resulted in reduced cell proliferation and colony formation, and in enhanced EV secretion. EVs from YIPF6

  2. 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

    2015-08-27

    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.

  3. 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

  4. A novel variant of the putative demethylase gene, s-JMJD1C, is a coactivator of the AR.

    PubMed

    Wolf, Siegmund S; Patchev, Vladimir K; Obendorf, Maik

    2007-04-01

    Evidence is accumulating in support of the view that tissue-specific effects of steroid hormones depend on the recruitment of nuclear receptor comodulator proteins. The latter interact directly with the hormone receptors and modify their transcriptional effects on specific target genes. The mechanisms of comodulator influence on nuclear receptor-controlled gene transcription is only partially understood. Here, we describe the discovery of a new AR coactivator which belongs to the JmjC containing enzyme family as a novel variant of JMJD1C (jumonji domain-containing 1C). By using a fragment of the human AR (aa 325-919) as bait in a yeast two-hybrid screen, a region of the human JMJD1C gene was identified as interacting with AR. A novel splice variant s-JMJD1C was amplified by RACE, and the binding to AR was analysed by GST-pull-down and mammalian one-hybrid experiments. As a nuclear-localized protein, the s-JMJD1C gene is expressed in a variety of human tissues. In the brain, this protein is present in several, but not confined to, AR-expressing neuronal populations and its abundance varies with the hormonal status in a region-specific fashion. Interestingly, the expression of s-JMJD1C is reduced in breast cancer tumors and significantly higher in normal breast tissues indicating a putative role in tumor suppression. As s-JMJD1C has putative demethylase activity, removal of methylation seems to be important for nuclear receptor-based gene regulation.

  5. Vav3 enhances androgen receptor splice variant activity and is critical for castration-resistant prostate cancer growth and survival.

    PubMed

    Peacock, Stephanie O; Fahrenholtz, Cale D; Burnstein, Kerry L

    2012-12-01

    Advanced or metastatic prostate cancer is treated by androgen deprivation; however, patients inevitably relapse with castration-resistant prostate cancer (CRPC). CRPC remains dependent on androgen receptor (AR) signaling, which may include constitutive, ligand-independent action of naturally occurring AR splice variants. For example, the AR splice variant AR3 (also termed AR-V7) is expressed in CRPC and is linked to poor prognosis. Vav3, a Rho GTPase guanine nucleotide exchange factor, is an AR coactivator that is up-regulated in human prostate cancer compared with benign tissue and in preclinical models of CRPC. Vav3 confers castration-resistant growth to androgen-dependent human prostate cancer cells. Despite the importance of AR coactivators in promoting CRPC, the potential role of these regulatory proteins in modulating AR splice variant activity is unknown. We examined the contributions of Vav3 to AR activity in two CRPC cell lines that naturally express relatively high levels of Vav3 and AR3. Vav3 or AR3 knockdown greatly attenuated cell proliferation, soft agar growth, and ligand-independent AR activity. Vav3 potently enhanced the transcriptional activity of AR3 and another clinically relevant AR splice variant, ARv567es. Vav3 knockdown resulted in lowered nuclear AR3 levels, whereas total AR3 levels remained similar. Conversely, overexpression of Vav3 resulted in increased nuclear AR3. Coimmunoprecipitation revealed that AR3 and Vav3 interact. These novel data demonstrating physical and functional interactions between Vav3, a unique AR coactivator, and an AR splice variant provide insights into the mechanisms by which Vav3 exploits and enhances AR signaling in the progression to CRPC.

  6. FOXO1 Binds to the TAU5 Motif and Inhibits Constitutively Active Androgen Receptor Splice Variants

    PubMed Central

    Bohrer, Laura R.; Liu, Ping; Zhong, Jian; Pan, Yunqian; Angstman, James; Brand, Lucas J.; Dehm, Scott M.; Huang, Haojie

    2014-01-01

    BACKGROUND Aberrant activation of the androgen receptor (AR) is a major factor highly relevant to castration-resistant progression of prostate cancer (PCa). FOXO1, a key downstream effector of PTEN, inhibits androgen-independent activation of the AR. However, the underlying mechanism remains elusive. METHODS The inhibitory effect of FOXO1 on full-length and constitutively active splice variants of the AR was examined by luciferase reporter assays and real-time reverse transcription polymerase chain reaction (RT-qPCR). In vitro protein binding assays and western blot analyses were used to determine the regions in FOXO1 and AR responsible for their interaction. RESULTS We found that a putative transcription repression domain in the NH2-terminus of FOXO1 is dispensable for FOXO1 inhibition of the AR. In vitro protein binding assays showed that FOXO1 binds to the transcription activation unit 5 (TAU5) motif in the AR NH2-terminal domain (NTD), a region required for recruitment of p160 activators including SRC-1. Ectopic expression of SRC-1 augmented transcriptional activity of some, but not all AR splice variants examined. Forced expression of FOXO1 blocked the effect of SRC-1 on AR variants’ transcriptional activity by decreasing the binding of SRC-1 to the AR NTD. Ectopic expression of FOXO1 inhibited expression of endogenous genes activated primarily by alternatively spliced AR variants in human castration-resistant PCa 22Rv1 cells. CONCLUSIONS FOXO1 binds to the TAU5 motif in the AR NTD and inhibits ligand-independent activation of AR splice variants, suggesting the PTEN/FOXO1 pathway as a potential therapeutic target for inhibition of aberrant AR activation and castration-resistant PCa growth. PMID:23389878

  7. Androgen Receptor Splice Variants are Resistant to Inhibitors of Hsp90 and FKBP52, which alter Androgen Receptor Activity and Expression

    PubMed Central

    Shafi, Ayesha A.; Cox, Marc B.; Weigel, Nancy L.

    2013-01-01

    Androgen ablation therapy is the most common treatment for advanced prostate cancer (PCa), but most patients will develop castration-resistant prostate cancer (CRPC), which has no cure. CRPC is androgen-depletion resistant but androgen receptor (AR) dependent. AR is a nuclear receptor whose transcriptional activity is regulated by hormone binding to the ligand-binding domain (LBD). Constitutively active AR splice variants that lack LBDs often are expressed in CRPC. The expression of these variants indicates that methods to inhibit AR activity that do not rely on inactivating the LBD are needed. Heat shock protein 90 (Hsp90), a potential therapeutic target in PCa, is an AR chaperone crucial for proper folding, hormone binding and transcriptional activity of AR. We generated LNCaP cell lines with regulated expression of the AR-V7 variant as well as a cell line expressing artificially truncated AR (termed AR-NTD) to characterize splice variant function. Using an Hsp90 inhibitor, Geldanamycin (GA), and an AR-Hsp90-FKBP52 specific inhibitor, MJC13, we sought to determine if the AR variants also require Hsp90 and associated co-chaperone, FKBP52, for their activity. GA inhibits AR transcriptional activity but has little effect on AR-V7 activity. Moreover, GA decreases the stability of AR protein, with no effect on AR-V7 levels. Full-length AR activity is strongly inhibited by MJC13 while AR-V7 is unaffected. Thus, the variants are resistant to inhibitors of the Hsp90-AR heterocomplex. Although Hsp90 inhibitors will continue to inhibit growth promoting kinases and signaling through activated full-length AR in CRPC, AR signaling through variants will be retained. PMID:23380368

  8. 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

  9. Cooperative Dynamics of AR and ER Activity in Breast Cancer.

    PubMed

    D'Amato, Nicholas C; Gordon, Michael A; Babbs, Beatrice; Spoelstra, Nicole S; Carson Butterfield, Kiel T; Torkko, Kathleen C; Phan, Vernon T; Barton, Valerie N; Rogers, Thomas J; Sartorius, Carol A; Elias, Anthony; Gertz, Jason; Jacobsen, Britta M; Richer, Jennifer K

    2016-11-01

    Androgen receptor (AR) is expressed in 90% of estrogen receptor alpha-positive (ER(+)) breast tumors, but its role in tumor growth and progression remains controversial. Use of two anti-androgens that inhibit AR nuclear localization, enzalutamide and MJC13, revealed that AR is required for maximum ER genomic binding. Here, a novel global examination of AR chromatin binding found that estradiol induced AR binding at unique sites compared with dihydrotestosterone (DHT). Estradiol-induced AR-binding sites were enriched for estrogen response elements and had significant overlap with ER-binding sites. Furthermore, AR inhibition reduced baseline and estradiol-mediated proliferation in multiple ER(+)/AR(+) breast cancer cell lines, and synergized with tamoxifen and fulvestrant. In vivo, enzalutamide significantly reduced viability of tamoxifen-resistant MCF7 xenograft tumors and an ER(+)/AR(+) patient-derived model. Enzalutamide also reduced metastatic burden following cardiac injection. Finally, in a comparison of ER(+)/AR(+) primary tumors versus patient-matched local recurrences or distant metastases, AR expression was often maintained even when ER was reduced or absent. These data provide preclinical evidence that anti-androgens that inhibit AR nuclear localization affect both AR and ER, and are effective in combination with current breast cancer therapies. In addition, single-agent efficacy may be possible in tumors resistant to traditional endocrine therapy, as clinical specimens of recurrent disease demonstrate AR expression in tumors with absent or refractory ER. This study suggests that AR plays a previously unrecognized role in supporting E2-mediated ER activity in ER(+)/AR(+) breast cancer cells, and that enzalutamide may be an effective therapeutic in ER(+)/AR(+) breast cancers. Mol Cancer Res; 14(11); 1054-67. ©2016 AACR. ©2016 American Association for Cancer Research.

  10. Identification of an AR Mutation-Negative Class of Androgen Insensitivity by Determining Endogenous AR Activity

    PubMed Central

    Ukat, M.; Schweikert, H. U.; Hiort, O.; Werner, R.; Drop, S. L. S.; Cools, M.; Hughes, I. A.; Audi, L.; Ahmed, S. F.; Demiri, J.; Rodens, P.; Worch, L.; Wehner, G.; Kulle, A. E.; Dunstheimer, D.; Müller-Roßberg, E.; Reinehr, T.; Hadidi, A. T.; Eckstein, A. K.; van der Horst, C.; Seif, C.; Siebert, R.; Ammerpohl, O.; Holterhus, P.-M.

    2016-01-01

    Context: Only approximately 85% of patients with a clinical diagnosis complete androgen insensitivity syndrome and less than 30% with partial androgen insensitivity syndrome can be explained by inactivating mutations in the androgen receptor (AR) gene. Objective: The objective of the study was to clarify this discrepancy by in vitro determination of AR transcriptional activity in individuals with disorders of sex development (DSD) and male controls. Design: Quantification of DHT-dependent transcriptional induction of the AR target gene apolipoprotein D (APOD) in cultured genital fibroblasts (GFs) (APOD assay) and next-generation sequencing of the complete coding and noncoding AR locus. Setting: The study was conducted at a university hospital endocrine research laboratory. Patients: GFs from 169 individuals were studied encompassing control males (n = 68), molecular defined DSD other than androgen insensitivity syndrome (AIS; n = 18), AR mutation-positive AIS (n = 37), and previously undiagnosed DSD including patients with a clinical suspicion of AIS (n = 46). Intervention(s): There were no interventions. Main Outcome Measure(s): DHT-dependent APOD expression in cultured GF and AR mutation status in 169 individuals was measured. Results: The APOD assay clearly separated control individuals (healthy males and molecular defined DSD patients other than AIS) from genetically proven AIS (cutoff < 2.3-fold APOD-induction; 100% sensitivity, 93.3% specificity, P < .0001). Of 46 DSD individuals with no AR mutation, 17 (37%) fell below the cutoff, indicating disrupted androgen signaling. Conclusions: AR mutation-positive AIS can be reliably identified by the APOD assay. Its combination with next-generation sequencing of the AR locus uncovered an AR mutation-negative, new class of androgen resistance, which we propose to name AIS type II. Our data support the existence of cellular components outside the AR affecting androgen signaling during sexual differentiation with high

  11. Identification of an AR Mutation-Negative Class of Androgen Insensitivity by Determining Endogenous AR Activity.

    PubMed

    Hornig, N C; Ukat, M; Schweikert, H U; Hiort, O; Werner, R; Drop, S L S; Cools, M; Hughes, I A; Audi, L; Ahmed, S F; Demiri, J; Rodens, P; Worch, L; Wehner, G; Kulle, A E; Dunstheimer, D; Müller-Roßberg, E; Reinehr, T; Hadidi, A T; Eckstein, A K; van der Horst, C; Seif, C; Siebert, R; Ammerpohl, O; Holterhus, P-M

    2016-11-01

    Only approximately 85% of patients with a clinical diagnosis complete androgen insensitivity syndrome and less than 30% with partial androgen insensitivity syndrome can be explained by inactivating mutations in the androgen receptor (AR) gene. The objective of the study was to clarify this discrepancy by in vitro determination of AR transcriptional activity in individuals with disorders of sex development (DSD) and male controls. Quantification of DHT-dependent transcriptional induction of the AR target gene apolipoprotein D (APOD) in cultured genital fibroblasts (GFs) (APOD assay) and next-generation sequencing of the complete coding and noncoding AR locus. The study was conducted at a university hospital endocrine research laboratory. GFs from 169 individuals were studied encompassing control males (n = 68), molecular defined DSD other than androgen insensitivity syndrome (AIS; n = 18), AR mutation-positive AIS (n = 37), and previously undiagnosed DSD including patients with a clinical suspicion of AIS (n = 46). There were no interventions. DHT-dependent APOD expression in cultured GF and AR mutation status in 169 individuals was measured. The APOD assay clearly separated control individuals (healthy males and molecular defined DSD patients other than AIS) from genetically proven AIS (cutoff < 2.3-fold APOD-induction; 100% sensitivity, 93.3% specificity, P < .0001). Of 46 DSD individuals with no AR mutation, 17 (37%) fell below the cutoff, indicating disrupted androgen signaling. AR mutation-positive AIS can be reliably identified by the APOD assay. Its combination with next-generation sequencing of the AR locus uncovered an AR mutation-negative, new class of androgen resistance, which we propose to name AIS type II. Our data support the existence of cellular components outside the AR affecting androgen signaling during sexual differentiation with high clinical relevance.

  12. TBLR1 as an AR coactivator selectively activates AR target genes to inhibit prostate cancer growth

    PubMed Central

    Daniels, Garrett; Li, Yirong; Gellert, Lan Lin; Zhou, Albert; Melamed, Jonathan; Wu, Xinyu; Zhang, Xinming; Zhang, David; Meruelo, Daniel; Logan, Susan K.; Basch, Ross; Lee, Peng

    2014-01-01

    Androgen Receptor (AR), a steroid hormone receptor, is critical for prostate cancer growth. However, activation of AR by androgens can also lead to growth suppression and differentiation. Transcriptional cofactors play an important role in this switch between proliferative and anti-proliferative AR target gene programs. TBLR1, a core component of the nuclear receptor corepressor (NCoR) complex, shows both co-repressor and co-activator activities on nuclear receptors, but little is known about its effects on AR and prostate cancer. We characterized TBLR1 as a coactivator of AR in prostate cancer cells and the activation is both phosphorylation and 19S proteosome dependent. We showed that TBLR1 physically interacts with AR and directly occupies the androgen response elements of affected AR target genes in an androgen-dependent manner. TBLR1 is primarily localized in the nucleus in benign prostate cells and nuclear expression is significantly reduced in prostate cancer cells in culture. Similarly, in human tumor samples, the expression of TBLR1 in the nucleus is significantly reduced in the malignant glands compared to the surrounding benign prostatic glands (p<0.005). Stable ectopic expression of nuclear TBLR1 leads to androgen-dependent growth suppression of prostate cancer cells in vitro and in vivo by selective activation of androgen regulated genes associated with differentiation (e.g. KRT18) and growth suppression (e.g. NKX3.1), but not cell proliferation of the prostate. Understanding the molecular switches involved in the transition from AR dependent growth promotion to AR dependent growth suppression will lead to more successful prostate cancer treatments. PMID:24243687

  13. 76 FR 27077 - Agency Information Collection Activities: Form AR-11 and Form AR-11SR, Extension of an Existing...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-10

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form AR-11 and Form AR- 11SR, Extension of an Existing Information Collection; Comment Request ACTION: 60-Day Notice of Information Collection under Review: Form AR- 11 and Form AR-11SR, Alien's Change of Address Card...

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

    PubMed

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

    2016-08-04

    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.

  15. 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

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

    PubMed

    Jones, Dominic; Wade, Mark; Nakjang, Sirintra; Chaytor, Lewis; Grey, James; Robson, Craig N; Gaughan, Luke

    2015-10-06

    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.

  17. ELF5-Mediated AR Activation Regulates Prostate Cancer Progression

    PubMed Central

    Li, Kai; Guo, Yongmin; Yang, Xiong; Zhang, Zhihong; Zhang, Changwen; Xu, Yong

    2017-01-01

    The transcription factor E74-like factor 5 (ELF5) is a potent antioncogene that can prevent epithelial-mesenchymal transition (EMT) and metastasis in prostate cancer (PCa). However, little is known how it suppress the tumor growth and if it can interact with androgen receptor (AR). In this study, we find that the ELF5 is frequently expressed in AR activated PCa cells, where it binds to AR acting as a physiological partner and negatively regulates its transcriptional activity. In addition, the interaction between ELF5 and AR is androgen-dependent. Downregulation of ELF5 by shRNA increases the expression of AR-response genes and the progression of PCa. Moreover, ELF5 is a AR-dependent gene that its expression can be induced by androgen and suppressed by antiandrogen treatment. Notably, forced reduction of ELF5 in LNCaP cells facilitates the binding of AR to ARE in ELF5 gene and enabling its transcription, so that low level ELF5 can turn up its own expression by the negative feedback loop. PMID:28287091

  18. ELF5-Mediated AR Activation Regulates Prostate Cancer Progression.

    PubMed

    Li, Kai; Guo, Yongmin; Yang, Xiong; Zhang, Zhihong; Zhang, Changwen; Xu, Yong

    2017-03-13

    The transcription factor E74-like factor 5 (ELF5) is a potent antioncogene that can prevent epithelial-mesenchymal transition (EMT) and metastasis in prostate cancer (PCa). However, little is known how it suppress the tumor growth and if it can interact with androgen receptor (AR). In this study, we find that the ELF5 is frequently expressed in AR activated PCa cells, where it binds to AR acting as a physiological partner and negatively regulates its transcriptional activity. In addition, the interaction between ELF5 and AR is androgen-dependent. Downregulation of ELF5 by shRNA increases the expression of AR-response genes and the progression of PCa. Moreover, ELF5 is a AR-dependent gene that its expression can be induced by androgen and suppressed by antiandrogen treatment. Notably, forced reduction of ELF5 in LNCaP cells facilitates the binding of AR to ARE in ELF5 gene and enabling its transcription, so that low level ELF5 can turn up its own expression by the negative feedback loop.

  19. Real-time optical recording of β1-adrenergic receptor activation reveals supersensitivity of the Arg389 variant to carvedilol

    PubMed Central

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

    2007-01-01

    Antagonists of β-adrenergic receptors (β-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 β1-AR and downstream signaling. While the commonly used β-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 β1-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 β1-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 β1-AR in living cells in real time. It revealed that β1-AR variants dramatically differ in their responses to diverse beta blockers, with possible consequences for their clinical use. PMID:17200720

  20. Branched Chain RNA In Situ Hybridization for Androgen Receptor Splice Variant AR-V7 as a Prognostic Biomarker for Metastatic Castration-Sensitive Prostate Cancer.

    PubMed

    Saylor, Philip J; Lee, Richard J; Arora, Kshitij S; Deshpande, Vikram; Hu, Rong; Olivier, Kara; Meneely, Erika; Rivera, Miguel N; Ting, David T; Wu, Chin-Lee; Miyamoto, David T

    2017-01-15

    The androgen receptor (AR) mRNA splice variant AR-V7 has emerged as a predictive biomarker for response to AR-targeted therapies. There are currently no commercially available assays to detect AR splice variants. The branched chain RNA in situ hybridization (ISH) platform enables the highly sensitive detection of RNA transcripts in formalin-fixed, paraffin-embedded (FFPE) tissues. We designed a branched chain RNA ISH probe to target the unique cryptic exon CE3 of AR-V7 using multiple tiling probes. This automated ISH assay was applied to tumor tissue from two distinct clinical cohorts that we hypothesized would differ in AR-V7 status. We detected AR-V7 in all tumor samples from men with metastatic castration-resistant prostate cancer with tissue obtained after disease progression despite at least one subsequent line of hormonal therapy (abiraterone, enzalutamide, or bicalutamide; n = 12). We detected AR-V7 in just one tumor from men who had undergone prostatectomy for localized adenocarcinoma (n = 30; Gleason 4 + 5 = 9 in the AR-V7-positive sample). Given the apparent distinction between the above groups by AR-V7 signal, we analyzed pretreatment AR-V7 status as a predictive and prognostic biomarker in men with treatment-naïve metastatic disease. Patients with metastases but without detectable AR-V7 RNA at baseline had significantly longer overall survival (log-rank P = 0.044) and a trend toward superior progression-free survival (log-rank P = 0.055). Within an institutional cohort, the RNA ISH assay identified AR-V7 within FFPE tissue and may have prognostic value in metastatic castration-sensitive prostate cancer. These preliminary findings warrant further study in larger cohorts. Clin Cancer Res; 23(2); 363-9. ©2016 AACR. ©2016 American Association for Cancer Research.

  1. TBLR1 as an androgen receptor (AR) coactivator selectively activates AR target genes to inhibit prostate cancer growth.

    PubMed

    Daniels, Garrett; Li, Yirong; Gellert, Lan Lin; Zhou, Albert; Melamed, Jonathan; Wu, Xinyu; Zhang, Xinming; Zhang, David; Meruelo, Daniel; Logan, Susan K; Basch, Ross; Lee, Peng

    2014-02-01

    Androgen receptor (AR), a steroid hormone receptor, is critical for prostate cancer growth. However, activation of AR by androgens can also lead to growth suppression and differentiation. Transcriptional cofactors play an important role in this switch between proliferative and anti-proliferative AR target gene programs. Transducin β-like-related protein 1 (TBLR1), a core component of the nuclear receptor corepressor complex, shows both corepressor and coactivator activities on nuclear receptors, but little is known about its effects on AR and prostate cancer. We characterized TBLR1 as a coactivator of AR in prostate cancer cells and determined that the activation is dependent on both phosphorylation and 19S proteosome. We showed that TBLR1 physically interacts with AR and directly occupies the androgen-response elements of the affected AR target genes in an androgen-dependent manner. TBLR1 is primarily localized in the nucleus in benign prostate cells and nuclear expression is significantly reduced in prostate cancer cells in culture. Similarly, in human tumor samples, the expression of TBLR1 in the nucleus is significantly reduced in the malignant glands compared with the surrounding benign prostatic glands (P<0.005). Stable ectopic expression of nuclear TBLR1 leads to androgen-dependent growth suppression of prostate cancer cells in vitro and in vivo by selective activation of androgen-regulated genes associated with differentiation (e.g. KRT18) and growth suppression (e.g. NKX3-1), but not cell proliferation of the prostate cancer. Understanding the molecular switches involved in the transition from AR-dependent growth promotion to AR-dependent growth suppression will lead to more successful treatments for prostate cancer.

  2. 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. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Cell cycle-coupled expansion of AR activity promotes cancer progression.

    PubMed

    McNair, C; Urbanucci, A; Comstock, C E S; Augello, M A; Goodwin, J F; Launchbury, R; Zhao, S G; Schiewer, M J; Ertel, A; Karnes, J; Davicioni, E; Wang, L; Wang, Q; Mills, I G; Feng, F Y; Li, W; Carroll, J S; Knudsen, K E

    2017-03-23

    The androgen receptor (AR) is required for prostate cancer (PCa) survival and progression, and ablation of AR activity is the first line of therapeutic intervention for disseminated disease. While initially effective, recurrent tumors ultimately arise for which there is no durable cure. Despite the dependence of PCa on AR activity throughout the course of disease, delineation of the AR-dependent transcriptional network that governs disease progression remains elusive, and the function of AR in mitotically active cells is not well understood. Analyzing AR activity as a function of cell cycle revealed an unexpected and highly expanded repertoire of AR-regulated gene networks in actively cycling cells. New AR functions segregated into two major clusters: those that are specific to cycling cells and retained throughout the mitotic cell cycle ('Cell Cycle Common'), versus those that were specifically enriched in a subset of cell cycle phases ('Phase Restricted'). Further analyses identified previously unrecognized AR functions in major pathways associated with clinical PCa progression. Illustrating the impact of these unmasked AR-driven pathways, dihydroceramide desaturase 1 was identified as an AR-regulated gene in mitotically active cells that promoted pro-metastatic phenotypes, and in advanced PCa proved to be highly associated with development of metastases, recurrence after therapeutic intervention and reduced overall survival. Taken together, these findings delineate AR function in mitotically active tumor cells, thus providing critical insight into the molecular basis by which AR promotes development of lethal PCa and nominate new avenues for therapeutic intervention.

  4. A model for meteoritic and lunar 40Ar/39Ar age spectra: Addressing the conundrum of multi-activation energies

    NASA Astrophysics Data System (ADS)

    Boehnke, P.; Harrison, T. Mark; Heizler, M. T.; Warren, P. H.

    2016-11-01

    Results of whole-rock 40Ar/39Ar step-heating analyses of extra-terrestrial materials have been used to constrain the timing of impacts in the inner solar system, solidification of the lunar magma ocean, and development of planetary magnetic fields. Despite the importance of understanding these events, the samples we have in hand are non-ideal due to mixed provenance, isotopic disturbances from potentially multiple heating episodes, and laboratory artifacts such as nuclear recoil. Although models to quantitatively assess multi-domain, diffusive 40Ar* loss have long been applied to terrestrial samples, their use on extra-terrestrial materials has been limited. Here we introduce a multi-activation energy, multi-diffusion domain model and apply it to 40Ar/39Ar temperature-cycling, step-heating data for meteoritic and lunar samples. We show that age spectra of extra-terrestrial materials, the Jilin chondrite (K-4) and Apollo 16 lunar breccia (67514 , 43), yielding seemingly non-ideal behavior commonly interpreted as either laboratory artifacts or localized shock heating of pyroxene, are meaningful and can be understood in context of the presence of multi-diffusion domains containing multiple activation energies. Internally consistent results from both the meteoritic and lunar samples reveal high-temperature/short duration thermal episodes we interpret as due to moderate shock heating.

  5. 76 FR 43336 - Agency Information Collection Activities: Form AR-11, Extension of an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-20

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form AR-11... Collection under Review: Form AR- 11, Alien's Change of Address Card; OMB Control No. 1615-0007. The... applicable component of the Department of Homeland Security sponsoring this collection: Form AR-11. U.S...

  6. Comprehensive Profiling of the Androgen Receptor in Liquid Biopsies from Castration-resistant Prostate Cancer Reveals Novel Intra-AR Structural Variation and Splice Variant Expression Patterns.

    PubMed

    De Laere, Bram; van Dam, Pieter-Jan; Whitington, Tom; Mayrhofer, Markus; Diaz, Emanuela Henao; Van den Eynden, Gert; Vandebroek, Jean; Del-Favero, Jurgen; Van Laere, Steven; Dirix, Luc; Grönberg, Henrik; Lindberg, Johan

    2017-08-01

    Expression of the androgen receptor splice variant 7 (AR-V7) is associated with poor response to second-line endocrine therapy in castration-resistant prostate cancer (CRPC). However, a large fraction of nonresponding patients are AR-V7-negative. To investigate if a comprehensive liquid biopsy-based AR profile may improve patient stratification in the context of second-line endocrine therapy. Peripheral blood was collected from patients with CRPC (n=30) before initiation of a new line of systemic therapy. We performed profiling of circulating tumour DNA via low-pass whole-genome sequencing and targeted sequencing of the entire AR gene, including introns. Targeted RNA sequencing was performed on enriched circulating tumour cell fractions to assess the expression levels of seven AR splice variants (ARVs). Somatic AR variations, including copy-number alterations, structural variations, and point mutations, were combined with ARV expression patterns and correlated to clinicopathologic parameters. Collectively, any AR perturbation, including ARV, was detected in 25/30 patients. Surprisingly, intra-AR structural variation was present in 15/30 patients, of whom 14 expressed ARVs. The majority of ARV-positive patients expressed multiple ARVs, with AR-V3 the most abundantly expressed. The presence of any ARV was associated with progression-free survival after second-line endocrine treatment (hazard ratio 4.53, 95% confidence interval 1.424-14.41; p=0.0105). Six out of 17 poor responders were AR-V7-negative, but four carried other AR perturbations. Comprehensive AR profiling, which is feasible using liquid biopsies, is necessary to increase our understanding of the mechanisms underpinning resistance to endocrine treatment. Alterations in the androgen receptor are associated with endocrine treatment outcomes. This study demonstrates that it is possible to identify different types of alterations via simple blood draws. Follow-up studies are needed to determine the effect of

  7. Regulatory Activities of Four ArsR Proteins in Agrobacterium tumefaciens 5A

    PubMed Central

    Kang, Yoon-Suk; Brame, Keenan; Jetter, Jonathan; Bothner, Brian B.; Wang, Gejiao

    2016-01-01

    ABSTRACT ArsR is a well-studied transcriptional repressor that regulates microbe-arsenic interactions. Most microorganisms have an arsR gene, but in cases where multiple copies exist, the respective roles or potential functional overlap have not been explored. We examined the repressors encoded by arsR1 and arsR2 (ars1 operon) and by arsR3 and arsR4 (ars2 operon) in Agrobacterium tumefaciens 5A. ArsR1 and ArsR4 are very similar in their primary sequences and diverge phylogenetically from ArsR2 and ArsR3, which are also quite similar to one another. Reporter constructs (lacZ) for arsR1, arsR2, and arsR4 were all inducible by As(III), but expression of arsR3 (monitored by reverse transcriptase PCR) was not influenced by As(III) and appeared to be linked transcriptionally to an upstream lysR-type gene. Experiments using a combination of deletion mutations and additional reporter assays illustrated that the encoded repressors (i) are not all autoregulatory as is typically known for ArsR proteins, (ii) exhibit variable control of each other's encoding genes, and (iii) exert variable control of other genes previously shown to be under the control of ArsR1. Furthermore, ArsR2, ArsR3, and ArsR4 appear to have an activator-like function for some genes otherwise repressed by ArsR1, which deviates from the well-studied repressor role of ArsR proteins. The differential regulatory activities suggest a complex regulatory network not previously observed in ArsR studies. The results indicate that fine-scale ArsR sequence deviations of the reiterated regulatory proteins apparently translate to different regulatory roles. IMPORTANCE Given the significance of the ArsR repressor in regulating various aspects of microbe-arsenic interactions, it is important to assess potential regulatory overlap and/or interference when a microorganism carries multiple copies of arsR. This study explores this issue and shows that the four arsR genes in A. tumefaciens 5A, associated with two separate

  8. Cell-cycle coupled expansion of AR activity promotes cancer progression

    PubMed Central

    McNair, Christopher; Urbanucci, Alfonso; Comstock, Clay E.S.; Augello, Michael A.; Goodwin, Jonathan F.; Launchbury, Rosalind; Zhao, Shuang; Schiewer, Mathew J.; Ertel, Adam; Karnes, Jeffrey; Davicioni, Elai; Wang, Liguo; Wang, Qianben; Mills, Ian G.; Feng, Felix Y.; Li, Wei; Carroll, Jason S.; Knudsen, Karen E.

    2016-01-01

    The androgen receptor (AR) is required for prostate cancer (PCa) survival and progression, and ablation of AR activity is the first line of therapeutic intervention for disseminated disease. While initially effective, recurrent tumors ultimately arise for which there is no durable cure. Despite the dependence of PCa on AR activity throughout the course of disease, delineation of the AR-dependent transcriptional network that governs disease progression remains elusive, and the function of AR in mitotically active cells is not well understood. Analyzing AR activity as a function of cell cycle revealed an unexpected and highly expanded repertoire of AR-regulated gene networks in actively cycling cells. New AR functions segregated into two major clusters: those that are specific to cycling cells and retained throughout the mitotic cell cycle (“Cell Cycle Common”), versus those that were specifically enriched in a subset of cell cycle phases (“Phase Restricted”). Further analyses identified previously unrecognized AR functions in major pathways associated with clinical PCa progression. Illustrating the impact of these unmasked AR-driven pathways, dihydroceramide-desaturase 1 (DEGS1) was identified as an AR regulated gene in mitotically active cells that promoted pro-metastatic phenotypes, and in advanced PCa proved to be highly associated with development of metastases, recurrence after therapeutic intervention, and reduced overall survival. Taken together, these findings delineate AR function in mitotically active tumor cells, thus providing critical insight into the molecular basis by which AR promotes development of lethal PCa and nominate new avenues for therapeutic intervention. PMID:27669432

  9. ArsAB, a Novel Enzyme from Sporomusa ovata Activates Phenolic Bases for Adenosylcobamide Biosynthesis

    PubMed Central

    Chan, Chi Ho; Escalante-Semerena, Jorge C.

    2011-01-01

    Summary In the homoacetogenic bacterium Sporomusa ovata, phenol and p-cresol are converted into α-ribotides, which are incorporated into biologically active cobamides (Cbas) whose lower ligand bases do not form axial coordination bonds with the cobalt ion of the corrin ring. Here we report the identity of two S. ovata genes that encode an enzyme that transfers the phosphoribosyl group of nicotinate mononucleotide (NaMN) to phenol or p-cresol, yielding α-O-glycosidic ribotides. The alluded genes were named arsA and arsB (for alpha-ribotide synthesis), arsA and arsB were isolated from a genomic DNA library of S. ovata. A positive selection strategy using an Escherichia coli strain devoid of NaMN:5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (CobT) activity was used to isolate a fragment of S. ovata DNA that contained arsA and arsB, whose nucleotide sequences overlapped by 8 bp. SoArsAB was isolated to homogeneity, shown to be functional as a heterodimer, and to have highest activity at pH 9. SoArsAB also activated DMB to its α-N-glycosidic ribotide. Previously characterized CobT-like enzymes activate DMB but do not activate phenolics. NMR spectroscopy was used to confirm the incorporation of phenol into the cobamide, and mass spectrometry was used to identity of the SoArsAB products. PMID:21696461

  10. ArsAB, a novel enzyme from Sporomusa ovata activates phenolic bases for adenosylcobamide biosynthesis.

    PubMed

    Chan, Chi Ho; Escalante-Semerena, Jorge C

    2011-08-01

    In the homoacetogenic bacterium Sporomusa ovata, phenol and p-cresol are converted into α-ribotides, which are incorporated into biologically active cobamides (Cbas) whose lower ligand bases do not form axial co-ordination bonds with the cobalt ion of the corrin ring. Here we report the identity of two S. ovata genes that encode an enzyme that transfers the phosphoribosyl group of nicotinate mononucleotide (NaMN) to phenol or p-cresol, yielding α-O-glycosidic ribotides. The alluded genes were named arsA and arsB (for alpha-ribotide synthesis), arsA and arsB were isolated from a genomic DNA library of S. ovata. A positive selection strategy using an Escherichia coli strain devoid of NaMN:5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (CobT) activity was used to isolate a fragment of S. ovata DNA that contained arsA and arsB, whose nucleotide sequences overlapped by 8 bp. SoArsAB was isolated to homogeneity, shown to be functional as a heterodimer, and to have highest activity at pH 9. SoArsAB also activated DMB to its α-N-glycosidic ribotide. Previously characterized CobT-like enzymes activate DMB but do not activate phenolics. NMR spectroscopy was used to confirm the incorporation of phenol into the cobamide, and mass spectrometry was used to identify SoArsAB reaction products. © 2011 Blackwell Publishing Ltd.

  11. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Semaphorin 7A protein variants differentially regulate T-cell activity.

    PubMed

    Gras, Christiane; Eiz-Vesper, Britta; Seltsam, Axel; Immenschuh, Stephan; Blasczyk, Rainer; Figueiredo, Constança

    2013-02-01

    Semaphorin 7A (Sema7A) carries the John-Milton-Hagen human blood group antigen on red blood cells and shows molecular diversity. It is known that Sema7A has immunomodulatory functions, but its regulatory effects on T-cell activation are not completely understood. In this study, the functional role of the R461C Sema7A polymorphism on T-cell responses was investigated. Soluble recombinant wild-type Sema7A (Sema7A_wt) and its R461C variant (Sema7A_R461C) were produced in human embryonic kidney cells. Specific assays were performed to determine the effects of Sema7A_wt and Sema7A_R461C on T-cell activation in terms of proliferation, phenotypic alterations, granzyme B transcript levels, and secretion of proinflammatory cytokines. Sema7A_wt did not affect T-cell activity, but Sema7A_R461C led to marked antigen-independent activation of T cells. In the presence of antigen stimulation, Sema7A_R461C had a major costimulatory effect on T-cell response. Upon Sema7A_R461C stimulation, CD4+ T cells strongly proliferated and exhibited a cytotoxic phenotype with significant up regulation of granzyme B transcripts (up to 220-fold), even in the absence of antigen stimulation. Antibody blocking studies indicated that Sema7A_R461C-mediated T-cell activation is largely β1 integrin dependent. These data demonstrate that Sema7A_R461C, unlike wild-type Sema7A, causes differential regulation of T-cell responses. Since Sema7A has important immunomodulatory functions in inflammatory responses, it might play a key role in autoimmune diseases and other major disorders. Further studies are needed to elucidate the regulatory role of Sema7A and its variants. © 2012 American Association of Blood Banks.

  13. 40Ar/39Ar and unspiked 40K-40Ar dating of upper Pleistocene volcanic activity in the Bas-Vivarais (Ardèche, France)

    NASA Astrophysics Data System (ADS)

    Sasco, Romain; Guillou, Hervé; Nomade, Sébastien; Scao, Vincent; Maury, René C.; Kissel, Catherine; Wandres, Camille

    2017-07-01

    Fifteen basanitic and tephritic flows from Bas-Vivarais, the youngest volcanic field in the French Massif Central together with the Chaîne des Puys, were dated by 40Ar/39Ar and 40K-40Ar on separated groundmass, and studied for paleomagnetism. An almost systematic discrepancy between the two types of ages is observed, the 40K-40Ar method providing ages up to 8.5 times the 40Ar/39Ar ones. Microscopic observations and geochemical analyses lead us to conclude that most of the K-Ar ages measured on Bas-Vivarais samples are in error due to extraneous argon originating from contamination by xenocrysts from disintegrated crustal and mantle xenoliths. However, 40Ar/39Ar experiments do not evidence any excess argon, suggesting two possibilities: 1, the extraneous argon contribution was eliminated during the pre-degassing of the samples at 600 °C prior to the step heating experiments, 2 - K-Ar ages may be older because larger quantities of xenocrysts, potential carriers of extraneous argon were involved in the K-Ar experiments than in the 40Ar/39Ar ones. 40Ar/39Ar ages are thus little or not affected by contamination and provide reliable ages for the studied volcanoes. Combined 40Ar/39Ar datings and magnetic directions for each flow point out to three successive stages in the volcanic evolution of Bas-Vivarais. Stage 1, limited to the northern part of the field, has a mean age of 187.3 ± 19.0 ka. In its southern part, Stages 2 and 3 emplaced magmas at 31.1 ± 3.9 ka and 23.9 ± 8.1 ka, respectively. These two last stages are consistent with available 14C dates but not with previous thermoluminescence data.

  14. Unique dominant negative mutation in the N-terminal mitochondrial targeting sequence of StAR, causing a variant form of congenital lipoid adrenal hyperplasia.

    PubMed

    Baquedano, María Sonia; Guercio, Gabriela; Marino, Roxana; Berensztein, Esperanza; Costanzo, Mariana; Bailez, Marcela; Vaiani, Elisa; Maceiras, Mercedes; Ramirez, Pablo; Chaler, Eduardo; Rivarola, Marco A; Belgorosky, Alicia

    2013-01-01

    Steroid acute regulatory (StAR) protein is a mitochondria-targeted protein that is part of the transduceosome complex crucial for transport of cholesterol to mitochondria. Recessive mutations cause classic and nonclassic congenital lipoid adrenal hyperplasia. The aim of this study was to report the clinical, hormonal, genetic, and functional data of a novel heterozygous mutation in the StAR gene found in a 46,XY patient with ambiguous genitalia and neonatal severe steroidogenic deficiency. Undetectable serum steroids with high ACTH and plasma renin activity but normal acute GnRH response were found in infancy. After gonadectomy (at 3 yr of age), serum LH and testosterone were undetectable, whereas FSH was normal but increased slowly afterward. Estrogen replacement therapy, started at 10.2 yr of age, suppressed gonadotropins (for 2 yr). However, after 1 month off estrogens, the patient showed castrated levels. At 11.9 yr old, after fludrocortisone withdrawal because of hypertension, plasma renin activity and aldosterone remained normal, suggesting mineralocorticoid recovery by a StAR-independent mechanism. We found a de novo heterozygous IVS-2A>G StAR mutation and the reported heterozygous p.G146A SF1 polymorphism with normal CYP11A1, FDXR, FDX1, VDAC1, and TSPO genes. The mutant StAR transcript lacked exon 2, resulting in the in-frame loss of amino acids 22 to 59 in the N-terminal mitochondrial targeting signal. In vitro, the mutant protein exhibited reduced StAR activity in a dominant-negative manner and almost no mitochondria localization. A misfolded p.G22_L59del StAR might interfere with wild-type StAR activity by blocking the transduceosome complex, causing an autosomal dominant form of StAR deficiency, explaining the clinical phenotype. We speculated that estrogen might have modulated mineralocorticoid function and pubertal maturation in a human natural model lacking endogenous steroid production.

  15. Inactivation of ID4 promotes a CRPC phenotype with constitutive AR activation through FKBP52.

    PubMed

    Joshi, Jugal Bharat; Patel, Divya; Morton, Derrick J; Sharma, Pankaj; Zou, Jin; Hewa Bostanthirige, Dhanushka; Gorantla, Yamini; Nagappan, Peri; Komaragiri, Shravan Kumar; Sivils, Jeffrey C; Xie, Huan; Palaniappan, Ravi; Wang, Guangdi; Cox, Marc B; Chaudhary, Jaideep

    2017-04-01

    Castration-resistant prostate cancer (CRPC) is the emergence of prostate cancer cells that have adapted to the androgen-depleted environment of the prostate. In recent years, targeting multiple chaperones and co-chaperones (e.g., Hsp27, FKBP52) that promote androgen receptor (AR) signaling and/or novel AR regulatory mechanisms have emerged as promising alternative treatments for CRPC. We have shown that inactivation of inhibitor of differentiation 4 (ID4), a dominant-negative helix loop helix protein, promotes de novo steroidogenesis and CRPC with a gene expression signature that resembles constitutive AR activity in castrated mice. In this study, we investigated the underlying mechanism through which loss of ID4 potentiates AR signaling. Proteomic analysis between prostate cancer cell line LNCaP (L+ns) and LNCaP lacking ID4 (L(-)ID4) revealed elevated levels of Hsp27 and FKBP52, suggesting a role for these AR-associated co-chaperones in promoting constitutively active AR signaling in L(-)ID4 cells. Interestingly, protein interaction studies demonstrated a direct interaction between ID4 and the 52-kDa FK506-binding protein (FKBP52) in vitro, but not with AR. An increase in FKBP52-dependent AR transcriptional activity was observed in L(-)ID4 cells. Moreover, pharmacological inhibition of FKBP52-AR signaling, by treatment with MJC13, attenuated the tumor growth, weight, and volume in L(-)ID4 xenografts. Together, our results demonstrate that ID4 selectively regulates AR activity through direct interaction with FKBP52, and its loss, promotes CRPC through FKBP52-mediated AR signaling. © 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  16. TALEN-engineered AR gene rearrangements reveal endocrine uncoupling of androgen receptor in prostate cancer

    PubMed Central

    Nyquist, Michael D.; Li, Yingming; Hwang, Tae Hyun; Manlove, Luke S.; Vessella, Robert L.; Silverstein, Kevin A. T.; Voytas, Daniel F.; Dehm, Scott M.

    2013-01-01

    Androgen receptor (AR) target genes direct development and survival of the prostate epithelial lineage, including prostate cancer (PCa). Thus, endocrine therapies that inhibit the AR ligand-binding domain (LBD) are effective in treating PCa. AR transcriptional reactivation is central to resistance, as evidenced by the efficacy of AR retargeting in castration-resistant PCa (CRPC) with next-generation endocrine therapies abiraterone and enzalutamide. However, resistance to abiraterone and enzalutamide limits this efficacy in most men, and PCa remains the second-leading cause of male cancer deaths. Here we show that AR gene rearrangements in CRPC tissues underlie a completely androgen-independent, yet AR-dependent, resistance mechanism. We discovered intragenic AR gene rearrangements in CRPC tissues, which we modeled using transcription activator-like effector nuclease (TALEN)-mediated genome engineering. This modeling revealed that these AR gene rearrangements blocked full-length AR synthesis, but promoted expression of truncated AR variant proteins lacking the AR ligand-binding domain. Furthermore, these AR variant proteins maintained the constitutive activity of the AR transcriptional program and a CRPC growth phenotype independent of full-length AR or androgens. These findings demonstrate that AR gene rearrangements are a unique resistance mechanism by which AR transcriptional activity can be uncoupled from endocrine regulation in CRPC. PMID:24101480

  17. Plasma parameters and active species kinetics in CF4/O2/Ar gas mixture: effects of CF4/O2 and O2/Ar mixing ratios

    NASA Astrophysics Data System (ADS)

    Lee, Junmyung; Kwon, Kwang-Ho; Efremov, A.

    2016-12-01

    The effects of both CF4/O2 and O2/Ar mixing ratios in three-component CF4/O2/Ar mixture on plasma parameters, densities and fluxes of active species determining the dry etching kinetics were analyzed. The investigation combined plasma diagnostics by Langmuir probes and zero-dimensional plasma modeling. It was found that the substitution of CF4 for O2 at constant fraction of Ar in a feed gas produces the non-monotonic change in F atom density, as it was repeatedly reported for the binary CF4/O2 gas mixtures. At the same time, the substitution of Ar for O2 at constant fraction of CF4 results in the monotonic increase in F atom density toward more oxygenated plasmas. The natures of these phenomena as well as theirs possible impacts on the etching/polymerization kinetics were discussed in details.

  18. 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.

  19. Hormonal activation of a kinase cascade localized at the mitochondria is required for StAR protein activity.

    PubMed

    Poderoso, Cecilia; Maloberti, Paula; Duarte, Alejandra; Neuman, Isabel; Paz, Cristina; Cornejo Maciel, Fabiana; Podesta, Ernesto J

    2009-03-05

    It is known that ERK1/2 and MEK1/2 participate in the regulation of Star gene transcription. However, their role in StAR protein post-transcriptional regulation is not described yet. In this study we analyzed the relationship between the MAPK cascade and StAR protein phosphorylation and function. We have demonstrated that (a) steroidogenesis in MA-10 Leydig cells depends on the specific of ERK1/2 activation at the mitochondria; (b) ERK1/2 phosphorylation is driven by mitochondrial PKA and constitutive MEK1/2 in this organelle; (c) active ERK1/2 interacts with StAR protein, leads to StAR protein phosphorylation at Ser(232) only in the presence of cholesterol; (d) directed mutagenesis of Ser(232) (S232A) inhibited in vitro StAR protein phosphorylation by ERK1; (e) transient transfection of MA-10 cells with StAR S232A cDNA markedly reduced the yield of progesterone production. We show that StAR protein is a substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric complex that regulates cholesterol transport.

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

    SciTech Connect

    Aehle, Wolfgang; Bott, Richard R.; Bower, Benjamin S.; Caspi, Jonathan; Goedegebuur, Frits; Hommes, Ronaldus Wilhelmus Joannes; Kaper, Thijs; Kelemen, Bradley R.; Kralj, Slavko; Van Lieshout, Johannes Franciscus Thomas; Nikolaev, Igor; Wallace, Louise; Van Stigt Thans, Sander; Vogtentanz, Gudrun; Sandgren, Mats

    2016-12-20

    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.

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

    DOEpatents

    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.

  2. Succinate/NLRP3 Inflammasome Induces Synovial Fibroblast Activation: Therapeutical Effects of Clematichinenoside AR on Arthritis

    PubMed Central

    Li, Yi; Zheng, Jia-Yi; Liu, Jian-Qun; Yang, Jie; Liu, Yang; Wang, Chen; Ma, Xiao-Nan; Liu, Bao-Lin; Xin, Gui-Zhong; Liu, Li-Fang

    2016-01-01

    Clematichinenoside AR (C-AR) is a triterpene saponin isolated from the root of Clematis manshurica Rupr., which is a herbal medicine used in traditional Chinese medicine for the treatment of arthritis. C-AR exerts anti-inflammatory and immunosuppressive properties, but little is known about its action in the suppression of fibroblast activation. Low oxygen tension and transforming growth factor-β (TGF-β1) induction in the synovium contribute to fibrosis in arthritis. This study was designed to investigate the effect of C-AR on synovial fibrosis from the aspects of hypoxic TGF-β1 and hypoxia-inducible transcription factor-1α (HIF-1α) induction. In the synovium of rheumatoid arthritis (RA) rats, hypoxic TGF-β1 induction increased succinate accumulation due to the reversal of succinate dehydrogenase (SDH) activation and induced NLRP3 inflammasome activation in a manner dependent on HIF-1α induction. In response to NLRP3 inflammasome activation, the released IL-1β further increased TGF-β1 induction, suggesting the forward cycle between inflammation and fibrosis in myofibroblast activation. In the synovium of RA rats, C-AR inhibited hypoxic TGF-β1 induction and suppressed succinate-associated NLRP3 inflammasome activation by inhibiting SDH activity, and thereby prevented myofibroblast activation by blocking the cross-talk between inflammation and fibrosis. Taken together, these results showed that succinate worked as a metabolic signaling, linking inflammation with fibrosis through NLRP3 inflammasome activation. These findings suggested that synovial succinate accumulation and HIF-1α induction might be therapeutical targets for the prevention of fibrosis in arthritis. PMID:28003810

  3. ARS racks

    NASA Image and Video Library

    2009-09-22

    ISS020-E-041651 (22 Sept. 2009) --- NASA astronaut Michael Barratt works with the Atmosphere Revitalization System (ARS) rack in the Destiny laboratory of the International Space Station. Barratt, Canadian Space Agency astronaut Robert Thirsk (out of frame) and European Space Agency astronaut Frank De Winne (out of frame), all Expedition 20 flight engineers, spent several hours with the extensive dual-rack swap/install activity, to move Destiny?s ARS rack to the Kibo laboratory and install in Destiny in its place the newly-delivered ARS rack for Node-3.

  4. ARS racks

    NASA Image and Video Library

    2009-09-22

    ISS020-E-041647 (22 Sept. 2009) --- NASA astronaut Michael Barratt works with the Atmosphere Revitalization System (ARS) rack in the Destiny laboratory of the International Space Station. Barratt, Canadian Space Agency astronaut Robert Thirsk (out of frame) and European Space Agency astronaut Frank De Winne (out of frame), all Expedition 20 flight engineers, spent several hours with the extensive dual-rack swap/install activity, to move Destiny?s ARS rack to the Kibo laboratory and install in Destiny in its place the newly-delivered ARS rack for Node-3.

  5. Neutron activation and prompt gamma intensity in Ar/CO2-filled neutron detectors at the European Spallation Source.

    PubMed

    Dian, E; Kanaki, K; Hall-Wilton, R J; Zagyvai, P; Czifrus, Sz

    2017-10-01

    Monte Carlo simulations using MCNP6.1 were performed to study the effect of neutron activation in Ar/CO2 neutron detector counting gas. A general MCNP model was built and validated with simple analytical calculations. Simulations and calculations agree that only the (40)Ar activation can have a considerable effect. It was shown that neither the prompt gamma intensity from the (40)Ar neutron capture nor the produced (41)Ar activity have an impact in terms of gamma dose rate around the detector and background level. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. 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.

  7. 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.

  8. Unconditioned- and Conditioned- Stimuli Induce Differential Memory Reconsolidation and β-AR-Dependent CREB Activation.

    PubMed

    Huang, Bing; Zhu, Huiwen; Zhou, Yiming; Liu, Xing; Ma, Lan

    2017-01-01

    Consolidated long-term fear memories become labile and reconsolidated upon retrieval by the presentation of conditioned stimulus (CS) or unconditioned stimulus (US). Whether CS-retrieval or US-retrieval will trigger different memory reconsolidation processes is unknown. In this study, we introduced a sequential fear conditioning paradigm in which footshock (FS) was paired with two distinct sounds (CS-A and CS-B). The treatment with propranolol, a β-adrenergic receptor (β-AR) antagonist, after US (FS)-retrieval impaired freezing behavior evoked by either CS-A or CS-B. Betaxolol, a selective β1-AR antagonist, showed similar effects. However, propranolol treatment after retrieval by one CS (e.g., CS-A) only inhibited freezing behavior evoked by the same CS (i.e., CS-A), not the other CS (CS-B). These data suggest that β-AR is critically involved in reconsolidation of fear memory triggered by US- and CS-retrieval, whereas β-AR blockade after US-retrieval disrupts more CS-US associations than CS-retrieval does. Furthermore, significant CREB activation in almost the whole amygdala and hippocampus was observed after US-retrieval, but CS-retrieval only stimulated CREB activation in the lateral amygdala and the CA3 of hippocampus. In addition, propranolol treatment suppressed memory retrieval-induced CREB activation. These data indicate that US-retrieval activates more memory traces than CS-retrieval does, leading to memory reconsolidation of more CS-US associations.

  9. Eocene-Miocene igneous activity in Provence (SE France): 40Ar/39Ar data, geochemical-petrological constraints and geodynamic implications

    NASA Astrophysics Data System (ADS)

    Lustrino, Michele; Fedele, Lorenzo; Agostini, Samuele; Di Vincenzo, Gianfranco; Morra, Vincenzo

    2017-09-01

    Provence (SE France) was affected by two main phases of sporadic igneous activity during the Cenozoic. New 40Ar/39Ar laser step-heating data constrain the beginning of the oldest phase to late Eocene (40.82 ± 0.73 Ma), with activity present until early Miocene ( 20 Ma). The products are mainly andesites, microdiorites, dacites and basaltic andesites mostly emplaced in the Agay-Estérel area. Major- and trace-element constraints, together with Srsbnd Ndsbnd Pb isotopic ratios suggest derivation from a sub-continental lithosphere mantle source variably modified by subduction-related metasomatic processes. The compositions of these rocks overlap those of nearly coeval (emplaced 38-15 Ma) late Eocene-middle Miocene magmatism of Sardinia. The genesis of dacitic rocks cannot be accounted for by simple fractional crystallization alone, and may require interaction of evolved melts with lower crustal lithologies. The youngest phase of igneous activity comprises basaltic volcanic rocks with mildly sodic alkaline affinity emplaced in the Toulon area 10 Myr after the end of the previous subduction-related phase. These rocks show geochemical and isotopic characteristics akin to magmas emplaced in intraplate tectonic settings, indicating a sub-lithospheric HiMu + EM-II mantle source for the magmas, melting approximately in the spinel/garnet-lherzolite transition zone. New 40Ar/39Ar laser step-heating ages place the beginning of the volcanic activity in the late Miocene-Pliocene (5.57 ± 0.09 Ma). The emplacement of ;anorogenic; igneous rocks a few Myr after rocks of orogenic character is a common feature in the Cenozoic districts of the Central-Western Mediterranean area. The origin of such ;anorogenic; rocks can be explained with the activation of different mantle sources not directly modified by subduction-related metasomatic processes, possibly located in the sub-lithospheric mantle, and thus unrelated to the shallower lithospheric mantle source of the ;orogenic; magmatism.

  10. Antinociceptive activity of fruits extracts and "arrope" of Geoffroea decorticans (chañar).

    PubMed

    Reynoso, M A; Vera, N; Aristimuño, M E; Daud, A; Sánchez Riera, A

    2013-01-09

    Geoffroea decorticans (chañar) fruits and their derivate product (arrope) have been traditionally used as food and a folk medicine for the treatment of a wide variety of diseases including bronchopulmonary disorders and to relieve dolorous process. In order to evaluate the pharmacology action of this plant, studies were performed of antinociceptive and antioxidant activities. The aqueous and ethanolic extracts and arrope of chañar were evaluated in various established pain models, including chemical nociception induced by subplantar formalin and intraperitoneal acetic acid and thermal nociception method, such as tail immersion test in rats. To examine the possible connection of the opioid receptor to the antinociceptive activity of extracts and arrope it was performed a combination test with naloxone, a non-selective opioid receptor antagonist. The aqueous extract and arrope (1000 mg/kg) caused an inhibition of the pain in formalin test in the first phase, similar to morphine and decrease in the second phase. In a combination test using naloxone, diminished analgesic activity of aqueous extract and arrope were observed, indicating that antinociceptive activity is connected with the opioid receptor. The aqueous extract and arrope, caused an inhibition of the writhing response induced by acetic acid. Central involvement in analgesic profile was confirmed by the tail immersion test, in which the aqueous extract and arrope showed a significant analgesic activity by increasing latency time. The aqueous extract showed higher antioxidant activity than the arrope, it may be due to the cooking process. This study has shown that the aqueous extract and arrope of Geoffroea decorticans (chañar) fruits, does possess significant antinociceptive effects. It is further concluded that aqueous extract with maximum inhibition of free radical is the most potent extract amount tested extracts. At the oral doses tested the aqueous extract and arrope were non-toxic. The present

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

    PubMed

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. High-resolution 40Ar/ 39Ar chronostratigraphy of the post-caldera (<20 ka) volcanic activity at Pantelleria, Sicily Strait

    NASA Astrophysics Data System (ADS)

    Scaillet, Stéphane; Rotolo, Silvio G.; La Felice, Sonia; Vita-Scaillet, Grazia

    2011-09-01

    The island of Pantelleria (Sicily Strait), the type locality for pantellerite, has been the locus of major caldera-forming eruptions that culminated, ca. 50 ka ago, in the formation of the Cinque Denti caldera produced by the Green Tuff eruption. The post-caldera silicic activity since that time has been mostly confined inside the caldera and consists of smaller-energy eruptions represented by more than twenty coalescing pantelleritic centers structurally controlled by resurgence and trapdoor faulting of the caldera floor. A high-resolution 40Ar/ 39Ar study was conducted on key units spanning the recent (post-20 ka) intracaldera activity to better characterize the present-day status (and forecast the short-term behavior of) the system based on the temporal evolution of the latest eruptions. The new 40Ar/ 39Ar data capture a long-term (> 15 ka) decline in eruption frequency with a shift in eruptive pace from 3.5 ka -1 to 0.8 ka -1 associated with a prominent paleosol horizon marking the only recognizable volcanic stasis around 12-14 ka. This shift in extraction frequency occurs without major changes in eruptive style, and is paralleled by a subtle trend of decreasing melt differentiation index. We speculate that this decline probably occurred (i) without short-term variations in melt production/differentiation rate in a steady-state compositionally-zoned silicic reservoir progressively tapped deeper through the sequence, and (ii) that it was possibly modulated by outboard eustatic forcing due to the 140 m sea level rise over the past 21 ka. The intracaldera system is experiencing a protracted stasis since 7 ka. Coupled with recent geodetic evidence of deflation and subsidence of the caldera floor, the system appears today to be on a wane with no temporal evidence for a short-term silicic eruption.

  13. Influence of excitation parameters and active medium on the efficiency of an electric-discharge excimer ArF laser

    SciTech Connect

    Razhev, A M; Zhupikov, A A; Shchedrin, A I; Kalyuzhnaya, Anna G

    2005-09-30

    The kinetic model of processes occurring in the plasma of an electric-discharge 193-nm excimer ArF laser operating on mixtures of He and Ne buffer gases is developed. The influence of excitation and active medium parameters on the lasing energy and total efficiency of the electric-discharge excimer ArF laser is studied theoretically and experimentally. It is shown that a specific pump power of {approx}4.5-5.0 MW cm{sup -3} is required for attaining the maximum lasing energy for the highest efficiency of an ArF laser operating on a He-Ar-F{sub 2} mixture. For the first time, the pulse energy of 1.3 J at an efficiency of 2.0% is attained for an ArF laser with a specific pump power of 5.0 MW cm{sup -3} using mixtures with helium as a buffer gas. (lasers)

  14. Computationally designed variants of Escherichia coli chorismate mutase show altered catalytic activity.

    PubMed

    Lassila, Jonathan Kyle; Keeffe, Jennifer R; Oelschlaeger, Peter; Mayo, Stephen L

    2005-04-01

    Computational protein design methods were used to predict five variants of monofunctional Escherichia coli chorismate mutase expected to maintain catalytic activity. The variants were tested experimentally and three active site mutants exhibited catalytic activity similar to or greater than the wild-type enzyme. One mutant, Ala32Ser, showed increased catalytic efficiency.

  15. Synthesis and biochemical evaluation of novel inhibitors of aromatase (AR) using an enhanced representation of the active site of AR derived from the consideration of the reaction mechanism.

    PubMed

    Ahmed, S; Amanuel, Y

    2000-01-07

    A novel molecular modeling study, involving inhibitors bound to the iron of cytochrome P450 heme, is described for nonsteroidal inhibitors of aromatase (AR). Study of compounds such as aminoglutethimide (AG) suggests that it utilizes hydrogen bonding group(s) at the active site which would usually H-bond to the steroid C(17) carbonyl group. Interaction between AG's carbonyl groups and the area of the active site corresponding to the substrate C(3)==O group is not possible due to steric interaction. Possible reasons for the difference in activity of enantiomers of alternative inhibitors is also suggested, as well as the mode of action of the new AR inhibitor, Arimidex-whose inhibitory activity previously has not been rationalized. The present study proposes that it is able to use hydrogen bonding groups at the active site corresponding to the steroid C(17)==O and C(3)==O area, contradicting a previous study where it is postulated that azole-type compounds only use polar groups at the active site corresponding to the steroid D ring. Using the hypotheses of the modeling study, we designed and synthesized a number of novel (enantiomerically pure) inhibitors, which upon biochemical evaluation were found to be good inhibitors; the N-nonyl derivative of the S-enantiomer was found to possess 39% inhibition at 100 microM inhibitor concentration (using androstenedione as the substrate), under similar conditions, and AG possessed 20% inhibition. Copyright 2000 Academic Press.

  16. Scaffold protein enigma homolog activates CREB whereas a short splice variant prevents CREB activation in cardiomyocytes.

    PubMed

    Ito, Jumpei; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2015-12-01

    Enigma Homolog (ENH1 or Pdlim5) is a scaffold protein composed of an N-terminal PDZ domain and three LIM domains at the C-terminal end. The enh gene encodes for several splice variants with opposing functions. ENH1 promotes cardiomyocytes hypertrophy whereas ENH splice variants lacking LIM domains prevent it. ENH1 interacts with various Protein Kinase C (PKC) isozymes and Protein Kinase D1 (PKD1). In addition, the binding of ENH1's LIM domains to PKC is sufficient to activate the kinase without stimulation. The downstream events of the ENH1-PKC/PKD1 complex remain unknown. PKC and PKD1 are known to phosphorylate the transcription factor cAMP-response element binding protein (CREB). We tested whether ENH1 could play a role in the activation of CREB. We found that, in neonatal rat ventricular cardiomyocytes, ENH1 interacts with CREB, is necessary for the phosphorylation of CREB at ser133, and the activation of CREB-dependent transcription. On the contrary, the overexpression of ENH3, a LIM-less splice variant, inhibited the phosphorylation of CREB. ENH3 overexpression or shRNA knockdown of ENH1 prevented the CREB-dependent transcription. Our results thus suggest that ENH1 plays an essential role in CREB's activation and dependent transcription in cardiomyocytes. At the opposite, ENH3 prevents the CREB transcriptional activity. In conclusion, these results provide a first molecular explanation to the opposing functions of ENH splice variants.

  17. 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

  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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Resonant proton scattering on 46Ar using the Active-Target Time Projection Chamber

    NASA Astrophysics Data System (ADS)

    Bradt, J.; Ahn, T.; Ayyad Limonge, Y.; Bazin, D.; Beceiro Novo, S.; Carpenter, L.; Kuchera, M. P.; Lynch, W.; Mittig, W.; Rost, S.; Watwood, N.; Barney, J.; Datta, U.; Estee, J.; Gillibert, A.; Manfredi, J.; Morfouace, P.; Perez Loureiro, D.; Pollacco, E.; Sammut, J.; Sweany, S.

    2016-09-01

    A well-known technique for studying the single-particle properties of neutron-rich nuclei is to use resonant proton scattering on a parent nucleus to populate the isobaric analog states of the corresponding neutron-rich nucleus. The locations and amplitudes of these resonances are directly related to the structure of the nucleus of interest by isospin symmetry. We performed an experiment of this type at the National Superconducting Cyclotron Laboratory to commission the recently completed Active-Target Time Projection Chamber (AT-TPC). A 4.6-MeV/u radioactive beam of 46Ar was injected into the AT-TPC. The detector was filled with isobutane gas-which provided the protons for the reaction and served as the tracking medium-and placed inside a 2-T magnetic field. We will present preliminary results from this experiment and discuss the benefits of the active-target method for this type of measurement.

  20. The Celecoxib Derivative AR-12 Has Broad-Spectrum Antifungal Activity In Vitro and Improves the Activity of Fluconazole in a Murine Model of Cryptococcosis

    PubMed Central

    Koselny, Kristy; Green, Julianne; DiDone, Louis; Halterman, Justin P.; Fothergill, Annette W.; Wiederhold, Nathan P.; Patterson, Thomas F.; Cushion, Melanie T.; Rappelye, Chad; Wellington, Melanie

    2016-01-01

    Only one new class of antifungal drugs has been introduced into clinical practice in the last 30 years, and thus the identification of small molecules with novel mechanisms of action is an important goal of current anti-infective research. Here, we describe the characterization of the spectrum of in vitro activity and in vivo activity of AR-12, a celecoxib derivative which has been tested in a phase I clinical trial as an anticancer agent. AR-12 inhibits fungal acetyl coenzyme A (acetyl-CoA) synthetase in vitro and is fungicidal at concentrations similar to those achieved in human plasma. AR-12 has a broad spectrum of activity, including activity against yeasts (e.g., Candida albicans, non-albicans Candida spp., Cryptococcus neoformans), molds (e.g., Fusarium, Mucor), and dimorphic fungi (Blastomyces, Histoplasma, and Coccidioides) with MICs of 2 to 4 μg/ml. AR-12 is also active against azole- and echinocandin-resistant Candida isolates, and subinhibitory AR-12 concentrations increase the susceptibility of fluconazole- and echinocandin-resistant Candida isolates. Finally, AR-12 also increases the activity of fluconazole in a murine model of cryptococcosis. Taken together, these data indicate that AR-12 represents a promising class of small molecules with broad-spectrum antifungal activity. PMID:27645246

  1. The Celecoxib Derivative AR-12 Has Broad-Spectrum Antifungal Activity In Vitro and Improves the Activity of Fluconazole in a Murine Model of Cryptococcosis.

    PubMed

    Koselny, Kristy; Green, Julianne; DiDone, Louis; Halterman, Justin P; Fothergill, Annette W; Wiederhold, Nathan P; Patterson, Thomas F; Cushion, Melanie T; Rappelye, Chad; Wellington, Melanie; Krysan, Damian J

    2016-12-01

    Only one new class of antifungal drugs has been introduced into clinical practice in the last 30 years, and thus the identification of small molecules with novel mechanisms of action is an important goal of current anti-infective research. Here, we describe the characterization of the spectrum of in vitro activity and in vivo activity of AR-12, a celecoxib derivative which has been tested in a phase I clinical trial as an anticancer agent. AR-12 inhibits fungal acetyl coenzyme A (acetyl-CoA) synthetase in vitro and is fungicidal at concentrations similar to those achieved in human plasma. AR-12 has a broad spectrum of activity, including activity against yeasts (e.g., Candida albicans, non-albicans Candida spp., Cryptococcus neoformans), molds (e.g., Fusarium, Mucor), and dimorphic fungi (Blastomyces, Histoplasma, and Coccidioides) with MICs of 2 to 4 μg/ml. AR-12 is also active against azole- and echinocandin-resistant Candida isolates, and subinhibitory AR-12 concentrations increase the susceptibility of fluconazole- and echinocandin-resistant Candida isolates. Finally, AR-12 also increases the activity of fluconazole in a murine model of cryptococcosis. Taken together, these data indicate that AR-12 represents a promising class of small molecules with broad-spectrum antifungal activity.

  2. β2-AR activation induces chemoresistance by modulating p53 acetylation through upregulating Sirt1 in cervical cancer cells.

    PubMed

    Chen, Hongyu; Zhang, Wei; Cheng, Xiang; Guo, Liang; Xie, Shuai; Ma, Yuanfang; Guo, Ning; Shi, Ming

    2017-07-01

    It has been suggested that β2-adrenergic receptor (β2-AR)-mediated signaling induced by catecholamines regulates the degradation of p53. However, the underlying molecular mechanisms were not known. In the present study, we demonstrated that catecholamines upregulated the expression of silent information regulator 1 (Sirt1) through activating β2-AR-mediated signaling pathway, since selective β2-AR antagonist ICI 118, 551 and non-selective β-blocker proprenolol effectively repressed isoproterenol (ISO)-induced Sirt1 expression. Catecholamines inhibited doxorubicin (DOX)-induced p53 acetylation and transcription-activation activities by inducing the expression of Sirt1. Knockdown of the Sirt1 expression by the specific siRNA remarkably blocked the inhibitory effects of ISO on DOX-induced p53 acetylation. In addition, we demonstrated that catecholamines induced resistance of cervical cancer cells to chemotherapeutics both in vitro and in vivo and that β2-AR was overexpressed in cervical cancer tissues. Our data suggest that the p53-dependent, chemotherapeutics-induced cytotoxicity in cervical cancer cells may be compromised by catecholamines-induced upregulation of the Sirt1 expression through activating the β2-AR signaling. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  3. BRCA1 inhibits AR-mediated proliferation of breast cancer cells through the activation of SIRT1.

    PubMed

    Zhang, Wenwen; Luo, Jiayan; Yang, Fang; Wang, Yucai; Yin, Yongmei; Strom, Anders; Gustafsson, Jan Åke; Guan, Xiaoxiang

    2016-02-23

    Breast cancer susceptibility gene 1 (BRCA1) is a tumor suppressor protein that functions to maintain genomic stability through critical roles in DNA repair, cell-cycle arrest, and transcriptional control. The androgen receptor (AR) is expressed in more than 70% of breast cancers and has been implicated in breast cancer pathogenesis. However, little is known about the role of BRCA1 in AR-mediated cell proliferation in human breast cancer. Here, we report that a high expression of AR in breast cancer patients was associated with shorter overall survival (OS) using a tissue microarray with 149 non-metastatic breast cancer patient samples. We reveal that overexpression of BRCA1 significantly inhibited expression of AR through activation of SIRT1 in breast cancer cells. Meanwhile, SIRT1 induction or treatment with a SIRT1 agonist, resveratrol, inhibits AR-stimulated proliferation. Importantly, this mechanism is manifested in breast cancer patient samples and TCGA database, which showed that low SIRT1 gene expression in tumor tissues compared with normal adjacent tissues predicts poor prognosis in patients with breast cancer. Taken together, our findings suggest that BRCA1 attenuates AR-stimulated proliferation of breast cancer cells via SIRT1 mediated pathway.

  4. Glycogen synthase kinase-3 inhibitors suppress the AR-V7-mediated transcription and selectively inhibit cell growth in AR-V7-positive prostate cancer cells.

    PubMed

    Nakata, Daisuke; Koyama, Ryokichi; Nakayama, Kazuhide; Kitazawa, Satoshi; Watanabe, Tatsuya; Hara, Takahito

    2017-06-01

    Recent evidence suggests that androgen receptor (AR) splice variants, including AR-V7, play a pivotal role in resistance to androgen blockade in prostate cancer treatment. The development of new therapeutic agents that can suppress the transcriptional activities of AR splice variants has been anticipated as the next generation treatment of castration-resistant prostate cancer. High-throughput screening of AR-V7 signaling inhibitors was performed using an AR-V7 reporter system. The effects of a glycogen synthase kinase-3 (GSK3) inhibitor, LY-2090314, on endogenous AR-V7 signaling were evaluated in an AR-V7-positive cell line, JDCaP-hr, by quantitative reverse transcription polymerase chain reaction. The relationship between AR-V7 signaling and β-catenin signaling was assessed using RNA interference. The effect of LY-2090314 on cell growth in various prostate cancer cell lines was also evaluated. We identified GSK3 inhibitors as transcriptional suppressors of AR-V7 using a high-throughput screen with an AR-V7 reporter system. LY-2090314 suppressed the reporter activity and endogenous AR-V7 activity in JDCaP-hr cells. Because silencing of β-catenin partly rescued the suppression, it was evident that the suppression was mediated, at least partially, via the activation of β-catenin signaling. AR-V7 signaling and β-catenin signaling reciprocally regulate each other in JDCaP-hr cells, and therefore, GSK3 inhibition can repress AR-V7 transcriptional activity by accumulating intracellular β-catenin. Notably, LY-2090314 selectively inhibited the growth of AR-V7-positive prostate cancer cells in vitro. Our findings demonstrate the potential of GSK3 inhibitors in treating advanced prostate cancer driven by AR splice variants. In vivo evaluation of AR splice variant-positive prostate cancer models will help illustrate the overall significance of GSK3 inhibitors in treating prostate cancer. © 2017 Wiley Periodicals, Inc.

  5. Castration resistance in human prostate cancer is conferred by a frequently occurring androgen receptor splice variant

    PubMed Central

    Sun, Shihua; Sprenger, Cynthia C.T.; Vessella, Robert L.; Haugk, Kathleen; Soriano, Kathryn; Mostaghel, Elahe A.; Page, Stephanie T.; Coleman, Ilsa M.; Nguyen, Holly M.; Sun, Huiying; Nelson, Peter S.; Plymate, Stephen R.

    2010-01-01

    Progression of prostate cancer following castration is associated with increased androgen receptor (AR) expression and signaling despite AR blockade. Recent studies suggest that these activities are due to the generation of constitutively active AR splice variants, but the mechanisms by which these splice variants could mediate such effects are not fully understood. Here we have identified what we believe to be a novel human AR splice variant in which exons 5, 6, and 7 are deleted (ARv567es) and demonstrated that this variant can contribute to cancer progression in human prostate cancer xenograft models in mice following castration. We determined that, in human prostate cancer cell lines, ARv567es functioned as a constitutively active receptor, increased expression of full-length AR (ARfl), and enhanced the transcriptional activity of AR. In human xenografts, human prostate cancer cells transfected with ARv567es cDNA formed tumors that were resistant to castration. Furthermore, the ratio of ARv567es to ARfl expression within the xenografts positively correlated with resistance to castration. Importantly, we also detected ARv567es frequently in human prostate cancer metastases. In summary, these data indicate that constitutively active AR splice variants can contribute to the development of castration-resistant prostate cancers and may serve as biomarkers for patients who are likely to suffer from early recurrence and are candidates for therapies directly targeting the AR rather than ligand. PMID:20644256

  6. A Novel Androgen Receptor Splice Variant Is Upregulated during Prostate Cancer Progression and Promotes Androgen-depletion-resistant Growth

    PubMed Central

    Guo, Zhiyong; Yang, Xi; Sun, Feng; Jiang, Richeng; Linn, Douglas E.; Chen, Hege; Chen, Hegang; Kong, Xiangtian; Melamed, Jonathan; Tepper, Clifford G.; Kung, Hsing-Jien; Brodie, Angela M. H.; Edwards, Joanne; Qiu, Yun

    2009-01-01

    The androgen receptor (AR) plays a key role in progression to incurable androgen-ablation resistant prostate cancer (PCA). We have identified three novel AR splice variants lacking the ligand binding domain (designated as AR3, AR4 and AR5) in hormone insensitive PCA cells. AR3, one of the major splice variants expressed in human prostate tissues, is constitutively active and its transcriptional activity is not regulated by androgens or antiandrogens. Immunohistochemistry analysis on tissue microarrays containing 429 human prostate tissue samples shows that AR3 is significantly upregulated during PCA progression and AR3 expression level is correlated with the risk of tumor recurrence after radical prostatectomy. Overexpression of AR3 confers ablation-independent growth of PCA cells while specific knock-down of AR3 expression (without altering AR level) in hormone resistant PCA cells attenuates their growth under androgen-depleted conditions in both cell culture and xenograft models, suggesting an indispensable role of AR3 in ablation-independent growth of PCA cells. Furthermore, AR3 may play a distinct yet essential role in ablation-independent growth through regulating a unique set of genes including AKT1, which are not regulated by the prototype AR. Our data suggest that aberrant expression of AR splice variants may be a novel mechanism underlying ablation-independence during PCA progression and AR3 may serve as a prognostic marker to predict patient outcome in response to hormonal therapy. Given that these novel AR splice variants are not inhibited by currently available anti-androgen drugs, development of new drugs targeting these AR isoforms may potentially be effective for treatment of ablation-resistant PCA. PMID:19244107

  7. Streptokinase variants from Streptococcus pyogenes isolates display altered plasminogen activation characteristics - implications for pathogenesis.

    PubMed

    Cook, Simon M; Skora, Amanda; Gillen, Christine M; Walker, Mark J; McArthur, Jason D

    2012-12-01

    Streptococcus pyogenes (group A streptococcus, GAS) secretes streptokinase, a potent plasminogen activating protein. Among GAS isolates, streptokinase gene sequences (ska) are polymorphic and can be grouped into two distinct sequence clusters (termed cluster type-1 and cluster type-2) with cluster type-2 being further divided into sub-clusters type-2a and type-2b. In this study, far-UV circular dichroism spectroscopy indicated that purified streptokinase variants of each type displayed similar secondary structure. Type-2b streptokinase variants could not generate an active site in Glu-plasminogen through non-proteolytic mechanisms while all other variants had this capability. Furthermore, when compared with other streptokinase variants, type-2b variants displayed a 29- to 35-fold reduction in affinity for Glu-plasminogen. All SK variants could activate Glu-plasminogen when an activator complex was preformed with plasmin; however, type-2b and type-1 complexes were inhibited by α(2) -antiplasmin. Exchanging ska(type-2a) in the M1T1 GAS strain 5448 with ska(type-2b) caused a reduction in virulence while exchanging ska(type-2a) with ska(type-1) into 5448 produced an increase in virulence when using a mouse model of invasive disease. These findings suggest that streptokinase variants produced by GAS isolates utilize distinct plasminogen activation pathways, which directly affects the pathogenesis of this organism.

  8. Receptor function, dominant negative activity and phenotype correlations for MC1R variant alleles.

    PubMed

    Beaumont, Kimberley A; Shekar, Sri N; Shekar, Sri L; Newton, Richard A; James, Michael R; Stow, Jennifer L; Duffy, David L; Sturm, Richard A

    2007-09-15

    The human melanocortin-1 receptor (MC1R) is a G-protein coupled receptor involved in the regulation of pigmentation. Several MC1R variant alleles are associated with red hair, fair skin and increased skin cancer risk. We have performed a systematic functional analysis of nine common MC1R variants and correlated these results with the strength of the genetic association of each variant allele with pigmentation phenotypes. In vitro expression studies revealed that variant receptors with reduced cell surface expression, including V60L, D84E, R151C, I155T, R160W and R163Q, showed a corresponding impairment in cAMP coupling. The R142H and D294H variants demonstrated normal cell surface expression, but had reduced functional responses, indicating that altered G-protein coupling may be responsible for this loss of function. The V92M variant cAMP activation was equal to or higher than that for wild-type MC1R. In co-expression studies, the D84E, R151C, I155T and R160W variants showed a dominant negative effect on wild-type receptor cell surface expression, which was reflected in a decreased ability to elevate intracellular cAMP levels. The D294H variant also demonstrated a dominant negative effect on wild-type MC1R cAMP signalling, but had no effect on wild-type surface expression. Importantly, comparison of the in vitro receptor characteristics with skin and hair colour data of individuals both homozygous and heterozygous for MC1R variant alleles revealed parallels between variant MC1R cell surface expression, functional ability, dominant negative activity and their effects on human pigmentation. These findings show the first direct correlations between variant MC1R biochemical properties and pigmentation phenotype.

  9. Activation of Variants of Hypoxanthine-Guanine Phosphoribosyl Transferase by the Normal Enzyme

    PubMed Central

    Bakay, Bohdan; Nyhan, William L.

    1972-01-01

    Deficient hypoxanthine-guanine phosphoribosyl transferase (HGPRT; EC 2.4.2.8) enzymes from erythrocytes of patients with hyperuricemia and with the Lesch-Nyhan syndrome migrate 15% faster in polyacrylamide gel disc electrophoresis than the normal enzyme. A half-sister of two males with partial deficiency, who had 34% of normal HGPRT activity in her erythrocytes, yielded profiles containing two distinct zones of activity; one corresponded to the enzyme found in normal individuals and one to the variant of her half-brothers. However, in her profile her variant enzyme showed notably greater activity than that observed in her half-brothers. This increase was due to an activation of the variant by normal enzyme. Electrophoresis of mixtures of normal enzyme with partially deficient enzymes from patients with hyperuricemia and with the Lesch-Nyhan syndrome also led to activation of deficient HGPRT variants by normal enzymes. Deficient variants were also activated by normal enzyme on filtration through Sephadex G-25. Experiments in which deficient variant enzymes were activated with purified normal enzyme labeled with 125I indicated that deficient enzymes incorporate components of the normal enzyme. No such activation of deficient enzymes was ever obtained when mixtures of deficient and normal enzymes were put together in a test tube. Images PMID:4341698

  10. Novel histone deacetylase inhibitor AR-42 exhibits antitumor activity in pancreatic cancer cells by affecting multiple biochemical pathways.

    PubMed

    Chen, Yi-Jin; Wang, Wen-Hung; Wu, Wan-Yu; Hsu, Chia-Chi; Wei, Ling-Rung; Wang, Sheng-Fan; Hsu, Ya-Wen; Liaw, Chih-Chuang; Tsai, Wan-Chi

    2017-01-01

    Pancreatic cancer is one of the most lethal types of cancer with a 5-year survival rate of ~5%. Histone deacetylases (HDACs) participate in many cellular processes, including carcinogenesis, and pharmacological inhibition of HDACs has emerged as a potential therapeutic strategy. In this study, we explored antitumor activity of the novel HDAC inhibitor AR-42 in pancreatic cancer. Human pancreatic cancer cell lines BxPC-3 and PANC-1 were used in this study. Real-time PCR, RT-PCR, and western blotting were employed to investigate expression of specific genes and proteins, respectively. Translocation of apoptosis-inducing factor was investigated by immunofluorescence and subcellular fractionation. The number of apoptotic cells, cell cycle stages, and reactive oxygen species (ROS) generation levels were determined by flow cytometry. Cell invasiveness was examined by the Matrigel invasion assay. Efficacy of AR-42 in vivo was evaluated by utilizing BxPC-3 xenograft mouse model. AR-42 inhibited pancreatic cancer cell proliferation by causing G2/M cell cycle arrest via regulating expression levels of genes and proteins involved in cell cycle. AR-42 also induced ROS generation and DNA damage, triggering apoptosis of pancreatic cancer cells via both caspase-3-dependent and caspase-3-independent pathways. In addition, AR-42 increased expression levels of negative regulators of p53 (miR-125b, miR-30d, and miR33), which could contribute to lower expression level of mutant p53 in pancreatic cancer cells. Cell invasion assay showed that AR-42 reduced cancer cell aggressiveness and significantly diminished BxPC-3 xenograft tumor growth in vivo. AR-42, a novel HDAC inhibitor, inhibited pancreatic cancer cells by regulating p53 expression, inducing cell cycle arrest, particularly at the G2/M stage, and activating multiple apoptosis pathways. Additionally, AR-42 inhibited cell invasiveness and potently suppressed pancreatic cancer tumors in vivo. We conclude that by virtue of its

  11. 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.

  12. 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.

  13. Genetic analysis of a transcriptional activation pathway by using hepatoma cell variants.

    PubMed Central

    Bulla, G A; Fournier, R E

    1994-01-01

    A hierarchy of liver-enriched transcription factors plays an important role in activating expression of many hepatic genes. In particular, hepatocyte nuclear factor 4 (HNF-4) is a major activator of the gene encoding HNF-1, and HNF-1 itself activates expression of more than 20 liver genes. To dissect this activation pathway genetically, we prepared somatic cell variants that were deficient in expression of the liver-specific alpha 1-antitrypsin (alpha 1AT) gene, which requires both HNF-1 and HNF-4 for high-level gene activity. This was accomplished in two steps. First, hepatoma transfectants that stably expressed two selectable markers under alpha 1AT promoter control were prepared; second, variant sublines that could no longer express either transgene were isolated by direct selection. In this report, we demonstrate that the variants contain defects in the HNF-4/HNF-1 activation pathway. These defects functioned in trans, as expression of many liver genes was affected, but the variant phenotypes were recessive to wild type in somatic cell hybrids. Three different variant classes could be discriminated by their phenotypic responses to ectopic expression of either HNF-4 or HNF-1. Two variant clones appeared specifically deficient in HNF-4 expression, as transfection with an HNF-4 expression cassette fully restored their hepatic phenotypes. Another line activated HNF-1 in response to forced HNF-4 expression, but activation of downstream genes failed to occur. One clone was unresponsive to either HNF-1 or HNF-4. Using the variants, we demonstrate further that the chromosomal genes encoding alpha 1AT, aldolase B, and alpha-fibrinogen display strict requirements for HNF-1 activation in vivo, while other liver genes were unaffected by the presence or absence of HNF-1 or HNF-4. We also provide evidence for the existence of an autoregulatory loop in which HNF-1 regulates its own expression through activation of HNF-4. Images PMID:7935424

  14. Influence of nitrogen impurities on the formation of active species in Ar-O2 plasmas

    NASA Astrophysics Data System (ADS)

    Guerra, V.; Kutasi, K.; Sá, P. A.; Lino da Silva, M.

    2011-11-01

    A self-consistent kinetic model was developed in order to study the production of active species in Ar-O2 surface-wave microwave plasmas with a relatively small N2 addition. It is shown that the Ar-O2-N2 mixture produces efficiently the same active species as an Ar-O2 discharge, including oxygen atoms, metastable O2(a1∆g) molecules and the VUV emitting Ar(4s) states. Furthermore, active N-containing species are additionally produced, in particular N atoms and NO ground-state and excited molecules, which makes the ternary mixture very interesting for numerous plasma applications.

  15. Predicting enhancer activity and variant impact using gkm-SVM.

    PubMed

    Beer, Michael A

    2017-01-25

    We participated in the Critical Assessment of Genome Interpretation eQTL challenge to further test computational models of regulatory variant impact and their association with human disease. Our prediction model is based on a discriminative gapped-kmer SVM (gkm-SVM) trained on genome-wide chromatin accessibility data in the cell type of interest. The comparisons with massively parallel reporter assays (MPRA) in lymphoblasts show that gkm-SVM is among the most accurate prediction models even though all other models used the MPRA data for model training, and gkm-SVM did not. In addition, we compare gkm-SVM with other MPRA datasets and show that gkm-SVM is a reliable predictor of expression and that deltaSVM is a reliable predictor of variant impact in K562 cells and mouse retina. We further show that DHS (DNase-I hypersensitive sites) and ATAC-seq (assay for transposase-accessible chromatin using sequencing) data are equally predictive substrates for training gkm-SVM, and that DHS regions flanked by H3K27Ac and H3K4me1 marks are more predictive than DHS regions alone.

  16. 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.

  17. 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.

  18. A Rare Myelin Protein Zero (MPZ) Variant Alters Enhancer Activity In Vitro and In Vivo

    PubMed Central

    Burzynski, Grzegorz; Huynh, Jimmy; Maduro, Valerie; Hodonsky, Chani J.; Khajavi, Mehrdad; Szigeti, Kinga; Mukkamala, Sandeep; Bessling, Seneca L.; Pavan, William J.; McCallion, Andrew S.; Lupski, James R.; Green, Eric D.

    2010-01-01

    Background Myelin protein zero (MPZ) is a critical structural component of myelin in the peripheral nervous system. The MPZ gene is regulated, in part, by the transcription factors SOX10 and EGR2. Mutations in MPZ, SOX10, and EGR2 have been implicated in demyelinating peripheral neuropathies, suggesting that components of this transcriptional network are candidates for harboring disease-causing mutations (or otherwise functional variants) that affect MPZ expression. Methodology We utilized a combination of multi-species sequence comparisons, transcription factor-binding site predictions, targeted human DNA re-sequencing, and in vitro and in vivo enhancer assays to study human non-coding MPZ variants. Principal Findings Our efforts revealed a variant within the first intron of MPZ that resides within a previously described SOX10 binding site is associated with decreased enhancer activity, and alters binding of nuclear proteins. Additionally, the genomic segment harboring this variant directs tissue-relevant reporter gene expression in zebrafish. Conclusions This is the first reported MPZ variant within a cis-acting transcriptional regulatory element. While we were unable to implicate this variant in disease onset, our data suggests that similar non-coding sequences should be screened for mutations in patients with neurological disease. Furthermore, our multi-faceted approach for examining the functional significance of non-coding variants can be readily generalized to study other loci important for myelin structure and function. PMID:21179557

  19. Systemic endothelial function is preserved in men with both active and inactive variant angina pectoris.

    PubMed

    Ito, K; Akita, H; Kanazawa, K; Yamada, S; Shiga, N; Terashima, M; Matsuda, Y; Takai, E; Iwai, C; Takaoka, H; Yokoyama, M

    1999-12-01

    To test the hypothesis that coronary spasm could be a coronary manifestation of systemic endothelial dysfunction and that the activity of coronary spasm could influence systemic endothelial function, we examined brachial flow-mediated, endothelium-dependent vasodilation and nitroglycerin-induced endothelium-independent vasodilation with high-resolution ultrasound in 11 men with variant angina pectoris (6 active and 5 inactive) without established coronary atherosclerosis. Endothelium-dependent vasodilation in peripheral circulation was preserved in men with active and inactive variant angina pectoris, suggesting that systemic endothelial dysfunction is not involved in either the pathogenesis or the activity of coronary spasm.

  20. Refolded Recombinant Human Paraoxonase 1 Variant Exhibits Prophylactic Activity Against Organophosphate Poisoning.

    PubMed

    Bajaj, Priyanka; Tripathy, Rajan K; Aggarwal, Geetika; Datusalia, Ashok K; Sharma, Shyam S; Pande, Abhay H

    2016-09-01

    Organophosphate (OP) compounds are neurotoxic chemicals, and current treatments available for OP-poisoning are considered as unsatisfactory and inadequate. There is an urgent need for the development of more effective treatment(s) for OP-poisoning. Human paraoxonase 1 (h-PON1) is known to hydrolyze a variety of OP-compounds and is a leading candidate for the development of prophylactic and therapeutic agent against OP-poisoning in humans. Non-availability of effective system(s) for the production of recombinant h-PON1 (rh-PON1) makes it hard to produce improved variant(s) of this enzyme and analyze their in vivo efficacy in animal models. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop variant(s) of h-PON1. Recently, we have developed a procedure to produce active rh-PON1 enzymes by using E. coli expression system. In this study, we have characterized the OP-hydrolyzing properties of refolded rh-PON1(wt) and rh-PON1(H115W;R192K) variant. Our results show that refolded rh-PON1(H115W;R192K) variant exhibit enhanced OP-hydrolyzing activity in in vitro and ex vivo assays and exhibited prophylactic activity in mouse model of OP-poisoning, suggesting that refolded rh-PON1 can be developed as a therapeutic candidate.

  1. 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.

  2. AR-v7 protein expression is regulated by protein kinase and phosphatase

    PubMed Central

    Li, Yinan; Xie, Ning; Gleave, Martin E.; Rennie, Paul S.; Dong, Xuesen

    2015-01-01

    Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. PMID:26378044

  3. AR-v7 protein expression is regulated by protein kinase and phosphatase.

    PubMed

    Li, Yinan; Xie, Ning; Gleave, Martin E; Rennie, Paul S; Dong, Xuesen

    2015-10-20

    Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked.

  4. Tectonic events, continental intraplate volcanism, and mantle plume activity in northern Arabia: Constraints from geochemistry and Ar-Ar dating of Syrian lavas

    NASA Astrophysics Data System (ADS)

    Krienitz, M.-S.; Haase, K. M.; Mezger, K.; van den Bogaard, P.; Thiemann, V.; Shaikh-Mashail, M. A.

    2009-04-01

    New 40Ar/39Ar ages combined with chemical and Sr, Nd, and Pb isotope data for volcanic rocks from Syria along with published data of Syrian and Arabian lavas constrain the spatiotemporal evolution of volcanism, melting regime, and magmatic sources contributing to the volcanic activity in northern Arabia. Several volcanic phases occurred in different parts of Syria in the last 20 Ma that partly correlate with different tectonic events like displacements along the Dead Sea Fault system or slab break-off beneath the Bitlis suture zone, although the large volume of magmas and their composition suggest that hot mantle material caused volcanism. Low Ce/Pb (<20), Nb/Th (<10), and Sr, Nd, and Pb isotope variations of Syrian lavas indicate the role of crustal contamination in magma genesis, and contamination of magmas with up to 30% of continental crustal material can explain their 87Sr/86Sr. Fractionation-corrected major element compositions and REE ratios of uncontaminated lavas suggest a pressure-controlled melting regime in western Arabia that varies from shallow and high-degree melt formation in the south to increasingly deeper regions and lower extents of the beginning melting process northward. Temperature estimates of calculated primary, crustally uncontaminated Arabian lavas indicate their formation at elevated mantle temperatures (Texcess ˜ 100-200°C) being characteristic for their generation in a plume mantle region. The Sr, Nd, and Pb isotope systematic of crustally uncontaminated Syrian lavas reveal a sublithospheric and a mantle plume source involvement in their formation, whereas a (hydrous) lithospheric origin of lavas can be excluded on the basis of negative correlations between Ba/La and K/La. The characteristically high 206Pb/204Pb (˜19.5) of the mantle plume source can be explained by material entrainment associated with the Afar mantle plume. The Syrian volcanic rocks are generally younger than lavas from the southern Afro-Arabian region, indicating

  5. The A645D mutation in the hinge region of the human androgen receptor (AR) gene modulates AR activity, depending on the context of the polymorphic glutamine and glycine repeats.

    PubMed

    Werner, Ralf; Holterhus, Paul-Martin; Binder, Gerhard; Schwarz, Hans-Peter; Morlot, Michel; Struve, Dagmar; Marschke, Christine; Hiort, Olaf

    2006-09-01

    Sufficient androgen receptor (AR) activity is crucial for normal male sexual differentiation. Here we report on two unrelated 46, XY patients suffering from undervirilization and genital malformations. Both patients had a short polyglycine (polyG) repeat of 10 residues and a relatively long polyglutamine (polyQ) repeat of 28 and 30 residues within the transactivation domain of the AR. In addition, they also harbor a rare A645D substitution. We made a set of AR expression plasmid constructs with varying polyQ and polyG tract sizes in context with or without the A645D substitution and analyzed their in vitro transactivation capacity in transfected CHO cells. We found that a short polyG repeat downmodulated AR activity to approximately 60-65% of the wild-type receptor. This effect was aggravated by A645D in context of a long polyQ repeat to less than 50% activity. In contrast, in the context of a short polyQ and a short polyG repeat, the A645D mutation rescues AR activity to almost wild-type levels, demonstrating a contradictory effect of this mutation, depending on the size of the polymorphic repeats. A combination of a short polyG repeat with a long polyQ repeat and an A645D substitution might contribute to the development of virilization disorders and explain the observed phenotypes of our patients as a form of androgen insensitivity. The whole recreation of AR sequence variations including individual polymorphic repeat sizes could unravel possible interference of mutations and variations on AR activity by in vitro transfection.

  6. A Whole Blood Assay for AR-V7 and AR(v567es) in Patients with Prostate Cancer.

    PubMed

    Liu, Xichun; Ledet, Elisa; Li, Dongying; Dotiwala, Ary; Steinberger, Allie; Feibus, Allison; Li, Jianzhuo; Qi, Yanfeng; Silberstein, Jonathan; Lee, Benjamin; Dong, Yan; Sartor, Oliver; Zhang, Haitao

    2016-12-01

    Most prostate cancer mortality can be attributed to metastatic castration resistant prostate cancer, an advanced stage that remains incurable despite recent advances. The AR (androgen receptor) signaling axis remains active in castration resistant prostate cancer. Recent studies suggest that expression of the AR-V (AR splice variant) AR-V7 may underlie resistance to abiraterone and enzalutamide. However, controversy exists over the optimal assay. Our objective was to develop a fast and sensitive assay for AR-Vs in patients. Two approaches were assessed in this study. The first approach was based on depletion of leukocytes and the second one used RNA purified directly from whole blood preserved in PAXgene® tubes. Transcript expression was analyzed by quantitative reverse transcription-polymerase chain reaction. Through a side-by-side comparison we found that the whole blood approach was suitable to detect AR-Vs. The specificity of the assay was corroborated in a cancer-free cohort. Using the PAXgene assay samples from a cohort of 46 patients with castration resistant prostate cancer were analyzed. Overall, AR-V7 and AR(v567es) were detected in 67.53% and 29.87% of samples, respectively. Statistical analysis revealed a strong association of AR-V positivity with a history of second line hormonal therapies. To our knowledge this is the first study to demonstrate that PAXgene preserved whole blood can be used to obtain clinically relevant information regarding the expression of 2 AR-Vs. These data on a castration resistant prostate cancer cohort support a role for AR-Vs in resistance to therapies targeting the AR ligand-binding domain. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  7. Differences in Transcriptional Activity of Human Papillomavirus Type 6 Molecular Variants in Recurrent Respiratory Papillomatosis

    PubMed Central

    Measso do Bonfim, Caroline; Simão Sobrinho, João; Lacerda Nogueira, Rodrigo; Salgado Kupper, Daniel; Cardoso Pereira Valera, Fabiana; Lacerda Nogueira, Maurício; Villa, Luisa Lina; Rahal, Paula; Sichero, Laura

    2015-01-01

    A significant proportion of recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6). The long control region (LCR) contains cis-elements for regulation of transcription. Our aim was to characterize LCR HPV-6 variants in RRP cases, compare promoter activity of these isolates and search for cellular transcription factors (TFs) that could explain the differences observed. The complete LCR from 13 RRP was analyzed. Transcriptional activity of 5 variants was compared using luciferase assays. Differences in putative TFs binding sites among variants were revealed using the TRANSFAC database. Chromatin immunoprecipation (CHIP) and luciferase assays were used to evaluate TF binding and impact upon transcription, respectively. Juvenile-onset RRP cases harbored exclusively HPV-6vc related variants, whereas among adult-onset cases HPV-6a variants were more prevalent. The HPV-6vc reference was more transcriptionally active than the HPV-6a reference. Active FOXA1, ELF1 and GATA1 binding sites overlap variable nucleotide positions among isolates and influenced LCR activity. Furthermore, our results support a crucial role for ELF1 on transcriptional downregulation. We identified TFs implicated in the regulation of HPV-6 early gene expression. Many of these factors are mutated in cancer or are putative cancer biomarkers, and must be further studied. PMID:26151558

  8. Identification of androgen receptor variants in testis from humans and other vertebrates.

    PubMed

    Laurentino, S S; Pinto, P I S; Tomás, J; Cavaco, J E; Sousa, M; Barros, A; Power, D M; Canário, A V M; Socorro, S

    2013-06-01

    The androgen receptor (AR) is a ligand-activated transcription factor member of the nuclear receptor superfamily. The existence of alternatively spliced variants is well recognised for several members of this superfamily, most of them having functional importance. For example, several testicular oestrogen receptor variants have been suggested to play a role in the regulation of spermatogenesis. However, information on AR variants is mostly related to cancer and androgen insensitivity syndrome (AIS) cases. The objective of this study was to investigate the expression of AR variants in the testis from humans and other vertebrates. Four AR variants [ARΔ2(Stop) , ARΔ2(23Stop) , ARΔ3 and ARΔ4(120)] were identified in human testis. ARΔ2(Stop) and ARΔ3, with exon 2 or 3 deleted, respectively, were also expressed in human liver, lung, kidney and heart. In addition, ARΔ2(Stop) was expressed in rat and gilthead seabream testis, while an ARΔ3 was detected in African clawed frog testis. This is the first report revealing the existence of AR variants in the testis of evolutionarily distant vertebrate species and in nonpathological tissues. These data suggest the functional importance of these novel AR forms and demonstrate a complexity in AR signalling that is not exclusive of pathological conditions.

  9. A single cell level measurement of StAR expression and activity in adrenal cells.

    PubMed

    Lee, Jinwoo; Yamazaki, Takeshi; Dong, Hui; Jefcoate, Colin

    2017-02-05

    The Steroidogenic acute regulatory protein (StAR) directs mitochondrial cholesterol uptake through a C-terminal cholesterol binding domain (CBD) and a 62 amino acid N-terminal regulatory domain (NTD) that contains an import sequence and conserved sites for inner membrane metalloproteases. Deletion of the NTD prevents mitochondrial import while maintaining steroidogenesis but with compromised cholesterol homeostasis. The rapid StAR-mediated cholesterol transfer in adrenal cells depends on concerted mRNA translation, p37 StAR phosphorylation and controlled NTD cleavage. The NTD controls this process with two cAMP-inducible modulators of, respectively, transcription and translation SIK1 and TIS11b/Znf36l1. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA resolves slow RNA splicing at the gene loci in cAMP-induced Y-1 cells and transfer of individual 3.5 kB mRNA molecules to mitochondria. StAR transcription depends on the CREB coactivator CRTC2 and PKA inhibition of the highly inducible suppressor kinase SIK1 and a basal counterpart SIK2. PKA-inducible TIS11b/Znf36l1 binds specifically to highly conserved elements in exon 7 thereby suppressing formation of mRNA and subsequent translation. Co-expression of SIK1, Znf36l1 with 3.5 kB StAR mRNA may limit responses to pulsatile signaling by ACTH while regulating the transition to more prolonged stress. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. In vitro residual activity of phenylalanine hydroxylase variants and correlation with metabolic phenotypes in PKU.

    PubMed

    Trunzo, Roberta; Santacroce, Rosa; Shen, Nan; Jung-Klawitter, Sabine; Leccese, Angelica; De Girolamo, Giuseppe; Margaglione, Maurizio; Blau, Nenad

    2016-12-05

    Hyperphenylalaninemias (HPAs) are genetic diseases predominantly caused by a wide range of variants in the phenylalanine hydroxylase (PAH) gene. In vitro expression analysis of PAH variants offers the opportunity to elucidate the molecular mechanisms involved in HPAs and to clarify whether a disease-associated variant is genuinely pathogenic, while investigating the severity of a metabolic phenotype, and determining how a variant exerts its deleterious effects on the PAH enzyme. To study the effects of gene variants on PAH activity, we investigated eight variants: c.611A>G (p.Y204C), c.635T>C (p.L212P), c.746T>C (p.L249P), c.745C>T (p.L249F), c.809G>A (p.R270K), c.782G>C (p.R261P), c.587C>A (p.S196Y) and c.1139C>T (p.T380M), associated with different phenotypic groups. Transient expression of mutant full-length cDNAs in COS-7 cells yielded PAH proteins with PAH activity levels between 7% and 51% compared to the wild-type enzyme. With one exception (p.Y204C, which had no significant impact on PAH function), lower PAH activity was associated with a more severe phenotype (e.g. p.L249P with 7% PAH activity, 100% of classic PKU and no BH4 responsiveness), while higher activity correlated with milder phenotypes (e.g. p.T380M with 28% PAH activity, 97% of mild HPA and 83% of BH4 responsiveness). The results of the in vitro residual PAH activity have major implications, both for our understanding of genotype-phenotype correlations, and thereby existing inconsistencies, but also for the elucidation of the molecular basis of tetrahydrobiopterin (BH4) responsiveness.

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

    USDA-ARS?s Scientific Manuscript database

    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....

  12. The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces systemic resistance in Arabidopsis thaliana by simultaneously activating salicylate- and jasmonate/ethylene-dependent signaling pathways.

    PubMed

    Niu, Dong-Dong; Liu, Hong-Xia; Jiang, Chun-Hao; Wang, Yun-Peng; Wang, Qing-Ya; Jin, Hai-Ling; Guo, Jian-Hua

    2011-05-01

    Bacillus cereus AR156 is a plant growth-promoting rhizobacterium that induces resistance against a broad spectrum of pathogens including Pseudomonas syringae pv. tomato DC3000. This study analyzed AR156-induced systemic resistance (ISR) to DC3000 in Arabidopsis ecotype Col-0 plants. Compared with mock-treated plants, AR156-treated ones showed an increase in biomass and reductions in disease severity and pathogen density in the leaves. The defense-related genes PR1, PR2, PR5, and PDF1.2 were concurrently expressed in the leaves of AR156-treated plants, suggesting simultaneous activation of the salicylic acid (SA)- and the jasmonic acid (JA)- and ethylene (ET)-dependent signaling pathways by AR156. The above gene expression was faster and stronger in plants treated with AR156 and inoculated with DC3000 than that in plants only inoculated with DC3000. Moreover, the cellular defense responses hydrogen peroxide accumulation and callose deposition were induced upon challenge inoculation in the leaves of Col-0 plants primed by AR156. Also, pretreatment with AR156 led to a higher level of induced protection against DC3000 in Col-0 than that in the transgenic NahG, the mutant jar1 or etr1, but the protection was absent in the mutant npr1. Therefore, AR156 triggers ISR in Arabidopsis by simultaneously activating the SA- and JA/ET-signaling pathways in an NPR1-dependent manner that leads to an additive effect on the level of induced protection.

  13. 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

  14. Ar and Kr concentrations in nitrogen as a measure of the 39Ar and 85Kr activities in connection with the solar neutrino experiment BOREXINO.

    PubMed

    Zuzel, G; Simgen, H; Heusser, G

    2004-01-01

    Among other radionuclides, 39Ar and 85Kr are potential background sources in the solar neutrino detector BOREXINO. The expected low event rate requires that the nitrogen used in the experiment needs to have lower concentrations than 0.5 microBq/m3 for 39Ar and 0.2 microBq/m3 for 85Kr, corresponding to volume concentrations in N2 of 0.4 x 10(-6)m3/m3 for Ar and 0.2 x 10(-12)m3/m3 for Kr. Applying gas mass spectrometry and special care in the sampling technique we succeeded in finding the required purity. In addition, we studied the possibility to purify nitrogen from Kr by the adsorption method.

  15. Altered motor activity of alternative splice variants of the mammalian kinesin-3 protein KIF1B.

    PubMed

    Matsushita, Masafumi; Yamamoto, Ruri; Mitsui, Keiji; Kanazawa, Hiroshi

    2009-11-01

    Several mammalian kinesin motor proteins exist as multiple isoforms that arise from alternative splicing of a single gene. However, the roles of many motor protein splice variants remain unclear. The kinesin-3 motor protein KIF1B has alternatively spliced isoforms distinguished by the presence or absence of insertion sequences in the conserved amino-terminal region of the protein. The insertions are located in the loop region containing the lysine-rich cluster, also known as the K-loop, and in the hinge region adjacent to the motor domain. To clarify the functions of these alternative splice variants of KIF1B, we examined the biochemical properties of recombinant KIF1B with and without insertion sequences. In a microtubule-dependent ATPase assay, KIF1B variants that contained both insertions had higher activity and affinity for microtubules than KIF1B variants that contained no insertions. Mutational analysis of the K-loop insertion revealed that variants with a longer insertion sequence at this site had higher activity. However, the velocity of movement in motility assays was similar between KIF1B with and without insertion sequences. Our results indicate that splicing isoforms of KIF1B that vary in their insertion sequences have different motor activities.

  16. 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

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

    PubMed

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

    2016-03-29

    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.

  18. Differential activity of bacteriocins and cefotaxime against Serratia marcescens clinical isolate SMG40 and its pigmented variant.

    PubMed

    Gratia, J P; Crenier, L

    1992-02-01

    Strain Serratia marcescens SMG40 was nonpigmented but yielded pigmented variants at low frequency. Of 15 tested bacteriocins, 10 were active against the original strain, but only 7 were active against the pigmented variant Pdg+. Moreover, active cephalosporins, e.g., cefotaxime, were more active against the Pdg+ variant than against strain SMG40. These reciprocal differences in susceptibility to some bacteriocins and to cefotaxime were maintained in subclones derived from Pdg+/- sectored colonies. However, mutants of pigmentation isolated from the Pdg+ variant were not modified with regard to bacteriocins and cefotaxime. Other clinical isolates of Serratia were examined for comparison, but strain SMG40 appeared to be unique.

  19. Basal adenosine modulates the functional properties of AMPA receptors in mouse hippocampal neurons through the activation of A1R A2AR and A3R

    PubMed Central

    Di Angelantonio, Silvia; Bertollini, Cristina; Piccinin, Sonia; Rosito, Maria; Trettel, Flavia; Pagani, Francesca; Limatola, Cristina; Ragozzino, Davide

    2015-01-01

    Adenosine is a widespread neuromodulator within the CNS and its extracellular level is increased during hypoxia or intense synaptic activity, modulating pre- and postsynaptic sites. We studied the neuromodulatory action of adenosine on glutamatergic currents in the hippocampus, showing that activation of multiple adenosine receptors (ARs) by basal adenosine impacts postsynaptic site. Specifically, the stimulation of both A1R and A3R reduces AMPA currents, while A2AR has an opposite potentiating effect. The effect of ARs stimulation on glutamatergic currents in hippocampal cultures was investigated using pharmacological and genetic approaches. A3R inhibition by MRS1523 increased GluR1-Ser845 phosphorylation and potentiated AMPA current amplitude, increasing the apparent affinity for the agonist. A similar effect was observed blocking A1R with DPCPX or by genetic deletion of either A3R or A1R. Conversely, impairment of A2AR reduced AMPA currents, and decreased agonist sensitivity. Consistently, in hippocampal slices, ARs activation by AR agonist NECA modulated glutamatergic current amplitude evoked by AMPA application or afferent fiber stimulation. Opposite effects of AR subtypes stimulation are likely associated to changes in GluR1 phosphorylation and represent a novel mechanism of physiological modulation of glutamatergic transmission by adenosine, likely acting in normal conditions in the brain, depending on the level of extracellular adenosine and the distribution of AR subtypes. PMID:26528137

  20. 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.

  1. {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.

  2. Tobramycin variants with enhanced ribosome-targeting activity

    PubMed Central

    Fosso, Marina Y.; Zhu, Hongkun; Green, Keith D.

    2015-01-01

    With the increased evolution of aminoglycoside (AG)-resistant bacterial strains, the need to develop AGs with (i) enhanced antimicrobial activity, (ii) the ability to evade resistance mechanisms, and (iii) the capability of targeting the ribosome with higher efficiency, is more and more pressing. The chemical derivatization of the naturally occurring tobramycin (TOB) by attachment of 37 different thioethers groups at the 6″-position led to the identification of generally poorer substrates of TOB-targeting AG-modifying enzymes (AMEs). Thirteen of these displayed better antibacterial activity than the parental TOB while retaining ribosome-targeting specificity. Analysis of these compounds in vitro shed light on the mechanism by which they act and revealed three with clearly enhanced ribosome-targeting activity. PMID:26033429

  3. Ribosomal DNA is active in different B chromosome variants of the grasshopper Eyprepocnemis plorans.

    PubMed

    Ruíz-Estévez, Mercedes; López-León, M Dolores; Cabrero, Josefa; Camacho, Juan Pedro M

    2013-09-01

    B chromosomes are considered to be genetically inert elements. However, some of them are able to show nucleolus organizer region (NOR) activity, as detected by both cytological and molecular means. The grasshopper Eyprepocnemis plorans shows a B chromosome polymorphism characterized by the existence of many B variants. One of them, B24, shows NOR activity in about half of B-carrying males in the Torrox population. Molecular data have suggested the recent origin for B chromosomes in this species, and on this basis it would be expected that NOR activity was widespread among the different B variants. Here we test this hypothesis in four different B chromosome variants (B1, B2, B5, and B24) from 11 natural populations of the grasshopper E. plorans covering the south and east of the Iberian Peninsula plus the Balearic Islands. We used two different approaches: (1) the cytological observation of nucleoli attached to the distal region of the B chromosome (where the rDNA is located), and (2) the molecular detection of the rDNA transcripts carrying an adenine insertion characteristic of B chromosome ITS2 sequences. The results showed NOR expression not only for B24 but also for the B1 and B2 variants. However, the level of B-NOR expression in these latter variants, measured by the proportion of cells showing nucleoli attached to the B chromosomes, was much lower than that previously reported for B24. This suggests the possibility that structural or genetic background conditions are enhancing the expressivity of the rDNA in the B24 variant.

  4. Cellulase variants

    DOEpatents

    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.

  5. The hTERTα Splice Variant is a Dominant Negative Inhibitor of Telomerase Activity1

    PubMed Central

    Colgin, Lorel M; Wilkinson, Christen; Englezou, Anna; Kilian, Andrzej; Robinson, Murray O; Redder, Roger R

    2000-01-01

    Abstract The telomerase catalytic subunit (hTERT) is an essential component of the holoenzyme complex that adds telomeric repeats to the ends of human chromosomes. Maintenance of telomeres by telomerase or another mechanism is required for cell immortalization, and loss of telomeric DNA has been proposed as a trigger for cellular senescence. Available evidence suggests that regulation of telomerase activity primarily depends on transcriptional control of hTERT. However, several human tissues as well as some normal cell strains have been shown to express low levels of hTERT mRNA even though they lack telomerase activity. We have previously identified six splice variants of hTERT, including a “deletion” variant (hTERTα) that is missing conserved residues from the catalytic core of the protein. Several of the deletion variants have been detected in normal and developing human tissues. We now show that hTERTα inhibits endogenous telomerase activity, which results in telomere shortening and chromosome end-to-end fusions. Telomerase inhibition induced a senescence-like state in HT1080 cells and apoptosis in a jejunal fibroblast cell line. These results suggest a possible role for hTERT splice variants in the regulation of telomerase activity. PMID:11191109

  6. Niclosamide inhibits androgen receptor variants expression and overcomes enzalutamide resistance in castration-resistant prostate cancer.

    PubMed

    Liu, Chengfei; Lou, Wei; Zhu, Yezi; Nadiminty, Nagalakshmi; Schwartz, Chad T; Evans, Christopher P; Gao, Allen C

    2014-06-15

    Enzalutamide, a second-generation antiandrogen, was recently approved for the treatment of castration-resistant prostate cancer (CRPC) in patients who no longer respond to docetaxel. Despite these advances that provide temporary respite, resistance to enzalutamide occurs frequently. Androgen receptor (AR) splice variants such as AR-V7 have recently been shown to drive castration-resistant growth and resistance to enzalutamide. This study was designed to identify inhibitors of AR variants and test its ability to overcome resistance to enzalutamide. The drug screening was conducted using luciferase activity assay to determine the activity of AR-V7 after treatment with the compounds in the Prestwick Chemical Library, which contains about 1,120 FDA-approved drugs. The effects of the identified inhibitors on AR-V7 activity and enzalutamide sensitivity were characterized in CRPC and enzalutamide-resistant prostate cancer cells in vitro and in vivo. Niclosamide, an FDA-approved antihelminthic drug, was identified as a potent AR-V7 inhibitor in prostate cancer cells. Niclosamide significantly downregulated AR-V7 protein expression by protein degradation through a proteasome-dependent pathway. Niclosamide also inhibited AR-V7 transcription activity and reduced the recruitment of AR-V7 to the PSA promoter. Niclosamide inhibited prostate cancer cell growth in vitro and tumor growth in vivo. Furthermore, the combination of niclosamide and enzalutamide resulted in significant inhibition of enzalutamide-resistant tumor growth, suggesting that niclosamide enhances enzalutamide therapy and overcomes enzalutamide resistance in CRPC cells. Niclosamide was identified as a novel inhibitor of AR variants. Our findings offer preclinical validation of niclosamide as a promising inhibitor of AR variants to treat, either alone or in combination with current antiandrogen therapies, patients with advanced prostate cancer, especially those resistant to enzalutamide. ©2014 American

  7. Conformational stability and catalytic activity of PTEN variants linked to cancers and autism spectrum disorders.

    PubMed

    Johnston, Sean B; Raines, Ronald T

    2015-02-24

    Phosphoinositides are membrane components that play critical regulatory roles in mammalian cells. The enzyme PTEN, which catalyzes the dephosphorylation of the phosphoinositide PIP3, is damaged in most sporadic tumors. Mutations in the PTEN gene have also been linked to autism spectrum disorders and other forms of delayed development. Here, human PTEN is shown to be on the cusp of unfolding under physiological conditions. Variants of human PTEN linked to somatic cancers and disorders on the autism spectrum are shown to be impaired in their conformational stability, catalytic activity, or both. Those variants linked only to autism have activity higher than the activity of those linked to cancers. PTEN-L, which is a secreted trans-active isoform, has conformational stability greater than that of the wild-type enzyme. These data indicate that PTEN is a fragile enzyme cast in a crucial role in cellular metabolism and suggest that PTEN-L is a repository for a critical catalytic activity.

  8. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Activation of the Wnt pathway through use of AR79, a glycogen synthase kinase 3β inhibitor, promotes prostate cancer growth in soft tissue and bone

    PubMed Central

    Jiang, Yuan; Dai, Jinlu; Zhang, Honglai; Sottnik, Joe L.; Keller, Jill M.; Escott, Katherine J.; Sanganee, Hitesh J.; Yao, Zhi; McCauley, Laurie K.; Keller, Evan T.

    2013-01-01

    Due to its bone anabolic activity, methods to increase Wnt activity, such as inhibitors of dickkopf-1 and sclerostin, are being clinically explored. Glycogen synthase kinase (GSK3β) inhibits Wnt signaling through inducing β-catenin degradation. Therefore, AR79, an inhibitor of GSK3β, is being evaluated as a bone anabolic agent. However, Wnt activation has potential to promote tumor growth. The goal of this study was to determine if AR79 impacted progression of prostate cancer (PCa). PCa tumors were established in subcutaneous and bone sites of mice followed by AR79 administration. Tumor growth, β-catenin activation, proliferation (Ki67 expression) and apoptosis (caspase 3 activity) were measured. Additionally, PCa and osteoblast cell lines were treated with AR79 and β-catenin status, proliferation (with β-catenin knocked down in some cases) and proportion of the ALDH+CD133+ stem-like cells was determined. AR79 promoted PCa growth, decreased phospho-β-catenin expression and increased total and nuclear β-catenin expression in tumors and increased tumor-induced bone remodeling. Additionally, it decreased caspase 3 and increased Ki67 expression. In addition, AR79 increased bone formation in normal mouse tibiae. AR79 inhibited β-catenin phosphorylation, increased nuclear β-catenin accumulation in PCa and osteoblast cell lines and increased proliferation of PCa cells in vitro through β-catenin. Furthermore, AR79 increased the ALDH+CD133+ cancer stem cell-like proportion of the PCa cell lines. We conclude that AR79, while being bone anabolic, promotes PCa cell growth through Wnt pathway activation. PMID:24088787

  10. The strength and time course of lexical activation of pronunciation variants.

    PubMed

    Pitt, Mark A

    2009-06-01

    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. In Experiment 1, the Ganong Paradigm (W. F. Ganong, 1980) was used to measure the strength of activation of /t/-deleted variants, comparing labeling and response time results with their citation counterparts. Phonemic restoration was used in Experiment 2 to generalize the results. In Experiment 3, Experiment 1 was replicated with a large number of trials so that the time course of activation could be mapped. Results show that lexical influences on labeling begin sooner and reach a higher level for the citation than for the /t/-deleted variant, although the overall shapes of their activation profiles are similar.

  11. 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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. 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

  13. 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

  14. Variant for estimating the activity of tropical cyclone groups in the world ocean

    NASA Astrophysics Data System (ADS)

    Yaroshevich, M. I.

    2016-12-01

    It is especially important to know the character and the intensity level of tropical cyclone (TC) activity when the system for estimating the cyclonic danger and risk is formed. During seasons of increased cyclonic activity, when several TCs are simultaneously active, the total energy effect of the cyclone group joint action is not estimated numerically. Cyclonic activity is as a rule characterized by the number of TCs that occur in the considered zone. A variant of the criterion, according to which relative cyclonic activity is estimated, is presented.

  15. FOXA1 modulates EAF2 regulation of AR transcriptional activity, cell proliferation, and migration in prostate cancer cells.

    PubMed

    Guo, Wenhuan; Keener, Anne L; Jing, Yifeng; Cai, Liquan; Ai, Junkui; Zhang, Jian; Fisher, A L; Fu, Guohui; Wang, Zhou

    2015-06-15

    ELL-associated factor 2 (EAF2) is an androgen-regulated tumor suppressor in the prostate. However, the mechanisms underlying tumor suppressive function of EAF2 are still largely unknown. Identification of factors capable of modulating EAF2 function will help elucidate the mechanisms underlying EAF2 tumor suppressive function. Using eaf-1(the ortholog of EAF2) mutant C. elegans model, RNAi screen was used to identify factors on the basis of their knockdown to synergistically enhance the reduced fertility phenotype of the eaf-1 mutant C. elegans. In human cells, the interaction of EAF2 with FOXA1 and the effect of EAF2 on the FOXA1 protein levels were determined by co-immunoprecipitation and protein stability assay. The effect of EAF2 and/or FOXA1 knockdown on the expression of AR-target genes was determined by real-time RT-PCR and luciferase reporter assays. The effect of EAF2 and/or FOXA1 knockdown on LNCaP human prostate cancer cell proliferation and migration was tested using BrdU assay and transwell migration assay. RNAi screen identified pha-4, the C. elegans ortholog of mammalian FOXA1, on the basis of its knockdown to synergistically enhance the reduced fertility phenotype of the eaf-1 mutant C. elegans causing sterility. EAF2 co-immunoprecipitated with FOXA1. EAF2 knockdown enhanced endogenous FOXA1 protein level, whereas transfected GFP-EAF2 down-regulated the FOXA1 protein. Also, EAF2 knockdown enhanced the expression of AR-target genes, cell proliferation, and migration in LNCaP cells. However, FOXA1 knockdown inhibited the effect of EAF2 knockdown on AR-target gene expression, cell proliferation, and migration in LNCaP cells, suggesting that FOXA1 can modulate EAF2 regulation of AR transcriptional activation, cell proliferation, and migration. These findings suggest that regulation of the AR signaling pathway, cell proliferation, and migration through FOXA1 represents an important mechanism of EAF2 suppression of prostate carcinogenesis. © 2015 Wiley

  16. Modeling Truncated AR Expression in a Natural Androgen Responsive Environment and Identification of RHOB as a Direct Transcriptional Target

    PubMed Central

    Tsai, Hui-Chi; Boucher, David L.; Martinez, Anthony; Tepper, Clifford G.; Kung, Hsing-Jien

    2012-01-01

    Recent studies identifying putative truncated androgen receptor isoforms with ligand-independent activity have shed new light on the acquisition of androgen depletion independent (ADI) growth of prostate cancer. In this study, we present a model system in which a C-terminally truncated variant of androgen receptor (TC-AR) is inducibly expressed in LNCaP, an androgen-dependent cell line, which expresses little truncated receptor. We observed that when TC-AR is overexpressed, the endogenous full length receptor (FL-AR) is transcriptionally downmodulated. This in essence allows us to “replace” FL-AR with TC-AR and compare their individual properties in exactly the same genetic and cellular background, which has not been performed before. We show that the TC-AR translocates to the nucleus, activates transcription of AR target genes in the absence of DHT and is sufficient to confer ADI growth to the normally androgen dependent LNCaP line. We also show that while there is significant overlap in the genes regulated by FL- and TC-AR there are also differences in the respective suites of target genes with each AR form regulating genes that the other does not. Among the genes uniquely activated by TC-AR is RHOB which is shown to be involved in the increased migration and morphological changes observed in LN/TC-AR, suggesting a role of RHOB in the regulation of androgen-independent behavior of prostate cancer cells. PMID:23209612

  17. 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

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

    2015-09-29

    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.

  18. 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

  19. Rare Variant of GM2 Gangliosidosis through Activator-Protein Deficiency.

    PubMed

    Brackmann, Florian; Kehrer, Christiane; Kustermann, Wibke; Böhringer, Judith; Krägeloh-Mann, Ingeborg; Trollmann, Regina

    2017-04-01

    GM2 gangliosidosis, AB variant, is a very rare form of GM2 gangliosidosis due to a deficiency of GM2 activator protein. We report on two patients with typical clinical features suggestive of GM2 gangliosidosis, but normal results for hexosaminidase A and hexosaminidase B as well as their corresponding genes. Genetic analysis of the gene encoding the activator protein, the GM2A gene, elucidated the cause of the disease, adding a novel mutation to the spectrum of GM2 AB variant. This report points out that in typical clinical constellations with normal enzyme results, genetic diagnostic for activator protein defects should be performed. Georg Thieme Verlag KG Stuttgart · New York.

  20. 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. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. 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.

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

    PubMed Central

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

    2015-01-01

    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

  3. 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

  4. Quillaja Saponin Variants with Central Glycosidic Linkage Modifications Exhibit Distinct Conformations and Adjuvant Activities

    PubMed Central

    Walkowicz, William E.; Fernández-Tejada, Alberto; George, Constantine; Corzana, Francisco; Jiménez-Barbero, Jesús; Gin, David Y.

    2016-01-01

    Immunological adjuvants such as the saponin natural product QS-21 help stimulate the immune response to co-administered antigens and have become increasingly important in the development of prophylactic and therapeutic vaccines. However, clinical use of QS21 is encumbered by chemical instability, dose-limiting toxicity, and low-yielding purification from the natural source. Previous studies of structure–activity relationships in the four structural domains of QS-21 have led to simplified, chemically stable variants that retain potent adjuvant activity and low toxicity in mouse vaccination models. However, modification of the central glycosyl ester linkage has not yet been explored. Herein, we describe the design, synthesis, immunologic evaluation, and molecular dynamics analysis of a series of novel QS-21 variants with different linker lengths, stereochemistry, and flexibility to investigate the role of this linkage in saponin adjuvant activity and conformation. Despite relatively conservative structural modifications, these variants exhibit striking differences in in vivo adjuvant activity that correlate with specific conformational preferences. These results highlight the junction of the triterpene and linear oligosaccharide domains as playing a critical role in the immunoadjuvant activity of the Quillaja saponins and also suggest a mechanism of action involving interaction with a discrete macromolecular target, in contrast to the non-specific mechanisms of emulsion-based adjuvants. PMID:27014435

  5. CD147 modulates androgen receptor activity through the Akt/Gsk-3β/β-catenin/AR pathway in prostate cancer cells

    PubMed Central

    Fang, Fang; Qin, Yingxin; Hao, Feng; Li, Qiang; Zhang, Wei; Zhao, Chen; Chen, Shuang; Zhao, Liangzhong; Wang, Liguo; Cai, Jianhui

    2016-01-01

    The androgen signaling pathway serves an important role in the development of prostate cancer. β-Catenin is an androgen receptor (AR) cofactor and augments AR signaling. Glycogen synthase kinase-3β (GSK-3β), a target of phosphorylated serine/threonine protein kinase B (p-Akt), regulates β-catenin stability. In addition, β-catenin, a coregulator of AR, physically interacts with AR and enhances AR-mediated target gene transcription. The multifunctional glycoprotein cluster of differentiation (CD) 147 is highly expressed on the cell surface of the majority of cancer cells, and it promotes tumor invasion, metastasis and growth. In the present study, the molecular effects of CD147 on the Akt/GSK-3β/β-catenin/AR signaling network were investigated in LNCaP cells. Using short hairpin-mediated RNA knockdown of CD147 in LNCaP cells, it was demonstrated that downregulation of CD147 resulted in inhibitory phosphorylation of GSK-3β, and then promoted degeneration of β-catenin and reduced nuclear accumulation of β-catenin. In addition, immunoprecipitation studies demonstrated that CD147 downregulation decreased the formation of a complex between β-catenin and AR. It was shown that CD147 knockdown suppressed the expression of the AR target gene prostate-specific antigen and the growth of AR-positive LNCaP cells. Furthermore, inhibition of PI3K/Akt with LY294002 augmented CD147-mediated function. The present study indicates that the PI3K/Akt pathway may facilitate CD147-mediated activation of the AR pathway. PMID:27446405

  6. CD147 modulates androgen receptor activity through the Akt/Gsk-3β/β-catenin/AR pathway in prostate cancer cells.

    PubMed

    Fang, Fang; Qin, Yingxin; Hao, Feng; Li, Qiang; Zhang, Wei; Zhao, Chen; Chen, Shuang; Zhao, Liangzhong; Wang, Liguo; Cai, Jianhui

    2016-08-01

    The androgen signaling pathway serves an important role in the development of prostate cancer. β-Catenin is an androgen receptor (AR) cofactor and augments AR signaling. Glycogen synthase kinase-3β (GSK-3β), a target of phosphorylated serine/threonine protein kinase B (p-Akt), regulates β-catenin stability. In addition, β-catenin, a coregulator of AR, physically interacts with AR and enhances AR-mediated target gene transcription. The multifunctional glycoprotein cluster of differentiation (CD) 147 is highly expressed on the cell surface of the majority of cancer cells, and it promotes tumor invasion, metastasis and growth. In the present study, the molecular effects of CD147 on the Akt/GSK-3β/β-catenin/AR signaling network were investigated in LNCaP cells. Using short hairpin-mediated RNA knockdown of CD147 in LNCaP cells, it was demonstrated that downregulation of CD147 resulted in inhibitory phosphorylation of GSK-3β, and then promoted degeneration of β-catenin and reduced nuclear accumulation of β-catenin. In addition, immunoprecipitation studies demonstrated that CD147 downregulation decreased the formation of a complex between β-catenin and AR. It was shown that CD147 knockdown suppressed the expression of the AR target gene prostate-specific antigen and the growth of AR-positive LNCaP cells. Furthermore, inhibition of PI3K/Akt with LY294002 augmented CD147-mediated function. The present study indicates that the PI3K/Akt pathway may facilitate CD147-mediated activation of the AR pathway.

  7. A splicing variant of the androgen receptor detected in a metastatic prostate cancer exhibits exclusively cytoplasmic actions.

    PubMed

    Jagla, Monika; Fève, Marie; Kessler, Pascal; Lapouge, Gaëlle; Erdmann, Eva; Serra, Sebastian; Bergerat, Jean-Pierre; Céraline, Jocelyn

    2007-09-01

    The androgen receptor (AR) is a ligand-activated transcription factor that displays genomic actions characterized by binding to androgen-response elements in the promoter of target genes as well as nongenomic actions that do not require nuclear translocation and DNA binding. In this study, we report exclusive cytoplasmic actions of a splicing variant of the AR detected in a metastatic prostate cancer. This AR variant, named AR23, results from an aberrant splicing of intron 2, wherein the last 69 nucleotides of the intronic sequence are retained, leading to the insertion of 23 amino acids between the two zinc fingers in the DNA-binding domain. We show that the nuclear entry of AR23 upon dihydrotestosterone (DHT) stimulation is impaired. Alternatively, DHT-activated AR23 forms cytoplasmic and perinuclear aggregates that partially colocalize with the endoplasmic reticulum and are devoid of genomic actions. However, in LNCaP cells, this cytoplasmic DHT-activated AR23 remains partially active as evidenced by the activation of transcription from androgen-responsive promoters, the stimulation of NF-kappaB transcriptional activity and by the decrease of AP-1 transcriptional activity. Our data reveal novel cytoplasmic actions for this splicing AR variant, suggesting a contribution in prostate cancer progression.

  8. Niclosamide inhibits androgen receptor variants expression and overcomes enzalutamide resistance in castration resistant prostate cancer

    PubMed Central

    Zhu, Yezi; Nadiminty, Nagalakshmi; Schwartz, Chad T.; Evans, Christopher P.; Gao, Allen C.

    2014-01-01

    Purpose Enzalutamide, a second-generation antiandrogen, was recently approved for the treatment of castration-resistant prostate cancer (CRPC) in patients who no longer respond to docetaxel. Despite these advances that provide temporary respite, resistance to enzalutamide occurs frequently. AR splice variants such as AR-V7 have recently been shown to drive castration resistant growth and resistance to enzalutamide. This study was designed to identify inhibitors of AR variants and test its ability to overcome resistance to enzalutamide. Experimental Design The drug screening was conducted using luciferase activity assay to determine the activity of AR-V7 after treatment with the compounds in the Prestwick Chemical Library, which contains about 1120 FDA-approved drugs. The effects of the identified inhibitors on AR-V7 activity and enzalutamide sensitivity were characterized in CRPC and enzalutamide-resistant prostate cancer cells in vitro and in vivo. Results Niclosamide, an FDA-approved anti-helminthic drug, was identified as a potent AR-V7 inhibitor in prostate cancer cells. Niclosamide significantly downregulated AR-V7 protein expression by protein degradation through a proteasome dependent pathway. Niclosamide also inhibited AR-V7 transcription activity and reduced the recruitment of AR-V7 to the PSA promoter. Niclosamide inhibited prostate cancer cell growth in vitro and tumor growth in vivo. Furthermore, the combination of niclosamide and enzalutamide resulted in significantly inhibition of enzalutamide-resistant tumor growth, suggesting that Niclosamide enhances enzalutamide therapy and overcomes enzalutamide resistance in castration resistant prostate cancer cells. Conclusions Niclosamide was identified as a novel inhibitor of AR variants. Our findings offer preclinical validation of niclosamide as a promising inhibitor of androgen receptor variants to treat, either alone or in combination with current antiandrogen therapies, advanced prostate cancer patients

  9. Functional significance of UDP-glucuronosyltransferase variants in the metabolism of active tamoxifen metabolites.

    PubMed

    Blevins-Primeau, Andrea S; Sun, Dongxiao; Chen, Gang; Sharma, Arun K; Gallagher, Carla J; Amin, Shantu; Lazarus, Philip

    2009-03-01

    Tamoxifen (TAM) is a selective estrogen receptor modulator widely used in the prevention and treatment of breast cancer. A major mode of metabolism of the major active metabolites of TAM, 4-OH-TAM and endoxifen, is by glucuronidation via the UDP-glucuronosyltransferase (UGT) family of enzymes. To examine whether polymorphisms in the UGT enzymes responsible for the glucuronidation of active TAM metabolites play an important role in interindividual differences in TAM metabolism, cell lines overexpressing wild-type or variant UGTs were examined for their activities against TAM metabolites in vitro. For variants of active extrahepatic UGTs, the UGT1A8(173Ala/277Tyr) variant exhibited no detectable glucuronidation activity against the trans isomers of either 4-OH-TAM or endoxifen. Little or no difference in TAM glucuronidating activity was observed for the UGT1A8(173Gly/277Cys) or UGT1A10(139Lys) variants compared with their wild-type counterparts. For active hepatic UGTs, the UGT2B7(268Tyr) variant exhibited significant (P < 0.01) 2- and 5-fold decreases in activity against the trans isomers of 4-OH-TAM and endoxifen, respectively, compared with wild-type UGT2B7(268His). In studies of 111 human liver microsomal specimens, the rate of O-glucuronidation against trans-4-OH-TAM and trans-endoxifen was 28% (P < 0.001) and 27% (P = 0.002) lower, respectively, in individuals homozygous for the UGT2B7 Tyr(268)Tyr genotype compared with subjects with the UGT2B7 His(268)His genotype, with a significant (P < 0.01) trend of decreasing activity against both substrates with increasing numbers of the UGT2B7(268His) allele. These results suggest that functional polymorphisms in TAM-metabolizing UGTs, including UGT2B7 and potentially UGT1A8, may be important in interindividual variability in TAM metabolism and response to TAM therapy.

  10. Role of androgen receptor splice variants in prostate cancer metastasis

    PubMed Central

    Xu, Jin; Qiu, Yun

    2017-01-01

    Prostate cancer (PCa) is one of the most lethal cancers in western countries. Androgen receptor (AR) signaling pathway plays a key role in PCa progression. Despite the initial effectiveness of androgen deprivation therapy (ADT) for treatment of patients with advanced PCa, most of them will develop resistance to ADT and progress to metastatic castration resistant prostate cancer (mCRPC). Constitutively transcriptional activated AR splice variants (AR-Vs) have emerged as critical players in the development and progression of mCRPC. Among AR-Vs identified to date, AR-V7 (a.k.a. AR3) is one of the most abundant and frequently found in both PCa cell lines and in human prostate tissues. Most of functional studies have been focused on AR-V7/AR3 and revealed its role in regulation of survival, growth, differentiation and migration in prostate cells. In this review, we will summarize our current understanding of regulation of expression and activity of AR-Vs in mCRPC. PMID:28239558

  11. Growth Inhibition by Testosterone in an Androgen Receptor Splice Variant-Driven Prostate Cancer Model.

    PubMed

    Nakata, Daisuke; Nakayama, Kazuhide; Masaki, Tsuneo; Tanaka, Akira; Kusaka, Masami; Watanabe, Tatsuya

    2016-12-01

    Castration resistance creates a significant problem in the treatment of prostate cancer. Constitutively active splice variants of androgen receptor (AR) have emerged as drivers for resistance to androgen deprivation therapy, including the next-generation androgen-AR axis inhibitors abiraterone and enzalutamide. In this study, we describe the characteristics of a novel castration-resistant prostate cancer (CRPC) model, designated JDCaP-hr (hormone refractory). JDCaP-hr was established from an androgen-dependent JDCaP xenograft model after surgical castration. The expression of AR and its splice variants in JDCaP-hr was evaluated by immunoblotting and quantitative reverse transcription-polymerase chain reaction. The effects of AR antagonists and testosterone on JDCaP-hr were evaluated in vivo and in vitro. The roles of full-length AR (AR-FL) and AR-V7 in JDCaP-hr cell growth were evaluated using RNA interference. JDCaP-hr acquired a C-terminally truncated AR protein during progression from the parental JDCaP. The expression of AR-FL and AR-V7 mRNA was upregulated by 10-fold in JDCaP-hr compared with that in JDCaP, indicating that the JDCaP and JDCaP-hr models simulate castration resistance with some clinical features, such as overexpression of AR and its splice variants. The AR antagonist bicalutamide did not affect JDCaP-hr xenograft growth, and importantly, testosterone induced tumor regression. In vitro analysis demonstrated that androgen-independent prostate-specific antigen secretion and cell proliferation of JDCaP-hr were predominantly mediated by AR-V7. JDCaP-hr cell growth displayed a bell-shaped dependence on testosterone, and it was suppressed by physiological concentrations of testosterone. Testosterone induced rapid downregulation of both AR-FL and AR-V7 expression at physiological concentrations and suppressed expression of the AR target gene KLK3. Our findings support the clinical value of testosterone therapy, including bipolar androgen therapy, in the

  12. NF-kappaB2/p52:c-Myc:hnRNPA1 pathway regulates expression of androgen receptor splice variants and enzalutamide sensitivity in prostate cancer

    PubMed Central

    Nadiminty, Nagalakshmi; Tummala, Ramakumar; Liu, Chengfei; Lou, Wei; Evans, Christopher P.; Gao, Allen C.

    2015-01-01

    Castration resistant prostate cancer (CRPC) remains dependent on androgen receptor (AR) signaling. Alternative splicing of the AR to generate constitutively active, ligand-independent variants is one of the principal mechanisms that promote the development of resistance to next-generation anti-androgens such as enzalutamide. Here, we demonstrate that the splicing factor heterogeneous nuclear RNA-binding protein A1 (hnRNPA1) plays a pivotal role in the generation of AR splice variants such as AR-V7. HnRNPA1 is overexpressed in prostate tumors compared to benign prostates and its expression is regulated by NF-kappaB2/p52 and c-Myc. CRPC cells resistant to enzalutamide exhibit higher levels of NF-kappaB2/p52, c-Myc, hnRNPA1, and AR-V7. Levels of hnRNPA1 and of AR-V7 are positively correlated with each other in PCa. The regulatory circuit involving NF-kappaB2/p52, c-Myc and hnRNPA1 plays a central role in the generation of AR splice variants. Downregulation of hnRNPA1 and consequently of AR-V7 resensitizes enzalutamide-resistant cells to enzalutamide, indicating that enhanced expression of hnRNPA1 may confer resistance to AR-targeted therapies by promoting the generation of splice variants. These findings may provide a rationale for co-targeting these pathways to achieve better efficacy through AR blockade. PMID:26056150

  13. [Sensitivity of different morphological variants of Leptospira to the leptospirocidal activity of normal animal sera].

    PubMed

    Anan'ina, Iu V; Chernukha, Iu G

    1984-10-01

    The leptospirocidal activity of normal animal sera with respect to 23 Leptospira strains was experimentally studied in vitro. 91.3% of the strains under study proved to be sensitive to the lytic action of cattle serum and 86.9%, to sheep serum. The uncinate variants of the pathogenic strains showed resistance to the action of the above sera, and their nonuncinate analogs were subject to agglutination with subsequent lysis, similarly to saprophytes.

  14. 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

  15. K-Ar constraints on fluid-rock interaction and dissolution-precipitation events within the actively creeping shear zones from SAFOD cores

    NASA Astrophysics Data System (ADS)

    Ali, S.; Hemming, S. R.; Torgersen, T.; Fleisher, M. Q.; Cox, S. E.; Stute, M.

    2009-12-01

    The San Andreas Fault Observatory at Depth (SAFOD) was drilled to study the physical and chemical processes responsible for faulting and earthquake generation along an active, plate-bounding fault at depth. SAFOD drill cores show multiple zones of alteration and deformation due to fluid-rock interaction in the fault rocks(Schleicher et al. 2008). In context of fluid studies in the SAFZ, noble gas and potassium measurements were performed on solid samples of sedimentary rocks obtained from drill cores across the fault (3050-4000m-MD). We used a combination of 40Ar/39Ar and K-Ar methods on crushed samples of mudrock with variable amounts of visible slickensides to constrain the degree of resetting of the K-Ar system across the San Andreas Fault zone. 40Ar/39Ar was analyzed from small fragments (sand sized grains) while K-Ar was measured in crushed bulk rock samples (100-250 mg for Ar, and 5-10 mg for K analyses). The apparent 40Ar/39Ar ages based on single step laser fusion of small fragments corresponding to the detrital component in the coarse fraction, show varying ages ranging from the provenance age to <13Ma. Although more data are needed to make detailed comparisons, the apparent K-Ar ages of bulk samples in the fault zone are biased toward authigenic materials contained in the fine fraction, similar to the 40Ar/39Ar ages reported for mineralogical separates from very fine size fractions of samples obtained from 3065.98m-MD and 3294.89m-MD (Schleicher et al., submitted to Geology). The small samples measured for 40Ar/39Ar show scatter in the apparent ages, generally bracketing the bulk ages. However they are picked from sieved portions of the samples, and it is likely that there may be a loss of the younger (finer) material. Detrital provenance ages appear to be 50-60Ma in the Pacific Plate, and 100Ma in the North American Plate. 40Ar/39Ar ages within the SAFZ, as defined by geophysical logs (3200-3400m MD), are dominated by apparent detrital ages of ˜100Ma

  16. Environmental Xenoestrogens Super-Activate a Variant Murine ER Beta in Cholangiocytes

    PubMed Central

    Meyer, Stephanie K.; Probert, Philip M. E.; Lakey, Anne K.; Leitch, Alastair C.; Blake, Lynsay I.; Jowsey, Paul A.; Cooke, Martin P.; Blain, Peter G.

    2017-01-01

    High systemic levels of oestrogens are cholestatic and primary biliary cholangitis (PBC)—which is characterized by hepatic ductular inflammation—is thought to be triggered by exposure to xenobiotics such as those around landfill sites. Xenoestrogens may be a component of this chemical trigger. We therefore hypothesized that xenoestrogens are present at higher levels in the proximity of landfill sites. To test this hypothesis, soil samples were collected, extracts prepared and biological oestrogenic activity examined using cell-based reporter gene assays. Extracts from several sample sites around a landfill site contained a chemical(s) which activated the human ERα in a dose-dependent manner. Extracts from 3 separate control sampling sites were absent of any detectable activity. The mouse ERα and 2 variant mouse ERβ cDNAs were cloned and extracts from sample sites around a landfill site also activated these receptors. One variant murine ERβ was constitutively active when expressed in cholangiocytes, was readily inactivated by ICI182780 and activated in a dose-responsive, ICI182780-inhibitable manner by oestrogen. However, when this receptor was activated by extracts from landfill site soils, ICI182780 failed to antagonize activation. ERβ was readily detectable in murine cholangiocytes and exposing mice acutely to a pooled ER activating soil extracts also gave rise to a mild cholestatic injury. These data indicate that the environment around landfill sites may contain higher levels of xenoestrogens; that these chemicals have “super-activating” characteristics with a variant ERβ and therefore these chemicals could be a component of a xenobiotic insult that triggers PBC. PMID:28013213

  17. 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.

  18. The risk variant in ODZ4 for bipolar disorder impacts on amygdala activation during reward processing.

    PubMed

    Heinrich, Angela; Lourdusamy, Anbarasu; Tzschoppe, Jelka; Vollstädt-Klein, Sabine; Bühler, Mira; Steiner, Sabina; Bach, Christiane; Poustka, Luise; Banaschewski, Tobias; Barker, Gareth; Büchel, Christian; Conrod, Patricia; Garavan, Hugh; Gallinat, Jürgen; Heinz, Andreas; Ittermann, Bernd; Loth, Eva; Mann, Karl; Martinot, Jean-Luc; Paus, Tomáš; Pausova, Zdenka; Smolka, Michael; Ströhle, Andreas; Struve, Maren; Witt, Stephanie; Flor, Herta; Schumann, Gunter; Rietschel, Marcella; Nees, Frauke

    2013-06-01

    Bipolar disorder is a severe mood disorder, which normally begins during adolescence or early adulthood and has a heritability of up to 80%. The largest genome-wide association analysis of bipolar disorder recently identified a new genome-wide associated variant in OZD4 (rs12576775). The aim of the present study was to further elucidate the role of this risk variant in the disease process using an imaging genetics approach. As increased amygdala and striatal responses during the processing of reward and emotion are characteristic for bipolar disorder patients, it was tested whether the risk variant has an influence on this endophenotype in healthy adolescents. We examined the impact of the risk variant rs12576775 on functional magnetic resonance imaging data in an adolescent sample (N = 485). Differential activation between carriers of the risk allele (G-allele) and homozygous A-allele carriers in the amygdala and the striatum during a modification of the monetary incentive delay task (examining reward) and a face task (examining emotion) was analyzed. Carriers of the risk allele showed an increased blood oxygen level-dependent response in the amygdala during reward sensitivity (p = 0.05) and reward expectation (p < 0.05) but not during the face task. No significant group differences were found in the striatum during both reward and emotion processing. Our results indicate that the ODZ4 risk variant influences reward processing in the amygdala. Alterations in the processing of emotion may have different underlying mechanisms and need to be further examined. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. 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

  20. Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2-8) in Normal and Cancerous Breast and Prostate Cells.

    PubMed

    Hu, Dong Gui; McKinnon, Ross A; Hulin, Julie-Ann; Mackenzie, Peter I; Meech, Robyn

    2016-12-27

    Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2-8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers (TOX3, ST8SIA4, and SLITRK3), and genes that are diagnostic/prognostic biomarkers of prostate cancer (GRINA3, and BCHE).

  1. Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2–8) in Normal and Cancerous Breast and Prostate Cells

    PubMed Central

    Hu, Dong Gui; McKinnon, Ross A.; Hulin, Julie-Ann; Mackenzie, Peter I.; Meech, Robyn

    2016-01-01

    Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2–8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers (TOX3, ST8SIA4, and SLITRK3), and genes that are diagnostic/prognostic biomarkers of prostate cancer (GRINA3, and BCHE). PMID:28035996

  2. 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.

  3. The antiandrogen bicalutamide activates the androgen receptor (AR) with a mutation in codon 741 through the mitogen activated protein kinase (MARK) pathway in human prostate cancer PC3 cells.

    PubMed

    Terakawa, Tomoaki; Miyake, Hideaki; Kumano, Masafumi; Sakai, Iori; Fujisawa, Masato

    2010-11-01

    The objective of this study was to assess the effect of antiandrogen on the activation of mutated androgen receptor (AR) and its signaling pathway in prostate cancer. We transfected the AR gene with a point mutation at codon 741 (tryptophan to leucine; W741L) into human androgen-independent prostate cancer PC3 cells lacking the expression of AR, and established PC3 cells overexpressing mutant type AR (PC3/W741L). Changes in the phenotype in these cells were compared to those in PC3 cells transfected with wild- type AR (PC3/Wild) and control vector alone (PC3/Co). There was no significant differences in the growth among PC3/Co, PC3/Wild and PC3/W741L cells. A transactivation assay using these cells showed that bicalutamide activated W741L mutant type AR, but not wild-type AR, while hydroxyflutamide failed to activate either type of ARs. Treatment with specific inhibitors of the MAPK or STST3 pathway (UO126 or AG490, respectively), in contrast to treatment with the Akt pathway inhibitor LY294002, significantly inhibited the dihydrotestosterone-induced activation of both wild-type and mutant ARs; however, activation of W741L mutant AR by bicalutamide was significantly inhibited by treatment with UO126, in contrast to treatment with AG490 or LY294002. Furthermore, treatment of PC3/W741L with bicalutamide, in contrast to treatment with hydroxyflutamide, resulted in significant upregulation of phosphorylated p44/42 MAPK. These findings suggest that the MAPK pathway might be involved in the activation of the AR with a point mutation at codon 741 induced by treatment with the antiandrogen bicalutamide.

  4. LASERS: Influence of excitation parameters and active medium on the efficiency of an electric-discharge excimer ArF laser

    NASA Astrophysics Data System (ADS)

    Razhev, A. M.; Shchedrin, A. I.; Kalyuzhnaya, Anna G.; Zhupikov, A. A.

    2005-09-01

    The kinetic model of processes occurring in the plasma of an electric-discharge 193-nm excimer ArF laser operating on mixtures of He and Ne buffer gases is developed. The influence of excitation and active medium parameters on the lasing energy and total efficiency of the electric-discharge excimer ArF laser is studied theoretically and experimentally. It is shown that a specific pump power of ~4.5-5.0 MW cm-3 is required for attaining the maximum lasing energy for the highest efficiency of an ArF laser operating on a He—Ar—F2 mixture. For the first time, the pulse energy of 1.3 J at an efficiency of 2.0% is attained for an ArF laser with a specific pump power of 5.0 MW cm-3 using mixtures with helium as a buffer gas.

  5. Site-restricted plasminogen activation mediated by group A streptococcal streptokinase variants.

    PubMed

    Cook, Simon M; Skora, Amanda; Walker, Mark J; Sanderson-Smith, Martina L; McArthur, Jason D

    2014-02-15

    SK (streptokinase) is a secreted plasminogen activator and virulence factor of GAS (group A Streptococcus). Among GAS isolates, SK gene sequences are polymorphic and are grouped into two sequence clusters (cluster type-1 and cluster type-2) with cluster type-2 being further classified into subclusters (type-2a and type-2b). In the present study, we examined the role of bacterial and host-derived cofactors in SK-mediated plasminogen activation. All SK variants, apart from type-2b, can form an activator complex with Glu-Plg (Glu-plasminogen). Specific ligand-binding-induced conformational changes in Glu-Plg mediated by fibrinogen, PAM (plasminogen-binding group A streptococcal M protein), fibrinogen fragment D or fibrin, were required for type-2b SK to form a functional activator complex with Glu-Plg. In contrast with type-1 and type-2a SK, type-2b SK activator complexes were inhibited by α2-antiplasmin unless bound to fibrin or to the GAS cell-surface via PAM in combination with fibrinogen. Taken together, these data suggest that type-2b SK plasminogen activation may be restricted to specific microenvironments within the host such as fibrin deposits or the bacterial cell surface through the action of α2-antiplasmin. We conclude that phenotypic SK variation functionally underpins a pathogenic mechanism whereby SK variants differentially focus plasminogen activation, leading to specific niche adaption within the host.

  6. Selective Activation of Shoulder, Trunk, and Arm Muscles: A Comparative Analysis of Different Push-Up Variants

    PubMed Central

    Marcolin, Giuseppe; Petrone, Nicola; Moro, Tatiana; Battaglia, Giuseppe; Bianco, Antonino; Paoli, Antonio

    2015-01-01

    Context The push-up is a widely used exercise for upper limb strengthening that can be performed with many variants. A comprehensive analysis of muscle activation during the ascendant phase (AP) and descendant phase (DP) in different variants could be useful for trainers and rehabilitators. Objective To obtain information on the effect of different push-up variants on the electromyography (EMG) of a large sample of upper limb muscles and to investigate the role of the trunk and abdomen muscles during the AP and DP. Design Cross-sectional study. Setting University laboratory. Patients or Other Participants Eight healthy, young volunteers without a history of upper extremity or spine injury. Intervention(s) Participants performed a set of 10 repetitions for each push-up variant: standard, wide, narrow, forward (FP), and backward (BP). Surface EMG of 12 selected muscles and kinematics data were synchronously recorded to describe the AP and DP. Main Outcome Measure(s) Mean EMG activity of the following muscles was analyzed: serratus anterior, deltoideus anterior, erector spinae, latissimus dorsi, rectus abdominis, triceps brachii caput longus, triceps brachii caput lateralis, obliquus externus abdominis, pectoralis major sternal head, pectoralis major clavicular head, trapezius transversalis, and biceps brachii. Results The triceps brachii and pectoralis major exhibited greater activation during the narrow-base variant. The highest activation of abdomen and back muscles was recorded for the FP and BP variants. The DP demonstrated the least electrical activity across all muscles, with less marked differences for the abdominal and erector spinae muscles because of their role as stabilizers. Conclusions Based on these findings, we suggest the narrow-base variant to emphasize triceps and pectoralis activity and the BP variant for total upper body strength conditioning. The FP and BP variants should be implemented carefully in participants with low back pain because of the

  7. Selective Activation of Shoulder, Trunk, and Arm Muscles: A Comparative Analysis of Different Push-Up Variants.

    PubMed

    Marcolin, Giuseppe; Petrone, Nicola; Moro, Tatiana; Battaglia, Giuseppe; Bianco, Antonino; Paoli, Antonio

    2015-11-01

    The push-up is a widely used exercise for upper limb strengthening that can be performed with many variants. A comprehensive analysis of muscle activation during the ascendant phase (AP) and descendant phase (DP) in different variants could be useful for trainers and rehabilitators. To obtain information on the effect of different push-up variants on the electromyography (EMG) of a large sample of upper limb muscles and to investigate the role of the trunk and abdomen muscles during the AP and DP. Cross-sectional study. University laboratory. Eight healthy, young volunteers without a history of upper extremity or spine injury. Participants performed a set of 10 repetitions for each push-up variant: standard, wide, narrow, forward (FP), and backward (BP). Surface EMG of 12 selected muscles and kinematics data were synchronously recorded to describe the AP and DP. Mean EMG activity of the following muscles was analyzed: serratus anterior, deltoideus anterior, erector spinae, latissimus dorsi, rectus abdominis, triceps brachii caput longus, triceps brachii caput lateralis, obliquus externus abdominis, pectoralis major sternal head, pectoralis major clavicular head, trapezius transversalis, and biceps brachii. The triceps brachii and pectoralis major exhibited greater activation during the narrow-base variant. The highest activation of abdomen and back muscles was recorded for the FP and BP variants. The DP demonstrated the least electrical activity across all muscles, with less marked differences for the abdominal and erector spinae muscles because of their role as stabilizers. Based on these findings, we suggest the narrow-base variant to emphasize triceps and pectoralis activity and the BP variant for total upper body strength conditioning. The FP and BP variants should be implemented carefully in participants with low back pain because of the greater activation of abdominal and back muscles.

  8. 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.

  9. Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR).

    PubMed

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Isaac, Sara; Eden, Amir; Lauria, Ines; Langer, Thomas; Orly, Joseph

    2015-06-15

    High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. Many disease-associated variants of hTERT retain high telomerase enzymatic activity

    PubMed Central

    Zaug, Arthur J.; Crary, Sharon M.; Jesse Fioravanti, Matthew; Campbell, Kristina; Cech, Thomas R.

    2013-01-01

    Mutations in the gene for telomerase reverse transcriptase (hTERT) are associated with diseases including dyskeratosis congenita, aplastic anemia, pulmonary fibrosis and cancer. Understanding the molecular basis of these telomerase-associated diseases requires dependable quantitative measurements of telomerase enzyme activity. Furthermore, recent findings that the human POT1-TPP1 chromosome end-binding protein complex stimulates telomerase activity and processivity provide incentive for testing variant telomerases in the presence of these factors. In the present work, we compare multiple disease-associated hTERT variants reconstituted with the RNA subunit hTR in two systems (rabbit reticulocyte lysates and human cell lines) with respect to telomerase enzymatic activity, processivity and activation by telomere proteins. Surprisingly, many of the previously reported disease-associated hTERT alleles give near-normal telomerase enzyme activity. It is possible that a small deficit in telomerase activity is sufficient to cause telomere shortening over many years. Alternatively, mutations may perturb functions such as the recruitment of telomerase to telomeres, which are essential in vivo but not revealed by simple enzyme assays. PMID:23901009

  11. 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.

  12. Electrostatic modification of the active site of myoglobin: characterization of the proximal Ser92Asp variant.

    PubMed

    Lloyd, E; Burk, D L; Ferrer, J C; Maurus, R; Doran, J; Carey, P R; Brayer, G D; Mauk, A G

    1996-09-10

    The structural and functional consequences of the introduction of a negatively charged amino acid into the active site of horse heart myoglobin have been investigated by replacement of the proximal Ser92 residue (F7) with an aspartyl residue (Ser92Asp). UV-visible absorption maxima of various ferrous and ferric derivatives and low-temperature EPR spectra of the metaquo (metMb) derivative indicate that the active site coordination geometry has not been perturbed significantly in the variant. 1H-NMR spectroscopy provides direct evidence for the existence of a distal water molecule as the sixth ligand in the oxidized form of the variant at pD 5.7. Spectrophotometric pH titration of the Ser92Asp variant is consistent with this finding and with a pKa = 8.90 +/- 0.02 [25.0 degrees C, mu = 0.10 M (NaCl)] for titration of the distal water molecule, identical to the value reported for the wild-type protein. X-ray crystallography of the metMb derivative indicates that the heme substituents conserve their orientations in the variant protein, except for a slight reorientation of the pyrrole A propionate group to which Ser92 normally hydrogen bonds and reorientation of the carboxyl end of the pyrrole D propionate group. No change is observed in conformation of the proximal (His93) or distal (Wat156) heme ligands. 1H-NMR spectroscopy of the metMbCN form of the protein indicates that a slight rotation of the proximal His93 ligand has occurred in this derivative. Resonance Raman experiments indicate increased conformational heterogeneity in the proximal pocket of the variant. Failure to detect electron density for the Asp residue in the X-ray diffraction map of the variant protein and high average thermal factors for the pyrrole A propionate substituent are consistent with this observation. The variant exhibits novel pH-dependent behavior in the metMb form, as shown by 1H-NMR spectroscopy, and provides evidence for a heme-linked titratable group with a pKa of 5.4 in this

  13. Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation.

    PubMed

    Hawley, Simon A; Ross, Fiona A; Chevtzoff, Cyrille; Green, Kevin A; Evans, Ashleigh; Fogarty, Sarah; Towler, Mhairi C; Brown, Laura J; Ogunbayo, Oluseye A; Evans, A Mark; Hardie, D Grahame

    2010-06-09

    A wide variety of agents activate AMPK, but in many cases the mechanisms remain unclear. We generated isogenic cell lines stably expressing AMPK complexes containing AMP-sensitive (wild-type, WT) or AMP-insensitive (R531G) gamma2 variants. Mitochondrial poisons such as oligomycin and dinitrophenol only activated AMPK in WT cells, as did AICAR, 2-deoxyglucose, hydrogen peroxide, metformin, phenformin, galegine, troglitazone, phenobarbital, resveratrol, and berberine. Excluding AICAR, all of these also inhibited cellular energy metabolism, shown by increases in ADP:ATP ratio and/or by decreases in cellular oxygen uptake measured using an extracellular flux analyzer. By contrast, A769662, the Ca(2+) ionophore, A23187, osmotic stress, and quercetin activated both variants to varying extents. A23187 and osmotic stress also increased cytoplasmic Ca(2+), and their effects were inhibited by STO609, a CaMKK inhibitor. Our approaches distinguish at least six different mechanisms for AMPK activation and confirm that the widely used antidiabetic drug metformin activates AMPK by inhibiting mitochondrial respiration.

  14. Engineered factor Xa variants retain procoagulant activity independent of direct factor Xa inhibitors.

    PubMed

    Verhoef, Daniël; Visscher, Koen M; Vosmeer, C Ruben; Cheung, Ka Lei; Reitsma, Pieter H; Geerke, Daan P; Bos, Mettine H A

    2017-09-13

    The absence of an adequate reversal strategy to prevent and stop potential life-threatening bleeding complications is a major drawback to the clinical use of the direct oral inhibitors of blood coagulation factor Xa. Here we show that specific modifications of the substrate-binding aromatic S4 subpocket within the factor Xa active site disrupt high-affinity engagement of the direct factor Xa inhibitors. These modifications either entail amino-acid substitution of S4 subsite residues Tyr99 and/or Phe174 (chymotrypsinogen numbering), or extension of the 99-loop that borders the S4 subsite. The latter modifications led to the engineering of a factor Xa variant that is able to support coagulation in human plasma spiked with (supra-)physiological concentrations of direct factor Xa inhibitors. As such, this factor Xa variant has the potential to be employed to bypass the direct factor Xa inhibitor-mediated anticoagulation in patients that require restoration of blood coagulation.A major drawback in the clinical use of the oral anticoagulants that directly inhibit factor Xa in order to prevent blood clot formation is the potential for life threatening bleeding events. Here the authors describe factor Xa variants that are refractory to inhibition by these anticoagulants and could serve as rescue agents in treated patients.

  15. 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

  16. A splice variant in the ACSL5 gene relates migraine with fatty acid activation in mitochondria

    PubMed Central

    Matesanz, Fuencisla; Fedetz, María; Barrionuevo, Cristina; Karaky, Mohamad; Catalá-Rabasa, Antonio; Potenciano, Victor; Bello-Morales, Raquel; López-Guerrero, Jose-Antonio; Alcina, Antonio

    2016-01-01

    Genome-wide association studies (GWAS) in migraine are providing the molecular basis of this heterogeneous disease, but the understanding of its aetiology is still incomplete. Although some biomarkers have currently been accepted for migraine, large amount of studies for identifying new ones is needed. The migraine-associated variant rs12355831:A>G (P=2 × 10−6), described in a GWAS of the International Headache Genetic Consortium, is localized in a non-coding sequence with unknown function. We sought to identify the causal variant and the genetic mechanism involved in the migraine risk. To this end, we integrated data of RNA sequences from the Genetic European Variation in Health and Disease (GEUVADIS) and genotypes from 1000 GENOMES of 344 lymphoblastoid cell lines (LCLs), to determine the expression quantitative trait loci (eQTLs) in the region. We found that the migraine-associated variant belongs to a linkage disequilibrium block associated with the expression of an acyl-coenzyme A synthetase 5 (ACSL5) transcript lacking exon 20 (ACSL5-Δ20). We showed by exon-skipping assay a direct causality of rs2256368-G in the exon 20 skipping of approximately 20 to 40% of ACSL5 RNA molecules. In conclusion, we identified the functional variant (rs2256368:A>G) affecting ACSL5 exon 20 skipping, as a causal factor linked to the migraine-associated rs12355831:A>G, suggesting that the activation of long-chain fatty acids by the spliced ACSL5-Δ20 molecules, a mitochondrial located enzyme, is involved in migraine pathology. PMID:27189022

  17. A pH-dependent molten globule transition is required for activity of the steroidogenic acute regulatory protein, StAR.

    PubMed

    Baker, Bo Y; Yaworsky, Dustin C; Miller, Walter L

    2005-12-16

    The steroidogenic acute regulatory protein (StAR) simulates steroid biosynthesis by increasing the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner membrane. StAR acts exclusively on the OMM, and only StAR's carboxyl-terminal alpha-helix (C-helix) interacts with membranes. Biophysical studies have shown that StAR becomes a molten globule at acidic pH, but a physiologic role for this structural transition has been controversial. Molecular modeling shows that the C-helix, which forms the floor of the sterol-binding pocket, is stabilized by hydrogen bonding to adjacent loops. Molecular dynamics simulations show that protonation of the C-helix and adjacent loops facilitates opening and closing the sterol-binding pocket. Two disulfide mutants, S100C/S261C (SS) and D106C/A268C (DA), designed to limit the mobility of the C-helix but not disrupt overall conformation, were prepared in bacteria, and their correct folding and positioning of the disulfide bonds was confirmed. The SS mutant lost half, and the DA mutant lost all cholesterol binding capacity and steroidogenic activity with isolated mitochondria in vitro, but full binding and activity was restored to each mutant by disrupting the disulfide bonds with dithiothreitol. These data strongly support the model that StAR activity requires a pH-dependent molten globule transition on the OMM.

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

    PubMed Central

    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

  19. Co-targeting AR and HSP90 suppresses prostate cancer cell growth and prevents resistance mechanisms.

    PubMed

    Centenera, Margaret M; Carter, Sarah L; Gillis, Joanna L; Marrocco-Tallarigo, Deborah L; Grose, Randall H; Tilley, Wayne D; Butler, Lisa M

    2015-10-01

    Persistent androgen receptor (AR) signaling in castration resistant prostate cancer (CRPC) underpins the urgent need for therapeutic strategies that better target this pathway. Combining classes of agents that target different components of AR signaling has the potential to delay resistance and improve patient outcomes. Many oncoproteins, including the AR, rely on the molecular chaperone heat shock protein 90 (Hsp90) for functional maturation and stability. In this study, enhanced anti-proliferative activity of the Hsp90 inhibitors 17-allylamino-demethoxygeldanamycin (17-AAG) and AUY922 in androgen-sensitive and CRPC cells was achieved when the agents were used in combination with AR antagonists bicalutamide or enzalutamide. Moreover, significant caspase-dependent cell death was achieved using sub-optimal agent doses that individually have no effect. Expression profiling demonstrated regulation of a broadened set of AR target genes with combined 17-AAG and bicalutamide compared with the respective single agent treatments. This enhanced inhibition of AR signaling was accompanied by impaired chromatin binding and nuclear localization of the AR. Importantly, expression of the AR variant AR-V7 that is implicated in resistance to AR antagonists was not induced by combination treatment. Likewise, the heat shock response that is typically elicited with therapeutic doses of Hsp90 inhibitors, and is a potential mediator of resistance to these agents, was significantly reduced by combination treatment. In summary, the co-targeting strategy in this study more effectively inhibits AR signaling than targeting AR or HSP90 alone and prevents induction of key resistance mechanisms in prostate cancer cells. These findings merit further evaluation of this therapeutic strategy to prevent CRPC growth.

  20. NF-κB and Androgen Receptor Variant Expression Correlate with Human BPH progression

    PubMed Central

    Austin, David C; Strand, Douglas W; Love, Harold L; Franco, Omar E; Jang, Alex; Grabowska, Magdalena M; Miller, Nicole L; Hameed, Omar; Clark, Peter E; Fowke, Jay H; Matusik, Robert J; Jin, Ren J; Hayward, Simon W

    2016-01-01

    Background Benign prostatic hyperplasia (BPH) is a common, chronic progressive disease. Inflammation is associated with prostatic enlargement and resistance to 5α-reductase inhibitor (5ARI) therapy. Activation of the nuclear factor-kappa B (NF-κB) pathway is linked to both inflammation and ligand-independent prostate cancer progression. Methods NF-κB activation and androgen receptor variant (AR-V) expression were quantified in transition zone tissue samples from patients with a wide range of AUASS from incidental BPH in patients treated for low grade, localized peripheral zone prostate cancer to advanced disease requiring surgical intervention. To further investigate these pathways, human prostatic stromal and epithelial cell lines were transduced with constitutively active or kinase dead forms of IKK2 to regulate canonical NF-κB activity. The effects on AR full length (AR-FL) and androgen-independent AR-V expression as well as cellular growth and differentiation were assessed. Results Canonical NF-κB signaling was found to be upregulated in late versus early stage BPH, and to be strongly associated with non-insulin dependent diabetes mellitus. Elevated expression of AR-variant 7 (AR-V7), but not other AR variants, was found in advanced BPH samples. Expression of AR-V7 significantly correlated with the patient AUASS and TRUS volume. Forced activation of canonical NF-κB in human prostatic epithelial and stromal cells resulted in elevated expression of both AR-FL and AR-V7, with concomitant ligand-independent activation of AR reporters. Activation of NF-κB and over expression of AR-V7 in human prostatic epithelial cells maintained cell viability in the face of 5ARI treatment. Conclusion Activation of NF-κB and AR-V7 in the prostate is associated with increased disease severity. AR-V7 expression is inducible in human prostate cells by forced activation of NF-κB resulting in resistance to 5ARI treatment, suggesting a potential mechanism by which patients may

  1. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  4. The Role of the Kink Instability of a Long-Lived Active Region AR 9604

    NASA Astrophysics Data System (ADS)

    Tian, Lirong; Liu, Yang; Yang, Jing; Alexander, David

    2005-07-01

    We have traced the long-term evolution of a non-Hale active region composed of NOAA 9604 9632 9672 9704 9738, which displayed strong transient activity with associated geomagnetic effects from September to December, 2001. By studying the development of spot-group and line-of-sight magnetic field together with the evolution of Hα filaments, the EUV and X-ray corona (TRACE 171 Å, Yohkoh/SXT), we have found that the magnetic structure of the active region exhibited a continuous clockwise rotation throughout its entire life. Vector magnetic data obtained from Huairou Solar Observing Station (HSOS) and full-disk line-of-sight magnetograms from SOHO/MDI allowed the determination of the best-fit force-free parameter (proxy of twist), αbest, and the systematic tilt angle (proxy of writhe) which were both found to take positive values. Soft X-ray coronal loops from Yohkoh/SXT displayed a pronounced forward-sigmoid structure in period of NOAA 9704. These observations imply that the magnetic flux tube (loops) with the same handedness (right) of the writhe and the twist rotated clockwise in the solar atmosphere for a long time. We argue that the continuous clockwise rotation of the long-lived active region may be a manifestation that a highly right-hand twisted and kinked flux tube was emerging through the photosphere and chromosphere into the corona.

  5. RHESSI AND TRACE OBSERVATIONS OF MULTIPLE FLARE ACTIVITY IN AR 10656 AND ASSOCIATED FILAMENT ERUPTION

    SciTech Connect

    Joshi, Bhuwan; Kushwaha, Upendra; Cho, K.-S.; Veronig, Astrid M.

    2013-07-01

    We present Reuven Ramaty High Energy Solar Spectroscopic Imager (RHESSI) and Transition Region and Coronal Explorer (TRACE) observations of multiple flare activity that occurred in the NOAA active region 10656 over a period of 2 hr on 2004 August 18. Out of four successive flares, three were class C events, and the final event was a major X1.8 solar eruptive flare. The activities during the pre-eruption phase, i.e., before the X1.8 flare, are characterized by three localized episodes of energy release occurring in the vicinity of a filament that produces intense heating along with non-thermal emission. A few minutes before the eruption, the filament undergoes an activation phase during which it slowly rises with a speed of {approx}12 km s{sup -1}. The filament eruption is accompanied by an X1.8 flare, during which multiple hard X-ray (HXR) bursts are observed up to 100-300 keV energies. We observe a bright and elongated coronal structure simultaneously in E(UV) and 50-100 keV HXR images underneath the expanding filament during the period of HXR bursts, which provides strong evidence for ongoing magnetic reconnection. This phase is accompanied by very high plasma temperatures of {approx}31 MK, followed by the detachment of the prominence from the solar source region. From the location, timing, strength, and spectrum of HXR emission, we conclude that the prominence eruption is driven by the distinct events of magnetic reconnection occurring in the current sheet below the erupting prominence. These multi-wavelength observations also suggest that the localized magnetic reconnections associated with different evolutionary stages of the filament in the pre-eruption phase play an important role in destabilizing the active-region filament through the tether-cutting process, leading to large-scale eruption and X-class flare.

  6. Genetic variants of peroxisome proliferator-activated receptor δ are associated with gastric cancer.

    PubMed

    Jeon, Christie; Chang, Shen-Chih; Mu, Lina; Zhao, Jinkou; Rao, Jian-Yu; Lu, Qing-Yi; Zhang, Zuo-Feng

    2013-10-01

    Peroxisome proliferator-activated receptors (PPAR) are implicated in pathogenesis of insulin resistance and cancers of the digestive system. We investigated the associations of single nucleotide polymorphisms (SNPs) of PPAR δ and γ with gastric cancer and explored interactions with risk factors of gastric cancer. We conducted our analysis in a case-control study of 196 gastric cancer patients and 397 controls residing in the Taixing region of Jiangsu, China. Six SNPs in the PPARδ (rs2076167, rs3734254) and PPARγ genes (rs10865710, rs1801282, rs3856806, rs13306747) were genotyped. We employed logistic regression to evaluate the association between each genotype and gastric cancer and tested for gene-environment interaction with Helicobacter pylori (H. pylori) infection, smoking status, and meat and salt intake. We found that the G/G variant rs2076167, in tight linkage disequilibrium with rs3734254 (R (2) = 0.97), was associated with increased risk of gastric cancer in a recessive model (OR 2.20, 95 % CI 1.12, 4.32). The association between G/G variant of rs2016167 and gastric cancer was particularly strong among those with higher salt intake (OR 5.11, 95 % CI 1.11, 23.5), but did not vary by H. pylori infection or smoking status. We found that genetic variants of PPARδ were associated with gastric cancer. If the association is confirmed in larger studies, it may implicate a role for PPARδ activators, such as insulin-sensitizing agents, in prevention of gastric cancer.

  7. Calcineurin Selectively Docks with the Dynamin Ixb Splice Variant to Regulate Activity-dependent Bulk Endocytosis

    PubMed Central

    Xue, Jing; Graham, Mark E.; Novelle, Aimee E.; Sue, Nancy; Gray, Noah; McNiven, Mark A.; Smillie, Karen J.; Cousin, Michael A.; Robinson, Phillip J.

    2011-01-01

    Depolarization of nerve terminals stimulates rapid dephosphorylation of two isoforms of dynamin I (dynI), mediated by the calcium-dependent phosphatase calcineurin (CaN). Dephosphorylation at the major phosphorylation sites Ser-774/778 promotes a dynI-syndapin I interaction for a specific mode of synaptic vesicle endocytosis called activity-dependent bulk endocytosis (ADBE). DynI has two main splice variants at its extreme C terminus, long or short (dynIxa and dynIxb) varying only by 20 (xa) or 7 (xb) residues. Recombinant GST fusion proteins of dynIxa and dynIxb proline-rich domains (PRDs) were used to pull down interacting proteins from rat brain nerve terminals. Both bound equally to syndapin, but dynIxb PRD exclusively bound to the catalytic subunit of CaNA, which recruited CaNB. Binding of CaN was increased in the presence of calcium and was accompanied by further recruitment of calmodulin. Point mutations showed that the entire C terminus of dynIxb is a CaN docking site related to a conserved CaN docking motif (PXIXI(T/S)). This sequence is unique to dynIxb among all other dynamin variants or genes. Peptide mimetics of the dynIxb tail blocked CaN binding in vitro and selectively inhibited depolarization-evoked dynI dephosphorylation in nerve terminals but not of other dephosphins. Therefore, docking to dynIxb is required for the regulation of both dynI splice variants, yet it does not regulate the phosphorylation cycle of other dephosphins. The peptide blocked ADBE, but not clathrin-mediated endocytosis of synaptic vesicles. Our results indicate that Ca2+ influx regulates assembly of a fully active CaN-calmodulin complex selectively on the tail of dynIxb and that the complex is recruited to sites of ADBE in nerve terminals. PMID:21730063

  8. 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

  9. OBSERVATIONS OF MULTIPLE SURGES ASSOCIATED WITH MAGNETIC ACTIVITIES IN AR 10484 ON 2003 OCTOBER 25

    SciTech Connect

    Uddin, Wahab; Srivastava, Abhishek K.; Schmieder, B.; Chandra, R.; Bisht, S.; Kumar, Pankaj

    2012-06-10

    We present a multi-wavelength study of recurrent surges observed in H{alpha}, UV (Solar and Heliospheric Observatory (SOHO)/EIT), and Radio (Learmonth, Australia) from the super-active region NOAA 10484 on 2003 October 25. Several bright structures visible in H{alpha} and UV corresponding to subflares are also observed at the base of each surge. Type III bursts are triggered and RHESSI X-ray sources are evident with surge activity. The major surge consists of bunches of ejective paths forming a fan-shaped region with an angular size of ( Almost-Equal-To 65 Degree-Sign ) during its maximum phase. The ejection speed reaches up to {approx}200 km s{sup -1}. The SOHO/Michelson Doppler Imager magnetograms reveal that a large dipole emerges from the east side of the active region on 2003 October 18-20, a few days before the surges. On 2003 October 25, the major sunspots were surrounded by 'moat regions' with moving magnetic features (MMFs). Parasitic fragmented positive polarities were pushed by the ambient dispersion motion of the MMFs and annihilated with negative polarities at the borders of the moat region of the following spot to produce flares and surges. A topology analysis of the global Sun using Potential Field Source Surface shows that the fan structures visible in the EIT 171 A images follow magnetic field lines connecting the present active region to a preceding active region in the southeast. Radio observations of Type III bursts indicate that they are coincident with the surges, suggesting that magnetic reconnection is the driver mechanism. The magnetic energy released by the reconnection is transformed into plasma heating and provides the kinetic energy for the ejections. A lack of a radio signature in the high corona suggests that the surges are confined to follow the closed field lines in the fans. We conclude that these cool surges may have some local heating effects in the closed loops, but probably play a minor role in global coronal heating and the

  10. An Artemis polymorphic variant reduces Artemis activity and confers cellular radiosensitivity.

    PubMed

    Woodbine, Lisa; Grigoriadou, Sofia; Goodarzi, Aaron A; Riballo, Enriqueta; Tape, Christopher; Oliver, Antony W; van Zelm, Menno C; Buckland, Matthew S; Davies, E Graham; Pearl, Laurence H; Jeggo, Penny A

    2010-09-04

    Artemis is required for V(D)J recombination and the repair of a subset of radiation-induced DNA double strand breaks (DSBs). Artemis-null patients display radiosensitivity (RS) and severe combined immunodeficiency (SCID), classified as RS-SCID. Strongly impacting hypomorphic Artemis mutations confer marked infant immunodeficiency and a predisposition for EBV-associated lymphomas. Here, we provide evidence that a polymorphic Artemis variant (c.512C > G: p.171P > R), which has a world-wide prevalence of 15%, is functionally impacting. The c.512C > G mutation causes an approximately 3-fold decrease in Artemis endonuclease activity in vitro. Cells derived from a patient who expressed a single Artemis allele with the polymorphic mutational change, showed radiosensitivity and a DSB repair defect in G2 phase, with Artemis cDNA expression rescuing both phenotypes. The c.512C > G change has an additive impact on Artemis function when combined with a novel C-terminal truncating mutation (p.436C > X), which also partially inactivates Artemis activity. Collectively, our findings provide strong evidence that monoallelic expression of the c.512C > G variant impairs Artemis function causing significant radiosensitivity and a G2 phase DSB repair defect. The patient exhibiting monoallelic c.512C > G-Artemis expression showed immunodeficiency only in adulthood, developed bilateral carcinoma of the nipple and myelodysplasia raising the possibility that modestly decreased Artemis function can impact clinically. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  11. Structural characterization of single nucleotide variants at ligand binding sites and enzyme active sites of human proteins

    PubMed Central

    Yamada, Kazunori D.; Nishi, Hafumi; Nakata, Junichi; Kinoshita, Kengo

    2016-01-01

    Functional sites on proteins play an important role in various molecular interactions and reactions between proteins and other molecules. Thus, mutations in functional sites can severely affect the overall phenotype. Progress of genome sequencing projects has yielded a wealth of information on single nucleotide variants (SNVs), especially those with less than 1% minor allele frequency (rare variants). To understand the functional influence of genetic variants at a protein level, we investigated the relationship between SNVs and protein functional sites in terms of minor allele frequency and the structural position of variants. As a result, we observed that SNVs were less abundant at ligand binding sites, which is consistent with a previous study on SNVs and protein interaction sites. Additionally, we found that non-rare variants tended to be located slightly apart from enzyme active sites. Examination of non-rare variants revealed that most of the mutations resulted in moderate changes of the physico-chemical properties of amino acids, suggesting the existence of functional constraints. In conclusion, this study shows that the mapping of genetic variants on protein structures could be a powerful approach to evaluate the functional impact of rare genetic variations. PMID:27924270

  12. CALCULATING ENERGY STORAGE DUE TO TOPOLOGICAL CHANGES IN EMERGING ACTIVE REGION NOAA AR 11112

    SciTech Connect

    Tarr, Lucas; Longcope, Dana

    2012-04-10

    The minimum current corona model provides a way to estimate stored coronal energy using the number of field lines connecting regions of positive and negative photospheric flux. This information is quantified by the net flux connecting pairs of opposing regions in a connectivity matrix. Changes in the coronal magnetic field, due to processes such as magnetic reconnection, manifest themselves as changes in the connectivity matrix. However, the connectivity matrix will also change when flux sources emerge or submerge through the photosphere, as often happens in active regions. We have developed an algorithm to estimate the changes in flux due to emergence and submergence of magnetic flux sources. These estimated changes must be accounted for in order to quantify storage and release of magnetic energy in the corona. To perform this calculation over extended periods of time, we must additionally have a consistently labeled connectivity matrix over the entire observational time span. We have therefore developed an automated tracking algorithm to generate a consistent connectivity matrix as the photospheric source regions evolve over time. We have applied this method to NOAA Active Region 11112, which underwent a GOES M2.9 class flare around 19:00 on 2010 October 16th, and calculated a lower bound on the free magnetic energy buildup of {approx}8.25 Multiplication-Sign 10{sup 30} erg over 3 days.

  13. Stilbene Induced Inhibition of Androgen Receptor Dimerization: Implications for AR and ARΔLBD-Signalling in Human Prostate Cancer Cells

    PubMed Central

    Streicher, Wolfgang; Luedeke, Manuel; Azoitei, Anca; Zengerling, Friedemann; Herweg, Alexander; Genze, Felicitas; Schrader, Mark G.; Schrader, Andres J.; Cronauer, Marcus V.

    2014-01-01

    Background Advanced castration resistant prostate cancer (CRPC) is often characterized by an increase of C-terminally truncated, constitutively active androgen receptor (AR) variants. Due to the absence of a ligand binding domain located in the AR-C-terminus, these receptor variants (also termed ARΔLBD) are unable to respond to all classical forms of endocrine treatments like surgical/chemical castration and/or application of anti-androgens. Methodology In this study we tested the effects of the naturally occurring stilbene resveratrol (RSV) and (E)-4-(2, 6-Difluorostyryl)-N, N-dimethylaniline, a fluorinated dialkylaminostilbene (FIDAS) on AR- and ARΔLBD in prostate cancer cells. The ability of the compounds to modulate transcriptional activity of AR and the ARΔLBD-variant Q640X was shown by reporter gene assays. Expression of endogenous AR and ARΔLBD mRNA and protein levels were determined by qRT-PCR and Western Blot. Nuclear translocation of AR-molecules was analyzed by fluorescence microscopy. AR and ARΔLBD/Q640X homo-/heterodimer formation was assessed by mammalian two hybrid assays. Biological activity of both compounds in vivo was demonstrated using a chick chorioallantoic membrane xenograft assay. Results The stilbenes RSV and FIDAS were able to significantly diminish AR and Q640X-signalling. Successful inhibition of the Q640X suggests that RSV and FIDAS are not interfering with the AR-ligand binding domain like all currently available anti-hormonal drugs. Repression of AR and Q640X-signalling by RSV and FIDAS in prostate cancer cells was caused by an inhibition of the AR and/or Q640X-dimerization. Although systemic bioavailability of both stilbenes is very low, both compounds were also able to downregulate tumor growth and AR-signalling in vivo. Conclusion RSV and FIDAS are able to inhibit the dimerization of AR and ARΔLBD molecules suggesting that stilbenes might serve as lead compounds for a novel generation of AR-inhibitors. PMID:24887556

  14. AR Signaling in Breast Cancer

    PubMed Central

    Rahim, Bilal; O’Regan, Ruth

    2017-01-01

    Androgen receptor (AR, a member of the steroid hormone receptor family) status has become increasingly important as both a prognostic marker and potential therapeutic target in breast cancer. AR is expressed in up to 90% of estrogen receptor (ER) positive breast cancer, and to a lesser degree, human epidermal growth factor 2 (HER2) amplified tumors. In the former, AR signaling has been correlated with a better prognosis given its inhibitory activity in estrogen dependent disease, though conversely has also been shown to increase resistance to anti-estrogen therapies such as tamoxifen. AR blockade can mitigate this resistance, and thus serves as a potential target in ER-positive breast cancer. In HER2 amplified breast cancer, studies are somewhat conflicting, though most show either no effect or are associated with poorer survival. Much of the available data on AR signaling is in triple-negative breast cancer (TNBC), which is an aggressive disease with inferior outcomes comparative to other breast cancer subtypes. At present, there are no approved targeted therapies in TNBC, making study of the AR signaling pathway compelling. Gene expression profiling studies have also identified a luminal androgen receptor (LAR) subtype that is dependent on AR signaling in TNBC. Regardless, there seems to be an association between AR expression and improved outcomes in TNBC. Despite lower pathologic complete response (pCR) rates with neoadjuvant therapy, patients with AR-expressing TNBC have been shown to have a better prognosis than those that are AR-negative. Clinical studies targeting AR have shown somewhat promising results. In this paper we review the literature on the biology of AR in breast cancer and its prognostic and predictive roles. We also present our thoughts on therapeutic strategies. PMID:28245550

  15. AR Signaling in Breast Cancer.

    PubMed

    Rahim, Bilal; O'Regan, Ruth

    2017-02-24

    Androgen receptor (AR, a member of the steroid hormone receptor family) status has become increasingly important as both a prognostic marker and potential therapeutic target in breast cancer. AR is expressed in up to 90% of estrogen receptor (ER) positive breast cancer, and to a lesser degree, human epidermal growth factor 2 (HER2) amplified tumors. In the former, AR signaling has been correlated with a better prognosis given its inhibitory activity in estrogen dependent disease, though conversely has also been shown to increase resistance to anti-estrogen therapies such as tamoxifen. AR blockade can mitigate this resistance, and thus serves as a potential target in ER-positive breast cancer. In HER2 amplified breast cancer, studies are somewhat conflicting, though most show either no effect or are associated with poorer survival. Much of the available data on AR signaling is in triple-negative breast cancer (TNBC), which is an aggressive disease with inferior outcomes comparative to other breast cancer subtypes. At present, there are no approved targeted therapies in TNBC, making study of the AR signaling pathway compelling. Gene expression profiling studies have also identified a luminal androgen receptor (LAR) subtype that is dependent on AR signaling in TNBC. Regardless, there seems to be an association between AR expression and improved outcomes in TNBC. Despite lower pathologic complete response (pCR) rates with neoadjuvant therapy, patients with AR-expressing TNBC have been shown to have a better prognosis than those that are AR-negative. Clinical studies targeting AR have shown somewhat promising results. In this paper we review the literature on the biology of AR in breast cancer and its prognostic and predictive roles. We also present our thoughts on therapeutic strategies.

  16. Sintokamide A Is a Novel Antagonist of Androgen Receptor That Uniquely Binds Activation Function-1 in Its Amino-terminal Domain*

    PubMed Central

    Banuelos, Carmen A.; Tavakoli, Iran; Tien, Amy H.; Caley, Daniel P.; Mawji, Nasrin R.; Li, Zhenzhen; Wang, Jun; Yang, Yu Chi; Imamura, Yusuke; Yan, Luping; Wen, Jian Guo; Andersen, Raymond J.; Sadar, Marianne D.

    2016-01-01

    Androgen receptor (AR) is a validated drug target for all stages of prostate cancer including metastatic castration-resistant prostate cancer (CRPC). All current hormone therapies for CRPC target the C-terminal ligand-binding domain of AR and ultimately all fail with resumed AR transcriptional activity. Within the AR N-terminal domain (NTD) is activation function-1 (AF-1) that is essential for AR transcriptional activity. Inhibitors of AR AF-1 would potentially block most AR mechanisms of resistance including constitutively active AR splice variants that lack the ligand-binding domain. Here we provide evidence that sintokamide A (SINT1) binds AR AF-1 region to specifically inhibit transactivation of AR NTD. Consistent with SINT1 targeting AR AF-1, it attenuated transcriptional activities of both full-length AR and constitutively active AR splice variants, which correlated with inhibition of growth of enzalutamide-resistant prostate cancer cells expressing AR splice variants. In vivo, SINT1 caused regression of CRPC xenografts and reduced expression of prostate-specific antigen, a gene transcriptionally regulated by AR. Inhibition of AR activity by SINT1 was additive to EPI-002, a known AR AF-1 inhibitor that is in clinical trials (NCT02606123). This implies that SINT1 binds to a site on AF-1 that is unique from EPI. Consistent with this suggestion, these two compounds showed differences in blocking AR interaction with STAT3. This work provides evidence that the intrinsically disordered NTD of AR is druggable and that SINT1 analogs may provide a novel scaffold for drug development for the treatment of prostate cancer or other diseases of the AR axis. PMID:27576691

  17. Triptolide Inhibits the AR Signaling Pathway to Suppress the Proliferation of Enzalutamide Resistant Prostate Cancer Cells

    PubMed Central

    Han, Yangyang; Huang, Weiwei; Liu, Jiakuan; Liu, Dandan; Cui, Yangyan; Huang, Ruimin; Yan, Jun; Lei, Ming

    2017-01-01

    Enzalutamide is a second-generation androgen receptor (AR) antagonist for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Unfortunately, AR dysfunction means that resistance to enzalutamide will eventually develop. Thus, novel agents are urgently needed to treat this devastating disease. Triptolide (TPL), a key active compound extracted from the Chinese herb Thunder God Vine (Tripterygium wilfordii Hook F.), possesses anti-cancer activity in human prostate cancer cells. However, the effects of TPL against CRPC cells and the underlying mechanism of any such effect are unknown. In this study, we found that TPL at low dose inhibits the transactivation activity of both full-length and truncated AR without changing their protein levels. Interestingly, TPL inhibits phosphorylation of AR and its CRPC-associated variant AR-V7 at Ser515 through XPB/CDK7. As a result, TPL suppresses the binding of AR to promoter regions in AR target genes along with reduced TFIIH and RNA Pol II recruitment. Moreover, TPL at low dose reduces the viability of prostate cancer cells expressing AR or AR-Vs. Low-dose TPL also shows a synergistic effect with enzalutamide to inhibit CRPC cell survival in vitro, and enhances the anti-cancer effect of enzalutamide on CRPC xenografts with minimal side effects. Taken together, our data demonstrate that TPL targets the transactivation activity of both full-length and truncated ARs. Our results also suggest that TPL is a potential drug for CRPC, and can be used in combination with enzalutamide to treat CRPC. PMID:28638477

  18. Triptolide Inhibits the AR Signaling Pathway to Suppress the Proliferation of Enzalutamide Resistant Prostate Cancer Cells.

    PubMed

    Han, Yangyang; Huang, Weiwei; Liu, Jiakuan; Liu, Dandan; Cui, Yangyan; Huang, Ruimin; Yan, Jun; Lei, Ming

    2017-01-01

    Enzalutamide is a second-generation androgen receptor (AR) antagonist for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Unfortunately, AR dysfunction means that resistance to enzalutamide will eventually develop. Thus, novel agents are urgently needed to treat this devastating disease. Triptolide (TPL), a key active compound extracted from the Chinese herb Thunder God Vine (Tripterygium wilfordii Hook F.), possesses anti-cancer activity in human prostate cancer cells. However, the effects of TPL against CRPC cells and the underlying mechanism of any such effect are unknown. In this study, we found that TPL at low dose inhibits the transactivation activity of both full-length and truncated AR without changing their protein levels. Interestingly, TPL inhibits phosphorylation of AR and its CRPC-associated variant AR-V7 at Ser515 through XPB/CDK7. As a result, TPL suppresses the binding of AR to promoter regions in AR target genes along with reduced TFIIH and RNA Pol II recruitment. Moreover, TPL at low dose reduces the viability of prostate cancer cells expressing AR or AR-Vs. Low-dose TPL also shows a synergistic effect with enzalutamide to inhibit CRPC cell survival in vitro, and enhances the anti-cancer effect of enzalutamide on CRPC xenografts with minimal side effects. Taken together, our data demonstrate that TPL targets the transactivation activity of both full-length and truncated ARs. Our results also suggest that TPL is a potential drug for CRPC, and can be used in combination with enzalutamide to treat CRPC.

  19. Clinical Relevance of Androgen Receptor Splice Variants in Castration-Resistant Prostate Cancer.

    PubMed

    Maughan, Benjamin L; Antonarakis, Emmanuel S

    2015-12-01

    Metastatic castration-resistant prostate cancer (mCRPC) currently benefits from a wealth of treatment options, yet still remains lethal in the vast majority of patients. It is becoming increasingly understood that this disease entity continues to evolve over time, acquiring additional and diverse resistance mechanisms with each subsequent therapy used. This dynamic relationship between treatment pressure and disease resistance can be challenging for the managing clinician. The recent discovery of alternate splice variants of the androgen receptor (AR) is one potential mechanism of escape in mCRPC, and recognizing this resistance mechanism might be important for optimal treatment selection for our patients. AR-V7 appears to be the most relevant AR splice variant, and early clinical data suggest that it is a negative prognostic marker in mCRPC. Emerging evidence also suggests that detection of AR-V7 may be associated with resistance to novel hormonal therapy (abiraterone and enzalutamide) but may be compatible with sensitivity to taxane chemotherapy (docetaxel and cabazitaxel). Adding to this complexity is the observation that AR-V7 is a dynamic marker whose status may change across time and depending on selective pressures induced by different therapies. Finally, it is possible that AR-V7 may represent a therapeutic target in mCRPC if drugs can be designed that degrade or inhibit AR splice variants or block their transcriptional activity. Several such agents (including galeterone, EPI-506, and bromodomain/BET inhibitors) are now in clinical development.

  20. Biologic and Clinical Significance of Androgen Receptor Variants in Castration Resistant Prostate Cancer

    PubMed Central

    Ware, Kathryn E; Garcia-Blanco, Mariano A; Armstrong, Andrew J; Dehm, Scott M.

    2014-01-01

    As prostate cancer progresses to the lethal castration resistant and metastatic form, genetic and epigenetic adaptation, clonal selection, and evolution of the tumor microenvironment contribute to the emergence of unique biologic characteristics under the selective pressure of external stresses. These stresses include the therapies applied in the clinic or laboratory and the exposures of cancers to hormonal, paracrine, or autocrine stimuli in the context of the tumor micro- and macro-environment. The androgen receptor (AR) is a key gene involved in prostate cancer etiology and oncogenesis, including disease development, progression, response to initial hormonal therapies, and subsequent resistance to hormonal therapies. Alterations in the AR signaling pathway have been observed in certain selection contexts and contribute to the resistance to agents that target hormonal regulation of the AR, including standard androgen deprivation therapy (ADT), anti-androgens such as enzalutamide, and androgen synthesis inhibition with abiraterone acetate. One such resistance mechanism is the synthesis of constitutively active AR variants lacking the canonical ligand binding domain. This review focuses on the etiology, characterization, biologic properties, and emerging data contributing to the clinical characteristics of AR variants, and suggests approaches to full-length AR and AR variant biomarker validation, assessment, and systemic targeting in the clinic. PMID:24859991

  1. Increased anti-leukemic activity of decitabine via AR-42-induced upregulation of miR-29b: A novel epigenetic-targeting approach in acute myeloid leukemia

    PubMed Central

    Mims, Alice; Walker, Alison R.; Huang, Xiaomeng; Sun, Jin; Wang, Hongyan; Santhanam, Ramasamy; Dorrance, Adrienne M.; Walker, Chris; Hoellerbauer, Pia; Tarighat, Somayeh S.; Chan, Kenneth K.; Klisovic, Rebecca B.; Perrotti, Danilo; Caligiuri, Michael A.; Byrd, John C.; Chen, Ching-Shih; Lee, L. James; Jacob, Samson; Mrózek, Krzysztof; Bloomfield, Clara D.; Blum, William; Garzon, Ramiro; Schwind, Sebastian; Marcucci, Guido

    2013-01-01

    Histone deacetylase (HDAC) inhibitors either alone or in combination with hypomethylating agents have limited clinical effect in acute myeloid leukemia (AML). Previously we demonstrated that AML patients with higher miR-29b expression had better response to the hypomethylating agent decitabine. Therefore, an increase in miR-29b expression preceding decitabine treatment may provide a therapeutic advantage. We previously showed that miR-29b expression is suppressed by a repressor complex that includes HDACs. Thus, HDAC inhibition may increase miR-29b expression. We hypothesized that priming AML cells with the novel HDAC inhibitor (HDACI) AR-42 would result in increased response to decitabine treatment via upregulation of miR-29b. Here we show that AR-42 is a potent HDACI in AML, increasing miR-29b levels and leading to downregulation of known miR-29b targets (i.e., SP1, DNMT1, DNMT3A, and DNMT3B). We then demonstrated that the sequential administration of AR-42 followed by decitabine resulted in a stronger anti-leukemic activity in vitro and in vivo than decitabine followed by AR-42 or either drug alone. These preclinical results with AR-42 priming before decitabine administration represents a promising, novel treatment approach and a paradigm shift with regard to the combination of epigenetic-targeting compounds in AML, where decitabine has been traditionally given before HDAC inhibitors. PMID:23178755

  2. Fluorinated analogues of marsanidine, a highly α2-AR/imidazoline I1 binding site-selective hypotensive agent. Synthesis and biological activities.

    PubMed

    Wasilewska, Aleksandra; Sączewski, Franciszek; Hudson, Alan L; Ferdousi, Mehnaz; Scheinin, Mika; Laurila, Jonne M; Rybczyńska, Apolonia; Boblewski, Konrad; Lehmann, Artur

    2014-11-24

    The aim of these studies was to establish the influence of fluorination of the indazole ring on the pharmacological properties of two selective α2-adrenoceptor (α2-AR) agonists: 1-[(imidazolidin-2-yl)imino]-1H-indazole (marsanidine, A) and its methylene analogue 1-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-1H-indazole (B). Introduction of fluorine into the indazole ring of A and B reduced both binding affinity and α2-AR/I1 imidazoline binding site selectivity. The most α2-AR-selective ligands were 6-fluoro-1-[(imidazolidin-2-yl)imino]-1H-indazole (6c) and 7-fluoro-1-[(imidazolidin-2-yl)imino]-1H-indazole (6d). The in vivo cardiovascular properties of fluorinated derivatives of A and B revealed that in both cases the C-7 fluorination leads to compounds with the highest hypotensive and bradycardic activities. The α2-AR partial agonist 6c was prepared as a potential lead compound for development of a radiotracer for PET imaging of brain α2-ARs.

  3. Angiotensin II-induced protein kinase D activates the ATF/CREB family of transcription factors and promotes StAR mRNA expression.

    PubMed

    Olala, Lawrence O; Choudhary, Vivek; Johnson, Maribeth H; Bollag, Wendy B

    2014-07-01

    Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.

  4. 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.

  5. 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.

  6. 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

  7. Methylmercury exposure, PON1 gene variants and serum paraoxonase activity in Eastern James Bay Cree adults.

    PubMed

    Drescher, Olivia; Dewailly, Eric; Diorio, Caroline; Ouellet, Nathalie; Sidi, Elhadji Anassour Laouan; Abdous, Belkacem; Valera, Beatriz; Ayotte, Pierre

    2014-11-01

    There is growing evidence that cardiovascular health can be affected by exposure to methylmercury (MeHg), by a mechanism involving oxidative stress. Paraoxonase 1 (PON1) is a high-density lipoprotein-bound enzyme that hydrolyzes toxic oxidized lipids and protects against cardiovascular diseases. Evidence from in vitro studies indicates that MeHg can inhibit PON1 activity but little is known regarding this effect in humans. We investigated whether increased blood mercury levels are associated with decreased serum PON1 activity in Cree people who are exposed to MeHg by fish consumption. We conducted a multi-community study of 881 Cree adults living in Eastern James Bay communities (Canada). Multivariate analyses considered sociodemographic, anthropometric, clinical, dietary and lifestyle variables and six PON1 gene variants (rs705379 (-108C/T), rs662 (Q192R), rs854560 (L55M), rs854572 (-909C/G), rs854571 (-832C/T) and rs705381 (-162C/T)). In a multiple regression model adjusted for all potential confounding factors and the rs854560 PON1 variant, a statistically significant MeHg*rs705379 interaction was observed. Blood mercury levels were inversely associated with serum PON1 activities in individual homozygous for the -108T allele (P=0.009). Our results suggest a gene-environment interaction between the rs705379 polymorphism and MeHg exposure on PON1 activity levels in this aboriginal population. This finding will need to be replicated in other population studies.

  8. 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.

  9. Epstein-Barr Virus LMP-1 Natural Sequence Variants Differ in Their Potential To Activate Cellular Signaling Pathways

    PubMed Central

    Fielding, Ceri A.; Sandvej, Kristian; Mehl, Anja; Brennan, Paul; Jones, Matthew; Rowe, Martin

    2001-01-01

    The latent membrane protein 1 (LMP-1) oncogene of Epstein-Barr virus (EBV) is believed to contribute to the development of many EBV-associated tumors, and there is evidence that sequence variation can affect some functions of LMP-1. Most studies have been restricted to the prototype B95.8 LMP-1 gene and genes isolated from EBV of nasopharyngeal carcinoma (NPC) patients. Here, we analyzed the signaling functions of LMP-1 from a panel of nine EBV isolates, including representatives of four defined groups of European LMP-1 variants (groups A to D [K. Sandvej, J. W. Gratama, M. Munch, X. G. Zhou, R. L. Bolhuis, B. S. Andresen, N. Gregersen, and S. Hamilton-Dutoit, Blood 90:323–330, 1997]) and Chinese NPC-derived LMP-1. Chinese and group D variants activated the transcription factor NF-κB two- to threefold more efficiently than B95.8 LMP-1, while Chinese, group B, and group D variants similarly activated activator protein 1 (AP-1) transcription more efficiently than did B95.8 LMP-1. However, there were no amino acid substitutions in the core binding regions for tumor necrosis factor receptor-associated adapter proteins known to mediate NF-κB and AP-1 activation. In contrast, despite sequence variation in the proposed Janus kinase 3 binding region, STAT activation was remarkably constant among the panel of LMP-1 variants. Analysis of the induction of CD54 (intercellular adhesion molecule 1) protein expression by the LMP-1 variants showed differences that did not correlate with either NF-κB or AP-1. Therefore, while the defined sequence variant groups do correlate with LMP-1 function, the results highlight the fact that the relationship between sequence variation and signaling function is extremely complex. It appears unlikely that one particular amino acid substitution or deletion will define a disease-associated variant of LMP-1. PMID:11533177

  10. Protein Profiling and Histone Deacetylation Activities in Somaclonal Variants of Oil Palm (Elaeis guineensis Jacq.)

    PubMed Central

    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

  11. The activity-dependent histone variant H2BE modulates the life span of olfactory neurons

    PubMed Central

    Santoro, Stephen W; Dulac, Catherine

    2012-01-01

    We have identified a replication-independent histone variant, Hist2h2be (referred to herein as H2be), which is expressed exclusively by olfactory chemosensory neurons. Levels of H2BE are heterogeneous among olfactory neurons, but stereotyped according to the identity of the co-expressed olfactory receptor (OR). Gain- and loss-of-function experiments demonstrate that changes in H2be expression affect olfactory function and OR representation in the adult olfactory epithelium. We show that H2BE expression is reduced by sensory activity and that it promotes neuronal cell death, such that inactive olfactory neurons display higher levels of the variant and shorter life spans. Post-translational modifications (PTMs) of H2BE differ from those of the canonical H2B, consistent with a role for H2BE in altering transcription. We propose a physiological function for H2be in modulating olfactory neuron population dynamics to adapt the OR repertoire to the environment. DOI: http://dx.doi.org/10.7554/eLife.00070.001 PMID:23240083

  12. 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.

  13. 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.

  14. 1914G variant of BCHE gene associated with enzyme activity, obesity and triglyceride levels.

    PubMed

    Lima, Jovana Karoline; Leite, Neiva; Turek, Luciane Viater; Souza, Ricardo Lehtonen Rodrigues; da Silva Timossi, Luciana; Osiecki, Ana Claudia Vecchi; Osiecki, Raul; Furtado-Alle, Lupe

    2013-12-10

    Polymorphisms of butyrylcholinesterase (BChE) have been reported to be associated to weight, BMI variance and hypertriglyceridemia in adults and adolescents. The aim of the present study was to investigate the association of -116A (SNP: G/A; rs1126680) and 1914G (SNP: A/G; rs3495) variants of BCHE gene with anthropometric and biochemical variables associated with obesity in population sample of 115 individuals, from Southern Brazil. Participants were grouped in two categories: obese (BMI≥30) and non-obese (BMI<30). The 1914G allele showed significantly higher frequency in the obese group, and carriers of 1914G allele showed lower mean BChE activity when compared to 1914A carriers (p=0.006). Higher means of BMI (p=0.02) and triglyceride (TG; p=0.01) were found in 1914G carriers (BMI=27.57 kg/m(2); TG=150.8 mg/dL) when compared to 1914A homozygotes (BMI=25.55 kg/m(2); TG=107.9 mg/dL). Carriers of the -116A allele showed lower mean BChE activity than usual homozygotes, and the -116A variant was found in cis with 1914G (p<0.0001; D'=1). The region of BCHE gene that contains the 1914G mutation site is target of microRNAs (miRs) and the response of BChE to glucocorticoids is especially influenced by these miRs. Therefore, it is possible that the 1914G allele can be interfering in gluconeogenesis, hyperglycemia, lipolysis and body fat distribution. This lower activity may cause an imbalance in lipid metabolism, which may lead to an increased predisposition to obesity and to a lower ability to maintain metabolic homeostasis. © 2013 Elsevier B.V. All rights reserved.

  15. Quantification of Hepatic UDP Glucuronosyltransferase 1A Splice Variant Expression and Correlation of UDP Glucuronosyltransferase 1A1 Variant Expression with Glucuronidation Activity

    PubMed Central

    Jones, Nathan R.; Sun, Dongxiao; Freeman, Willard M.

    2012-01-01

    The UDP glucuronosyltransferase (UGT) 1A gene cluster encodes nine UGT1A family members via splicing of individual first exons to common exons 2 through 5. Each of these nine UGT1As can also undergo alternative splicing at their 3′ ends by using an alternate exon 5, resulting in 27 different UGT1A mRNA species with each UGT1A gene encoding three different combinations of 5A and 5B UGT1A exons. To examine the importance of UGT1A exon 5 splice variants on overall UGT1A activity, a nested quantitative polymerase chain reaction assay was developed to accurately assess the combined expression of exon 5 splice variants (termed v2/v3) versus the expression of wild-type (termed v1) for each specific UGT1A. v1 expression was 16-, 17-, 57- and 29-fold higher than that observed for the levels of v2/v3 for UGTs 1A1, 1A4, 1A6, and 1A9, respectively, in normal human liver specimens. In a series of 58 normal human liver specimens, the expression of both UGT1A1 v1 and v2/v3 mRNAs was positively correlated with raloxifene glucuronidation activity in corresponding microsomes prepared from the same specimens (p < 0.0001, r2 = 0.720; p = 0.0002, r2 = 0.241, respectively), with expression of both variants lower in individuals homozygous for the UGT1A1*28 allele (42% for v1, p = 0.041; 53% for v2/v3, p = 0.0075). The expression of UGT1A1 v2/v3 was 1.6-fold higher than v1 (p = 0.03) in HepG2 cells, and short interfering RNA knockdown of HepG2 v2/v3 increased raloxifene glucuronidation activity by 83%. Together, these data suggest that hepatic UGT1A v2/v3 mRNA species are minor form variants in human livers from most individuals. PMID:22661630

  16. 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.

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

    PubMed Central

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

    2016-01-01

    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

  18. Differential activity of conditional MYC and its variant MYC-S in human mortal fibroblasts.

    PubMed

    Hirst, S K; Grandori, C

    2000-10-26

    We have explored the effects of the conditional MYC-estrogen receptor fusion protein, MYC-ERTM, in human mortal fibroblasts, WI38, on cell-cycle entry, apoptosis and gene expression. The results indicate that activation of MYC-ERTM in WI38 cells is sufficient to cause S phase entry of quiescent cells, which is preceded by phosphorylation of Rb and activation of the Cdk2-associated kinase. We also analysed the MYC protein variant, MYC-S, which lacks part of the transcriptional activation domain but includes the conserved MYC box II and 26 amino acids N-terminal to it. MYC-S was previously shown to promote proliferation and apoptosis of immortalized rodent cell lines. The results indicate that MYC-S has undetectable activity as an inducer of S phase or apoptosis of quiescent WI38 cells. However, Myc-S stimulates proliferation of WI38 cells in the presence of 10% fetal calf serum. Surprisingly, we found that MYC-S, previously considered solely a repressor of specific reporter genes, is instead a weak transactivator of endogenous target genes both in mortal and immortalized cells. In addition, MYC-S exhibit a weak repressor activity upon an endogenous target gene only in immortalized cells. MYC-S transcriptional properties suggest that MYC box II and the adjacent N-terminal amino acids, while not sufficient for full repression function, participate in transactivation of endogenous target genes.

  19. 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.

  20. 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.

  1. Crystal structure of an activated variant of small heat shock protein Hsp16.5.

    PubMed

    McHaourab, Hassane S; Lin, Yi-Lun; Spiller, Benjamin W

    2012-06-26

    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 α-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 α-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 α-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 β-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.

  2. Nuclear and nucleolar activity of linker histone variant H1.0.

    PubMed

    Kowalski, Andrzej

    2016-01-01

    Histone H1.0 belongs to the class of linker histones (H1), although it is substantially distinct from other histone H1 family members. The differences can be observed in the chromosomal location and organization of the histone H1.0 encoding gene, as well as in the length and composition of its amino acid chain. Whereas somatic (H1.1-H1.5) histone H1 variants are synthesized in the cell cycle S-phase, histone H1.0 is synthesized throughout the cell cycle. By replacing somatic H1 variants during cell maturation, histone H1.0 is gradually deposited in low dividing cells and achieves the highest level of expression in the terminally differentiated cells. Compared to other differentiation-specific H1 histone (H5) characteristic for unique tissue and organisms, the distribution of histone H1.0 remains non-specific. Classic investigations emphasize that histone H1.0 is engaged in the organization of nuclear chromatin accounting for formation and maintenance of its nucleosomal and higher-order structure, and thus influences gene expression. However, the recent data confirmed histone H1.0 peculiar localization in the nucleolus and unexpectedly revealed its potential for regulation of nucleolar, RNA-dependent, activity via interaction with other proteins. According to such findings, histone H1.0 participates in the formation of gene-coded information through its control at both transcriptional and translational levels. In order to reappraise the biological significance of histone H1.0, both aspects of its activity are presented in this review.

  3. Lack of association of fatness-related FTO gene variants with energy expenditure or physical activity.

    PubMed

    Berentzen, T; Kring, S I I; Holst, C; Zimmermann, E; Jess, T; Hansen, T; Pedersen, O; Toubro, S; Astrup, A; Sørensen, T I A

    2008-07-01

    A common variant in the first intron of FTO (rs9939609, T/A) is associated with fatness in Caucasians. FTO may regulate energy homeostasis through the hypothalamus, and we hypothesized that AA-genotypes of rs9939609 FTO have lower energy expenditure and/or a lower level of physical activity. The study population included all obese young men (body mass index > or = 31 kg/m(2)) at the mandatory draft board examinations in the Copenhagen area from 1943 to 1977 and a randomly selected control group from this population. Subgroups of 234 obese and 323 controls were examined in 1998-2000 (median age 48 yr). Fat mass (FM), lean body mass (LBM), leisure-time physical activity (LTPA), maximum oxygen uptake (VO(2)max), resting energy expenditure (REE), and glucose-induced thermogenesis (GIT) were measured. The FTO rs9939609 variant was genotyped. A recessive transmission mode fit the data best. Logistic regression was used to assess the odds ratios of the AA-genotype in relation to LTPA, VO(2)max, REE, and GIT. The AA-genotype of FTO rs9939609 had higher REE in the age-adjusted model, but the association was eliminated when adjusting for FM and LBM. The AA-genotype was not associated with LTPA, VO(2)max, or GIT. This was not influenced by adjustment for age, FM, or LBM. The AA-genotype had increased FM, even with adjustment for age, LBM, REE, GIT, VO(2)max, and LTPA. Results were similar for FTO rs8050136 and rs7193144. Homozygous carriers of the A-allele of rs9939609 FTO do not have lower REE, GIT, VO(2)max, or LTPA but higher FM, irrespective of LBM, REE, GIT, VO(2)max, and LTPA.

  4. Metrologically-Calibrated 40Ar Concentrations and Ages of Mineral Standards Used in 40Ar/39Ar Geochronology

    NASA Astrophysics Data System (ADS)

    Morgan, L. E.; Davidheiser-Kroll, B.; Kuiper, K.; Wijbrans, J. R.; Mark, D. F.

    2015-12-01

    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 present results for the 40Ar concentrations and ages of mineral standards determined based on a modern gas delivery system (Morgan et al. 2011), which delivers metrologically-traceable amounts of 40Ar and thus allows for the sensitivity calibration of noble gas mass spectrometers.

  5. The histone variant H2A.Z is an important regulator of enhancer activity

    PubMed Central

    Brunelle, Mylène; Nordell Markovits, Alexei; Rodrigue, Sébastien; Lupien, Mathieu; Jacques, Pierre-Étienne; Gévry, Nicolas

    2015-01-01

    Gene regulatory programs in different cell types are largely defined through cell-specific enhancers activity. The histone variant H2A.Z has been shown to play important roles in transcription mainly by controlling proximal promoters, but its effect on enhancer functions remains unclear. Here, we demonstrate by genome-wide approaches that H2A.Z is present at a subset of active enhancers bound by the estrogen receptor alpha (ERα). We also determine that H2A.Z does not influence the local nucleosome positioning around ERα enhancers using ChIP sequencing at nucleosomal resolution and unsupervised pattern discovery. We further highlight that H2A.Z-enriched enhancers are associated with chromatin accessibility, H3K122ac enrichment and hypomethylated DNA. Moreover, upon estrogen stimulation, the enhancers occupied by H2A.Z produce enhancer RNAs (eRNAs), and recruit RNA polymerase II as well as RAD21, a member of the cohesin complex involved in chromatin interactions between enhancers and promoters. Importantly, their recruitment and eRNAs production are abolished by H2A.Z depletion, thereby revealing a novel functional link between H2A.Z occupancy and enhancer activity. Taken together, our findings suggest that H2A.Z acts as an important player for enhancer functions by establishing and maintaining a chromatin environment required for RNA polymerase II recruitment, eRNAs transcription and enhancer-promoters interactions, all essential attributes of enhancer activity. PMID:26319018

  6. Variants of glycerol dehydrogenase having D-lactate dehydrogenase activity and uses thereof

    DOEpatents

    Wang, Qingzhao; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

    2017-08-29

    The present invention provides methods of designing and generating glycerol dehydrogenase (GlyDH) variants that have altered function as compared to a parent polypeptide. The present invention further provides nucleic acids encoding GlyDH polypeptide variants having altered function as compared to the parent polypeptide. Host cells comprising polynucleotides encoding GlyDH variants and methods of producing lactic acids are also provided in various aspects of the invention.

  7. 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

  8. Prenatal diagnosis of GM2 gangliosidosis with high residual hexosaminidase A activity (variant B1; pseudo AB variant).

    PubMed

    Conzelmann, E; Nehrkorn, H; Kytzia, H J; Sandhoff, K; Macek, M; Lehovský, M; Elleder, M; Jirásek, A; Kobilková, J

    1985-11-01

    A case of infantile GM2 gangliosidosis with high residual beta-hexosaminidase A activity toward the synthetic substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside was diagnosed prenatally. Extracts from cultured amniotic fluid cells of the fetus had a hexosaminidase A activity of 27% of total hexosaminidase but were almost completely unable to degrade [3H]ganglioside GM2 (less than 0.5% of control values) when assayed in the presence of the natural activator protein. These results were confirmed by analyses of fetal muscle fibroblasts, liver, and brain. All tissues examined showed a profound deficiency of ganglioside GM2 galactosaminidase despite hexosaminidase A levels in the heterozygote range. In brain tissue, ganglioside GM2 content was elevated more than 4-fold. Hydrolysis of p-nitrophenyl glucosaminide-6-sulfate, a substrate specific for hexosaminidases A and S, by tissue extracts was also markedly reduced but the residual activities found (5% in liver, 12% in fibroblasts, and 16% in brain) were much higher than those with the physiological lipid substrate, ganglioside GM2.

  9. Variants of the protein PRDM9 differentially regulate a set of human meiotic recombination hotspots highly active in African populations.

    PubMed

    Berg, Ingrid L; Neumann, Rita; Sarbajna, Shriparna; Odenthal-Hesse, Linda; Butler, Nicola J; Jeffreys, Alec J

    2011-07-26

    PRDM9 is a major specifier of human meiotic recombination hotspots, probably via binding of its zinc-finger repeat array to a DNA sequence motif associated with hotspots. However, our view of PRDM9 regulation, in terms of motifs defined and hotspots studied, has a strong bias toward the PRDM9 A variant particularly common in Europeans. We show that population diversity can reveal a second class of hotspots specifically activated by PRDM9 variants common in Africans but rare in Europeans. These African-enhanced hotspots nevertheless share very similar properties with their counterparts activated by the A variant. The specificity of hotspot activation is such that individuals with differing PRDM9 genotypes, even within the same population, can use substantially if not completely different sets of hotspots. Each African-enhanced hotspot is activated by a distinct spectrum of PRDM9 variants, despite the fact that all are predicted to bind the same sequence motif. This differential activation points to complex interactions between the zinc-finger array and hotspots and identifies features of the array that might be important in controlling hotspot activity.

  10. Spectroscopic and modeling investigations of the gas-phase chemistry and composition in microwave plasma activated B2H6/Ar/H2 mixtures.

    PubMed

    Ma, Jie; Richley, James C; Davies, David R W; Cheesman, Andrew; Ashfold, Michael N R; Mankelevich, Yuri A

    2010-02-25

    This paper describes a three-pronged study of microwave (MW) activated B(2)H(6)/Ar/H(2) plasmas as a precursor to diagnosis of the B(2)H(6)/CH(4)/Ar/H(2) plasmas used for the chemical vapor deposition of B-doped diamond. Absolute column densities of B atoms and BH radicals have been determined by cavity ring-down spectroscopy as a function of height (z) above a molybdenum substrate and of the plasma process conditions (B(2)H(6) and Ar partial pressures, total pressure, and supplied MW power). Optical emission spectroscopy has been used to explore variations in the relative densities of electronically excited BH, H, and H(2) species as a function of the same process conditions and of time after introducing B(2)H(6) into a pre-existing Ar/H(2) plasma. The experimental measurements are complemented by extensive 2-D(r, z) modeling of the plasma chemistry, which results in refinements to the existing B/H chemistry and thermochemistry and demonstrates the potentially substantial loss of gas-phase BH(x) species through reaction with trace quantities of air/O(2) in the process gas mixture and heterogeneous processes occurring at the reactor wall.

  11. Acid-Induced Activation of the Urease Promoters Is Mediated Directly by the ArsRS Two-Component System of Helicobacter pylori

    PubMed Central

    Pflock, Michael; Kennard, Simone; Delany, Isabel; Scarlato, Vincenzo; Beier, Dagmar

    2005-01-01

    The nickel-containing enzyme urease is an essential colonization factor of the human gastric pathogen Helicobacter pylori which enables the bacteria to survive the low-pH conditions of the stomach. Transcription of the urease genes is positively controlled in response to increasing concentrations of nickel ions and acidic pH. Here we demonstrate that acid-induced transcription of the urease genes is mediated directly by the ArsRS two-component system. Footprint analyses identify binding sites of the phosphorylated ArsR response regulator within the ureA and ureI promoters. Furthermore, deletion of a distal upstream ArsR binding site of the ureA promoter demonstrates its role in acid-dependent activation of the promoter. In addition, acid-induced transcription of the ureA gene is unaltered in a nikR mutant, providing evidence that pH-responsive regulation and nickel-responsive regulation of the ureA promoter are mediated by independent mechanisms involving the ArsR response regulator and the NikR protein. PMID:16177315

  12. 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.

  13. NOS3 variants, physical activity, and blood pressure in the European Youth Heart Study.

    PubMed

    Grøntved, Anders; Andersen, Lars B; Franks, Paul W; Verhage, Bas; Wareham, Nicholas J; Ekelund, Ulf; Loos, Ruth J F; Brage, Søren

    2011-04-01

    In this study, we examined the influence of genetic variation in NOS3 on resting blood pressure (BP) in children and adolescents from the European Youth Heart Study (EYHS). Because the NOS3 gene expression is altered by physical activity (PA), we also tested for interaction between habitual PA and NOS3 variants on BP. A cross-sectional, random sample of 8-10-year old children (n = 1,214) and 14-16-year old adolescents (n = 1,141) from Denmark and Estonia were genotyped for four NOS3 tagging polymorphisms (rs1800783, rs1799983 (Glu298Asp), rs3918227, rs743507). PA was measured objectively using a hip-mounted accelerometer and through self-reported bicycling and TV-viewing. Permutation testing was used to correct for multiple testing, yielding an α level of 0.006. Glu298Asp showed age-group-dependent associations with BP. In adolescents, Asp298 allele homozygotes had 0.19 s.d. (95% confidence interval (CI): 0.06; 0.13, P = 0.004) higher diastolic BP (DBP) and 0.25 s.d. (95% CI: 0.05; 0.46, P = 0.015) higher systolic BP (SBP), compared to Glu298 allele carriers. None of the three other single-nucleotide polymorphisms (SNPs) were associated with BP in adolescents. In children, none of the SNPs were associated with BP. No evidence of interaction between Glu298Asp and objectively measured PA was observed. Both self-reported bicycling and TV-viewing nominally modified the association between Glu298Asp and BP in adolescents (P < 0.05), the genetic effect being most apparent in inactive individuals. However, none of the interactions persisted after correcting for multiple testing. The NOS3 Glu298Asp variant may associate with resting BP in adolescence but not in childhood, an effect that could be modified by PA.

  14. Systematic and functional characterization of novel androgen receptor variants arising from alternative splicing in the ligand-binding domain

    PubMed Central

    Uo, T; Dvinge, H; Sprenger, C C; Bradley, R K; Nelson, P S; Plymate, S R

    2017-01-01

    The presence of intact ligand-binding domain (LBD) ensures the strict androgen-dependent regulation of androgen receptor (AR): binding of androgen induces structural reorganization of LBD resulting in release of AR from HSP90, suppression of nuclear export which otherwise dominates over import and nuclear translocation of AR as a transcription factor. Thus, loss or defects of the LBD abolish constraint from un-liganded LBD as exemplified by constitutively active AR variants (AR-Vs), which are associated with emerging resistance mechanism to anti-AR therapy in castration-resistant prostate cancer (mCRPC). Recent analysis of the AR splicing landscapes revealed mCRPC harboring multiple AR-Vs with diverse patterns of inclusion/exclusion of exons (exons 4–8) corresponding to LBD to produce namely exon-skipping variants. In silico construction for these AR-Vs revealed four novel AR-Vs having unique features: Exclusion of specified exons introduces a frameshift in variants v5es, v6es and v7es. ARv56es maintains the reading frame resulting in the inclusion of the C-terminal half of the LBD. We systematically characterized these AR-Vs regarding their subcellular localization, affinity for HSP90 and transactivation capability. Notably, ARv5es was free from HSP90, exclusively nuclear, and constitutively active similarly as previously reported for v567es. In contrast, v6es and v7es were similar in that they are cytoplasmic, transcriptionally inactive and bind HSP90, ARv56es was present in both nucleus and cytoplasm, does not bind HSP90 and is transcriptionally inactive. Converting these transcriptionally inactive AR-Vs into active forms, we identified the two separate elements that allosterically suppress otherwise constitutively active AR-Vs; one in exon 5 for v6es and v7es and the other in exon 8 for v56es. Our findings identify a novel constitutively active AR-V, ARv5es and establish a method to predict potential activities of AR-Vs carrying impaired LBD. PMID:27694897

  15. Analytic Validation of RNA In Situ Hybridization (RISH) for AR and AR-V7 Expression in Human Prostate Cancer

    PubMed Central

    Guedes, Liana B.; Morais, Carlos L.; Almutairi, Fawaz; Haffner, Michael C.; Zheng, Qizhi; Isaacs, John T.; Antonarakis, Emmanuel S.; Lu, Changxue; Tsai, Harrison; Luo, Jun; De Marzo, Angelo M.; Lotan, Tamara L.

    2016-01-01

    Purpose RNA expression of androgen receptor splice variants may be a biomarker of resistance to novel androgen deprivation therapies in castrate resistant prostate cancer (CRPC). We analytically validated an RNA in situ hybridization (RISH) assay for total AR and AR-V7 for use in formalin fixed paraffin embedded (FFPE) prostate tumors. Experimental Design We used prostate cell lines and xenografts to validate chromogenic RISH to detect RNA containing AR exon 1 (AR-E1, surrogate for total AR RNA species) and cryptic exon 3 (AR-CE3, surrogate for AR-V7 expression). RISH signals were quantified in FFPE primary tumors and CRPC specimens, comparing to known AR and AR-V7 status by immunohistochemistry and RT-PCR. Results The quantified RISH results correlated significantly with total AR and AR-V7 levels by RT-PCR in cell lines, xenografts and autopsy metastases. Both AR-E1 and AR-CE3 RISH signals were localized in nuclear punctae in addition to the expected cytoplasmic speckles. Compared to admixed benign glands, AR-E1 expression was significantly higher in primary tumor cells with a median fold increase of 3.0 and 1.4 in two independent cohorts (p<0.0001 and p=0.04, respectively). While AR-CE3 expression was detectable in primary prostatic tumors, levels were substantially higher in a subset of CRPC metastases and cell lines, and were correlated with AR-E1 expression. Conclusions RISH for AR-E1 and AR-CE3 is an analytically valid method to examine total AR and AR-V7 RNA levels in FFPE tissues. Future clinical validation studies are required to determine whether AR RISH is a prognostic or predictive biomarker in specific clinical contexts. PMID:27166397

  16. Analytic Validation of RNA In Situ Hybridization (RISH) for AR and AR-V7 Expression in Human Prostate Cancer.

    PubMed

    Guedes, Liana B; Morais, Carlos L; Almutairi, Fawaz; Haffner, Michael C; Zheng, Qizhi; Isaacs, John T; Antonarakis, Emmanuel S; Lu, Changxue; Tsai, Harrison; Luo, Jun; De Marzo, Angelo M; Lotan, Tamara L

    2016-09-15

    RNA expression of androgen receptor splice variants may be a biomarker of resistance to novel androgen deprivation therapies in castrate-resistant prostate cancer (CRPC). We analytically validated an RNA in situ hybridization (RISH) assay for total AR and AR-V7 for use in formalin-fixed paraffin-embedded (FFPE) prostate tumors. We used prostate cell lines and xenografts to validate chromogenic RISH to detect RNA containing AR exon 1 (AR-E1, surrogate for total AR RNA species) and cryptic exon 3 (AR-CE3, surrogate for AR-V7 expression). RISH signals were quantified in FFPE primary tumors and CRPC specimens, comparing to known AR and AR-V7 status by IHC and RT-PCR. The quantified RISH results correlated significantly with total AR and AR-V7 levels by RT-PCR in cell lines, xenografts, and autopsy metastases. Both AR-E1 and AR-CE3 RISH signals were localized in nuclear punctae in addition to the expected cytoplasmic speckles. Compared with admixed benign glands, AR-E1 expression was significantly higher in primary tumor cells with a median fold increase of 3.0 and 1.4 in two independent cohorts (P < 0.0001 and P = 0.04, respectively). While AR-CE3 expression was detectable in primary prostatic tumors, levels were substantially higher in a subset of CRPC metastases and cell lines, and were correlated with AR-E1 expression. RISH for AR-E1 and AR-CE3 is an analytically valid method to examine total AR and AR-V7 RNA levels in FFPE tissues. Future clinical validation studies are required to determine whether AR RISH is a prognostic or predictive biomarker in specific clinical contexts. Clin Cancer Res; 22(18); 4651-63. ©2016 AACR. ©2016 American Association for Cancer Research.

  17. Biochemical Characterization of APOBEC3H Variants: Implications for Their HIV-1 Restriction Activity and mC Modification.

    PubMed

    Gu, Jiang; Chen, Qihan; Xiao, Xiao; Ito, Fumiaki; Wolfe, Aaron; Chen, Xiaojiang S

    2016-11-20

    APOBEC3H (A3H) is the most polymorphic member of the APOBEC3 family. Seven haplotypes (hap I-VII) and four mRNA splicing variants (SV) of A3H have been identified. The various haplotypes differ in anti-HIV activity, which is attributed to differences in protein stability, subcellular distribution, and/or RNA binding and virion packaging. Here, we report the first comparative biochemical studies of all the A3H variants using highly purified proteins. We show that all haplotypes were stably expressed and could be purified to homogeneity by Escherichia coli expression. Surprisingly, four out of the seven haplotypes showed high cytosine (C) deaminase activity, with hap V displaying extremely high activity that was comparable to the highly active A3A. Furthermore, all four haplotypes that were active in C deamination were also highly active on methylated C (mC), with hap II displaying almost equal deamination efficiency on both. The deamination activity of these A3H variants correlates well with their reported anti-HIV activity for the different haplotypes, suggesting that deaminase activity may be an important factor in determining their respective anti-HIV activities. Moreover, mC deamination of A3H displayed a strong preference for the sequence motif of T-mCpG-C/G, which may suggest a potential role in genomic mC modification at the characteristic "CpG" island motif.

  18. Variants of stretch-activated two-pore potassium channel TREK-1 associated with preterm labor in humans.

    PubMed

    Wu, Yi-Ying; Singer, Cherie A; Buxton, Iain L O

    2012-10-01

    Spontaneous preterm labor (PTL) is a uniquely human problem that results in preterm delivery of an underdeveloped fetus. The underlying cause remains elusive. The cost to societies in human suffering and treasure is enormous. The stretch-activated two pore potassium channel TREK-1 is up-regulated during gestation to term such that it may maintain uterine quiescence by hyperpolarizing the smooth muscle cell membrane. We have hypothesized that the human TREK-1 channel is involved in myometrial relaxation during pregnancy and that splice variants of the TREK-1 channel expressed in preterm myometrium are associated with preterm delivery by interaction with full-length TREK-1. We detected three wild-type human TREK-1 transcript isoforms in nonpregnant and pregnant human myometrium. Using RT-PCR, we identified five unique TREK-1 splice variants in myometrium from women in PTL. These myometrial TREK-1 variants lack either the pore or the transmembrane domains or both. In transiently transfected HEK293T cells, wild-type TREK-1 was predominantly expressed at the plasma membrane. However, individual splice variants were expressed uniformly throughout the cell. Wild-type TREK-1 was localized at the plasma membrane and cytoplasm close to the plasma membrane when coexpressed with each splice variant. Co-immunoprecipitation of FLAG epitope-tagged TREK-1 and six-His epitope-tagged splice variants using Ni bead columns successfully pulled down wild-type TREK-1. These results suggest that each of four TREK-1 splice variants interacts with full-length wild-type TREK-1 and that in vivo, such interactions may contribute to a PTL phenotype.

  19. Hypoxia reduces testosterone synthesis in mouse Leydig cells by inhibiting NRF1-activated StAR expression.

    PubMed

    Wang, Xueting; Pan, Longlu; Zou, Zhiran; Wang, Dan; Lu, Yapeng; Dong, Zhangji; Zhu, Li

    2017-03-07

    Male fertility disorders play a key role in half of all infertility cases. Reduction in testosterone induced by hypoxia might cause diseases in reproductive system and other organs. Hypoxic exposure caused a significant decrease of NRF1. Software analysis reported that the promoter region of steroidogenic acute regulatory protein (StAR) contained NRF1 binding sites, indicating NRF1 promoted testicular steroidogenesis. The purpose of this study is to determine NRF1 is involved in testosterone synthesis; and under hypoxia, the decrease of testosterone synthesis is caused by lower expression of NRF1. We designed both in vivo and in vitro experiments. Under hypoxia, the expressions of NRF1 in Leydig cells and testosterone level were significantly decreased both in vivo and in vitro. Overexpression and interference NRF1 could induced StAR and testosterone increased and decreased respectively. ChIP results confirmed the binding of NRF1 to StAR promoter region. In conclusion, decline of NRF1 expression downregulated the level of StAR, which ultimately resulted in a reduction in testosterone synthesis.

  20. 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

  1. Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responses.

    PubMed

    Zhang, Manchao; Deng, Youping; Tandon, Ruchi; Bai, Cheng; Riedel, Heimo

    2008-01-01

    The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin.

  2. Alzheimer's Disease Variants with the Genome-Wide Significance are Significantly Enriched in Immune Pathways and Active in Immune Cells.

    PubMed

    Jiang, Qinghua; Jin, Shuilin; Jiang, Yongshuai; Liao, Mingzhi; Feng, Rennan; Zhang, Liangcai; Liu, Guiyou; Hao, Junwei

    2017-01-01

    The existing large-scale genome-wide association studies (GWAS) datasets provide strong support for investigating the mechanisms of Alzheimer's disease (AD) by applying multiple methods of pathway analysis. Previous studies using selected single nucleotide polymorphisms (SNPs) with several thresholds of nominal significance for pathway analysis determined that the threshold chosen for SNPs can reflect the disease model. Presumably, then, pathway analysis with a stringent threshold to define "associated" SNPs would test the hypothesis that highly associated SNPs are enriched in one or more particular pathways. Here, we selected 599 AD variants (P < 5.00E-08) to investigate the pathways in which these variants are enriched and the cell types in which these variants are active. Our results showed that AD variants are significantly enriched in pathways of the immune system. Further analysis indicated that AD variants are significantly enriched for enhancers in a number of cell types, in particular the B-lymphocyte, which is the most substantially enriched cell type. This cell type maintains its dominance among the strongest enhancers. AD SNPs also display significant enrichment for DNase in 12 cell types, among which the top 6 significant signals are from immune cell types, including 4 B cells (top 4 significant signals) and CD14+ and CD34+ cells. In summary, our results show that these AD variants with P < 5.00E-08 are significantly enriched in pathways of the immune system and active in immune cells. To a certain degree, the genetic predisposition for development of AD is rooted in the immune system, rather than in neuronal cells.

  3. T-13910 DNA variant associated with lactase persistence interacts with Oct-1 and stimulates lactase promoter activity in vitro.

    PubMed

    Lewinsky, Rikke H; Jensen, Tine G K; Møller, Jette; Stensballe, Allan; Olsen, Jørgen; Troelsen, Jesper T

    2005-12-15

    Two phenotypes exist in the human population with regard to expression of lactase in adults. Lactase non-persistence (adult-type hypolactasia and lactose intolerance) is characterized by a decline in the expression of lactase-phlorizin hydrolase (LPH) after weaning. In contrast, lactase-persistent individuals have a high LPH throughout their lifespan. Lactase persistence and non-persistence are associated with a T/C polymorphism at position -13,910 upstream the lactase gene. A nuclear factor binds more strongly to the T-13,910 variant associated with lactase persistence than the C-13,910 variant associated with lactase non-persistence. Oct-1 and glyceraldehyde-3-phosphate dehydrogenase were co-purified by DNA affinity purification using the sequence of the T-13,910 variant. Supershift analyses show that Oct-1 binds directly to the T-13,910 variant, and we suggest that GAPDH is co-purified due to interactions with Oct-1. Expression of Oct-1 stimulates reporter gene expression from the T and the C-13,910 variant/LPH promoter constructs only when it is co-expressed with HNF1alpha. Binding sites for other intestinal transcription factors (GATA-6, HNF4alpha, Fox and Cdx-2) were identified in the region of the -13,910 T/C polymorphism. Three of these sites are required for the enhancer activity of the -13,910 region. The data suggest that the binding of Oct-1 to the T-13,910 variant directs increased lactase promoter activity and this might provide an explanation for the lactase persistence phenotype in the human population.

  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. Alzheimer disease-related presenilin-1 variants exert distinct effects on monoamine oxidase-A activity in vitro.

    PubMed

    Pennington, Paul R; Wei, Zelan; Rui, Lewei; Doig, Jennifer A; Graham, Brett; Kuski, Kelly; Gabriel, Geraldine G; Mousseau, Darrell D

    2011-07-01

    Monoamine oxidase-A (MAO-A) has been associated with both depression and Alzheimer disease (AD). Recently, carriers of AD-related presenilin-1 (PS-1) alleles have been found to be at higher risk for developing clinical depression. We chose to examine whether PS-1 could influence MAO-A function in vitro. Overexpression of selected AD-related PS-1 variants (wildtype, Y115H, ΔEx9 and M146V) in mouse hippocampal HT-22 cells affects MAO-A catalytic activity in a variant-specific manner. The ability of the PS-1 substrate-competitor DAPT to induce MAO-A activity in cells expressing either PS-1 wildtype or PS-1(M146V) suggests the potential for a direct influence of PS-1 on MAO-A function. In support of this, we were able to co-immunoprecipitate MAO-A with FLAG-tagged PS-1 wildtype and M146V proteins. This potential for a direct protein-protein interaction between PS-1 and MAO-A is not specific for HT-22 cells as we were also able to co-immunoprecipitate MAO-A with FLAG-PS-1 variants in N2a mouse neuroblastoma cells and in HEK293 human embryonic kidney cells. Finally, we demonstrate that the two PS-1 variants reported to be associated with an increased incidence of clinical depression [e.g., A431E and L235V] both induce MAO-A activity in HT-22 cells. A direct influence of PS-1 variants on MAO-A function could provide an explanation for the changes in monoaminergic tone observed in several neurodegenerative processes including AD. The ability to induce MAO-A catalytic activity with a PS-1/γ-secretase inhibitor should also be considered when designing secretase inhibitor-based therapeutics.

  6. Specific activation of the Bacillus quorum-sensing systems by isoprenylated pheromone variants.

    PubMed

    Ansaldi, Mireille; Marolt, Darja; Stebe, Tina; Mandic-Mulec, Ines; Dubnau, David

    2002-06-01

    Natural genetic competence in Bacillus subtilis is controlled by quorum-sensing (QS). The ComP- ComA two-component system detects the signalling molecule ComX, and this signal is transduced by a conserved phosphotransfer mechanism. ComX is synthesized as an inactive precursor and is then cleaved and modified by ComQ before export to the extracellular environment. The comQXP' loci of a set of natural Bacillus isolates have been sequenced and shown to possess a striking polymorphism that determines specific patterns of both activation and inhibition of the quorum-sensing response. We have developed a simple purification method for the modified peptide signalling pheromones allowing the characterization of four distinct ComX molecules representing different pherotypes. Genetic and biochemical evidence demonstrate that all the ComX variants are isoprenylated by the post-translational modification of a conserved tryptophan residue and that the modifications on the ComX peptide backbones vary in mass among the various pherotypes. These results give new insights into peptidemediated quorum-sensing signalling in Gram-positive bacteria and emphasize the role of isoprenylation in bacterial signal transduction.

  7. 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.

  8. Independent CMEs from a Single Solar Active Region - The Case of the Super-Eruptive NOAA AR11429

    NASA Astrophysics Data System (ADS)

    Chintzoglou, Georgios; Patsourakos, Spiros; Vourlidas, Angelos

    2014-06-01

    In this investigation we study AR 11429, the origin of the twin super-fast CME eruptions of 07-Mar-2012. This AR fulfills all the requirements for the 'perfect storm'; namely, Hale's law incompatibility and a delta-magnetic configuration. In fact, during its limb-to-limb transit, AR 11429 spawned several eruptions which caused geomagnetic storms, including the biggest in Cycle 24 so far. Magnetic Flux Ropes (MFRs) are twisted magnetic structures in the corona, best seen in ~10MK hot plasma emission and are often considered as the culprit causing such super-eruptions. However, their 'dormant' existence in the solar atmosphere (i.e. prior to eruptions), is a matter of strong debate. Aided by multi-wavelength and multi-spacecraft observations (SDO/HMI & AIA, HINODE/SOT/SP, STEREO B/EUVI) and by using a Non-Linear Force-Free (NLFFF) model for the coronal magnetic field, our work shows two separate, weakly-twisted magnetic flux systems which suggest the existence of possible pre-eruption MFRs.

  9. Identification of Novel Crk-associated Substrate (p130Cas) Variants with Functionally Distinct Focal Adhesion Kinase Binding Activities*

    PubMed Central

    Kumbrink, Joerg; Soni, Shefali; Laumbacher, Barbara; Loesch, Barbara; Kirsch, Kathrin H.

    2015-01-01

    Elevated levels of p130Cas (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1 gene) are associated with aggressiveness of breast tumors. Following phosphorylation of its substrate domain, p130Cas promotes the integration of protein complexes involved in multiple signaling pathways and mediates cell proliferation, adhesion, and migration. In addition to the known BCAR1-1A (wild-type) and 1C variants, we identified four novel BCAR1 mRNA variants, generated by alternative first exon usage (1B, 1B1, 1D, and 1E). Exons 1A and 1C encode for four amino acids (aa), whereas 1D and 1E encode for 22 aa and 1B1 encodes for 50 aa. Exon 1B is non-coding, resulting in a truncated p130Cas protein (Cas1B). BCAR1-1A, 1B1, and variant 1C mRNAs were ubiquitously expressed in cell lines and a survey of human tissues, whereas 1B, 1D, and 1E expression was more restricted. Reconstitution of all isoforms except for 1B in p130Cas-deficient murine fibroblasts induced lamellipodia formation and membrane ruffling, which was unrelated to the substrate domain phosphorylation status. The longer isoforms exhibited increased binding to focal adhesion kinase (FAK), a molecule important for migration and adhesion. The shorter 1B isoform exhibited diminished FAK binding activity and significantly reduced migration and invasion. In contrast, the longest variant 1B1 established the most efficient FAK binding and greatly enhanced migration. Our results indicate that the p130Cas exon 1 variants display altered functional properties. The truncated variant 1B and the longer isoform 1B1 may contribute to the diverse effects of p130Cas on cell biology and therefore will be the target of future studies. PMID:25805500

  10. Identification of Novel Crk-associated Substrate (p130Cas) Variants with Functionally Distinct Focal Adhesion Kinase Binding Activities.

    PubMed

    Kumbrink, Joerg; Soni, Shefali; Laumbacher, Barbara; Loesch, Barbara; Kirsch, Kathrin H

    2015-05-08

    Elevated levels of p130(Cas) (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1 gene) are associated with aggressiveness of breast tumors. Following phosphorylation of its substrate domain, p130(Cas) promotes the integration of protein complexes involved in multiple signaling pathways and mediates cell proliferation, adhesion, and migration. In addition to the known BCAR1-1A (wild-type) and 1C variants, we identified four novel BCAR1 mRNA variants, generated by alternative first exon usage (1B, 1B1, 1D, and 1E). Exons 1A and 1C encode for four amino acids (aa), whereas 1D and 1E encode for 22 aa and 1B1 encodes for 50 aa. Exon 1B is non-coding, resulting in a truncated p130(Cas) protein (Cas1B). BCAR1-1A, 1B1, and variant 1C mRNAs were ubiquitously expressed in cell lines and a survey of human tissues, whereas 1B, 1D, and 1E expression was more restricted. Reconstitution of all isoforms except for 1B in p130(Cas)-deficient murine fibroblasts induced lamellipodia formation and membrane ruffling, which was unrelated to the substrate domain phosphorylation status. The longer isoforms exhibited increased binding to focal adhesion kinase (FAK), a molecule important for migration and adhesion. The shorter 1B isoform exhibited diminished FAK binding activity and significantly reduced migration and invasion. In contrast, the longest variant 1B1 established the most efficient FAK binding and greatly enhanced migration. Our results indicate that the p130(Cas) exon 1 variants display altered functional properties. The truncated variant 1B and the longer isoform 1B1 may contribute to the diverse effects of p130(Cas) on cell biology and therefore will be the target of future studies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. The major reverse-transcriptase-incompetent splice variant of the human telomerase protein inhibits telomerase activity but protects from apoptosis

    PubMed Central

    Listerman, Imke; Sun, Jie; Gazzaniga, Francesca S.; Lukas, Jason L.; Blackburn, Elizabeth H.

    2013-01-01

    hTERT (TERT), the catalytic protein subunit of telomerase, is subjected to numerous alternative splicing events, but the regulation and function of these splice variants is obscure. Full-length hTERT includes conserved domains that encode reverse transcriptase activity, RNA binding and other functions. The major splice variant termed α+β− or β-deletion is highly expressed in stem and cancer cells, where it codes for a truncated protein lacking most of the reverse transcriptase domain but retaining the known RNA binding motifs. In a breast cancer cell panel, we found that β-deletion was the hTERT transcript that was most highly expressed. Splicing of this transcript was controlled by the splice regulators SRSF11, HNRNPH2 and HNRNPL and the β-deletion transcript variant was associated with polyribosomes in cells. When ectopically overexpressed, β-deletion protein competed for binding to hTR (TERC) RNA, thereby inhibiting endogenous telomerase activity. Overexpressed β-deletion protein localized to the nucleus and mitochondria and it protected breast cancer cells from cisplatin-induced apoptosis. Our results reveal that a major hTERT splice variant can confer a growth advantage to cancer cells independent of telomere maintenance, suggesting hTERT makes multiple contributions to cancer pathophysiology. PMID:23610451

  12. Discrimination of differentially inhibited cysteine proteases by activity-based profiling using cystatin variants with tailored specificities.

    PubMed

    Sainsbury, Frank; Rhéaume, Ann-Julie; Goulet, Marie-Claire; Vorster, Juan; Michaud, Dominique

    2012-12-07

    Recent research has shown the possibility of tailoring the inhibitory specificity of plant cystatins toward cysteine (Cys) proteases by single mutations at positively selected amino acid sites. Here we devised a cystatin activity-based profiling approach to assess the impact of such mutations at the proteome scale using single variants of tomato cystatin SlCYS8 and digestive Cys proteases of the herbivorous insect, Colorado potato beetle, as a model. Biotinylated forms of SlCYS8 and SlCYS8 variants were used to capture susceptible Cys proteases in insect midgut protein extracts by biotin immobilization on avidin-embedded beads. A quantitative LC-MS/MS analysis of the captured proteins was performed to compare the inhibitory profile of different SlCYS8 variants. The approach confirmed the relevance of phylogenetic inferences categorizing the insect digestive Cys proteases into six functionally distinct families. It also revealed significant variation in protease family profiles captured with N-terminal variants of SlCYS8, in line with in silico structural models for Cys protease-SlCYS8 interactions suggesting a functional role for the N-terminal region. Our data confirm overall the usefulness of cystatin activity-based protease profiling for the monitoring of Cys protease-inhibitor interactions in complex biological systems. They also illustrate the potential of biotinylated cystatins to identify recombinant cystatin candidates for the inactivation of specific Cys protease targets.

  13. Cardiac glycoside ouabain induces activation of ATF-1 and StAR expression by interacting with the α4 isoform of the sodium pump in Sertoli cells.

    PubMed

    Dietze, Raimund; Konrad, Lutz; Shihan, Mazen; Kirch, Ulrike; Scheiner-Bobis, Georgios

    2013-03-01

    Sertoli cells express α1 and α4 isoforms of the catalytic subunit of Na(+),K(+)-ATPase (sodium pump). Our recent findings demonstrated that interactions of the α4 isoform with cardiotonic steroids (CTS) like ouabain induce signaling cascades that resemble the so-called non-classical testosterone pathway characterized by activation of the c-Src/c-Raf/Erk1/2/CREB signaling cascade. Here we investigate a possible physiological significance of the activated cascade. The results obtained in the current investigation show that the ouabain-induced signaling cascade also leads to the activation of the CREB-related activating transcription factor 1 (ATF-1) in the Sertoli cell line 93RS2 in a concentration- and time-dependent manner, as demonstrated by detection of ATF-1 phosphorylated on Ser63 in western blots. The ouabain-activated ATF-1 protein was found to localize to the cell nuclei. The sodium pump α4 isoform mediates this activation, as it is ablated when cells are incubated with siRNA to the α4 isoform. Ouabain also leads to increased expression of steroidogenic acute regulator (StAR) protein, which has been shown to be a downstream consequence of CREB/ATF-1 activation. Taking into consideration that CTS are most likely produced endogenously, the demonstrated induction of StAR expression by ouabain establishes a link between CTS, the α4 isoform of the sodium pump, and steroidogenesis crucial for male fertility and reproduction.

  14. Tsukamurella paurometabola lipoglycan, a new lipoarabinomannan variant with pro-inflammatory activity.

    PubMed

    Gibson, Kevin J C; Gilleron, Martine; Constant, Patricia; Brando, Thérèse; Puzo, Germain; Besra, Gurdyal S; Nigou, Jérôme

    2004-05-28

    The genus Tsukamurella is a member of the phylogenetic group nocardioform actinomycetes and is closely related to the genus Mycobacterium. The mycobacterial cell envelope contains lipoglycans, and of particular interest is lipoarabinomannan, one of the most potent mycobacterial immunomodulatory molecules. We have investigated the presence of lipoglycans in Tsukamurella paurometabola and report here the isolation and structural characterization of a new lipoarabinomannan variant, designated TpaLAM. Matrix-assisted laser desorption ionization-mass spectrometric analysis revealed that TpaLAM had an average molecular mass of 12.5 kDa and consequently was slightly smaller than Mycobacterium tuberculosis lipoarabinomannan. Using a range of chemical degradations, NMR experiments, capillary electrophoresis, and mass spectrometry analyses, TpaLAM revealed an original carbohydrate structure. Indeed, TpaLAM contained a mannosylphosphatidyl-myo-inositol (MPI) anchor glycosylated by a linear (alpha1-->6)-Manp mannan domain, which is further substituted by an (alpha1-->5)-Araf chain. Half of the Araf units are further substituted at the O-2 position by a Manp-(alpha1-->2)-Manp-(alpha1--> dimannoside motif. Altogether, TpaLAM appears to be the most elaborated non-mycobacterial LAM molecule identified to date. TpaLAM was found to induce the pro-inflammatory cytokine tumor necrosis factor (TNF)-alpha when tested with either human or murine monocyte/macrophage cell lines. This induction was completely abrogated in the presence of an anti-toll-like receptor-2 (TLR-2) antibody, suggesting that TLR-2 participates in the mediation of TNF-alpha production in response to TpaLAM. Moreover, we established that the lipomannan core of TpaLAM is the primary moiety responsible for the observed TNF-alpha-inducing activity. This conclusively demonstrates that a linear (alpha1-->6)-Manp chain, linked to the MPI anchor, is sufficient in providing pro-inflammatory activity.

  15. Functional analysis of TMLH variants and definition of domains required for catalytic activity and mitochondrial targeting.

    PubMed

    Monfregola, Jlenia; Cevenini, Armando; Terracciano, Antonio; van Vlies, Naomi; Arbucci, Salvatore; Wanders, Ronald J A; D'Urso, Michele; Vaz, Frédéric M; Ursini, Matilde Valeria

    2005-09-01

    epsilon-N-Trimethyllysine hydroxylase (TMLH) (EC 1.14.11.8) is a non-heme-ferrous iron hydroxylase, Fe(++) and 2-oxoglutarate (2OG) dependent, catalyzing the first of four enzymatic reactions of the highly conserved carnitine biosynthetic pathway. Otherwise from all the other enzymes of carnitine biosynthesis, TMLH was found to be associated to the mitochondrial fraction. We here report molecular cloning of two alternative spliced forms of TMLH, which appear ubiquitously expressed in human adult and fetal tissues. The deduced proteins are designated TMLH-a and TMLH-b, and contain 421 and 399 amino acids, respectively. They share the first N-terminal 332 amino acids, including a mitochondrial targeting signal, but diverge at the C-terminal end. TMLH-a and TMLH-b exogenous expression in COS-1 cells shows that the first 15 amino acids are necessary and sufficient for mitochondrial import. Furthermore, comparative evolutionary analysis of the C-terminal portion of TMLH-a identifies a conserved domain characterized by a key triad of residues, His242-Glu244-His389 predicted to bind 2OG end. This sequence is conserved in the TMLH enzyme from all species but is partially substituted by a unique sequence in the TMLH-b variant. Indeed, TMLH-b is not functional by itself as well as a TMLH-H389L mutant produced by site directed mutagenesis. As great interest, we found that TMLH-b and TMLH-H389L, individually co-expressed with TMLH-a in COS-1 cells, negatively affect TMLH activity. Therefore, our studies on the TMLH alternative form provide relevant novel information, first that the C-terminal region of TMLH contains the main determinants for its enzymatic activity including a key H389 residue, and second that TMLH-b could act as a crucial physiological negative regulator of TMLH. Copyright 2005 Wiley-Liss, Inc.

  16. 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.

  17. ArArCALC—software for 40Ar/ 39Ar age calculations

    NASA Astrophysics Data System (ADS)

    Koppers, Anthony A. P.

    2002-06-01

    ArArCALC is a Microsoft Excel ® 97-2000-XP application for performing calculations in 40Ar/ 39Ar geochronology. It is coded in Visual Basic for Applications and can be used under the Windows ® 95/98/NT/2000/ME/XP operating systems. ArArCALC provides an easy-to-use graphical interface for the calculation of age plateaus, total fusion ages and isochrons following the regression of 40Ar/ 39Ar mass spectrometry data. Results are stored in single Excel workbooks including nine different data tables and four different diagrams. Analytical, internal and external errors are calculated based on error propagation of all input parameters, analytical data and applied corrections. Finally, the age calculation results can be recalibrated with reference to the primary K-Ar standards (e.g. GA-1550, MMhb-1) in order to obtain more consistent absolute40Ar/ 39Ar age determinations. ArArCALC is distributed as freeware.

  18. Induced Systemic Resistance against Botrytis cinerea by Bacillus cereus AR156 through a JA/ET- and NPR1-Dependent Signaling Pathway and Activates PAMP-Triggered Immunity in Arabidopsis.

    PubMed

    Nie, Pingping; Li, Xia; Wang, Shune; Guo, Jianhua; Zhao, Hongwei; Niu, Dongdong

    2017-01-01

    Induced resistance response is a potent and cost effective plant defense against pathogen attack. The effectiveness and underlying mechanisms of the suppressive ability by Bacillus cereus AR156 to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in Arabidopsis has been investigated previously; however, the strength of induced systemic resistance (ISR) activity against Botrytis cinerea remains unknown. Here, we show that root-drench application of AR156 significantly reduces disease incidence through activation of ISR. This protection is accompanied with multilayered ISR defense response activated via enhanced accumulation of PR1 protein expression in a timely manner, hydrogen peroxide accumulation and callose deposition, which is significantly more intense in plants with both AR156 pretreatment and B. cinerea inoculation than that in plants with pathogen inoculation only. Moreover, AR156 can trigger ISR in sid2-2 and NahG mutants, but not in jar1, ein2 and npr1 mutant plants. Our results indicate that AR156-induced ISR depends on JA/ET-signaling pathway and NPR1, but not SA. Also, AR156-treated plants are able to rapidly activate MAPK signaling and FRK1/WRKY53 gene expression, both of which are involved in pathogen associated molecular pattern (PAMP)-triggered immunity (PTI). The results indicate that AR156 can induce ISR by the JA/ET-signaling pathways in an NPR1-dependent manner and involves multiple PTI components.

  19. Induced Systemic Resistance against Botrytis cinerea by Bacillus cereus AR156 through a JA/ET- and NPR1-Dependent Signaling Pathway and Activates PAMP-Triggered Immunity in Arabidopsis

    PubMed Central

    Nie, Pingping; Li, Xia; Wang, Shune; Guo, Jianhua; Zhao, Hongwei; Niu, Dongdong

    2017-01-01

    Induced resistance response is a potent and cost effective plant defense against pathogen attack. The effectiveness and underlying mechanisms of the suppressive ability by Bacillus cereus AR156 to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in Arabidopsis has been investigated previously; however, the strength of induced systemic resistance (ISR) activity against Botrytis cinerea remains unknown. Here, we show that root-drench application of AR156 significantly reduces disease incidence through activation of ISR. This protection is accompanied with multilayered ISR defense response activated via enhanced accumulation of PR1 protein expression in a timely manner, hydrogen peroxide accumulation and callose deposition, which is significantly more intense in plants with both AR156 pretreatment and B. cinerea inoculation than that in plants with pathogen inoculation only. Moreover, AR156 can trigger ISR in sid2-2 and NahG mutants, but not in jar1, ein2 and npr1 mutant plants. Our results indicate that AR156-induced ISR depends on JA/ET-signaling pathway and NPR1, but not SA. Also, AR156-treated plants are able to rapidly activate MAPK signaling and FRK1/WRKY53 gene expression, both of which are involved in pathogen associated molecular pattern (PAMP)-triggered immunity (PTI). The results indicate that AR156 can induce ISR by the JA/ET-signaling pathways in an NPR1-dependent manner and involves multiple PTI components. PMID:28293243

  20. 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

  1. Characterization of antibacterial and hemolytic activity of synthetic pandinin 2 variants and their inhibition against Mycobacterium tuberculosis.

    PubMed

    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.

  2. 20(S)-Protopanaxadiol-aglycone Downregulation of the Full-length and Splice Variants of Androgen Receptor

    PubMed Central

    Cao, Bo; Liu, Xichun; Li, Jing; Liu, Shuang; Qi, Yanfeng; Xiong, Zhenggang; Zhang, Allen; Wiese, Thomas; Fu, Xueqi; Gu, Jingkai; Rennie, Paul S.; Sartor, Oliver; Lee, Benjamin R.; Ip, Clement; Zhao, Lijuan; Zhang, Haitao; Dong, Yan

    2012-01-01

    As a public health problem, prostate cancer engenders huge economic and life-quality burden. Developing effective chemopreventive regimens to alleviate the burden remains a major challenge. Androgen signaling is vital to the development and progression of prostate cancer. Targeting androgen signaling via blocking the production of the potent ligand dihydrotestosterone has been shown to decrease prostate cancer incidence. However, the potential of increasing the incidence of high-grade prostate cancers has been a concern. Mechanisms of disease progression after the intervention may include increased expression of androgen receptor (AR) in prostate tissue and expression of the constitutively-active AR splice variants (AR-Vs) lacking the ligand-binding domain. Thus, novel agents targeting the receptor, preferentially both the full-length and AR-Vs, are urgently needed. In the present study, we show that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) effectively downregulates the expression and activity of both the full-length AR and AR-Vs. The effects of PPD on AR and AR-Vs are manifested by an immediate drop in proteins followed by a reduction in transcripts, attributed to PPD induction of proteasome-mediated degradation and inhibition of the transcription of the AR gene. We further show that although PPD inhibits the growth as well as AR expression and activity in LNCaP xenograft tumors, the morphology and AR expression in normal prostates are not affected. This study is the first to show that PPD suppresses androgen signaling through downregulating both the full-length AR and AR-Vs, and provides strong rationale for further developing PPD as a promising agent for the prevention and/or treatment of prostate cancer. PMID:22907191

  3. H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions.

    PubMed

    Jin, Chunyuan; Zang, Chongzhi; Wei, Gang; Cui, Kairong; Peng, Weiqun; Zhao, Keji; Felsenfeld, Gary

    2009-08-01

    To understand how chromatin structure is organized by different histone variants, we have measured the genome-wide distribution of nucleosome core particles (NCPs) containing the histone variants H3.3 and H2A.Z in human cells. We find that a special class of NCPs containing both variants is enriched at 'nucleosome-free regions' of active promoters, enhancers and insulator regions. We show that preparative methods used previously in studying nucleosome structure result in the loss of these unstable double-variant NCPs. It seems likely that this instability facilitates the access of transcription factors to promoters and other regulatory sites in vivo. Other combinations of variants have different distributions, consistent with distinct roles for histone variants in the modulation of gene expression.

  4. A combination of maternal histone variants and chaperones promotes paternal genome activation and boosts somatic cell reprogramming

    PubMed Central

    Yang, Peng; Wu, Warren; Macfarlan, Todd S.

    2015-01-01

    The mammalian egg employs a wide spectrum of epigenome modification machinery to reprogram the sperm nucleus shortly after fertilization. This event is required for transcriptional activation of the paternal/zygotic genome and progression through cleavage divisions. Reprogramming of paternal nuclei requires replacement of sperm protamines with canonical and non-canonical histones, covalent modification of histone tails, and chemical modification of DNA (notably oxidative demethylation of methylated cytosines). In this essay we highlight the role maternal histone variants play during developmental reprogramming following fertilization. We discuss how reduced maternal histone variant incorporation in somatic nuclear transfer experiments may explain the reduced viability of resulting embryos and how knowledge of repressive and activating maternal factors may be used to improve somatic cell reprogramming. PMID:25328107

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

    PubMed

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

    2017-09-14

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

  6. Multimodal actions of the phytochemical sulforaphane suppress both AR and AR-V7 in 22Rv1 cells: Advocating a potent pharmaceutical combination against castration-resistant prostate cancer.

    PubMed

    Khurana, Namrata; Kim, Hogyoung; Chandra, Partha K; Talwar, Sudha; Sharma, Pankaj; Abdel-Mageed, Asim B; Sikka, Suresh C; Mondal, Debasis

    2017-08-30

    Prostate cancer (PCa) cells expressing full-length androgen receptor (AR-FL) are susceptible to androgen deprivation therapy (ADT). However, outgrowth of castration-resistant prostate cancer (CRPC) can occur due to the expression of constitutively active (ligand-independent) AR splice variants, particularly AR-V7. We previously demonstrated that sulforaphane (SFN), an isothiocyanate phytochemical, can decrease AR-FL levels in the PCa cell lines, LNCaP and C4-2B. Here, we examined the efficacy of SFN in targeting both AR-FL and AR-V7 in the CRPC cell line, CWR22Rv1 (22Rv1). MTT cell viability, wound-heal assay, and colony forming unit (CFU) measurements revealed that 22Rv1 cells are resistant to the anti-androgen, enzalutamide (ENZ). However, co-exposure to SFN sensitized these cells to the potent anticancer effects of ENZ (P<0.05). Immunoblot analyses showed that SFN (5-20 µM) rapidly decreases both AR-FL and AR-V7 levels, and immunofluorescence microscopy (IFM) depicted decreased AR in both cytoplasm and nucleus with SFN treatment. SFN increased both ubiquitination and proteasomal activity in 22Rv1 cells. Studies using a protein synthesis inhibitor (cycloheximide) or a proteasomal inhibitor (MG132) indicated that SFN increases both ubiquitin-mediated aggregation and subsequent proteasomal-degradation of AR proteins. Previous studies reported that SFN inhibits the chaperone activity of heat-shock protein 90 (Hsp90) and induces the nuclear factor erythroid-2-like 2 (Nrf2) transcription factor. Therefore, we investigated whether the Hsp90 inhibitor, ganetespib (G) or the Nrf2 activator, bardoxolone methyl (BM) can similarly suppress AR levels in 22Rv1 cells. Low doses of G and BM, alone or in combination, decreased both AR-FL and AR-V7 levels, and combined exposure to G+BM sensitized 22Rv1 cells to ENZ. Therefore, adjunct treatment with the phytochemical SFN or a safe pharmaceutical combination of G+BM may be effective against CRPC cells, especially those

  7. 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

  8. Red-cell GSH regeneration and glutathione reductase activity in G6PD variants in the Ferrara area.

    PubMed

    Anderson, B B; Carandina, G; Lucci, M; Perry, G M; Vullo, C

    1987-12-01

    Red-cell studies were carried out on three groups of G6PD-deficient subjects with different G6PD variants from the Ferrara area of Northern Italy. Red-cell GSH and activities of G6PD, glutathione reductase (GR), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were measured. A method was developed to measure red-cell GSH regeneration after oxidation of endogenous GSH in whole blood by diamide and only this clearly distinguished the variants from each other and from normal. Regeneration by 1 h was lowest in the Mediterranean variant, 0-10.2% in contrast to 93-98% in normal. A predisposition to a haemolytic crisis after ingestion of fava beans was not clearcut, but subjects appeared to be at risk if GSH regeneration at 1 h was less than 30% of the endogenous level, and red-cell FAD+ was very high indicated by high in vitro GR activity and inhibition by added FAD+. It is suggested that the most informative tests in G6PD deficiency are measurements of GSH regeneration in intact red cells plus GR activity and/or red-cell flavin compounds.

  9. Effect of cultivation conditions on the activity of the beromycin producer Streptomyces glomeratus 3980 and its spontaneous variants.

    PubMed

    Blumauerová, M; Podojil, M; Gauze, G F; Maksimova, T S; Panos, J; Vanĕk, Z

    1980-01-01

    Optimal conditions for the submerged cultivation of Streptomyces glomeratus 3980, producer of the anthracycline antibiotics beromycins, and its variants were sought in media with glucose, soybean meal and salts differing in the content of ammonium sulphate. As compared with the original activity of the strain the antibiotic titre of some variants increased about 12 times on increasing the glucose concenration from 3 to 5%, or on omitting CaCO3 from the medium (i.e. under conditions leading to an increased production of propionic acid and suppression of production of the melanin-like pigment). In melanin-less variants accumulating propionate even under standard conditions the activity increased about 18-40 times in the medium with 3% glucose and 0.2% CaCO3 under conditions of more intensive aeration (i.e. under conditions when no propionic acid accumulated). Individual strains also differed in the requirement for (NH4)2SO4 in the medium, their response to changes of volume of the vegetative inoculum and semsitivity to MgSO4 x 7H2O. The biosynthetic activity of all strains was inhibited by the addition of ZnSO4 x 7H2O or CaCl2 and substitution of glucose with starch, lactose or sucrose.

  10. 39Ar/Ar measurements using ultra-low background proportional counters

    SciTech Connect

    Hall, Jeter C.; Aalseth, Craig E.; Bonicalzi, Ricco; Brandenberger, Jill M.; Day, Anthony R.; Humble, Paul H.; Mace, Emily K.; Panisko, Mark E.; Seifert, Allen

    2016-01-08

    Age dating groundwater and seawater using 39Ar/Ar ratios is an important tool to understand water mass flow rates and mean residence time. For modern or contemporary argon, the 39Ar activity is 1.8 mBq per liter of argon. Radiation measurements at these activity levels require ultra low-background detectors. Low-background proportional counters have been developed at Pacific Northwest National Laboratory. These detectors use traditional mixtures of argon and methane as counting gas, and the residual 39Ar from commercial argon has become a predominant source of background activity in these detectors. We demonstrated sensitivity to 39Ar by using geological or ancient argon from gas wells in place of commercial argon. The low level counting performance of these proportional counters is then demonstrated for sensitivities to 39Ar/Ar ratios sufficient to date water masses as old as 1000 years.

  11. 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

  12. One-pot N-arylation of indoles directly from N-arylsulfonylindoles via consecutive deprotection and S(N)Ar Reactions with activated Aryl halides.

    PubMed

    Xu, Hui; Fan, Ling-Ling

    2009-03-01

    An efficient one-pot step by step t-BuOK-mediated procedure for the synthesis of N-arylindoles has been developed in moderate to good yields. The protocol involves the consecutive deprotection of N-arylsulfonylindoles as latent indoles and subsequent S(N)Ar reactions with activated aryl halides. This tandem reaction affords an efficient and convenient preparation of N-arylindoles that benefit from prior indoles protection by arylsulfonyl group, and can shorten a reaction sequence and improve synthetic efficiency.

  13. ACK1/TNK2 Regulates Histone H4 Tyr88-phosphorylation and AR Gene Expression in Castration-Resistant Prostate Cancer.

    PubMed

    Mahajan, Kiran; Malla, Pavani; Lawrence, Harshani R; Chen, Zhihua; Kumar-Sinha, Chandan; Malik, Rohit; Shukla, Sudhanshu; Kim, Jongphil; Coppola, Domenico; Lawrence, Nicholas J; Mahajan, Nupam P

    2017-06-12

    The androgen receptor (AR) is critical for the progression of prostate cancer to a castration-resistant (CRPC) state. AR antagonists are ineffective due to their inability to repress the expression of AR or its splice variant, AR-V7. Here, we report that the tyrosine kinase ACK1 (TNK2) phosphorylates histone H4 at tyrosine 88 upstream of the AR transcription start site. The WDR5/MLL2 complex reads the H4-Y88-phosphorylation marks and deposits the transcriptionally activating H3K4-trimethyl marks promoting AR transcription. Reversal of the pY88-H4 epigenetic marks by the ACK1 inhibitor (R)-9bMS-sensitized naive and enzalutamide-resistant prostate cancer cells and reduced AR and AR-V7 levels to mitigate CRPC tumor growth. Thus, a feedforward ACK1/pY88-H4/WDR5/MLL2/AR epigenetic circuit drives CRPC and is necessary for maintenance of the malignant state. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Genetic Characterization of Esterase Activity Variants Associated with an Esterase Gene Amplification in a Strain of Culex pipiens from California.

    PubMed

    Ferrari, James A

    2015-03-01

    In the Culex pipiens complex, a common mechanism of insecticide resistance is amplification of esterase genes leading to overproduction of detoxifying esterase enzymes. A number of electrophoretic esterase alleles have been identified, and in field populations individuals with the same esterase electromorph can exhibit a wide range of esterase enzyme activities. We isolated and characterized esterase activity variants associated with the esterase B1 electromorph from a field strain. A mating scheme was used to isolate chromosomes with esterase genes from the strain into 45 families. Twenty-six of the families received esterase genes from the field strain that conferred elevated esterase activity. Mean esterase activities in these families ranged from 43 to 695 nmoles α-naphthyl acetate hydrolyzed/min/mg protein. Variance components indicated that genetic variance (i.e., genetic differences among families) accounted for 77% of the total variation in esterase activity. A comparison of mean esterase activities indicated that there were at least 11 different esterase activity variants contributing to the observed genetic variation in esterase activity among the 26 families. The relevance of these results to understanding the dynamics of amplified esterase genes in populations is discussed.

  15. 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}.

  16. Differences in Transcriptional Activation by the Two Allelic (L162V Polymorphic) Variants of PPARα after Omega-3 Fatty Acids Treatment

    PubMed Central

    Rudkowska, Iwona; Verreault, Mélanie; Barbier, Olivier; Vohl, Marie-Claude

    2009-01-01

    Omega-3 fatty acids (FAs) have the potential to regulate gene expression via the peroxisome proliferator-activated receptor α (PPARα); therefore, genetic variations in this gene may impact its transcriptional activity on target genes. It is hypothesized that the transcriptional activity by wild-type L162-PPARα is enhanced to a greater extent than the mutated variant (V162-PPARα) in the presence of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or a mixture of EPA:DHA. To examine the functional difference of the two allelic variants on receptor activity, transient co-transfections were performed in human hepatoma HepG2 cells activated with EPA, DHA and EPA:DHA mixtures. Results indicate that the addition of EPA or DHA demonstrate potential to increase the transcriptional activity by PPARα with respect to basal level in both variants. Yet, the EPA:DHA mixtures enhanced the transcriptional activity to a greater extent than individual FAs indicating possible additive effects of EPA and DHA. Additionally, the V162 allelic form of PPARα demonstrated consistently lower transcriptional activation when incubated with EPA, DHA or EPA:DHA mixtures than, the wild-type variant. In conclusion, both allelic variants of the PPARα L162V are activated by omega-3 FAs; however, the V162 allelic form displays a lower transcriptional activity than the wild-type variant. PMID:19266045

  17. The concept and the variants of the activities deployment on the Moon research and exploration

    NASA Astrophysics Data System (ADS)

    Ivanov, Victor; Lukiyashchenko, Vasily; Uspenskiy, Georgy; Tselin, Andrey

    The article presents the concept and the variants of the advanced Lunar program realization for the period up to 2050, oriented to the Moon investigation and its further exploration. The basic problems are considered decided by space and Lunar means for the Moon study, their creation technical problems as well as the preliminary technical and economical cost estimations of Lunar research and exploration phases.

  18. Early stellate cell activation and veno-occlusive-disease (VOD)-like hepatotoxicity in dogs treated with AR-H047108, an imidazopyridine proton pump inhibitor.

    PubMed

    Berg, Anna-Lena; Böttcher, Gerhard; Andersson, Kjell; Carlsson, Enar; Lindström, Anna-Karin; Huby, Russell; Håkansson, Helen; Skånberg-Wilhelmsson, Inger; Hellmold, Heike

    2008-07-01

    Dogs treated with AR-H047108, an imidazopyridine potassium competitive acid blocker (P-CAB), developed clinical signs of hepatic dysfunction as well as morphologically manifest hepatotoxicity in repeat-dose toxicity studies. An investigative one-month study was performed, with interim euthanasia after one and two weeks. A detailed histopathological and immunohistochemical characterization of the liver lesions was conducted, including markers for fibrosis, Kupffer cell activation, apoptosis, and endothelial injury. In addition, hepatic retinoid and procollagen 1alpha2 mRNA levels in livers of dogs treated with AR-H047108 were analyzed. The results showed an early inflammatory process in central veins and centrilobular areas, present after one week of treatment. This inflammatory reaction was paralleled by activation of stellate/Ito cells to myofibroblasts and was associated with sinusoidal and centrivenular fibrosis. The early activation of stellate cells coincided with a significant decrease in retinyl ester levels, and a significant increase in procollagen 1alpha2 mRNA levels, in the liver. At later time points (three and six months), there was marked sinusoidal fibrosis in centrilobular areas, as well as occlusion of central veins resulting from a combination of fibrosis and increased thickness of smooth muscle bundles in the vessel wall. The pattern of lesions suggests a veno-occlusive-disease (VOD)-like scenario, possibly linked to the imidazopyridine chemical structure of the compound facilitated by specific morphological features of the dog liver.

  19. 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. Copyright © 2014. Published by Elsevier Inc.

  20. Functional analyses for tRNase Z variants: an aspartate and a histidine in the active site are essential for the catalytic activity.

    PubMed

    Elbarbary, Reyad A; Takaku, Hiroaki; Nashimoto, Masayuki

    2008-12-01

    We performed functional analyses for various single amino-acid substitution variants of Escherichia coli, Bacillus subtilis, and human tRNase Zs. The well-conserved six histidine, His(I)-His(VI), and two aspartate, Asp(I) and Asp(II), residues together with metal ions are thought to form the active site of tRNase Z. The Mn(2+)-rescue analysis for Thermotoga maritima tRNase Z(S) has suggested that Asp(I) and His(V) directly contribute the proton transfer for the catalysis, and a catalytic mechanism has been proposed. However, experimental evidence supporting the proposed mechanism was limited. Here we intensively examined E. coli and B. subtilis tRNase Z(S) variants and human tRNase Z(L) variants for cleavage activities on pre-tRNAs in the presence of Mg(2+) or Mn(2+) ions. We observed that the Mn(2+) ions cannot rescue the activities of Asp(I)Ala and His(V)Ala variants from each species, which are lost in the presence of Mg(2+). This observation may support the proposed catalytic mechanism.

  1. 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%.

  2. Genetic analysis of four novel peroxisome proliferator activated receptor-γ splice variants in monkey macrophages

    PubMed Central

    Zhou, Jiming; Wilson, Katina M.; Medh, Jheem D.

    2009-01-01

    Peroxisome proliferator activated receptor-γ(PPAR-γ) is abundantly expressed in atherosclerotic lesions and is implicated in atherogenesis. The existence of three splice variants, PPAR-γ1, PPAR-γ2, and PPAR-γ3 has been established. Using monocyte-derived macrophages from cynomolgus monkeys, we demonstrate here the identification of two new PPAR-γ exons, exon C and exon D, which splice together with already established exons A1, A2, and B in the 5′ terminal region to generate four novel PPAR-γ subtypes, PPAR-γ4, -γ5, -γ6, and -γ7. PPAR-γ4 and γ5 were detected only in macrophages whereas γ6 and γ7 were expressed both in macrophages and adipose tissues. None of these novel isoforms were detected in muscle, kidney, and spleen from monkeys. We found sequences identical to exons C and D in the human genome database. These and all PPAR-γ exons known to date are encoded by a single gene, located from region 10498 K to 10384 K on human chromosome 3. We cloned and expressed PPAR-γ1, PPAR-γ4, and PPAR-γ5 proteins in yeast using the expression vector pPICZB. As expected, all recombinant proteins showed a molecular weight of approximately 50 kDa. We also investigated the effect of a high-fat diet on the level of macrophage PPAR-γ expression in monkeys. RT-PCR showed a significant increase in total PPAR-γ and ABCA1 mRNA levels in macrophages of fat-fed monkeys (n = 7) compared to those maintained on a normal diet (n = 2). However, none of the novel isoforms seemed to be induced by fat-feeding. We used tetracycline-responsive expression vectors to obtain moderate expression of PPAR-γ4 and -γ5 in CHO cells. In these cells, expression of PPAR-γ5 but not -γ4 repressed the expression of ABCA1. Neither isoform modulated the expression of lipoprotein lipase. Our results suggest that individual PPAR-γ isoforms may be responsible for unique tissue-specific biological effects and that PPAR-γ4 and -γ5 may modulate macrophage function and atherogenesis. PMID

  3. Genetic variants in the ADAMTS13 and SUPT3H genes are associated with ADAMTS13 activity.

    PubMed

    de Vries, Paul S; Boender, Johan; Sonneveld, Michelle A H; Rivadeneira, Fernando; Ikram, M Arfan; Rottensteiner, Hanspeter; Hofman, Albert; Uitterlinden, André G; Leebeek, Frank W G; Franco, Oscar H; Dehghan, Abbas; de Maat, Moniek P M

    2015-06-18

    A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) cleaves von Willebrand factor, reducing its prothrombotic activity. The genetic determinants of ADAMTS13 activity remain unclear. We performed a genome-wide association study of ADAMTS13 activity in the Rotterdam Study, a population-based cohort study. We used imputed genotypes of common variants in a discovery sample of 3443 individuals and replication sample of 2025 individuals. We examined rare exonic variant associations in ADAMTS13 in 1609 individuals using an exome array. rs41314453 in ADAMTS13 was associated with ADAMTS13 activity in both our discovery (β, -20.2%; P = 1.3 × 10(-33)) and replication sample (P = 3.3 × 10(-34)), and explained 3.6% to 6.5% of the variance. In the combined analysis of our discovery and replication samples, there were 2 further independent associations at the ADAMTS13 locus: rs3118667 (β, 3.0; P = 9.6 × 10(-21)) and rs139911703 (β, -11.6; P = 3.6 × 10(-8)). In addition, rs10456544 in SUPT3H was associated with a 4.2 increase in ADAMTS13 activity (P = 1.13.6 × 10(-8)). Finally, we found 3 independent associations with rare coding variants in ADAMTS13: rs148312697 (β, -32.2%; P = 3.7 × 10(-6)), rs142572218 (β, -46.0%; P = 3.9 × 10(-5)), and rs36222275 (β, -13.9%; P = 2.9 × 10(-3)). In conclusion, we identified rs41314453 as the main genetic determinant of ADAMTS13 activity, and we present preliminary findings for further associations at the ADAMTS13 and SUPT3H loci.

  4. Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant.

    PubMed

    Hamasy, A; Wang, Q; Blomberg, K E M; Mohammad, D K; Yu, L; Vihinen, M; Berglöf, A; Smith, C I E

    2017-01-01

    Irreversible Bruton tyrosine kinase (BTK) inhibitors, ibrutinib and acalabrutinib have demonstrated remarkable clinical responses in multiple B-cell malignancies. Acquired resistance has been identified in a sub-population of patients in which mutations affecting BTK predominantly substitute cysteine 481 in the kinase domain for catalytically active serine, thereby ablating covalent binding of inhibitors. Activating substitutions in the BTK substrate phospholipase Cγ2 (PLCγ2) instead confers resistance independent of BTK. Herein, we generated all six possible amino acid substitutions due to single nucleotide alterations for the cysteine 481 codon, in addition to threonine, requiring two nucleotide substitutions, and performed functional analysis. Replacement by arginine, phenylalanine, tryptophan or tyrosine completely inactivated the catalytic activity, whereas substitution with glycine caused severe impairment. BTK with threonine replacement was catalytically active, similar to substitution with serine. We identify three potential ibrutinib resistance scenarios for cysteine 481 replacement: (1) Serine, being catalytically active and therefore predominating among patients. (2) Threonine, also being catalytically active, but predicted to be scarce, because two nucleotide changes are needed. (3) As BTK variants replaced with other residues are catalytically inactive, they presumably need compensatory mutations, therefore being very scarce. Glycine and tryptophan variants were not yet reported but likely also provide resistance.

  5. Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant

    PubMed Central

    Hamasy, A; Wang, Q; Blomberg, K E M; Mohammad, D K; Yu, L; Vihinen, M; Berglöf, A; Smith, C I E

    2017-01-01

    Irreversible Bruton tyrosine kinase (BTK) inhibitors, ibrutinib and acalabrutinib have demonstrated remarkable clinical responses in multiple B-cell malignancies. Acquired resistance has been identified in a sub-population of patients in which mutations affecting BTK predominantly substitute cysteine 481 in the kinase domain for catalytically active serine, thereby ablating covalent binding of inhibitors. Activating substitutions in the BTK substrate phospholipase Cγ2 (PLCγ2) instead confers resistance independent of BTK. Herein, we generated all six possible amino acid substitutions due to single nucleotide alterations for the cysteine 481 codon, in addition to threonine, requiring two nucleotide substitutions, and performed functional analysis. Replacement by arginine, phenylalanine, tryptophan or tyrosine completely inactivated the catalytic activity, whereas substitution with glycine caused severe impairment. BTK with threonine replacement was catalytically active, similar to substitution with serine. We identify three potential ibrutinib resistance scenarios for cysteine 481 replacement: (1) Serine, being catalytically active and therefore predominating among patients. (2) Threonine, also being catalytically active, but predicted to be scarce, because two nucleotide changes are needed. (3) As BTK variants replaced with other residues are catalytically inactive, they presumably need compensatory mutations, therefore being very scarce. Glycine and tryptophan variants were not yet reported but likely also provide resistance. PMID:27282255

  6. Differences between the internal energy depositions induced by collisional activation and by electron transfer of W(CO)62+ ions on collision with Ar and K targets

    NASA Astrophysics Data System (ADS)

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

    2006-06-01

    Doubly charged tungsten hexacarbonyl W(CO )62+ 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)2+ and W(CO )3+ 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, 7.8eV below the energy level of the W(CO )62+ 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 5.9×10-8cm between a W(CO )62+ ion and a K atom, and the coulombic repulsion between singly charged ions in the exit channel. A large cross section of 1.1×10-14cm2 was estimated for electron capture of the doubly charged W(CO )62+ 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 differences in the observed ions and in their intensities with Ar and K targets.

  7. PSM/SH2B1 splice variants: critical role in src catalytic activation and the resulting STAT3s-mediated mitogenic response.

    PubMed

    Zhang, Manchao; Deng, Youping; Riedel, Heimo

    2008-05-01

    A role of PSM/SH2B1 had been shown in mitogenesis and extending to phenotypic cell transformation, however, the underlying molecular mechanism remained to be established. Here, four alternative PSM splice variants and individual functional protein domains were compared for their role in the regulation of Src activity. We found that elevated cellular levels of PSM variants resulted in phenotypic cell transformation and potentiated cell proliferation and survival in response to serum withdrawal. PSM variant activity presented a consistent signature pattern for any tested response of highest activity observed for gamma, followed by delta, alpha, and beta with decreasing activity. PSM-potentiated cell proliferation was sensitive to Src inhibitor herbimycin and PSM and Src were found in the same immune complex. PSM variants were substrates of the Src Tyr kinase and potentiated Src catalytic activity by increasing the V(max) and decreasing the K(m) for ATP with the signature pattern of variant activity. Dominant-negative PSM peptide mimetics including the SH2 or PH domains inhibited Src catalytic activity as well as Src-mediated phenotypic cell transformation. Activation of major Src substrate STAT3 was similarly potentiated by the PSM variants in a Src-dependent fashion or inhibited by PSM domain-specific peptide mimetics. Expression of a dominant-negative STAT3 mutant blocked PSM variant-mediated phenotypic cell transformation. Our results implicate an essential role of the PSM variants in the activation of the Src kinase and the resulting mitogenic response--extending to phenotypic cell transformation and involving the established Src substrate STAT3.

  8. 4G/5G variant of plasminogen activator inhibitor-1 gene and severe pregnancy-induced hypertension: subgroup analyses of variants of angiotensinogen and endothelial nitric oxide synthase.

    PubMed

    Kobashi, Gen; Ohta, Kaori; Yamada, Hideto; Hata, Akira; Minakami, Hisanori; Sakuragi, Noriaki; Tamashiro, Hiko; Fujimoto, Seiichiro

    2009-01-01

    Pregnancy-induced hypertension (PIH) is a common cause of perinatal mortality. It is believed to result from the interaction of several factors, including those related to the blood coagulation system. We performed genotyping and subgroup analyses to determine if the 4G/5G genotypes of the plasminogen activator inhibitor-1 gene (PAI-1) play a role in the pathogenesis of PIH, and to evaluate possible interactions of the PAI-1 polymorphisms with those of the angiotensinogen gene (AGT) and the endothelial nitric oxide synthase gene (NOS3). An association study of PAI-1 polymorphism, and subgroup analyses of common variants of AGT and NOS3, among 128 patients with PIH and 376 healthy pregnant controls. No significant differences were found between the cases and controls in the frequencies of allele 4G or the 4G/4G genotype. In subgroup analyses, after adjustment for multiple comparison, a significant association with the AGT TT genotype was found among women with the PAI-1 4G/4G genotype, and an association with the NOS3 GA+AA genotype was found among women with the 5G/5G or 4G/5G genotypes. Our findings suggest that there are at least 2 pathways in the pathogenesis of severe PIH. However, with respect to early prediction and prevention of severe PIH, although the PAI-1 4G/4G genotype alone was not a risk factor for severe PIH, the fact that PAI-1 genotypes are associated with varying risks for severe PIH suggests that PAI-1 genotyping of pregnant women, in combination with other tests, may be useful in the development of individualized measures that may prevent severe PIH.

  9. Redesigning the active site of transaldolase TalB from Escherichia coli: new variants with improved affinity towards nonphosphorylated substrates.

    PubMed

    Schneider, Sarah; Gutiérrez, Mariana; Sandalova, Tatyana; Schneider, Gunter; Clapés, Pere; Sprenger, Georg A; Samland, Anne K

    2010-03-22

    Recently, we reported on a transaldolase B variant (TalB F178Y) that is able to use dihydroxyacetone (DHA) as donor in aldol reactions. In a second round of protein engineering, we aimed at improving the affinity of this variant towards nonphosphorylated acceptor aldehydes, that is, glyceraldehyde (GA). The anion binding site was identified in the X-ray structure of TalB F178Y where a sulfate ion from the buffer was bound in the active site. Therefore, we performed site-directed saturation mutagenesis at three residues forming the putative phosphate binding site, Arg181, Ser226 and Arg228. The focused libraries were screened for the formation of D-fructose from DHA and d,l-GA by using an adjusted colour assay. The best results with respect to the synthesis of D-fructose were achieved with the TalB F178Y/R181E variant, which exhibited an at least fivefold increase in affinity towards d,l-GA (K(M)=24 mM). We demonstrated that this double mutant can use D-GA, glycolaldehyde and the L-isomer, L-GA, as acceptor substrates. This resulted in preparative synthesis of D-fructose, D-xylulose and L-sorbose when DHA was used as donor. Hence, we engineered a DHA-dependent aldolase that can synthesise the formation of polyhydroxylated compounds from simple and cheap substrates at preparative scale.

  10. ACTH Action on StAR Biology

    PubMed Central

    Clark, Barbara J.

    2016-01-01

    Adrenocorticotropin hormone (ACTH) produced by the anterior pituitary stimulates glucocorticoid synthesis by the adrenal cortex. The first step in glucocorticoid synthesis is the delivery of cholesterol to the mitochondrial matrix where the first enzymatic reaction in the steroid hormone biosynthetic pathway occurs. A key response of adrenal cells to ACTH is activation of the cAMP-protein kinase A (PKA) signaling pathway. PKA activation results in an acute increase in expression and function of the Steroidogenic Acute Regulatory protein (StAR). StAR plays an essential role in steroidogenesis- it controls the hormone-dependent movement of cholesterol across the mitochondrial membranes. Currently StAR's mechanism of action remains a major unanswered question in the field. However, some insight may be gained from understanding the mechanism(s) controlling the PKA-dependent phosphorylation of StAR at S194/195 (mouse/human StAR), a modification that is required for function. This mini-review provides a background on StAR's biology with a focus on StAR phosphorylation. The model for StAR translation and phosphorylation at the outer mitochondrial membrane, the location for StAR function, is presented to highlight a unifying theme emerging from diverse studies. PMID:27999527

  11. ACTH Action on StAR Biology.

    PubMed

    Clark, Barbara J

    2016-01-01

    Adrenocorticotropin hormone (ACTH) produced by the anterior pituitary stimulates glucocorticoid synthesis by the adrenal cortex. The first step in glucocorticoid synthesis is the delivery of cholesterol to the mitochondrial matrix where the first enzymatic reaction in the steroid hormone biosynthetic pathway occurs. A key response of adrenal cells to ACTH is activation of the cAMP-protein kinase A (PKA) signaling pathway. PKA activation results in an acute increase in expression and function of the Steroidogenic Acute Regulatory protein (StAR). StAR plays an essential role in steroidogenesis- it controls the hormone-dependent movement of cholesterol across the mitochondrial membranes. Currently StAR's mechanism of action remains a major unanswered question in the field. However, some insight may be gained from understanding the mechanism(s) controlling the PKA-dependent phosphorylation of StAR at S194/195 (mouse/human StAR), a modification that is required for function. This mini-review provides a background on StAR's biology with a focus on StAR phosphorylation. The model for StAR translation and phosphorylation at the outer mitochondrial membrane, the location for StAR function, is presented to highlight a unifying theme emerging from diverse studies.

  12. Plasminogen activator inhibitor 1 4G/5G and -844G/A variants in idiopathic recurrent pregnancy loss.

    PubMed

    Magdoud, Kalthoum; Herbepin, Viviana G; Touraine, Renaud; Almawi, Wassim Y; Mahjoub, Touhami

    2013-09-01

    Plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolysis, and the common promoter region variants -675G/A (4G/5G) and -844G/A are associated with increased thrombotic risk. Despite evidence linking altered fibrinolysis with adverse pregnancy events, including idiopathic recurrent pregnancy loss (RPL), the contribution of PAI-1 variants to RPL risk remains controversial. We investigated the association between the PAI-1 -844G/A and 4G/5G (-675G/A) variants with altered risk of RPL. This was a case-control study involving 304 women with confirmed RPL and 371 age- and ethnically matched control women. PAI-1 genotyping was performed by PCR single-specific primer -675 (G/A) and real-time PCR (-844G/A) analysis. Minor allele frequency (MAF) of 4G/5G (P < 0.001), but not -844G/A (P = 0.507), was higher in RPL cases. PAI-1 4G/5G single-nucleotide polymorphism (SNP) was significantly associated with RPL under additive, dominant, and recessive genetic models; no association of -844G/A with RPL was seen irrespective of the genetic model tested. Taking common -844G/5G haplotype as reference (OR = 1.00), multivariate analysis confirmed the association of 4G-containing -844A/4G (P < 0.001) and -844G/4G (P = 0.011) haplotypes with increased RPL risk. 4G/5G, but not -844G/A, PAI-1 variant is associated with an increased risk of RPL. © 2013 John Wiley & Sons Ltd.

  13. 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

  14. Expression of Androgen Receptor Splice Variants in Prostate Cancer Bone Metastases is Associated with Castration-Resistance and Short Survival

    PubMed Central

    Hörnberg, Emma; Ylitalo, Erik Bovinder; Crnalic, Sead; Antti, Henrik; Stattin, Pär; Widmark, Anders; Bergh, Anders; Wikström, Pernilla

    2011-01-01

    Background Constitutively active androgen receptor variants (AR-V) lacking the ligand binding domain (LBD) may promote the development of castration-resistant prostate cancer (CRPC). The expression of AR-Vs in the clinically most important metastatic site, the bone, has, however, not been well documented. Our aim was therefore to compare levels of AR-Vs in hormone-naive (HN) and CRPC bone metastases in comparison to primary PC and non-malignant prostate tissue, as well as in relation to AR protein expression, whole-genome transcription profiles and patient survival. Methodology/Principal Findings Hormone-naïve (n = 10) and CRPC bone metastases samples (n = 30) were obtained from 40 patients at metastasis surgery. Non-malignant and malignant prostate samples were acquired from 13 prostatectomized men. Levels of full length AR (ARfl) and AR-Vs termed AR-V1, AR-V7, and AR-V567es mRNA were measured with RT-PCR and whole-genome transcription profiles with an Illumina Beadchip array. Protein levels were examined by Western blotting and immunohistochemistry. Transcripts for ARfl, AR-V1, and AR-V7 were detected in most primary tumors and metastases, and levels were significantly increased in CRPC bone metastases. The AR-V567es transcript was detected in 23% of the CRPC bone metastases only. A sub-group of CRPC bone metastases expressed LBD-truncated AR proteins at levels comparable to the ARfl. Detectable AR-V567es and/or AR-V7 mRNA in the upper quartile, seen in 1/3 of all CRPC bone metastases, was associated with a high nuclear AR immunostaining score, disturbed cell cycle regulation and short survival. Conclusions/Significance Expression of AR-Vs is increased in CRPC compared to HN bone metastases and associated with a particularly poor prognosis. Further studies are needed to test if patients expressing such AR-Vs in their bone metastases benefit more from drugs acting on or down-stream of these AR-Vs than from therapies inhibiting androgen synthesis. PMID

  15. Role of active species in surface cleaning by an Ar-N2 atmospheric pressure post-discharge

    NASA Astrophysics Data System (ADS)

    Belmonte, T.; Thiébaut, J. M.; Mézerette, D.

    2002-08-01

    Post-discharge cleaning at atmospheric pressure of oxidized iron foils rinsed by acetone and methanol is studied by XPS. The influence of the temperature (T<450 K) and the UV photons on cleaning is negligible. When pure rare gases (He, Ne, Ar) are used, metastable species in post-discharge are transported downstream the plasma and relax their energy on the surface. C-C bonds are mainly removed by this process. When nitrogen is added to the rare gas (from 0 to 4.6 vol.% ), a selective influence on the etching process of the surface contaminants by nitrogen atoms is observed. The removal kinetics of chemical groups analysed by XPS is determined by using an exponential decay function corresponding to a first-order abstraction process. The decrease of C(1s) in N-C, O(1s) in N-C-O and N(1s) XPS peaks is correlated with the nitrogen atom concentration in the carrier gas. Reactions between nitrogen atoms and specific carbon containing groups occur (probably C-OH bonds of methanol). Stronger bonds like C=O (probably from acetone) are not removed by the post-discharge.

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

    PubMed

    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.

  17. 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

  18. Autonomic control network active in Aplysia during locomotion includes neurons that express splice variants of R15-neuropeptides.

    PubMed

    Romanova, Elena V; McKay, Natasha; Weiss, Klaudiusz R; Sweedler, Jonathan V; Koester, John

    2007-01-01

    Splice-variant products of the R15 neuropeptide gene are differentially expressed within the CNS of Aplysia. The goal of this study was to test whether the neurons in the abdominal ganglion that express the peptides encoded by this gene are part of a common circuit. Expression of R15 peptides had been demonstrated previously in neuron R15. Using a combination of immunocytochemical and analytical methods, this study demonstrated that R15 peptides are also expressed in heart exciter neuron RB(HE), the two L9(G) gill motoneurons, and L40--a newly identified interneuron. Mass spectrometric profiling of individual neurons that exhibit R15 peptide-like immunoreactivity confirmed the mutually exclusive expression of two splice-variant forms of R15 peptides in different neurons. The L9(G) cells were found to co-express pedal peptide in addition to the R15 peptides. The R15 peptide-expressing neurons examined here were shown to be part of an autonomic control circuit that is active during fictive locomotion. Activity in this circuit contributes to implementing a central command that may help to coordinate autonomic activity with escape locomotion. Chronic extracellular nerve recording was used to determine the activity patterns of a subset of neurons of this circuit in vivo. These results demonstrate the potential utility of using shared patterns of neuropeptide expression as a guide for neural circuit identification.

  19. HES6 drives a critical AR transcriptional programme to induce castration-resistant prostate cancer through activation of an E2F1-mediated cell cycle network

    PubMed Central

    Ramos-Montoya, Antonio; Lamb, Alastair D; Russell, Roslin; Carroll, Thomas; Jurmeister, Sarah; Galeano-Dalmau, Nuria; Massie, Charlie E; Boren, Joan; Bon, Helene; Theodorou, Vasiliki; Vias, Maria; Shaw, Greg L; Sharma, Naomi L; Ross-Adams, Helen; Scott, Helen E; Vowler, Sarah L; Howat, William J; Warren, Anne Y; Wooster, Richard F; Mills, Ian G; Neal, David E

    2014-01-01

    Castrate-resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c-Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co-factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up-regulated in aggressive human prostate cancer and drives castration-resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6-associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient-specific therapeutic strategies. PMID:24737870

  20. A novel variant of androgen receptor is associated with idiopathic azoospermia.

    PubMed

    Mou, Lisha; Gui, Yaoting

    2016-10-01

    A variety of genetic variants can lead to abnormal human spermatogenesis. The androgen receptor (AR) is an important steroid hormone receptor that is critical for male sexual differentiation and the maintenance of normal spermatogenesis. In the present study, each exon of AR in 776 patients diagnosed with idiopathic azoospermia (IA) and 709 proven fertile men were sequenced using use panel re‑sequencing methods to examine whether AR is involved in the pathogenesis of IA. Two synonymous variants and seven missense variants were detected. Of the missense variants, a luciferase assay demonstrated that the R630W variant reduced the transcriptional regulatory function of AR. This novel variant (p. R630W) of AR is the first to be identified in association with IA, thereby highlighting the importance of AR during spermatogenesis.

  1. A novel variant of androgen receptor is associated with idiopathic azoospermia

    PubMed Central

    Mou, Lisha; Gui, Yaoting

    2016-01-01

    A variety of genetic variants can lead to abnormal human spermatogenesis. The androgen receptor (AR) is an important steroid hormone receptor that is critical for male sexual differentiation and the maintenance of normal spermatogenesis. In the present study, each exon of AR in 776 patients diagnosed with idiopathic azoospermia (IA) and 709 proven fertile men were sequenced using use panel re-sequencing methods to examine whether AR is involved in the pathogenesis of IA. Two synonymous variants and seven missense variants were detected. Of the missense variants, a luciferase assay demonstrated that the R630W variant reduced the transcriptional regulatory function of AR. This novel variant (p. R630W) of AR is the first to be identified in association with IA, thereby highlighting the importance of AR during spermatogenesis. PMID:27498682

  2. Active cigarette smoking, variants in carcinogen metabolism genes and breast cancer risk among pre- and postmenopausal women in Ontario, Canada.

    PubMed

    Cotterchio, Michelle; Mirea, Lucia; Ozcelik, Hilmi; Kreiger, Nancy

    2014-01-01

    Cigarette smoking is strongly associated with various diseases including many cancers; however, evidence regarding breast cancer risk remains inconclusive with some studies reporting no association, and others an increased risk with long duration and early initiation of smoking. Genetic variation in carcinogen-metabolizing enzymes may modify these associations. Breast cancer cases were identified from the Ontario Cancer Registry (OCR) during 2003-2004 and population controls through random digit dialing methods. All subjects completed self-administered questionnaires. Subsequently, saliva samples were obtained from cases (N = 1,776) and controls (N = 1,839) for deoxyribonucleic acid (DNA) extraction. Multivariate logistic regression was used to estimate odds ratio (OR) and 95% confidence intervals (CI) for active smoking variables, and interactions were assessed between smoking and 36 carcinogen-metabolizing candidate gene variants. No statistically significant association was found between active smoking and breast cancer risk among all women nor when stratified by menopausal status; however, nonsignificant increased premenopausal breast cancer risk was observed among current smokers and women smoking before first pregnancy. Several statistically significant interactions were observed between smoking and genetic variants (CYP1A2 1548C>T, CYP1A1 3801T>C, CYP1B1 4326G>C, NAT1 c.-85-1014T>A, UGT1A7 W208R 622T>C, SOD2 c.47T>C, GSTT1 deletion). However, in analyses stratified by these genotypes, smoking ORs had wide confidence intervals (and with few exceptions included 1.0) making interpretations difficult. Active smoking was not associated with breast cancer risk, although several significant interactions were observed between smoking, carcinogen-metabolizing genetic variants, and breast cancer risk. © 2014 Wiley Periodicals, Inc.

  3. AR-v7 liquid biopsy for treatment stratification in prostate cancer: how close are we?

    PubMed

    Fletcher, Claire

    2017-09-01

    Recent clinical introduction of the novel antiandrogen, Enzalutamide (Enza), CYP17 inhibitor, Abiraterone (Abi), and the second-generation chemotherapeutic, Cabazitaxel, has increased survival of patients with advanced, metastatic castration-resistant prostate cancer (mCRPC). However, de novo and acquired resistance rates are high. A liquid biopsy that can rapidly, sensitively and robustly identify which patients will respond to treatment in a minimally invasive manner is urgently required to permit switch to a potentially more efficacious drug regimen, thus increasing survival whilst avoiding debilitating side effects associated with unnecessary treatment. This review will highlight recent developments in detection of AR-v7 in circulating mRNA/whole blood and circulating tumour cells (CTCs) as a liquid biopsy for patient-stratification in mCRPC. Continued androgen receptor (AR) activity in mCRPC has been linked to the expression of a number of truncated but constitutively active AR isoforms. One such variant, AR-v7, can drive drug resistance in preclinical models and is correlated with disease progression whilst showing dynamic response to AR-targeting treatments when assessed in blood. It has thus been proposed as an Abi/Enza treatment-response biomarker. AR-v7 liquid biopsy has the potential to transform clinical management of mCRPC and increase patient survival. This review will explore recent efforts to validate AR-v7 as a robust, clinically informative biomarker. I will also address potential limitations of detection and quantification that could frustrate its adoption into routine clinical practise.

  4. 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.

  5. 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.

  6. Identification of a truncated alternative splicing variant of human PPAR{gamma}1 that exhibits dominant negative activity

    SciTech Connect

    Kim, Hyo Jung; Woo, Im Sun; Kang, Eun Sil; Eun, So Young; Kim, Hye Jung; Lee, Jae Heun; Chang, Ki Churl; Kim, Jin-Hoi; Seo, Han Geuk . E-mail: hgseo@gnu.ac.kr

    2006-09-01

    We have identified a novel variant of human peroxisome proliferator-activated receptor gamma (hPPAR{gamma}), derived from insertion of a novel exon 3'. Insertion leads to the introduction of a premature stop codon, resulting in the formation of a truncated splice variant of PPAR{gamma}1 (PPAR{gamma}1{sub tr}). Western blot analysis confirmed the presence of PPAR{gamma}1{sub tr} in tumor-derived cell lines. Although PPAR{gamma}1{sub tr} interfered with transcriptional activity of wild-type PPAR{gamma}1 (PPAR{gamma}1{sub wt}), activity could be rescued by cotransfection with a vector expressing p300. Overexpression of PPAR{gamma}1{sub tr} protein in CHO cells greatly enhanced their proliferation and anchorage-independent colony growth on soft agar. These data demonstrate that PPAR{gamma}1{sub tr} is an important physiologic isoform of PPAR{gamma} that modulates cellular functions of PPAR{gamma}1{sub wt}.

  7. Identification of a splice variant of mouse TRPA1 that regulates TRPA1 activity.

    PubMed

    Zhou, Yiming; Suzuki, Yoshiro; Uchida, Kunitoshi; Tominaga, Makoto

    2013-01-01

    Transient receptor potential ankyrin 1 (TRPA1) protein is a nonselective cation channel. Although many studies suggest that TRPA1 is involved in inflammatory and neuropathic pain, its mechanism remains unclear. Here we identify an alternative splice variant of the mouse Trpa1 gene. TRPA1a (full-length) and TRPA1b (splice variant) physically interact with each other and TRPA1b increases the expression of TRPA1a in the plasma membrane. TRPA1a and TRPA1b co-expression significantly increases current density in response to different agonists without affecting their single-channel conductance. Exogenous overexpression of Trpa1b gene in wild-type and TRPA1KO DRG neurons also increases TRPA1a-mediated AITC responses. Moreover, expression levels of Trpa1a and Trpa1b mRNAs change dynamically in two pain models (complete Freund's adjuvant-induced inflammatory pain and partial sciatic nerve ligation-induced neuropathic pain models). These results suggest that TRPA1 may be regulated through alternative splicing under these pathological conditions.

  8. Calpain and AR-V7: Two potential therapeutic targets to overcome acquired docetaxel resistance in castration-resistant prostate cancer cells.

    PubMed

    Liu, Lei; Lou, Ning; Li, Xiang; Xu, Guanghua; Ruan, Hailong; Xiao, Wen; Qiu, Bin; Bao, Lin; Yuan, Changfei; Huang, Xinmian; Wang, Keshan; Cao, Qi; Chen, Ke; Yang, Hongmei; Zhang, Xiaoping

    2017-06-01

    Docetaxel-based chemotherapy has been widely used as the first-line treatment for castration-resistant prostate cancer (CRPC) patients. However, the mechanisms of docetaxel-resistance remain unclear. In the present study with the establishment of 2 in vitro models of docetaxel-resistant CRPC cell sublines, we firstly reported that activation of calpain may play a promotional role in the resistance of docetaxel in prostate cancer, meanwhile using the calpain inhibitor combined with docetaxel improved the efficiency of docetaxel in docetaxel-resistant cell sublines. Moreover, we also found that the expression of androgen-independent constitutively and transcriptionally active androgen receptor splice variant-7 (AR-V7) remained high in the docetaxel-resistant CRPC cell subline Rv1-DR, and that it may be involved in acquired docetaxel-resistance of CRPC. However, a novel importin-β inhibitor (importazole) was only capable of slightly decreasing the transcriptional activity of the AR signaling pathway via blocking nuclear import of AR-FL and various non-specific AR-Vs, instead of AR-V7. These findings suggest that calpain and AR-V7 may serve as important biomarkers in the treatment of CRPC, and targeting calpain and AR-V7 may provide a new approach in overcoming docetaxel-resistance.

  9. Truncated Peroxisome Proliferator-Activated Receptor-γ Coactivator 1α Splice Variant Is Severely Altered in Huntington's Disease

    PubMed Central

    Johri, Ashu; Starkov, Anatoly A.; Chandra, Abhishek; Hennessey, Thomas; Sharma, Abhijeet; Orobello, Sara; Squitieri, Ferdinando; Yang, Lichuan; Beal, M. Flint

    2011-01-01

    Background Reduced peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) gene expression has been observed in striatal cell lines, transgenic mouse models of Huntington's disease (HD), and brain tissue from HD patients. As this protein is a key transcription regulator of the expression of many mitochondrial proteins, these observations strongly support the role of aberrant mitochondrial function in the pathogenesis of HD. The PGC1α protein undergoes posttranslational modifications that affect its transcriptional activity. The N-truncated splice variant of PGC1α (NT-PGC1α) is produced in tissues, but the role of truncated splice variants of PGC1α in HD and in the regulation of mitochondrial gene expression has not been elucidated. Objective To examine the expression and modulation of expression of NT-PGC1α levels in HD. Methods and Results We found that the NT-PGC1α protein, a splice variant of ∼38 kDa, but not full-length PGC1α is severely and consistently altered in human HD brain, human HD myoblasts, mouse HD models, and HD striatal cells. NT-PGC1α levels were significantly upregulated in HD cells and mouse brown fat by physiologically relevant stimuli that are known to upregulate PGC1α gene expression. This resulted in an increase in mitochondrial gene expression and cytochrome c content. Conclusion Our data suggest that NT-PGC1α is an important component of the PGC1α transcriptional network, which plays a significant role in the pathogenesis of HD. Copyright © 2011 S. Karger AG, Basel PMID:21757867

  10. Conditional trimerization and lytic activity of HIV-1 gp41 variants containing the membrane-associated segments.

    PubMed

    Dai, Zhou; Tao, Yisong; Liu, Nina; Brenowitz, Michael D; Girvin, Mark E; Lai, Jonathan R

    2015-03-03

    Fusion of host and viral membranes is a critical step during infection by membrane-bound viruses. The HIV-1 glycoproteins gp120 (surface subunit) and gp41 (fusion subunit) represent the prototypic system for studying this process; in the prevailing model, the gp41 ectodomain forms a trimeric six-helix bundle that constitutes a critical intermediate and provides the energetic driving force for overcoming barriers associated with membrane fusion. However, most structural studies of gp41 variants have been performed either on ectodomain constructs lacking one or more of the membrane-associated segments (the fusion peptide, FP, the membrane-proximal external region, MPER, and the transmembrane domain, TM) or on variants consisting of these isolated segments alone without the ectodomain. Several recent reports have suggested that the HIV-1 ectodomain, as well as larger construct containing the membrane-bound segments, dissociates from a trimer to a monomer in detergent micelles. Here we compare the properties of a series of gp41 variants to delineate the roles of the ectodomain, FP, and MPER and TM, all in membrane-mimicking environments. We find that these proteins are prone to formation of a monomer in detergent micelles. In one case, we observed exclusive monomer formation at pH 4 but conditional trimerization at pH 7 even at low micromolar (∼5 μM) protein concentrations. Liposome release assays demonstrate that these gp41-related proteins have the capacity to induce content leakage but that this activity is also strongly modulated by pH with much higher activity at pH 4. Circular dichroism, nuclear magnetic resonance, and binding assays with antibodies specific to the MPER provide insight into the structural and functional roles of the FP, MPER, and TM and their effect on structure within the larger context of the fusion subunit.

  11. Truncated peroxisome proliferator-activated receptor-γ coactivator 1α splice variant is severely altered in Huntington's disease.

    PubMed

    Johri, Ashu; Starkov, Anatoly A; Chandra, Abhishek; Hennessey, Thomas; Sharma, Abhijeet; Orobello, Sara; Squitieri, Ferdinando; Yang, Lichuan; Beal, M Flint

    2011-01-01

    Reduced peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) gene expression has been observed in striatal cell lines, transgenic mouse models of Huntington's disease (HD), and brain tissue from HD patients. As this protein is a key transcription regulator of the expression of many mitochondrial proteins, these observations strongly support the role of aberrant mitochondrial function in the pathogenesis of HD. The PGC1α protein undergoes posttranslational modifications that affect its transcriptional activity. The N-truncated splice variant of PGC1α (NT-PGC1α) is produced in tissues, but the role of truncated splice variants of PGC1α in HD and in the regulation of mitochondrial gene expression has not been elucidated. To examine the expression and modulation of expression of NT-PGC1α levels in HD. We found that the NT-PGC1α protein, a splice variant of ∼38 kDa, but not full-length PGC1α is severely and consistently altered in human HD brain, human HD myoblasts, mouse HD models, and HD striatal cells. NT-PGC1α levels were significantly upregulated in HD cells and mouse brown fat by physiologically relevant stimuli that are known to upregulate PGC1α gene expression. This resulted in an increase in mitochondrial gene expression and cytochrome c content. Our data suggest that NT-PGC1α is an important component of the PGC1α transcriptional network, which plays a significant role in the pathogenesis of HD. Copyright © 2011 S. Karger AG, Basel.

  12. 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.

  13. CX4945 suppresses the growth of castration-resistant prostate cancer cells by reducing AR-V7 expression.

    PubMed

    Deng, Chuangzhong; Chen, Jieping; Guo, Shengjie; Wang, Yanjun; Zhou, Qianghua; Li, Zaishang; Yang, Xingping; Yu, Xingsu; Zhang, Zhenfeng; Zhou, Fangjian; Han, Hui; Yao, Kai

    2017-08-01

    The aberrant expression of casein kinase 2 (CK2) has been reported to be involved in the tumorigenesis and progression of prostate cancer. The inhibition of CK2 activity represses androgen-dependent prostate cancer cells by attenuating the androgen receptor (AR) signaling pathway. In this study, we examined the effect of CK2 inhibition in castration-resistant prostate cancer (CRPC) cells, in which AR variants (ARVs) play a predominant role. A newly synthetic CK2 selective inhibitor CX4945 was utilized to study the effect of CK2 inhibition in CRPC cells by CCK8 assay and colony formation assay. Protein and mRNA levels of full-length AR (AR-FL) and AR-V7 were determined by qPCR and western blot, respectively. The nuclear translocation of p50 and p65 was assessed to reflect the activity of the NF-κB pathway. CX4945 reduced the proliferation of CRPC cells in a dose-dependent and time-dependent manner. AR-V7 rather than AR-FL was downregulated by CX4945 in both the mRNA and protein level. Furthermore, CX4945 could restore the sensitivity of CRPC cells to bicalutamide. The analysis of possible mechanisms demonstrated that the inhibition of CK2 diminished the phosphorylation of p65 at ser529 and thus attenuated the activity of the NF-κB pathway. The inhibition of CK2 by CX4945 can repress the viability of CRPC cells and restore their sensitivity to anti-androgen therapy by suppressing AR-V7. This finding presents a potential option for the treatment of prostate cancer, especially CRPC.

  14. Genetic variants in AVPR1A linked to autism predict amygdala activation and personality traits in healthy humans.

    PubMed

    Meyer-Lindenberg, A; Kolachana, B; Gold, B; Olsh, A; Nicodemus, K K; Mattay, V; Dean, M; Weinberger, D R

    2009-10-01

    In mammals, the neuropeptide vasopressin is a key molecule for complex emotional and social behaviours. Two microsatellite polymorphisms, RS1 and RS3, near the promoter of AVPR1A, encoding the receptor subtype most heavily implicated in behaviour regulation, have been linked to autism and behavioural traits. However, the impact of these variants on human brain function is unknown. Here we show that human amygdala function is strongly associated with genetic variation in AVPR1A. Using an imaging genetics approach in a sample of 121 volunteers studied with an emotional face-matching paradigm, we found that differential activation of amygdala is observed in carriers of risk alleles for RS3 and RS1. Alleles in RS1 previously reported to be significantly over- and undertransmitted to autistic probands showed opposing effects on amygdala activation. Furthermore, we show functional difference in human brain between short and long repeat lengths that mirror findings recently obtained in a corresponding variant in voles. Our results indicate a neural mechanism mediating genetic risk for autism through an impact on amygdala signalling and provide a rationale for exploring therapeutic strategies aimed at abnormal amygdala function in this disorder.

  15. A risk variant for alcoholism in the NMDA receptor affects amygdala activity during fear conditioning in humans.

    PubMed

    Cacciaglia, Raffaele; Nees, Frauke; Pohlack, Sebastian T; Ruttorf, Michaela; Winkelmann, Tobias; Witt, Stephanie H; Nieratschker, Vanessa; Rietschel, Marcella; Flor, Herta

    2013-09-01

    People at high risk for alcoholism show deficits in aversive learning, as indicated by impaired electrodermal responses during fear conditioning, a basic form of associative learning that depends on the amygdala. A positive family history of alcohol dependence has also been related to decreased amygdala responses during emotional processing. In the present study we report reduced amygdala activity during the acquisition of conditioned fear in healthy carriers of a risk variant for alcoholism (rs2072450) in the NR2A subunit-containing N-methyl-d-aspartate (NMDA)-receptor. These results indicate that rs2072450 might confer risk for alcohol dependence through deficient fear acquisition indexed by a diminished amygdala response during aversive learning, and provide a neural basis for a weak behavioral inhibition previously documented in individuals at high risk for alcohol dependence. Carriers of the risk variant additionally exhibit dampened insula activation, a finding that further strengthens our data, given the importance of this brain region in fear conditioning. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. MED1 mediates androgen receptor splice variant induced gene expression in the absence of ligand

    PubMed Central

    Liu, Gang; Sprenger, Cynthia; Wu, Pin-Jou; Sun, Shihua; Uo, Takuma; Haugk, Kathleen; Epilepsia, Kathryn Soriano; Plymate, Stephen

    2015-01-01

    The appearance of constitutively active androgen receptor splice variants (AR-Vs) has been proposed as one of the causes of castration-resistant prostate cancer (CRPC). However, the underlying mechanism of AR-Vs in CRPC transcriptional regulation has not been defined. A distinct transcriptome enriched with cell cycle genes, e.g. UBE2C, has been associated with AR-Vs, which indicates the possibility of an altered transcriptional mechanism when compared to full-length wild-type AR (ARfl). Importantly, a recent study reported the critical role of p-MED1 in enhancing UBE2C expression through a locus looping pattern, which only occurs in CRPC but not in androgen-dependent prostate cancer (ADPC). To investigate the potential correlation between AR-V and MED1, in the present study we performed protein co-immunoprecipitation, chromatin immunoprecipitation, and cell proliferation assays and found that MED1 is necessary for ARv567es induced UBE2C up-regulation and subsequent prostate cancer cell growth. Furthermore, p-MED1 is bound to ARv567es independent of full-length AR; p-MED1 has higher recruitment to UBE2C promoter and enhancer regions in the presence of ARv567es. Our data indicate that p-MED1 serves as a key mediator in ARv567es induced gene expression and suggests a mechanism by which AR-Vs promote the development and progression of CRPC. PMID:25481872

  17. Species-specific activity of HIV-1 Vpu and positive selection of tetherin transmembrane domain variants.

    PubMed

    McNatt, Matthew W; Zang, Trinity; Hatziioannou, Theodora; Bartlett, Mackenzie; Fofana, Ismael Ben; Johnson, Welkin E; Neil, Stuart J D; Bieniasz, Paul D

    2009-02-01

    Tetherin/BST-2/CD317 is a recently identified antiviral protein that blocks the release of nascent retrovirus, and other virus, particles from infected cells. An HIV-1 accessory protein, Vpu, acts as an antagonist of tetherin. Here, we show that positive selection is evident in primate tetherin sequences and that HIV-1 Vpu appears to have specifically adapted to antagonize variants of tetherin found in humans and chimpanzees. Tetherin variants found in rhesus macaques (rh), African green monkeys (agm) and mice were able to inhibit HIV-1 particle release, but were resistant to antagonism by HIV-1 Vpu. Notably, reciprocal exchange of transmembrane domains between human and monkey tetherins conferred sensitivity and resistance to Vpu, identifying this protein domain as a critical determinant of Vpu function. Indeed, differences between hu-tetherin and rh-tetherin at several positions in the transmembrane domain affected sensitivity to antagonism by Vpu. Two alterations in the hu-tetherin transmembrane domain, that correspond to differences found in rh- and agm-tetherin proteins, were sufficient to render hu-tetherin completely resistant to HIV-1 Vpu. Interestingly, transmembrane and cytoplasmic domain sequences in primate tetherins exhibit variation at numerous codons that is likely the result of positive selection, and some of these changes coincide with determinants of HIV-1 Vpu sensitivity. Overall, these data indicate that tetherin could impose a barrier to viral zoonosis as a consequence of positive selection that has been driven by ancient viral antagonists, and that the HIV-1 Vpu protein has specialized to target the transmembrane domains found in human/chimpanzee tetherin proteins.

  18. Species-Specific Activity of HIV-1 Vpu and Positive Selection of Tetherin Transmembrane Domain Variants

    PubMed Central

    McNatt, Matthew W.; Zang, Trinity; Hatziioannou, Theodora; Bartlett, Mackenzie; Fofana, Ismael Ben; Johnson, Welkin E.; Neil, Stuart J. D.; Bieniasz, Paul D.

    2009-01-01

    Tetherin/BST-2/CD317 is a recently identified antiviral protein that blocks the release of nascent retrovirus, and other virus, particles from infected cells. An HIV-1 accessory protein, Vpu, acts as an antagonist of tetherin. Here, we show that positive selection is evident in primate tetherin sequences and that HIV-1 Vpu appears to have specifically adapted to antagonize variants of tetherin found in humans and chimpanzees. Tetherin variants found in rhesus macaques (rh), African green monkeys (agm) and mice were able to inhibit HIV-1 particle release, but were resistant to antagonism by HIV-1 Vpu. Notably, reciprocal exchange of transmembrane domains between human and monkey tetherins conferred sensitivity and resistance to Vpu, identifying this protein domain as a critical determinant of Vpu function. Indeed, differences between hu-tetherin and rh-tetherin at several positions in the transmembrane domain affected sensitivity to antagonism by Vpu. Two alterations in the hu-tetherin transmembrane domain, that correspond to differences found in rh- and agm-tetherin proteins, were sufficient to render hu-tetherin completely resistant to HIV-1 Vpu. Interestingly, transmembrane and cytoplasmic domain sequences in primate tetherins exhibit variation at numerous codons that is likely the result of positive selection, and some of these changes coincide with determinants of HIV-1 Vpu sensitivity. Overall, these data indicate that tetherin could impose a barrier to viral zoonosis as a consequence of positive selection that has been driven by ancient viral antagonists, and that the HIV-1 Vpu protein has specialized to target the transmembrane domains found in human/chimpanzee tetherin proteins. PMID:19214216

  19. Catalase (CAT) promoter and 5'-UTR genetic variants lead to its altered expression and activity in vitiligo.

    PubMed

    Mansuri, M S; Jadeja, S D; Singh, M; Laddha, N C; Dwivedi, M; Begum, R

    2017-05-25

    Oxidative stress is considered to be the initial event during the course of vitiligo. Catalase is mainly involved in the defense against oxidizing agents in the cell through detoxifying H2 O2 . Aims of the present study were 1) to assess the erythrocyte catalase activity and LPO levels, as well as CAT mRNA expression in skin and blood; 2) to investigate CAT promoter rs7943316, rs1001179; 5'-UTR rs1049982 and exon (rs17886350, rs11032709, rs17880442, rs35677492) polymorphisms and; 3) to perform genotype-phenotype/ haplotype correlation analyses, in vitiligo patients and controls from Gujarat. Catalase activity and lipid peroxidation levels were measured spectrophotometrically. CAT mRNA levels were estimated using Real-time PCR by SYBR-Green method. SNP genotyping was performed using PCR-RFLP/ ARMS-PCR methods. Vitiligo patients showed significantly decreased CAT mRNA-expression in lesional, non-lesional skin and in blood with reduced catalase activity compared to that of controls. CAT -89A/T and -20T/C polymorphisms were significantly associated with patients, especially with active and generalized vitiligo whereas, no association was observed for -262G/A and exon polymorphisms. 'A-262 T-89 C-20 ' haplotype with variant alleles was found to be associated with 6.4-fold risk of vitiligo. Genotype/ haplotype-phenotype correlation analyses revealed that individuals with susceptible genotypes/ haplotype for CAT -89 A/T and -20 T/C polymorphisms showed significantly decreased catalase mRNA/activity, and only -89 A/T polymorphisms showed significantly increased lipid peroxidation levels, as compared to wild type genotypes/ haplotype. In conclusion, the present study proposes the crucial role of catalase and its allelic variants in oxidative stress mediated pathogenesis of vitiligo. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  20. Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity*

    PubMed Central

    Sorensen, Anders B.; Madsen, Jesper J.; Svensson, L. Anders; Pedersen, Anette A.; Østergaard, Henrik; Overgaard, Michael T.; Olsen, Ole H.; Gandhi, Prafull S.

    2016-01-01

    The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215–217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215–217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity. PMID:26694616

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

    PubMed Central

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

    2010-01-01

    Previous studies have shown association of single nucleotide polymorphisms (SNPs) in three 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 three genes. Haplotypes including SNPs spanning the PON genes were generally more significant than haplotypes comprising SNPs from one 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

  2. Human procaspase-1 variants with decreased enzymatic activity are associated with febrile episodes and may contribute to inflammation via RIP2 and NF-κB signaling.

    PubMed

    Heymann, Michael C; Winkler, Stefan; Luksch, Hella; Flecks, Silvana; Franke, Marcus; Ruß, Susanne; Ozen, Seza; Yilmaz, Engin; Klein, Christoph; Kallinich, Tilmann; Lindemann, Dirk; Brenner, Sebastian; Ganser, Gerd; Roesler, Joachim; Rösen-Wolff, Angela; Hofmann, Sigrun R

    2014-05-01

    The proinflammatory enzyme caspase-1 plays an important role in the innate immune system and is involved in a variety of inflammatory conditions. Rare naturally occurring human variants of the caspase-1 gene (CASP1) lead to different protein expression and structure and to decreased or absent enzymatic activity. Paradoxically, a significant number of patients with such variants suffer from febrile episodes despite decreased IL-1β production and secretion. In this study, we investigate how variant (pro)caspase-1 can possibly contribute to inflammation. In a transfection model, such variant procaspase-1 binds receptor interacting protein kinase 2 (RIP2) via Caspase activation and recruitment domain (CARD)/CARD interaction and thereby activates NF-κB, whereas wild-type procaspase-1 reduces intracellular RIP2 levels by enzymatic cleavage and release into the supernatant. We approach the protein interactions by coimmunoprecipitation and confocal microscopy and show that NF-κB activation is inhibited by anti-RIP2-short hairpin RNA and by the expression of a RIP2 CARD-only protein. In conclusion, variant procaspase-1 binds RIP2 and thereby activates NF-κB. This pathway could possibly contribute to proinflammatory signaling.

  3. In Vitro Functional Analyses of Infrequent Nucleotide Variants in the Lactase Enhancer Reveal Different Molecular Routes to Increased Lactase Promoter Activity and Lactase Persistence.

    PubMed

    Liebert, Anke; Jones, Bryony L; Danielsen, Erik Thomas; Olsen, Anders Krüger; Swallow, Dallas M; Troelsen, Jesper T

    2016-11-01

    The genetic trait that allows intestinal lactase to persist into adulthood in some 35% of humans worldwide operates at the level of transcription, the effect being caused by cis-acting nucleotide changes upstream of the lactase gene (LCT). A single nucleotide substitution, -13910 C>T, the first causal variant to be identified, accounts for lactase persistence over most of Europe. Located in a region shown to have enhancer function in vitro, it causes increased activity of the LCT promoter in Caco-2 cells, and altered transcription factor binding. Three other variants in close proximity, -13907 C>G, -13915 T>C and -14010 G>C, were later shown to behave in a similar manner. Here, we study four further candidate functional variants. Two, -14009 T>G and -14011 C>T, adjacent to the well-studied -14010 G>C variant, also have a clear effect on promoter activity upregulation as assessed by transfection assays, but notably are involved in different molecular interactions. The results for the two other variants (-14028 T>C, -13779 G>C) were suggestive of function, -14028*C showing a clear change in transcription factor binding, but no obvious effect in transfections, while -13779*G showed greater effect in transfections but less on transcription factor binding. Each of the four variants arose on independent haplotypic backgrounds with different geographic distribution. © 2016 The Authors. Annals of Human Genetics published by University College London (UCL) and John Wiley & Sons Ltd.

  4. In Vitro Functional Analyses of Infrequent Nucleotide Variants in the Lactase Enhancer Reveal Different Molecular Routes to Increased Lactase Promoter Activity and Lactase Persistence

    PubMed Central

    Liebert, Anke; Jones, Bryony L.; Danielsen, Erik Thomas; Olsen, Anders Krüger; Troelsen, Jesper T.

    2016-01-01

    Summary The genetic trait that allows intestinal lactase to persist into adulthood in some 35% of humans worldwide operates at the level of transcription, the effect being caused by cis‐acting nucleotide changes upstream of the lactase gene (LCT). A single nucleotide substitution, ‐13910 C>T, the first causal variant to be identified, accounts for lactase persistence over most of Europe. Located in a region shown to have enhancer function in vitro, it causes increased activity of the LCT promoter in Caco‐2 cells, and altered transcription factor binding. Three other variants in close proximity, ‐13907 C>G, ‐13915 T>C and ‐14010 G>C, were later shown to behave in a similar manner. Here, we study four further candidate functional variants. Two, ‐14009 T>G and ‐14011 C>T, adjacent to the well‐studied ‐14010 G>C variant, also have a clear effect on promoter activity upregulation as assessed by transfection assays, but notably are involved in different molecular interactions. The results for the two other variants (‐14028 T>C, ‐13779 G>C) were suggestive of function, ‐14028*C showing a clear change in transcription factor binding, but no obvious effect in transfections, while ‐13779*G showed greater effect in transfections but less on transcription factor binding. Each of the four variants arose on independent haplotypic backgrounds with different geographic distribution. PMID:27714771

  5. Antimitotic activity of DY131 and the estrogen-related receptor beta 2 (ERRβ2) splice variant in breast cancer

    PubMed Central

    Heckler, Mary M.; Zeleke, Tizita Zewde; Divekar, Shailaja D.; Fernandez, Aileen I.; Tiek, Deanna M.; Woodrick, Jordan; Farzanegan, Alexander; Roy, Rabindra; Üren, Aykut; Mueller, Susette C.; Riggins, Rebecca B.

    2016-01-01

    Breast cancer remains a leading cause of cancer-related death in women, and triple negative breast cancer (TNBC) lacks clinically actionable therapeutic targets. Death in mitosis is a tumor suppressive mechanism that occurs in cancer cells experiencing a defective M phase. The orphan estrogen-related receptor beta (ERRβ) is a key reprogramming factor in murine embryonic and induced pluripotent stem cells. In primates, ERRβ is alternatively spliced to produce several receptor isoforms. In cellular models of glioblastoma, short form (ERRβsf) and beta2 (ERRβ2) splice variants differentially regulate cell cycle progression in response to the synthetic agonist DY131, with ERRβ2 driving arrest in G2/M. The goals of the present study are to determine the cellular function(s) of ligand-activated ERRβ splice variants in breast cancer and evaluate the potential of DY131 to serve as an antimitotic agent, particularly in TNBC. DY131 inhibits growth in a diverse panel of breast cancer cell lines, causing cell death that involves the p38 stress kinase pathway and a bimodal cell cycle arrest. ERRβ2 facilitates the block in G2/M, and DY131 delays progression from prophase to anaphase. Finally, ERRβ2 localizes to centrosomes and DY131 causes mitotic spindle defects. Targeting ERRβ2 may therefore be a promising therapeutic strategy in breast cancer. PMID:27363015

  6. Androgen receptor (AR) coregulators: an overview.

    PubMed

    Heinlein, Cynthia A; Chang, Chawnshang

    2002-04-01

    The biological action of androgens is mediated through the androgen receptor (AR). Androgen-bound AR functions as a transcription factor to regulate genes involved in an array of physiological processes, most notably male sexual differentiation and maturation, and the maintenance of spermatogenesis. The transcriptional activity of AR is affected by coregulators that influence a number of functional properties of AR, including ligand selectivity and DNA binding capacity. As the promoter of target genes, coregulators participate in DNA modification, either directly through modification of histones or indirectly by the recruitment of chromatin-modifying complexes, as well as functioning in the recruitment of the basal transcriptional machinery. Aberrant coregulator activity due to mutation or altered expression levels may be a contributing factor in the progression of diseases related to AR activity, such as prostate cancer. AR demonstrates distinct differences in its interaction with coregulators from other steroid receptors due to differences in the functional interaction between AR domains, possibly resulting in alterations in the dynamic interactions between coregulator complexes.

  7. 40Ar/39Ar ages of the AD 79 eruption of Vesuvius, Italy

    USGS Publications Warehouse

    Lanphere, Marvin A.; Champion, Duane E.; Melluso, Leone; Morra, Vincenzo; Perrotta, Annamaria; Scarpati, Claudio; Tedesco, Dario; Calvert, Andrew T.

    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 (1σ 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.

  8. 40Ar/39Ar ages of the AD 79 eruption of Vesuvius, Italy

    NASA Astrophysics Data System (ADS)

    Lanphere, Marvin; Champion, Duane; Melluso, Leone; Morra, Vincenzo; Perrotta, Annamaria; Scarpati, Claudio; Tedesco, Dario; Calvert, Andrew

    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 (1σ 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.

  9. Flight activity of USDA-ARS Russian honey bees (Hymenoptera: Apidae) during pollination of lowbush blueberries in Maine.

    PubMed

    Danka, Robert G; Beaman, Lorraine D

    2007-04-01

    Flight activity was compared in colonies of Russian honey bees, Apis mellifera L. (Hymenoptera: Apidae), and Italian bees during commercial pollination of lowbush blueberries (principally Vaccinium angustifolium Aiton) in Washington Co., ME, in late May and early June in 2003 and 2004. Colonies of the two stocks were managed equally in Louisiana during autumn through early spring preceding observations in late spring each year. Resulting average populations of adult bees and of brood were similar in colonies of the two bee stocks during pollination. Flight during pollination was monitored hourly on 6 d each year by counting bees exiting each colony per minute; counts were made manually with flight cones on 17 colonies per stock in 2003 and electronically with ApiSCAN-Plus counters on 20 colonies per stock in 2004. Analysis of variance showed that temperature, colony size (population of adult bees or brood), and the interaction of these effects were the strongest regulators of flight activity in both years. Russian and Italian bees had similar flight activity at any given colony size, temperature, or time of day. Flight increased linearly with rising temperatures and larger colony sizes. Larger colonies, however, were more responsive than smaller colonies across the range of temperatures measured. In 2003, flight responses to varying temperatures were less in the afternoon and evening (1500-1959 hours) than they were earlier in the day. Russian colonies had flight activity that was suitable for late spring pollination of lowbush blueberries.

  10. Natural allelic variants of bovine ATP-binding cassette transporter ABCG2: increased activity of the Ser581 variant and development of tools for the discovery of new ABCG2 inhibitors.

    PubMed

    Merino, Gracia; Real, Rebeca; Baro, Marta F; Gonzalez-Lobato, Lucia; Prieto, Julio G; Alvarez, Ana I; Marques, Margarita M

    2009-01-01

    ATP-binding cassette transporter ABCG2 [breast cancer resistance protein (BCRP)] is a member of the ABC transporter superfamily that actively extrudes xenotoxins from cells and is a major determinant of the bioavailability of many compounds. ABCG2 expression is strongly induced during lactation in the mammary gland and is related to the active secretion of drugs into the milk. The presence of drug residues and environmental pollutants in milk is an outstanding problem for human milk consumption and milk industrial processes, involving important risks to public health and the dairy industry. In cows, a single nucleotide polymorphism (SNP) in this protein has been described previously (Tyr581) and is associated with higher fat and protein percentages and lower milk yield. However, whether this amino acid substitution affects ABCG2-mediated drug transport in cows, including milk secretion, required further exploration. We cloned the two variants of bovine ABCG2 and evaluated the effect of this SNP on mitoxantrone accumulation assays performed in ovine primary fibroblasts transiently expressing either of the variants. It is interesting to note that statistically significant differences in activity between both variants were observed, and the Ser581 variant was related with an increased efflux activity. In addition, we demonstrated that genistein is a very good inhibitor of bovine ABCG2 and identified new inhibitors of the transporter, such as the macrocyclic lactones, ivermectin, and selamectin. Moreover, the inhibitory effect of these compounds on human and murine ABCG2 homologs was confirmed using transduced Marbin-Dabin canine kidney II cells. These findings may have important implications regarding the presence of drug residues in milk and drug interactions affecting the pharmacological behavior of ABCG2 substrates.

  11. 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

  12. Efficient conversion of primary azides to aldehydes catalyzed by active site variants of myoglobin

    PubMed Central

    Giovani, Simone; Singh, Ritesh; Fasan, Rudi

    2015-01-01

    The oxidation of primary azides to aldehydes constitutes a convenient but underdeveloped transformation for which no efficient methods are available. Here, we demonstrate that engineered variants of the hemoprotein myoglobin can catalyze this transformation with high efficiency (up to 8,500 turnovers) and selectivity across a range of structurally diverse aryl-substituted primary azides. Mutagenesis of the 'distal' histidine residue was particularly effective in enhancing the azide oxidation reactivity of myoglobin, enabling these reactions to proceed in good to excellent yields (37-89%) and to be carried out at a synthetically useful scale. Kinetic isotope effect, isotope labeling, and substrate binding experiments support a mechanism involving heme-catalyzed decomposition of the organic azide followed by alpha hydrogen deprotonation to generate an aldimine which, upon hydrolysis, releases the aldehyde product. This work provides the first example of a biocatalytic azide-to-aldehyde conversion and expands the range of non-native chemical transformations accessible through hemoprotein-mediated catalysis. PMID:26900445

  13. A functional variant of TLR10 modifies the activity of NFkB and may help predict a worse prognosis in patients with rheumatoid arthritis.

    PubMed

    Torices, Silvia; Julia, Antonio; Muñoz, Pedro; Varela, Ignacio; Balsa, Alejandro; Marsal, Sara; Fernández-Nebro, Antonio; Blanco, Francisco; López-Hoyos, Marcos; Martinez-Taboada, Víctor; Fernández-Luna, Jose L

    2016-10-04

    Toll-like receptor (TLR) family members are key players in inflammation. TLR10 has been poorly studied in chronic inflammatory disorders, and its clinical relevance in rheumatoid arthritis (RA) is as yet unknown. We aimed at identifying TLR10 variants within all coding regions of the gene in patients with RA as well as studying their functional and clinical significance. TLR10 gene variants were studied by performing sequencing of 66 patients with RA and 30 control subjects. A selected variant, I473T, was then analyzed in 1654 patients and 1702 healthy control subjects. The capacity of this TLR10 variant to modify the transcriptional activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) was determined by using a luciferase reporter assay and analyzing the expression of NFkB target genes by quantitative polymerase chain reaction. Differences between groups were analyzed by using the Mann-Whitney U test and the unpaired two-tailed Student's t test. We detected ten missense variants in the TLR10 gene and focused on the I473T substitution based on allele frequencies and the predicted functional impact. I473T variant is not associated with susceptibility to RA, but it significantly correlates with erosive disease in patients seropositive for antibodies to citrullinated protein antigens (p = 0.017 in the total cohort and p = 0.0049 in female patients) and with a lower response to infliximab treatment as measured by the change in Disease Activity Score in 28 joints (p = 0.012) and by the European League Against Rheumatism criteria (p = 0.049). Functional studies showed that TLR10 reduced activation of the NFkB inflammatory pathway in hematopoietic cells, whereas the I473T variant lacked this inhibitory capacity. Consistently, after exposure to infliximab, cells expressing the I437T variant showed higher NFkB activity than cells carrying wild-type TLR10. A TLR10 allelic variant, I473T, has impaired NFkB inhibitory activity and is

  14. A DPYD variant (Y186C) in individuals of african ancestry is associated with reduced DPD enzyme activity.

    PubMed

    Offer, S M; Lee, A M; Mattison, L K; Fossum, C; Wegner, N J; Diasio, R B

    2013-07-01

    5-Fluorouracil (5-FU) is used to treat many aggressive cancers, such as those of the colon, breast, and head and neck. The responses to 5-FU, with respect to both toxicity and efficacy, vary among racial groups, potentially because of variability in the activity levels of the enzyme dihydropyrimidine dehydrogenase (DPD, encoded by the DPYD gene). In this study, the genetic associations between DPYD variations and circulating mononuclear-cell DPD enzyme activity were evaluated in 94 African-American and 81 European-American volunteers. The DPYD-Y186C variant was unique to individuals of African ancestry, and DPD activity was 46% lower in carriers as compared with noncarriers (279 ± 35 vs. 514 ± 168 pmol 5-FU min(-1) mg(-1); P = 0.00029). In this study, 26% of the African Americans with reduced DPD activity were carriers of Y186C. In the African-American cohort, after excluding Y186C carriers, homozygous carriers of C29R showed 27% higher DPD activity as compared with noncarriers (609 ± 152 and 480 ± 152 pmol 5-FU min(-1) mg(-1), respectively; P = 0.013).

  15. SAP155-mediated c-myc suppressor far-upstream element-binding protein-interacting repressor splicing variants are activated in colon cancer tissues.

    PubMed

    Kajiwara, Toshiko; Matsushita, Kazuyuki; Itoga, Sakae; Tamura, Mai; Tanaka, Nobuko; Tomonaga, Takeshi; Matsubara, Hisahiro; Shimada, Hideaki; Habara, Yasuaki; Matsuo, Masafumi; Nomura, Fumio

    2013-02-01

    The c-myc transcriptional suppressor, far-upstream element (FUSE)-binding protein (FBP)-interacting repressor (FIR), is alternatively spliced in colorectal cancer tissue (Matsushita et al., Cancer Res 2006). Recently, the knockdown of SAP155 pre-mRNA-splicing factor, a subunit of SF3b, was reported to disturb FIR pre-mRNA splicing and yield FIRΔexon2, an exon 2-spliced variant of FIR, which lacks c-myc repression activity. In the present study, novel splicing variants of FIR, Δ3 and Δ4, were also generated by SAP155 siRNA, and these variants were found to be activated in human colorectal cancer tissue. Furthermore, the expression levels of FIR variant mRNA were examined in the peripheral blood of colorectal cancer patients and healthy volunteers to assess its potency for tumor detection. As expected, circulating FIR variant mRNA in the peripheral blood of cancer patients were significantly overexpressed compared to that in healthy volunteers. In particular, the area under the receiving operating characteristic curve of FIR, FIRΔexon2 or FIRΔexon2/FIR, was greater than those of conventional carcinoembryonic antigen or carbohydrate antigen 19-9. In addition, FIRΔexon2 or FIR mRNA expression in the peripheral blood was significantly reduced after operative removal of colorectal tumors. Thus, circulating FIR and FIRΔexon2 mRNA are potential novel screening markers for colorectal cancer testing with conventional carcinoembryonic antigen and or carbohydrate antigen 19-9. Taken together, our results indicate that overexpression of FIR and its splicing variants in colorectal cancer directs feed-forward or addicted circuit c-myc transcriptional activation. Clinical implications for colorectal cancers of novel FIR splicing variants are also discussed in the present paper. © 2012 Japanese Cancer Association.

  16. 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

  17. Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry.

    PubMed

    Schulz, Danae; Mugnier, Monica R; Boothroyd, Catherine E; Papavasiliou, F Nina

    2016-10-19

    Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

  18. Analytical Validation of Androgen Receptor Splice Variant 7 Detection in a Clinical Laboratory Improvement Amendments (CLIA) Laboratory Setting.

    PubMed

    Lokhandwala, Parvez M; Riel, Stacy L; Haley, Lisa; Lu, Changxue; Chen, Yan; Silberstein, John; Zhu, Yezi; Zheng, Gang; Lin, Ming-Tseh; Gocke, Christopher D; Partin, Alan W; Antonarakis, Emmanuel S; Luo, Jun; Eshleman, James R

    2017-01-01

    Patients with castration-resistant prostate cancer (CRPC) often are treated with drugs that target the androgen receptor (AR) ligand-binding domain. Constitutively active AR splice variant 7 (AR-V7) lacks the ligand-binding domain and, if detected in circulating tumor cells, may be associated with resistance to these agents. We validated an AR-V7 assay in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory. Circulating tumor cells were isolated, and mRNA was reverse-transcribed into cDNA. Real-time quantitative PCR amplification of reference transcripts (beta-actin and glyceraldehyde-3-phosphate dehydrogenase), prostate-specific transcripts (prostate-specific membrane antigen, prostate-specific antigen, and AR-full length), and AR-V7 was performed. Specimens for validation included an AR-V7 expressing prostate cancer (LNCaP95), 38 peripheral blood controls, and 21 blood samples from CRPC patients. The assay detected as few as five LNCaP95 cells spiked into peripheral blood, showing high analytical sensitivity. Multiple inter-run and intrarun replicates of LNCaP95 cell line experiments yielded similar cycle threshold values for all genes, showing high analytical precision (AR-V7 cycle threshold CV of 0.67%). All 38 healthy control samples were negative for AR-V7, showing high diagnostic specificity (100%). The diagnostic accuracy was confirmed by concurrent testing of 21 CRPC samples in the research laboratory and the clinical diagnostic laboratory: concordance in AR-V7 status was achieved in all cases (positive in 4, negative in 17) (100% accuracy). This first validated clinical assay detects the AR-V7 with high analytical sensitivity, precision, specificity, and accuracy.

  19. Dysregulation of miR-212 Promotes Castration Resistance through hnRNPH1-Mediated Regulation of AR and AR-V7: Implications for Racial Disparity of Prostate Cancer.

    PubMed

    Yang, Yijun; Jia, Dingwu; Kim, Hogyoung; Abd Elmageed, Zakaria Y; Datta, Amrita; Davis, Rodney; Srivastav, Sudesh; Moroz, Krzysztof; Crawford, Byron E; Moparty, Krishnarao; Thomas, Raju; Hudson, Robert S; Ambs, Stefan; Abdel-Mageed, Asim B

    2016-04-01

    The causes of disproportionate incidence and mortality of prostate cancer among African Americans (AA) remain elusive. The purpose of this study was to investigate the mechanistic role and assess clinical utility of the splicing factor heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) in prostate cancer progression among AA men. We employed an unbiased functional genomics approach coupled with suppressive subtractive hybridization (SSH) and custom cDNA microarrays to identify differentially expressed genes in microdissected tumors procured from age- and tumor grade-matched AA and Caucasian American (CA) men. Validation analysis was performed in independent cohorts and tissue microarrays. The underlying mechanisms of hnRNPH1 regulation and its impact on androgen receptor (AR) expression and tumor progression were explored. Aberrant coexpression of AR and hnRNPH1 and downregulation of miR-212 were detected in prostate tumors and correlate with disease progression in AA men compared with CA men. Ectopic expression of miR-212 mimics downregulated hnRNPH1 transcripts, which in turn reduced expression of AR and its splice variant AR-V7 (or AR3) in prostate cancer cells. hnRNPH1 physically interacts with AR and steroid receptor coactivator-3 (SRC-3) and primes activation of androgen-regulated genes in a ligand-dependent and independent manner. siRNA silencing of hnRNPH1 sensitized prostate cancer cells to bicalutamide and inhibited prostate tumorigenesis in vivo Our findings define novel roles for hnRNPH1 as a putative oncogene, splicing factor, and an auxiliary AR coregulator. Targeted disruption of the hnRNPH1-AR axis may have therapeutic implications to improve clinical outcomes in patients with advanced prostate cancer, especially among AA men. ©2015 American Association for Cancer Research.

  20. Quantitative Structure Inter-Activity Relationship (QSInAR). Cytotoxicity Study of Some Hemisynthetic and Isolated Natural Steroids and Precursors on Human Fibrosarcoma Cells HT1080.

    PubMed

    Putz, Mihai V; Lazea, Marius; Sandjo, Louis P

    2011-08-05

    Combined experimental and quantitative structure inter-activity relationship (QSIAR) computation methods were advanced in order to establish the structural and mechanistic influences that steroids and triterpenes, either as newly synthesized or naturally isolated products, have on human HT1080 mammalian cancer cells. The main Hansch structural indicators such as hydrophobicity (LogP), polarizability (POL) and total energy (Etot) were considered and both the structure-projected as well as globally computed correlations were reported; while the inter-activity correlation of the global activity with those projected on structural information was revealed as equal to the direct structural-activity one for the trial sets of compounds, the prediction for the testing set of molecules reported even superior performances respecting those characteristic for the calibration set, validating therefore the present QSInAR models; accordingly, it follows that the LogP carries the most part of the cytotoxic signal, while POL has little influence on inhibiting tumor growth-A complementary behavior with their earlier known influence on genotoxic carcinogenesis. Regarding the newly hemisynthetic compounds it was found that stigmasta-4,22-dien-3-one is not adapted for cell membrane diffusion; it is recommended that aminocinnamyl chlorohydrate be further modified in order to acquire better steric influence, while aminocinnamyl-2,3,4,6-O-tétraacétyl-α-D-glucopyranoside was identified as being inhibited in the tumor cell by other molecular mechanisms-here not revealed-although it has a moderate-high anti-cancer structurally predicted activity.

  1. Characterization of Escherichia coli UmuC active-site loops identifies variants that confer UV hypersensitivity.

    PubMed

    Hawver, Lisa A; Gillooly, Caitlin A; Beuning, Penny J

    2011-10-01

    DNA is constantly exposed to chemical and environmental mutagens, causing lesions that can stall replication. In order to deal with DNA damage and other stresses, Escherichia coli utilizes the SOS response, which regulates the expression of at least 57 genes, including umuDC. The gene products of umuDC, UmuC and the cleaved form of UmuD, UmuD', form the specialized E. coli Y-family DNA polymerase UmuD'2C, or polymerase V (Pol V). Y-family DNA polymerases are characterized by their specialized ability to copy damaged DNA in a process known as translesion synthesis (TLS) and by their low fidelity on undamaged DNA templates. Y-family polymerases exhibit various specificities for different types of DNA damage. Pol V carries out TLS to bypass abasic sites and thymine-thymine dimers resulting from UV radiation. Using alanine-scanning mutagenesis, we probed the roles of two active-site loops composed of residues 31 to 38 and 50 to 54 in Pol V activity by assaying the function of single-alanine variants in UV-induced mutagenesis and for their ability to confer resistance to UV radiation. We find that mutations of the N-terminal residues of loop 1, N32, N33, and D34, confer hypersensitivity to UV radiation and to 4-nitroquinoline-N-oxide and significantly reduce Pol V-dependent UV-induced mutagenesis. Furthermore, mutating residues 32, 33, or 34 diminishes Pol V-dependent inhibition of recombination, suggesting that these mutations may disrupt an interaction of UmuC with RecA, which could also contribute to the UV hypersensitivity of cells expressing these variants.

  2. Trinucleotide repeat variants in the promoter of the thiopurine S-methyltransferase gene of patients exhibiting ultra-high enzyme activity.

    PubMed

    Roberts, Rebecca L; Gearry, Richard B; Bland, Michael V; Sies, Christiaan W; George, Peter M; Burt, Michael; Marinaki, Anthony M; Arenas, Monica; Barclay, Murray L; Kennedy, Martin A

    2008-05-01

    Thiopurine S-methyl transferase (TPMT) is a cytosolic enzyme that catalyses the S-methylation of the thiopurine immunosuppressants. To date, 22 variants have been identified that are predictive of decreased TPMT activity. In contrast, no molecular explanation has been found for the 1-2% of Caucasians who exhibit ultra-high TPMT activity. Here, we report the characterization of polymorphisms within a trinucleotide (GCC) repeat element of the TPMT promoter in two patients with inflammatory bowel disease exhibiting the highest TPMT activity from two testing centres. The first patient was heterozygous for a variant allele carrying seven GCC repeats [(GCC)7], whereas the second patient was heterozygous for a variant allele containing five GCC repeats [(GCC)5]. Fifty patients with inflammatory bowel disease with normal TPMT activity were all homozygous for six GCC repeats [(GCC)6]. Of 200 healthy controls, five were found to be heterozygous for the (GCC)7 variant. Within in vitro reporter gene assays, the mean luciferase activities of the (GCC)6, (GCC)7, and (GCC)5 constructs were 8.0+/-0.26, 13.2+/-0.10 and 12.3+/-0.12, respectively. The significant increase in activity observed for (GCC)5 and (GCC)7 compared with (GCC)6 (P-value activity observed in these patients.

  3. Alterations of structure and hydrolase activity of parkinsonism-associated human ubiquitin carboxyl-terminal hydrolase L1 variants.

    PubMed

    Nishikawa, Kaori; Li, Hang; Kawamura, Ryoichi; Osaka, Hitoshi; Wang, Yu-Lai; Hara, Yoko; Hirokawa, Takatsugu; Manago, Yoshimasa; Amano, Taiju; Noda, Mami; Aoki, Shunsuke; Wada, Keiji

    2003-04-25

    Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease.

  4. Effector activity of peanut allergens: a critical role for Ara h 2, Ara h 6, and their variants.

    PubMed

    Porterfield, H S; Murray, K S; Schlichting, D G; Chen, X; Hansen, K C; Duncan, M W; Dreskin, S C

    2009-07-01

    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. 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. 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 approximately 20 kDa and a range of 13-25 kDa. 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 kDa 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 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 >97% of the protein mass consisted of Ara h 2.0101, Ara h 2.0201, Ara h 6 isoforms, and variants of these proteins. Ara h 2 and Ara h 6 account for the majority of the effector activity found in a crude peanut extract.

  5. Assessing the effect of interaction between an FTO variant (rs9939609) and physical activity on obesity in 15,925 Swedish and 2,511 Finnish adults.

    PubMed

    Jonsson, A; Renström, F; Lyssenko, V; Brito, E C; Isomaa, B; Berglund, G; Nilsson, P M; Groop, L; Franks, P W

    2009-07-01

    Recent reports have suggested that genotypes at the FTO locus interact with physical activity to modify levels of obesity-related traits. We tested this hypothesis in two non-diabetic population-based cohorts, the first from southern Sweden and the second from the Botnia region of western Finland. In total 2,511 Finnish and 15,925 Swedish non-diabetic middle-aged adults were genotyped for the FTO rs9939609 variant. Physical activity was assessed by questionnaires and standard clinical procedures were conducted, including measures of height and weight and glucose regulation. Tests of gene x physical activity interaction were performed using linear interaction effects to determine whether the effect of this variant on BMI is modified by physical activity. The minor A allele at rs9939609 was associated with higher BMI in both cohorts, with the per allele difference in BMI being about 0.13 and 0.43 kg/m(2) in the Swedish and Finnish cohorts, respectively (p < 0.0001). The test of interaction between physical activity and the rs9939609 variant on BMI was not statistically significant after controlling for age and sex in either cohort (Sweden: p = 0.71, Finland: p = 0.18). The present report does not support the notion that physical activity modifies the effects of the FTO rs9939609 variant on obesity risk in the non-diabetic Swedish or Finnish adults studied here.

  6. Melanopsin Variants as Intrinsic Optogenetic On and Off Switches for Transient versus Sustained Activation of G Protein Pathways.

    PubMed

    Spoida, Katharina; Eickelbeck, Dennis; Karapinar, Raziye; Eckhardt, Tobias; Mark, Melanie D; Jancke, Dirk; Ehinger, Benedikt Valerian; König, Peter; Dalkara, Deniz; Herlitze, Stefan; Masseck, Olivia A

    2016-05-09

    G-protein-coupled receptors (GPCRs) represent the major protein family for cellular modulation in mammals. Therefore, various strategies have been developed to analyze the function of GPCRs involving pharmaco- and optogenetic approaches [1, 2]. However, a tool that combines precise control of the activation and deactivation of GPCR pathways and/or neuronal firing with limited phototoxicity is still missing. We compared the biophysical properties and optogenetic application of a human and a mouse melanopsin variant (hOpn4L and mOpn4L) on the control of Gi/o and Gq pathways in heterologous expression systems and mouse brain. We found that GPCR pathways can be switched on/off by blue/yellow light. The proteins differ in their kinetics and wavelength dependence to activate and deactivate G protein pathways. Whereas mOpn4L is maximally activated by very short light pulses, leading to sustained G protein activation, G protein responses of hOpn4L need longer light pulses to be activated and decline in amplitude. Based on the different biophysical properties, brief light activation of mOpn4L is sufficient to induce sustained neuronal firing in cerebellar Purkinje cells (PC), whereas brief light activation of hOpn4L induces AP firing, which declines in frequency over time. Most importantly, mOpn4L-induced sustained firing can be switched off by yellow light. Based on the biophysical properties, hOpn4L and mOpn4L represent the first GPCR optogenetic tools, which can be used to switch GPCR pathways/neuronal firing on an off with temporal precision and limited phototoxicity. We suggest to name these tools moMo and huMo for future optogenetic applications.

  7. 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

  8. Loss of Cytochrome c Oxidase Activity and Acquisition of Resistance to Quinone Analogs in a Laccase-Positive Variant of Azospirillum lipoferum

    PubMed Central

    Alexandre, Gladys; Bally, René; Taylor, Barry L.; Zhulin, Igor B.

    1999-01-01

    Laccase, a p-diphenol oxidase typical of plants and fungi, has been found recently in a proteobacterium, Azospirillum lipoferum. Laccase activity was detected in both a natural isolate and an in vitro-obtained phase variant that originated from the laccase-negative wild type. In this study, the electron transport systems of the laccase-positive variant and its parental laccase-negative forms were compared. During exponential (but not stationary) growth under fully aerobic (but not under microaerobic) conditions, the laccase-positive variant lost a respiratory branch that is terminated in a cytochrome c oxidase of the aa3 type; this was most likely due to a defect in the biosynthesis of a heme component essential for the oxidase. The laccase-positive variant was significantly less sensitive to the inhibitory action of quinone analogs and fully resistant to inhibitors of the bc1 complex, apparently due to the rearrangements of its respiratory system. We propose that the loss of the cytochrome c oxidase-containing branch in the variant is an adaptive strategy to the presence of intracellular oxidized quinones, the products of laccase activity. PMID:10542175

  9. GRS Measurements of Ar in Mars' Atmosphere

    NASA Astrophysics Data System (ADS)

    Sprague, A. L.; Boynton, W. V.; Kerry, K. E.; Janes, D. M.; Kelly, N. J.; Crombie, M. K.; Hunten, D. M.; Nelli, S. M.; Murphy, J. R.; Reedy, R. C.; Metzger, A. E.

    2005-08-01

    One and one half Mars years of atmospheric argon (Ar) measurements are described in the context of understanding how Ar, a minor constituent of Mars atmosphere that does not condense at Mars temperatures, can be used to study martian circulation and dynamics. There is a repeated factor of 6 enhancement of Ar measured over south polar latitudes. The maximum in Ar abundance occurs near the onset of southern winter. There is no similar strong enhancement of Ar over north-polar regions during northern winter; only modest evidence for an enhancement peak is present. Part of this difference is explained by the global topographic dichotomy and the fact that the duration of northern autumn and winter is shorter than southern autumn and winter. Rapid seasonal fluctuations in Ar abundance may indicate evidence for wave activity at the perimeter of the southern seasonal polar cap. The apparent lack of coincidence of Ar enhancement with the relatively cold, cryptic terrain or relatively warm, bright albedo regions, indirectly supports the conclusion that the low temperatures measured over the south polar region by IRTM are probably caused by the combination of low CO2 abundance over south polar night and low emissivity regions on the surface associated with small grain size.

  10. The activity of the integrase inhibitor dolutegravir against HIV-1 variants isolated from raltegravir-treated adults.

    PubMed

    Underwood, Mark R; Johns, Brian A; Sato, Akihiko; Martin, Jeffrey N; Deeks, Steven G; Fujiwara, Tamio

    2012-11-01

    Dolutegravir (DTG, S/GSK1349572) is an integrase inhibitor with low nanomolar potency. Susceptibility to dolutegravir and raltegravir was determined for raltegravir-resistant clinical isolates. Genotypic and phenotypic susceptibility to integrase inhibitors was examined using 39 clinical isolate samples obtained from 18 adults who had exhibited incomplete viral suppression on a raltegravir-based regimen. Of 39 samples evaluated, 30 had genotypic and phenotypic resistance to raltegravir. All samples lacking raltegravir resistance retained complete susceptibility to dolutegravir. Of the 30 samples with genotypic evidence of raltegravir resistance, the median level of phenotypic resistance to raltegravir was high (median fold change in inhibitory concentration at 50%, >81; range, 3.7 to >87), while the level of resistance to dolutegravir was close to that of wild-type variants (median fold change, 1.5; range, 0.9-19.0). Longitudinal samples from 5 subjects collected during long-term failure of raltegravir revealed time-dependent general decreases in phenotypic susceptibility to raltegravir, with minimal changes in phenotypic susceptibility to dolutegravir. The median fold change to dolutegravir for isolates containing changes at G140S + Q148H, G140S + Q148R, T97A + Y143R, and N155H (thus including raltegravir signature resistance codons) were 3.75, 13.3, 1.05, and 1.37, respectively. Dolutegravir retained in vitro activity against clinical isolates obtained from subjects who failed raltegravir-based therapy at near wild-type levels for variants containing the Y143 and N155 resistance mutations. Isolates with Q148 plus additional integrase mutations possessed a broader range of and more reduced susceptibility to dolutegravir.

  11. The Activity of the Integrase Inhibitor Dolutegravir Against HIV-1 Variants Isolated From Raltegravir-Treated Adults

    PubMed Central

    Underwood, Mark R.; Johns, Brian A.; Sato, Akihiko; Martin, Jeffrey N.; Deeks, Steven G.; Fujiwara, Tamio

    2013-01-01

    Background Dolutegravir (DTG, S/GSK1349572) is an integrase inhibitor with low nanomolar potency. Susceptibility to dolutegravir and raltegravir was determined for raltegravir-resistant clinical isolates. Methods Genotypic and phenotypic susceptibility to integrase inhibitors was examined using 39 clinical isolate samples obtained from 18 adults who had exhibited incomplete viral suppression on a raltegravir-based regimen. Results Of 39 samples evaluated, 30 had genotypic and phenotypic resistance to raltegravir. All samples lacking raltegravir resistance retained complete susceptibility to dolutegravir. Of the 30 samples with genotypic evidence of raltegravir resistance, the median level of phenotypic resistance to raltegravir was high (median fold change in inhibitory concentration at 50%, >81; range, 3.7 to >87) while the level of resistance to dolutegravir was close to that of wild-type variants (median fold change, 1.5; range, 0.9–19.0). Longitudinal samples from 5 subjects collected during long-term failure of raltegravir revealed time-dependent general decreases in phenotypic susceptibility to raltegravir, with minimal changes in phenotypic susceptibility to dolutegravir. The median fold change to dolutegravir for isolates containing changes at G140S + Q148H; G140S + Q148R; T97A + Y143R; and N155H (thus including raltegravir signature resistance codons) were 3.75, 13.3, 1.05, and 1.37, respectively. Conclusions Dolutegravir retained in vitro activity against clinical isolates obtained from subjects who failed raltegravir-based therapy at near wild-type levels for variants containing the Y143 and N155 resistance mutations. Isolates with Q148 plus additional integrase mutations possessed a broader range of and more reduced susceptibility to dolutegravir. PMID:22878423

  12. Palmitoylation of the β4-Subunit Regulates Surface Expression of Large Conductance Calcium-activated Potassium Channel Splice Variants*

    PubMed Central

    Chen, Lie; Bi, Danlei; Tian, Lijun; McClafferty, Heather; Steeb, Franziska; Ruth, Peter; Knaus, Hans Guenther; Shipston, Michael J.

    2013-01-01

    Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels. PMID:23504458

  13. Plasma Motions and Turbulent Magnetic Diffusivity of Active Region AR 12158 Using a Minimum Energy Functional and Non-Force-Free Reconstructions of Vector Magnetograms

    NASA Astrophysics Data System (ADS)

    Tremblay, Benoit; Vincent, Alain

    2017-01-01

    We present a generalization of the resistive minimum-energy fit (MEF-R: Tremblay and Vincent, Solar Phys. 290, 437, 2015) for non-force-free (NFF) magnetic fields. In MEF-R, an extremum principle is used to infer two-dimensional maps of plasma motions [boldsymbol{v}(x,y)] and magnetic eddy diffusivity [η _{eddy}(x,y)] at the photosphere. These reconstructions could be used as boundary conditions in data-driven simulations or in data assimilation. The algorithm is validated using the analytical model of a resistive expanding spheromak by Rakowski, Laming, and Lyutikov ( Astrophys. J. 730, 30, 2011). We study the flaring Active Region AR 12158 using a series of magnetograms and Dopplergrams provided by the Helioseismic and Magnetic Imager (HMI) onboard the Solar Dynamics Observatory (SDO). The results are discussed for a non-force-free magnetic-field reconstruction [boldsymbol{B}_{NFF}] (Hu and Dasgupta in Solar Phys. 247, 87, 2008). We found that the vertical plasma velocities [vz(x,y)] inferred using MEF-R are very similar to the observed Doppler velocities [vr(x,y)]. Finally, we study the potential spatial correlation between microturbulent velocities and significant values of η_{eddy}(x,y).

  14. 39Ar- 40Ar ages of martian nakhlites

    NASA Astrophysics Data System (ADS)

    Park, Jisun; Garrison, Daniel H.; Bogard, Donald D.

    2009-04-01

    We report 39Ar- 40Ar ages of whole rock (WR) and plagioclase and pyroxene mineral separates of nakhlites MIL 03346 and Y-000593, and of WR samples of nakhlites NWA 998 and Nakhla. All age spectra are complex and indicate variable degrees of 39Ar recoil and variable amounts of trapped 40Ar in the samples. Thus, we examine possible Ar-Ar ages in several ways. From consideration of both limited plateau ages and isochron ages, we prefer Ar-Ar ages of NWA 998 = 1334 ± 11 Ma, MIL 03346 = 1368 ± 83 Ma (mesostasis) and 1334 ± 54 Ma (pyroxene), Y-000593 = 1367 ± 7 Ma, and Nakhla = 1357 ± 11 Ma, (2 σ errors). For NWA 998 and MIL 03346 the Ar-Ar ages are within uncertainties of preliminary Rb-Sr isochron ages reported in the literature. These Ar-Ar ages for Y-000593 and Nakhla are several Ma older than Sm-Nd ages reported in the literature. We conclude that the major factor in producing Ar-Ar ages slightly too old is the presence of small amounts of trapped martian or terrestrial 40Ar on weathered grain surfaces that was degassed along with the first several percent of 39Ar. A total K- 40Ar isochron for WR and mineral data from five nakhlites analyzed by us, plus Lafayette data in the literature, gives an isochron age of 1325 ± 18 Ma (2 σ). We emphasize the precision of this isochron over the value of the isochron age. Our Ar-Ar data are consistent with a common formation age for nakhlites. The cosmic-ray exposure (CRE) age for NWA 998 of ˜12 Ma is also similar to CRE ages for other nakhlites.

  15. GBM heterogeneity as a function of variable epidermal growth factor receptor variant III activity

    PubMed Central

    Lindberg, Olle R.; McKinney, Andrew; Engler, Jane R.; Koshkakaryan, Gayane; Gong, Henry; Robinson, Aaron E.; Ewald, Andrew J.; Huillard, Emmanuelle; James, C. David; Molinaro, Annette M.; Shieh, Joseph T.; Phillips, Joanna J.

    2016-01-01

    Abnormal activation of the epidermal growth factor receptor (EGFR) due to a deletion of exons 2-7 of EGFR (EGFRvIII) is a common alteration in glioblastoma (GBM). While this alteration can drive gliomagenesis, tumors harboring EGFRvIII are heterogeneous. To investigate the role for EGFRvIII activation in tumor phenotype we used a neural progenitor cell-based murine model of GBM driven by EGFR signaling and generated tumor progenitor cells with high and low EGFRvIII activation, pEGFRHi and pEGFRLo. In vivo, ex vivo, and in vitro studies suggested a direct association between EGFRvIII activity and increased tumor cell proliferation, decreased tumor cell adhesion to the extracellular matrix, and altered progenitor cell phenotype. Time-lapse confocal imaging of tumor cells in brain slice cultures demonstrated blood vessel co-option by tumor cells and highlighted differences in invasive pattern. Inhibition of EGFR signaling in pEGFRHi promoted cell differentiation and increased cell-matrix adhesion. Conversely, increased EGFRvIII activation in pEGFRLo reduced cell-matrix adhesion. Our study using a murine model for GBM driven by a single genetic driver, suggests differences in EGFR activation contribute to tumor heterogeneity and aggressiveness. PMID:27738329

  16. SB 9200, a novel agonist of innate immunity, shows potent antiviral activity against resistant HCV variants.

    PubMed

    Jones, Meleri; Cunningham, Morven E; Wing, Peter; DeSilva, Sampath; Challa, Rupa; Sheri, Anjaneyulu; Padmanabhan, Seetharamaiyer; Iyer, Radhakrishnan P; Korba, Brent E; Afdhal, Nezam; Foster, Graham R

    2017-09-01

    SB 9200 is a novel, first-in-class oral modulator of innate immunity that is believed to act via the activation of the RIG-I and NOD2 pathways. SB 9200 has broad-spectrum antiviral activity against RNA viruses including hepatitis C virus (HCV), norovirus, respiratory syncytial virus, and influenza and has demonstrated activity against hepatitis B virus (HBV) in vitro and in vivo. In phase I clinical trials in chronically infected HCV patients, SB 9200 has been shown to reduce HCV RNA by up to 1.9 log10 . Here, we demonstrate the antiviral activity of SB 9200 against a HCV replicon system and patient derived virus. Using the HCV capture-fusion assay, we show that SB 9200 is active against diverse HCV genotypes and is also effective against HCV derived from patients who relapse following direct-acting antiviral treatment, including viruses containing known NS5A resistance-associated sequences. These data confirm the broad antiviral activity of SB 9200 and indicate that it may have clinical utility in HCV patients who have failed to respond to current antiviral regimens. © 2017 Wiley Periodicals, Inc.

  17. Structure-based activity prediction of CYP21A2 stability variants: A survey of available gene variations.

    PubMed

    Bruque, Carlos D; Delea, Marisol; Fernández, Cecilia S; Orza, Juan V; Taboas, Melisa; Buzzalino, Noemí; Espeche, Lucía D; Solari, Andrea; Luccerini, Verónica; Alba, Liliana; Nadra, Alejandro D; Dain, Liliana

    2016-12-14

    Congenital adrenal hyperplasia due to 21-hydroxylase deficiency accounts for 90-95% of CAH cases. In this work we performed an extensive survey of mutations and SNPs modifying the coding sequence of the CYP21A2 gene. Using bioinformatic tools and two plausible CYP21A2 structures as templates, we initially classified all known mutants (n = 343) according to their putative functional impacts, which were either reported in the literature or inferred from structural models. We then performed a detailed analysis on the subset of mutations believed to exclusively impact protein stability. For those mutants, the predicted stability was calculated and correlated with the variant's expected activity. A high concordance was obtained when comparing our predictions with available in vitro residual activities and/or the patient's phenotype. The predicted stability and derived activity of all reported mutations and SNPs lacking functional assays (n = 108) were assessed. As expected, most of the SNPs (52/76) showed no biological implications. Moreover, this approach was applied to evaluate the putative synergy that could emerge when two mutations occurred in cis. In addition, we propose a putative pathogenic effect of five novel mutations, p.L107Q, p.L122R, p.R132H, p.P335L and p.H466fs, found in 21-hydroxylase deficient patients of our cohort.

  18. A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties

    PubMed Central

    Millet, Jean Kaoru; Goldstein, Monty E; Labitt, Rachael N; Hsu, Hung-Lun; Daniel, Susan; Whittaker, Gary R

    2016-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) continues to circulate in both humans and camels, and the origin and evolution of the virus remain unclear. Here we characterize the spike protein of a camel-derived MERS-CoV (NRCE-HKU205) identified in 2013, early in the MERS outbreak. NRCE-HKU205 spike protein has a variant cleavage motif with regard to the S2′ fusion activation site—notably, a novel substitution of isoleucine for the otherwise invariant serine at the critical P1′ cleavage site position. The substitutions resulted in a loss of furin-mediated cleavage, as shown by fluorogenic peptide cleavage and western blot assays. Cell–cell fusion and pseudotyped virus infectivity assays demonstrated that the S2′ substitutions decreased spike-mediated fusion and viral entry. However, cathepsin and trypsin-like protease activation were retained, albeit with much reduced efficiency compared with the prototypical EMC/2012 human strain. We show that NRCE-HKU205 has more limited fusion activation properties possibly resulting in more restricted viral tropism and may represent an intermediate in the complex pattern of MERS-CoV ecology and evolution. PMID:27999426

  19. A Truncation Variant of the Cation Channel P2RX5 Is Upregulated during T Cell Activation

    PubMed Central

    Abramowski, Pierre; Ogrodowczyk, Christoph

    2014-01-01

    Members of the P2X family of ligand-gated cation channels (P2RX) are expressed by various cell types including neurons, smooth- and cardiac muscle cells, and leukocytes. The channels mediate signalling in response to extracellular ATP. Seven subunit isoforms (P2RX1-P2RX7) have been identified and these can assemble as homo- and heterotrimeric molecules. In humans, P2RX5 exists as a natural deletion mutant lacking amino acids 328–349 of exon 10, which are part of transmembrane (TM) 2 and pre-TM2 regions in other organisms like rat, chicken and zebrafish. We show that P2RX5 gene expression of human T lymphocytes is upregulated during activation. P2RX5 is recruited to the cell surface. P2RX5-siRNA-transfected CD4+ T cells produced twofold more IL-10 than controls. Surface and intracellular P2RX5 expression was upregulated in activated antigen-specific CD4+ T cell clones. These data indicate a functional role of the human P2RX5 splice variant in T cell activation and immunoregulation. PMID:25181038

  20. Bacterial expression and antibiotic activities of recombinant variants of human β-defensins on pathogenic bacteria and M. tuberculosis.

    PubMed

    Corrales-Garcia, Ligia; Ortiz, Ernesto; Castañeda-Delgado, Julio; Rivas-Santiago, Bruno; Corzo, Gerardo

    2013-05-01

    Five variants of human β-defensins (HBDs) were expressed in Escherichia coli using two vector systems (pET28a(+) and pQE30) with inducible expression by IPTG. The last vector has not been previously reported as an expression system for HBDs. The recombinant peptides were different in their lengths and overall charge. The HBDs were expressed as soluble or insoluble proteins depending on the expression system used, and the final protein yields ranged from 0.5 to 1.6 mg of peptide/g of wet weight cells, with purities higher than 90%. The recombinant HBDs demonstrated a direct correlation between antimicrobial activity and the number of basic charged residues; that is, their antimicrobial activity was as follows: HBD3-M-HBD2 > HBD3 = HBD3-M = HB2-KLK > HBD2 when assayed against E. coli, Staphylococcus aureus and Pseudomonas aeruginosa. Interestingly, HBD2 had the best antimicrobial activity against the Mycobacterium tuberculosis strain H37Rv (1.5 μM) and the heterologous tandem peptide, HBD3-M-HBD2, had the best minimal inhibitory concentration (MIC) value (2.7 μM) against a multidrug resistance strain (MDR) of M. tuberculosis, demonstrating the feasibility of the use of HBDs against pathogenic M. tuberculosis reported to be resistant to commercial antibiotics.

  1. The embryonic linker histone H1 variant of Drosophila, dBigH1, regulates zygotic genome activation.

    PubMed

    Pérez-Montero, Salvador; Carbonell, Albert; Morán, Tomás; Vaquero, Alejandro; Azorín, Fernando

    2013-09-30

    Histone H1 is an essential chromatin component. Metazoans usually contain multiple stage-specific H1s. In particular, specific variants replace somatic H1s during early embryogenesis. In this regard, Drosophila was an exception because a single dH1 was identified that, starting at cellularization, is detected throughout development in somatic cells. Here, we identify the embryonic H1 of Drosophila, dBigH1. dBigH1 is abundant before cellularization occurs, when somatic dH1 is absent and the zygotic genome is inactive. Upon cellularization, when the zygotic genome is progressively activated, dH1 replaces dBigH1 in the soma, but not in the primordial germ cells (PGCs) that have delayed zygotic genome activation (ZGA). In addition, a loss-of-function mutant shows premature ZGA in both the soma and PGCs. Mutant embryos die at cellularization, showing increased levels of active RNApol II and zygotic transcripts, along with DNA damage and mitotic defects. These results show an essential function of dBigH1 in ZGA regulation.

  2. Dual-Targeting of AR and Akt Pathways by Berberine in Castration-Resistant Prostate Cancer

    DTIC Science & Technology

    2015-08-01

    Berberine; natural compound; LuCaP86.2; castration resistant prostate cancer; AR splice variants. 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF...resistant prostate cancer (CRPC) driven by AR splice variants. 2. Keywords Prostate cancer; androgen receptor; berberine; natural compound; LuCaP86.2...5P20GM103518-10, Louisiana Cancer Research Consortium Fund, Oliver Sartor Prostate Cancer Research Fund, National Natural Science Foundation of China

  3. Identification of a novel K311 ubiquitination site critical for androgen receptor transcriptional activity.

    PubMed

    McClurg, Urszula L; Cork, David M W; Darby, Steven; Ryan-Munden, Claudia A; Nakjang, Sirintra; Mendes Côrtes, Leticia; Treumann, Achim; Gaughan, Luke; Robson, Craig N

    2016-11-29

    The androgen receptor (AR) is the main driver of prostate cancer (PC) development and progression, and the primary therapeutic target in PC. To date, two functional ubiquitination sites have been identified on AR, both located in its C-terminal ligand binding domain (LBD). Recent reports highlight the emergence of AR splice variants lacking the LBD that can arise during disease progression and contribute to castrate resistance. Here, we report a novel N-terminal ubiquitination site at lysine 311. Ubiquitination of this site plays a role in AR stability and is critical for its transcriptional activity. Inactivation of this site causes AR to accumulate on chromatin and inactivates its transcriptional function as a consequence of inability to bind to p300. Additionally, mutation at lysine 311 affects cellular transcriptome altering the expression of genes involved in chromatin organization, signaling, adhesion, motility, development and metabolism. Even though this site is present in clinically relevant AR-variants it can only be ubiquitinated in cells when AR retains LBD suggesting a role for AR C-terminus in E2/E3 substrate recognition. We report that as a consequence AR variants lacking the LBD cannot be ubiquitinated in the cellular environment and their protein turnover must be regulated via an alternate pathway.

  4. 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

  5. RNA Chaperone Activity of Human La Protein Is Mediated by Variant RNA Recognition Motif*

    PubMed Central

    Naeeni, Amir R.; Conte, Maria R.; Bayfield, Mark A.

    2012-01-01

    La proteins are conserved factors in eukaryotes that bind and protect the 3′ trailers of pre-tRNAs from exonuclease digestion via sequence-specific recognition of UUU-3′OH. La has also been hypothesized to assist pre-tRNAs in attaining their native fold through RNA chaperone activity. In addition to binding polymerase III transcripts, human La has also been shown to enhance the translation of several internal ribosome entry sites and upstream ORF-containing mRNA targets, also potentially through RNA chaperone activity. Using in vitro FRET-based assays, we show that human and Schizosaccharomyces pombe La proteins harbor RNA chaperone activity by enhancing RNA strand annealing and strand dissociation. We use various RNA substrates and La mutants to show that UUU-3′OH-dependent La-RNA binding is not required for this function, and we map RNA chaperone activity to its RRM1 motif including a noncanonical α3-helix. We validate the importance of this α3-helix by appending it to the RRM of the unrelated U1A protein and show that this fusion protein acquires significant strand annealing activity. Finally, we show that residues required for La-mediated RNA chaperone activity in vitro are required for La-dependent rescue of tRNA-mediated suppression via a mutated suppressor tRNA in vivo. This work delineates the structural elements required for La-mediated RNA chaperone activity and provides a basis for understanding how La can enhance the folding of its various RNA targets. PMID:22203678

  6. RNA chaperone activity of human La protein is mediated by variant RNA recognition motif.

    PubMed

    Naeeni, Amir R; Conte, Maria R; Bayfield, Mark A

    2012-02-17

    La proteins are conserved factors in eukaryotes that bind and protect the 3' trailers of pre-tRNAs from exonuclease digestion via sequence-specific recognition of UUU-3'OH. La has also been hypothesized to assist pre-tRNAs in attaining their native fold through RNA chaperone activity. In addition to binding polymerase III transcripts, human La has also been shown to enhance the translation of several internal ribosome entry sites and upstream ORF-containing mRNA targets, also potentially through RNA chaperone activity. Using in vitro FRET-based assays, we show that human and Schizosaccharomyces pombe La proteins harbor RNA chaperone activity by enhancing RNA strand annealing and strand dissociation. We use various RNA substrates and La mutants to show that UUU-3'OH-dependent La-RNA binding is not required for this function, and we map RNA chaperone activity to its RRM1 motif including a noncanonical α3-helix. We validate the importance of this α3-helix by appending it to the RRM of the unrelated U1A protein and show that this fusion protein acquires significant strand annealing activity. Finally, we show that residues required for La-mediated RNA chaperone activity in vitro are required for La-dependent rescue of tRNA-mediated suppression via a mutated suppressor tRNA in vivo. This work delineates the structural elements required for La-mediated RNA chaperone activity and provides a basis for understanding how La can enhance the folding of its various RNA targets.

  7. Pharmacological targeting of constitutively active truncated androgen receptor by nigericin and suppression of hormone-refractory prostate cancer cell growth.

    PubMed

    Mashima, Tetsuo; Okabe, Sachiko; Seimiya, Hiroyuki

    2010-11-01

    In prostate cancer, blockade of androgen receptor (AR) signaling confers a therapeutic benefit. Nevertheless, this standard therapy allows relapse of hormone-refractory prostate cancer (HRPC) with a poor prognosis. HRPC cells often express variant ARs, such as point-mutated alleles and splicing isoforms, resulting in androgen-independent cell growth and resistance to antiandrogen (e.g., flutamide). However, a pharmacological strategy to block such aberrant ARs remains to be established. Here, we established a reporter system that monitors AR-mediated activation of a prostate-specific antigen (PSA) promoter. Our chemical library screening revealed that the antibiotic nigericin inhibits AR-mediated activation of the PSA promoter and PSA production in prostate cancer cells. Nigericin suppressed the androgen-dependent LNCaP cell growth even though the cells expressed a flutamide-resistant mutant AR. These effects were caused by AR suppression at the mRNA and post-translational levels. In HRPC 22Rv1 cells, which express the full-length AR and the constitutively active, truncated ARs lacking the carboxyl-terminal ligand-binding domain, small interfering RNA-mediated knockdown of both AR isoforms efficiently suppressed the androgen-independent cell growth, whereas knockdown of the full-length AR alone had no significant effect. It is noteworthy that nigericin was able to mimic the knockdown of both AR isoforms: it reduced the expression of the full-length and the truncated ARs, and it induced G(1) cell-cycle arrest and apoptosis of 22Rv1 cells. These observations suggest that nigericin-like compounds that suppress AR expression at the mRNA level could be applied as new-type therapeutic agents that inhibit a broad spectrum of AR variants in HRPC.

  8. Biochemical characterization of three hamster melanoma variants--I. Tyrosinase activity and melanin content.

    PubMed

    Słomiński, A; Scisłowski, P W; Bomirski, A

    1984-01-01

    Tyrosinase activity in the soluble fraction of the cells and melanin content in the whole cells of the black-melanotic (Ma), brown-melanotic (MI) and amelanotic (Ab) hamster melanomas were studied. The activity of the soluble tyrosinase was highest in MI lower in Ma, and very low in Ab melanoma. Melanin content was greatest in the Ma, lower in MI, and none in Ab melanoma. Acrylamide gel electrophoretic pattern of the soluble tyrosinase consisted of 2 bands in Ma and MI melanomas, and of 1 band in Ab melanoma.

  9. 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.…

  10. 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.…

  11. Genetic variants in ABCA1 promoter affect transcription activity and plasma HDL level in pigs.

    PubMed

    Dang, Xiao-yong; Chu, Wei-wei; Shi, Heng-chuan; Yu, Shi-gang; Han, Hai-yin; Gu, Shu-Hua; Chen, Jie

    2015-01-25

    Excess accumulation of cholesterol in plasma may result in coronary artery disease. Numerous studies have demonstrated that ATP-binding cassette protein A1 (ABCA1) mediates the efflux of cholesterol and phospholipids to apolipoproteins, a process necessary for plasma high density lipoprotein (HDL) formation. Higher plasma levels of HDL are associated with lower risk for cardiovascular disease. Studies of human disease and animal models had shown that an increased hepatic ABCA1 activity relates to an enhanced plasma HDL level. In this study, we hypothesized that functional mutations in the ABCA1 promoter in pigs may affect gene transcription activity, and consequently the HDL level in plasma. The promoter region of ABCA1 was comparatively scanned by direct sequencing with pool DNA of high- and low-HDL groups (n=30 for each group). Two polymorphisms, c. - 608A>G and c. - 418T>A, were revealed with reverse allele distribution in the two groups. The two polymorphisms were completely linked and formed only G-A or A-T haplotypes when genotyped in a larger population (n=526). Furthermore, we found that the G-A/G-A genotype was associated with higher HDL and ABCA1 mRNA level than A-T/A-T genotype. Luciferase assay also revealed that G-A haplotype promoter had higher activity than A-T haplotype. Single-nucleotide mutant assay showed that c.-418T>A was the causal mutation for ABCA1 transcription activity alteration. Conclusively, we identified two completely linked SNPs in porcine ABCA1 promoter region which have influence on the plasma HDL level by altering ABCA1 gene transcriptional activity.

  12. FTO variant rs9939609 is associated with body mass index and waist circumference, but not with energy intake or physical activity in European- and African-American youth

    PubMed Central

    2010-01-01

    Background Genome-wide association studies found common variants in the fat mass and obesity-associated (FTO) gene associated with adiposity in Caucasians and Asians but the association was not confirmed in African populations. Association of FTO variants with insulin resistance and energy intake showed inconsistent results in previous studies. This study aimed to assess the influence of FTO variant rs9939609 on adiposity, insulin resistance, energy intake and physical activity in European - (EA) and African-American (AA) youth. Methods We conducted a cross-sectional study in EA and AA youths. One thousand, nine hundred and seventy-eight youths (48.2% EAs, 47.1% male, mean age 16.5 years) had measures of anthropometry. Percent body fat (%BF) was measured by dual-energy X-ray absorptiometry, visceral adipose tissue (VAT) and subcutaneous abdominal adipose tissue (SAAT) by magnetic resonance imaging. Energy intake and physical activity were based on self report from up to 7 24-hour recalls. Physical activity was also measured by accelerometry. Results FTO rs9939609 was significantly associated with body mass index (BMI) (P = 0.01), weight (P = 0.03) and waist circumference (P = 0.04), with per-allele effects of 0.4 kg/m2, 1.3 kg and 0.8 cm, respectively. No significant association was found between rs9939609 and %BF, VAT, SAAT or insulin resistance (P > 0.05), or between rs9939609 and energy intake or vigorous physical activity (P > 0.05). No significant interactions of rs9939609 with ethnicity, gender, energy intake or physical activity were observed (P > 0.05). Conclusions The FTO variant rs9939609 is modestly associated with BMI and waist circumference, but not with energy intake or physical activity. Moreover, these effects were similar for EAs and AAs. Improved understanding of the effect of the FTO variant will offer new insights into the etiology of excess adiposity. PMID:20377915

  13. FTO variant rs9939609 is associated with body mass index and waist circumference, but not with energy intake or physical activity in European- and African-American youth.

    PubMed

    Liu, Gaifen; Zhu, Haidong; Lagou, Vasiliki; Gutin, Bernard; Stallmann-Jorgensen, Inger S; Treiber, Frank A; Dong, Yanbin; Snieder, Harold

    2010-04-09

    Genome-wide association studies found common variants in the fat mass and obesity-associated (FTO) gene associated with adiposity in Caucasians and Asians but the association was not confirmed in African populations. Association of FTO variants with insulin resistance and energy intake showed inconsistent results in previous studies. This study aimed to assess the influence of FTO variant rs9939609 on adiposity, insulin resistance, energy intake and physical activity in European - (EA) and African-American (AA) youth. We conducted a cross-sectional study in EA and AA youths. One thousand, nine hundred and seventy-eight youths (48.2% EAs, 47.1% male, mean age 16.5 years) had measures of anthropometry. Percent body fat (%BF) was measured by dual-energy X-ray absorptiometry, visceral adipose tissue (VAT) and subcutaneous abdominal adipose tissue (SAAT) by magnetic resonance imaging. Energy intake and physical activity were based on self report from up to 7 24-hour recalls. Physical activity was also measured by accelerometry. FTO rs9939609 was significantly associated with body mass index (BMI) (P = 0.01), weight (P = 0.03) and waist circumference (P = 0.04), with per-allele effects of 0.4 kg/m2, 1.3 kg and 0.8 cm, respectively. No significant association was found between rs9939609 and %BF, VAT, SAAT or insulin resistance (P > 0.05), or between rs9939609 and energy intake or vigorous physical activity (P > 0.05). No significant interactions of rs9939609 with ethnicity, gender, energy intake or physical activity were observed (P > 0.05). The FTO variant rs9939609 is modestly associated with BMI and waist circumference, but not with energy intake or physical activity. Moreover, these effects were similar for EAs and AAs. Improved understanding of the effect of the FTO variant will offer new insights into the etiology of excess adiposity.

  14. 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

    2016-09-07

    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.

  15. Functional characterization of a constitutively active kinase variant of Arabidopsis phototropin 1.

    PubMed

    Petersen, Jan; Inoue, Shin-Ichiro; Kelly, Sharon M; Sullivan, Stuart; Kinoshita, Toshinori; Christie, John M

    2017-08-18

    Phototropins (phots) are plasma membrane-associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen-, or voltage-sensing (LOV) domains within their N terminus, with each binding one molecule of flavin mononucleotide as a UV/blue light-absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A'α-LOV2-Jα photosensory module. Mutations that disrupt interactions between the LOV2 core and its flanking helical segments can uncouple this mode of light regulation. However, the impact of these mutations on phot function in Arabidopsis has not been explored. Here we report that histidine substitution of Arg-472 located within the A'α-helix of Arabidopsis phot1 constitutively activates phot1 kinase activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo but unable to initiate phot1 signaling in the absence of light. Instead, we found that phot1 R472H is poorly functional under low-light conditions but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant in part by reducing its stability, whereas the activity of the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. SALSA, a variant of yeast SAGA, contains truncated Spt7, which correlates with activated transcription.

    PubMed

    Sterner, David E; Belotserkovskaya, Rimma; Berger, Shelley L

    2002-09-03

    Spt-Ada-Gcn5 acetyltransferase (SAGA) is a previously described histone acetyltransferase/transcriptional coactivator complex in yeast. At promoters of certain genes (HIS3 and TRP3), SAGA has an inhibitory function involving a nonproductive TATA-binding protein interaction mediated by the Spt3 and Spt8 subunits. Related to this, Spt8-less SAGA is a major form of the complex under activating conditions for these genes. In the present study, we purify this activation-specific complex, called SALSA (SAGA altered, Spt8 absent). Besides lacking Spt8, SALSA contains Spt7 subunit that is truncated. Examining the role of this subunit, we find that C-terminally truncated SPT7 resulted in derepressed HIS3 transcription. Furthermore, when grown in rich media (repressing conditions), wild-type cells yielded predominantly SAGA, but Spt7 C-terminal truncations resulted primarily in a form of complex similar to SALSA. Thus, SALSA-like structure and activating function can be partially recapitulated in yeast by truncating the C terminus of Spt7. Overall, these results lead to a model that for a subset of promoters SAGA is inhibitory through Spt3, Spt8, and an Spt8-interacting subdomain of Spt7, whereas SALSA is a form of complex for positive transcriptional regulation. These data clarify a mechanism by which a transcriptional regulatory complex can switch between positive and negative modulation.

  17. 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

  18. 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

  19. 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

  20. AR Sco observing campaign

    NASA Astrophysics Data System (ADS)

    Waagen, Elizabeth O.

    2016-08-01

    Dr. Thomas Marsh (University of Warwick) and colleagues have requested AAVSO coverage of the intriguing binary AR Sco in support of upcoming Newton-XMM observations scheduled for 2016 September 10 15:41 - September 11 02:26 UT. This fascinating binary system is the subject of an exciting paper in the July 2016 issue of Nature (Marsh et al., 2016Natur.537..374M; pre-print version at arXiv (http://arxiv.org/abs/1607.08265). Marsh writes of their research on AR Sco: "...it was down to [the amateurs [who are co-authors] on the paper that we got onto it in the first place. Coverage immediately before, after and (especially) during [the XMM observations] would be great. The most challenging aspect is the time resolution: ideally one wants a cadence < 29 seconds because of the strong harmonic of the basic 2 minute period, and the faster the better. Observers should use whatever filter (including clear/white light) is needed to allow them to match this constraint. Accurate timing is also essential - the centres of the exposures need to be known to better than ± 2 seconds, and preferably better." A page of materials on AR Sco related to the Nature paper may be found at http://deneb.astro.warwick.ac.uk/phsaap/arsco-info/ . Item #9 on that page is a YouTube video of a fascinating movie Dr. Marsh made of AR Sco from their data (https://www.youtube.com/watch?v=QJGAv2jCF4s&feature=youtu.be). Finder charts with sequence may be created using the AAVSO Variable Star Plotter (https://www.aavso.org/vsp). Observations should be submitted to the AAVSO International Database. See full Alert Notice for more details.

  1. 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.

  2. 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

  3. Expression of NKp46 Splice Variants in Nasal Lavage Following Respiratory Viral Infection: Domain 1-Negative Isoforms Predominate and Manifest Higher Activity

    PubMed Central

    Shemer-Avni, Yonat; Kundu, Kiran; Shemesh, Avishai; Brusilovsky, Michael; Yossef, Rami; Meshesha, Mesfin; Solomon-Alemayehu, Semaria; Levin, Shai; Gershoni-Yahalom, Orly; Campbell, Kerry S.; Porgador, Angel

    2017-01-01

    The natural killer (NK) cell activating receptor NKp46/NCR1 plays a critical role in elimination of virus-infected and tumor cells. The NCR1 gene can be transcribed into five different splice variants, but the functional importance and physiological distribution of NKp46 isoforms are not yet fully understood. Here, we shed light on differential expression of NKp46 splice variants in viral respiratory tract infections and their functional difference at the cellular level. NKp46 was the most predominantly expressed natural cytotoxicity receptor in the nasal lavage of patients infected with four respiratory viruses: respiratory syncytia virus, adenovirus, human metapneumovirus, or influenza A. Expression of NKp30 was far lower and NKp44 was absent in all patients. Domain 1-negative NKp46 splice variants (i.e., NKp46 isoform d) were the predominantly expressed isoform in nasal lavage following viral infections. Using our unique anti-NKp46 mAb, D2-9A5, which recognizes the D2 extracellular domain, and a commercial anti-NKp46 mAb, 9E2, which recognizes D1 domain, allowed us to identify a small subset of NKp46 D1-negative splice variant-expressing cells within cultured human primary NK cells. This NKp46 D1-negative subset also showed higher degranulation efficiency in term of CD107a surface expression. NK-92 cell lines expressing NKp46 D1-negative and NKp46 D1-positive splice variants also showed functional differences when interacting with targets. A NKp46 D1-negative isoform-expressing NK-92 cell line showed enhanced degranulation activity. To our knowledge, we provide the first evidence showing the physiological distribution and functional importance of human NKp46 splice variants under pathological conditions. PMID:28261217

  4. 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.

  5. Engineered Tissue Inhibitor of Metalloproteinases-3 Variants Resistant to Endocytosis Have Prolonged Chondroprotective Activity*

    PubMed Central

    Doherty, Christine M.; Visse, Robert; Dinakarpandian, Deendayal; Strickland, Dudley K.; Nagase, Hideaki; Troeberg, Linda

    2016-01-01

    Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a central inhibitor of matrix-degrading and sheddase families of metalloproteinases. Extracellular levels of the inhibitor are regulated by the balance between its retention on the extracellular matrix and its endocytic clearance by the scavenger receptor low density lipoprotein receptor-related protein 1 (LRP1). Here, we used molecular modeling to predict TIMP-3 residues potentially involved in binding to LRP1 based on the proposed LRP1 binding motif of 2 lysine residues separated by about 21 Å and mutated the candidate lysine residues to alanine individually and in pairs. Of the 22 mutants generated, 13 displayed a reduced rate of uptake by HTB94 chondrosarcoma cells. The two mutants (TIMP-3 K26A/K45A and K42A/K110A) with lowest rates of uptake were further evaluated and found to display reduced binding to LRP1 and unaltered inhibitory activity against prototypic metalloproteinases. TIMP-3 K26A/K45A retained higher affinity for sulfated glycosaminoglycans than K42A/K110A and exhibited increased affinity for ADAMTS-5 in the presence of heparin. Both mutants inhibited metalloproteinase-mediated degradation of cartilage at lower concentrations and for longer than wild-type TIMP-3, indicating that their increased half-lives improved their ability to protect cartilage. These mutants may be useful in treating connective tissue diseases associated with increased metalloproteinase activity. PMID:27582494

  6. Inherited genetic variants in autism-related CNTNAP2 show perturbed trafficking and ATF6 activation

    PubMed Central

    Falivelli, Giulia; De Jaco, Antonella; Favaloro, Flores Lietta; Kim, Hyuck; Wilson, Jennifer; Dubi, Noga; Ellisman, Mark H.; Abrahams, Brett S.; Taylor, Palmer; Comoletti, Davide

    2012-01-01

    Although genetic variations in several genes encoding for synaptic adhesion proteins have been found to be associated with autism spectrum disorders, one of the most consistently replicated genes has been CNTNAP2, encoding for contactin-associated protein-like 2 (CASPR2), a multidomain transmembrane protein of the neurexin superfamily. Using immunofluorescence confocal microscopy and complementary biochemical techniques, we compared wild-type CASPR2 to 12 point mutations identified in individuals with autism. In contrast to the wild-type protein, localized to the cell surface, some of the mutants show altered cellular disposition. In particular, CASPR2-D1129H is largely retained in the endoplasmic reticulum (ER) in HEK-293 cells and in hippocampal neurons. BiP/Grp78, Calnexin and ERp57, key ER chaperones, appear to be responsible for retention of this mutant and activation of one signaling pathway of the unfolded protein response (UPR). The presence of this mutation also lowers expression and activates proteosomal degradation. A frame-shift mutation that causes a form of syndromic epilepsy (CASPR2-1253*), results in a secreted protein with seemingly normal folding and oligomerization. Taken together, these data indicate that CASPR2-D1129H has severe trafficking abnormalities and CASPR2-1253* is a secreted soluble protein, suggesting that the structural or signaling functions of the membrane tethered form are lost. Our data support a complex genetic architecture in which multiple distinct risk factors interact with others to shape autism risk and presentation. PMID:22872700

  7. The -14010*C variant associated with lactase persistence is located between an Oct-1 and HNF1α binding site and increases lactase promoter activity.

    PubMed

    Jensen, Tine G K; Liebert, Anke; Lewinsky, Rikke; Swallow, Dallas M; Olsen, Jørgen; Troelsen, Jesper T

    2011-10-01

    In most people worldwide intestinal lactase expression declines in childhood. In many others, particularly in Europeans, lactase expression persists into adult life. The lactase persistence phenotype is in Europe associated with the -13910*T single nucleotide variant located 13,910 bp upstream the lactase gene in an enhancer region that affects lactase promoter activity. This variant falls in an Oct-1 binding site and shows greater Oct-1 binding than the ancestral variant and increases enhancer activity. Several other variants have been identified very close to the -13910 position, which are associated with lactase persistence in the Middle East and Africa. One of them, the -14010*C, is associated with lactase persistence in Africa. Here we show by deletion analysis that the -14010 position is located in a 144 bp region that reduces the enhancer activity. In transfections the -14010*C allele shows a stronger enhancer effect than the ancestral -4010*G allele. Binding sites for Oct-1 and HNF1α surrounding the -14010 position were identified by gel shift assays, which indicated that -14010*C has greater binding affinity to Oct-1 than -14010*G.

  8. Clinical Variants of the Native Class D β-Lactamase of Acinetobacter baumannii Pose an Emerging Threat through Increased Hydrolytic Activity against Carbapenems

    PubMed Central

    Schroder, Emma C.; Klamer, Zachary L.; Saral, Aysegul; Sugg, Kyle A.; June, Cynthia M.; Wymore, Troy; Szarecka, Agnieszka

    2016-01-01

    The threat posed by the chromosomally encoded class D β-lactamase of Acinetobacter baumannii (OXA-51/66) has been unclear, in part because of its relatively low affinity and turnover rate for carbapenems. Several hundred clinical variants of OXA-51/66 have been reported, many with substitutions of active-site residues. We determined the kinetic properties of OXA-66 and five clinical variants with respect to a wide variety of β-lactam substrates. The five variants displayed enhanced activity against carbapenems and in some cases against penicillins, late-generation cephalosporins, and the monobactam aztreonam. Molecular dynamics simulations show that in OXA-66, P130 inhibits the side-chain rotation of I129 and thereby prevents doripenem binding because of steric clash. A single amino acid substitution at this position (P130Q) in the variant OXA-109 greatly enhances the mobility of both I129 and a key active-site tryptophan (W222), thereby facilitating carbapenem binding. This expansion of substrate specificity represents a very worrisome development for the efficacy of β-lactams against this troublesome pathogen. PMID:27480863

  9. Alternative splicing of CARMA2/CARD14 transcripts generates protein variants with differential effect on NF-κB activation and endoplasmic reticulum stress-induced cell death.

    PubMed

    Scudiero, Ivan; Zotti, Tiziana; Ferravante, Angela; Vessichelli, Mariangela; Vito, Pasquale; Stilo, Romania

    2011-12-01

    The caspase recruitment domain (CARD)-containing proteins CARMA1-3 share high degree of sequence, structure and functional homology. Whereas CARMA1 and CARMA3 have been identified as crucial components of signal transduction pathways that lead to activation of NF-κB transcription factor, little is known about the function of CARMA2. Here we report the identification of two splice variants of CARMA2. One transcript, named CARMA2short (CARMA2sh), is predicted to encode for a CARMA2 polypeptide containing the CARD, coiled coil, and a PDZ domains, but lacking the SH3 and the GuK domains. The second variant, CARMA2cardless (CARMA2cl), encodes for a polypeptide lacking the CARD domain and containing only a portion of the coiled coil domain and a linker region. Expression analysis confirmed the presence of the CARMA2 alternatively spliced transcripts in both human cell lines and tissues. Fluorescence microscopy data show that both splice variants localize in the cytosol. Biochemical experiments indicate that CARMA2sh interacts with TRAF2 and activates NF-κB in a TRAF2-dependent manner. Finally, CARMA2sh variant protects cells from apoptosis induced by different stimuli. Taken together, these results demonstrate that multiple transcripts encoding several CARMA2 isoforms exist in vivo and regulate NF-κB activation and apoptosis.

  10. Alternative Splicing of CARMA2/CARD14 Transcripts Generates Protein Variants With Differential Effect on NF-κB Activation and Endoplasmic Reticulum Stress-Induced Cell Death

    PubMed Central

    Scudiero, Ivan; Zotti, Tiziana; Ferravante, Angela; Vessichelli, Mariangela; Vito, Pasquale; Stilo, Romania

    2011-01-01

    The caspase recruitment domain (CARD)-containing proteins CARMA1-3 share high degree of sequence, structure and functional homology. Whereas CARMA1 and CARMA3 have been identified as crucial components of signal transduction pathways that lead to activation of NF-κB transcription factor, little is known about the function of CARMA2. Here we report the identification of two splice variants of CARMA2. One transcript, named CARMA2short (CARMA2sh), is predicted to encode for a CARMA2 polypeptide containing the CARD, coiled coil, and a PDZ domains, but lacking the SH3 and the GuK domains. The second variant, CARMA2cardless (CARMA2cl), encodes for a polypeptide lacking the CARD domain and containing only a portion of the coiled coil domain and a linker region. Expression analysis confirmed the presence of the CARMA2 alternatively spliced transcripts in both human cell lines and tissues. Fluorescence microscopy data show that both splice variants localize in the cytosol. Biochemical experiments indicate that CARMA2sh interacts with TRAF2 and activates NF-κB in a TRAF2-dependent manner. Finally, CARMA2sh variant protects cells from apoptosis induced by different stimuli. Taken together, these results demonstrate that multiple transcripts encoding several CARMA2 isoforms exist in vivo and regulate NF-κB activation and apoptosis. J. Cell. Physiol. 226: 3121–3131, 2011. © 2011 Wiley Periodicals, Inc. PMID:21302310

  11. Time-variant power spectrum analysis for the detection of transient episodes in HRV signal.

    PubMed

    Bianchi, A M; Mainardi, L; Petrucci, E; Signorini, M G; Mainardi, M; Cerutti, S

    1993-02-01

    A time-variant algorithm of autoregressive (AR) identification is introduced and applied to the heart rate variability (HRV) signal. The power spectrum is calculated from the AR coefficients derived from each single RR interval considered. Time-variant AR coefficients are determined through adaptive parametric identification with a forgetting factor which obtains weighed values on a running temporal window of 50 preceding measurements. Power spectrum density (PSD) is hence obtained at each cardiac cycle, making it possible to follow the dynamics of the spectral parameters on a beat-by-beat basis. These parameters are mainly the LF (low frequency) and the HF (high frequency) powers, and their ratio LF/HF. These together account for the balanced sympatho-vagal control mechanism affecting the heart rate. This method is applied to subjects suffering from transient ischemic attacks. The time variant spectral parameters suggest an early activation of LF component in the HRV power spectrum. It precedes by approximately 1.5-2 min the tachycardia and the ST displacement, generally indicative of the onset of an ischemic episode. The results suggest an arousal of sympathetic system before the acute attack.

  12. 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

  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.

  14. 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

  15. Synthesis and structure-activity relationships of a new model of arylpiperazines. 8. Computational simulation of ligand-receptor interaction of 5-HT(1A)R agonists with selectivity over alpha1-adrenoceptors.

    PubMed

    López-Rodríguez, María L; Morcillo, Maria José; Fernández, Esther; Benhamú, Bellinda; Tejada, Ignacio; Ayala, David; Viso, Alma; Campillo, Mercedes; Pardo, Leonardo; Delgado, Mercedes; Manzanares, Jorge; Fuentes, José A

    2005-04-07

    We have designed and synthesized a new series of arylpiperazines V exhibiting high 5-HT(1A)R affinity and selectivity over alpha(1)-adrenoceptors. The new selective 5-HT(1A)R ligands contain a hydantoin (m = 0) or diketopiperazine (m = 1) moiety and an arylpiperazine moiety separated by one methylene unit (n = 1). The aryl substituent of the piperazine moiety (Ar) consists of different benzofused rings mimicking the favorable voluminous substituents at ortho and meta positions predicted by 3D-QSAR analysis in the previously reported series I. In particular, (S)-2-[[4-(naphth-1-yl)piperazin-1-yl]methyl]-1,4-dioxoperhydropyrrolo[1,2-a]pyrazine [(S)-9, CSP-2503] (5-HT(1A), K(i) = 4.1 nM; alpha(1), K(i) > 1000 nM) has been pharmacologically characterized as a 5-HT(1A)R agonist at somatodendritic and postsynaptic sites, endowed with anxiolytic properties. Ligand (S)-9 is predicted, in computer simulations, to bind Asp(3.32) in TMH 3, Thr(5.39) and Ser(5.42) in TMH 5, and Trp(6.48) in TMH 6. We propose that agonists modify, by means of an explicit hydrogen bond, the conformation of Trp(6.48) from pointing toward TMH 7, in the inactive gauche+ conformation, to pointing toward the ligand binding site, in the active trans conformation.

  16. High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.

    PubMed

    Xu, Bing; Chakraborty, Raja; Eilers, Markus; Dakshinamurti, Shyamala; O'Neil, Joe D; Smith, Steven O; Bhullar, Rajinder P; Chelikani, Prashen

    2013-01-01

    G protein-coupled receptors (GPCRs) exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM) of the dim-light receptor, rhodopsin. The structural characterizations of CAMs are impeded by the lack of proper expression systems. The thromboxane A2 receptor (TP) is a GPCR that mediates vasoconstriction and promotes thrombosis in response to the binding of thromboxane. Here, we report on the expression and purification of a genetic variant and CAM in TP, namely A160T, using tetracycline-inducible HEK293S-TetR and HEK293S (GnTI¯)-TetR cell lines. Expression of the TP and the A160T genes in these mammalian cell lines resulted in a 4-fold increase in expression to a level of 15.8 ±0.3 pmol of receptor/mg of membrane protein. The receptors expressed in the HEK293S (GnTI(-))-TetR cell line showed homogeneous glycosylation. The functional yield of the receptors using a single step affinity purification was 45 µg/10⁶ cells. Temperature- dependent secondary structure changes of the purified TP and A160T receptors were characterized using circular dichroism (CD) spectropolarimetry. The CD spectra shows that the loss of activity or thermal sensitivity that was previously observed for the A160T mutant, is not owing to large unfolding of the protein but rather to a more subtle effect. This is the first study to report on the successful high-level expression, purification, and biophysical characterization of a naturally occurring, diffusible ligand activated GPCR CAM.

  17. High-Level Expression, Purification and Characterization of a Constitutively Active Thromboxane A2 Receptor Polymorphic Variant

    PubMed Central

    Xu, Bing; Chakraborty, Raja; Eilers, Markus; Dakshinamurti, Shyamala; O’Neil, Joe D.; Smith, Steven O.; Bhullar, Rajinder P.; Chelikani, Prashen

    2013-01-01

    G protein-coupled receptors (GPCRs) exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM) of the dim-light receptor, rhodopsin. The structural characterizations of CAMs are impeded by the lack of proper expression systems. The thromboxane A2 receptor (TP) is a GPCR that mediates vasoconstriction and promotes thrombosis in response to the binding of thromboxane. Here, we report on the expression and purification of a genetic variant and CAM in TP, namely A160T, using tetracycline-inducible HEK293S-TetR and HEK293S (GnTI¯)-TetR cell lines. Expression of the TP and the A160T genes in these mammalian cell lines resulted in a 4-fold increase in expression to a level of 15.8 ±0.3 pmol of receptor/mg of membrane protein. The receptors expressed in the HEK293S (GnTI-)-TetR cell line showed homogeneous glycosylation. The functional yield of the receptors using a single step affinity purification was 45 µg/106 cells. Temperature- dependent secondary structure changes of the purified TP and A160T receptors were characterized using circular dichroism (CD) spectropolarimetry. The CD spectra shows that the loss of activity or thermal sensitivity that was previously observed for the A160T mutant, is not owing to large unfolding of the protein but rather to a more subtle effect. This is the first study to report on the successful high-level expression, purification, and biophysical characterization of a naturally occurring, diffusible ligand activated GPCR CAM. PMID:24086743

  18. Expression Levels and Activation of a PXR Variant Are Directly Related to Drug Resistance in Osteosarcoma Cell Lines

    PubMed Central

    Mensah-Osman, Edith J.; Thomas, Dafydd G.; Tabb, Michelle M.; Larios, Jose M.; Hughes, Dennis P.; Giordano, Thomas J.; Lizyness, Michelle L.; Rae, James M.; Blumberg, Bruce; Hollenberg, Paul F.; Baker, Laurence H.

    2011-01-01

    BACKGROUND Approximately 30% to 40% of all patients with osteosarcomas ultimately experience recurrence. The study investigated the hypothesis that the resistance of osteosarcoma to chemotherapy may be related to the expression of a pregnane xenobiotic receptor (PXR) variant protein and its role as the major inducer of P450 3A4 in these tumors. METHODS Polymerase chain reaction (PCR) and Western blot analysis were used to determine PXR mRNA and protein expression, respectively. Real-time PCR and CYP3A catalytic activity using 7-benzyl-trifluoromethyl coumarin (BFC) as the probe substrate were used to measure the induction of P450 3A4 or MDR1. siRNA transfections were performed for PXR and cytotoxicity determined by a colorimetric based assay or Annexin v-Fitc staining. RESULTS Differences were observed in the molecular size of the PXR protein expressed in sarcoma cell lines when compared with the wildtype PXR expressed in normal liver, kidney, or small intestine. A polyclonal PXR antibody raised against the N-terminus of the wildtype PXR did not detect PXR expressed in these sarcoma cell lines. In the osteosarcoma cell lines, etoposide and doxorubicin were better inducers of P450 3A4 and MDR1 than rifampin. siRNA against PXR down-regulated P450 3A4 expression only in the osteosarcoma cell line. Cytotoxicity assays showed that the resistance of the osteosarcoma cell lines to etoposide correlated with PXR protein expression levels and activation of P450 3A4 and could be prevented by ketoconazole. CONCLUSION The results suggest that PXR plays a critical role in the regulation of P450 3A4 expression in osteosarcoma and that its expression and activation in these tumors may influence the effect of chemotherapeutic agents on the induction of target genes implicated in drug resistance. PMID:17279585

  19. A Genetic Variant (COMT) Coding Dopaminergic Activity Predicts Personality Traits in Healthy Elderly.

    PubMed

    Kotyuk, Eszter; Duchek, Janet; Head, Denise; Szekely, Anna; Goate, Alison M; Balota, David A

    2015-08-01

    Association studies between the NEO five factor personality inventory and COMT rs4680 have focused on young adults and the results have been inconsistent. However, personality and cortical changes with age may put older adults in a more sensitive range for detecting a relationship. The present study examined associations of COMT rs4680 and personality in older adults. Genetic association analyses were carried out between the NEO and the targeted COMT rs4680 in a large, well-characterized sample of healthy, cognitively normal older adults (N = 616, mean age = 69.26 years). Three significant associations were found: participants with GG genotype showed lower mean scores on Neuroticism (p = 0.039) and higher scores on Agreeableness (p = 0.020) and Conscientiousness (p = 0.006) than participants with AA or AG genotypes. These results suggest that older adults with higher COMT enzymatic activity (GG), therefore lower dopamine level, have lower Neuroticism scores, and higher Agreeableness and Conscientiousness scores. This is consistent with a recent model of phasic and tonic dopamine release suggesting that even though GG genotype is associated with lower tonic dopamine release, the phasic release of dopamine might be optimal for a more adaptive personality profile.

  20. A Genetic Variant (COMT) Coding Dopaminergic Activity Predicts Personality Traits in Healthy Elderly

    PubMed Central

    Kotyuk, Eszter; Duchek, Janet; Head, Denise; Szekely, Anna; Goate, Alison M.; Balota, David A.

    2015-01-01

    Association studies between the NEO five factor personality inventory and COMT rs4680 have focused on young adults and the results have been inconsistent. However, personality and cortical changes with age may put older adults in a more sensitive range for detecting a relationship. The present study examined associations of COMT rs4680 and personality in older adults. Genetic association analyses were carried out between the NEO and the targeted COMT rs4680 in a large, well-characterized sample of healthy, cognitively normal older adults (N = 616, mean age = 69.26 years). Three significant associations were found: participants with GG genotype showed lower mean scores on Neuroticism (p = 0.039) and higher scores on Agreeableness (p = 0.020) and Conscientiousness (p = 0.006) than participants with AA or AG genotypes. These results suggest that older adults with higher COMT enzymatic activity (GG), therefore lower dopamine level, have lower Neuroticism scores, and higher Agreeableness and Conscientiousness scores. This is consistent with a recent model of phasic and tonic dopamine release suggesting that even though GG genotype is associated with lower tonic dopamine release, the phasic release of dopamine might be optimal for a more adaptive personality profile. PMID:25960587

  1. HNF1B variants associate with promoter methylation and regulate gene networks activated in prostate and ovarian cancer

    PubMed Central

    Ross-Adams, Helen; Ball, Stephen; Lawrenson, Kate; Halim, Silvia; Russell, Roslin; Wells, Claire; Strand, Siri H.; Ørntoft, Torben F.; Larson, Melissa; Armasu, Sebastian; Massie, Charles E.; Asim, Mohammad; Mortensen, Martin M.; Borre, Michael; Woodfine, Kathryn; Warren, Anne Y.; Lamb, Alastair D.; Kay, Jonathan; Whitaker, Hayley; Ramos-Montoya, Antonio; Murrell, Adele; Sørensen, Karina D.; Fridley, Brooke L.; Goode, Ellen L.; Gayther, Simon A.; Masters, John

    2016-01-01

    Two independent regions within HNF1B are consistently identified in prostate and ovarian cancer genome-wide association studies (GWAS); their functional roles are unclear. We link prostate cancer (PC) risk SNPs rs11649743 and rs3760511 with elevated HNF1B gene expression and allele-specific epigenetic silencing, and outline a mechanism by which common risk variants could effect functional changes that increase disease risk: functional assays suggest that HNF1B is a pro-differentiation factor that suppresses epithelial-to-mesenchymal transition (EMT) in unmethylated, healthy tissues. This tumor-suppressor activity is lost when HNF1B is silenced by promoter methylation in the progression to PC. Epigenetic inactivation of HNF1B in ovarian cancer also associates with known risk SNPs, with a similar impact on EMT. This represents one of the first comprehensive studies into the pleiotropic role of a GWAS-associated transcription factor across distinct cancer types, and is the first to describe a conserved role for a multi-cancer genetic risk factor. PMID:27732966

  2. A major EBNA1 variant from Asian EBV isolates shows enhanced transcriptional activity compared to prototype B95.8.

    PubMed

    Do, Nguyen-Van; Ingemar, Ernberg; Phi, Phan-Thi Phi; Jenny, Almqvist; Chinh, Tran-Thi; Zeng, Yixin; Hu, Lifu

    2008-03-01

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) has an instrumental role in maintaining EBV latent infection by controlling EBV episome replication and regulating viral transcription. It is a ubiquitously expressed protein during latent viral infection and in EBV-associated tumors. The EBNA1 C-terminus interacts functionally with the Qp and Cp that control viral gene expression in latency I/II and III, respectively. EBNA1 has been classified into five subtypes due to sequence variation in the DNA-interacting C-terminus. By DNA sequence analysis of its C-terminus, we detected a main sub-variant (V-val-v1) of EBNA1 with valine located in both positions 487 and 528 from matched samples including NPC biopsies and peripheral blood taken from Vietnamese (9), Chinese (12) NPC patients and healthy donors (5). In the FR-region of oriP from nine NPC biopsies from Vietnam we also frequently found substitutions, deletions and variable numbers of repeats. Using a luciferase reporter system, EBNA1 and FR both derived from Asian isolates induced higher transcriptional activity than those from B95-8 virus.

  3. Histone variant H2A.Z marks the 5' ends of both active and inactive genes in euchromatin.

    PubMed

    Raisner, Ryan M; Hartley, Paul D; Meneghini, Marc D; Bao, Marie Z; Liu, Chih Long; Schreiber, Stuart L; Rando, Oliver J; Madhani, Hiten D

    2005-10-21

    In S. cerevisiae, histone variant H2A.Z is deposited in euchromatin at the flanks of silent heterochromatin to prevent its ectopic spread. We show that H2A.Z nucleosomes are found at promoter regions of nearly all genes in euchromatin. They generally occur as two positioned nucleosomes that flank a nucleosome-free region (NFR) that contains the transcription start site. Astonishingly, enrichment at 5' ends is observed not only at actively transcribed genes but also at inactive loci. Mutagenesis of a typical promoter revealed a 22 bp segment of DNA sufficient to program formation of a NFR flanked by two H2A.Z nucleosomes. This segment contains a binding site of the Myb-related protein Reb1 and an adjacent dT:dA tract. Efficient deposition of H2A.Z is further promoted by a specific pattern of histone H3 and H4 tail acetylation and the bromodomain protein Bdf1, a component of the Swr1 remodeling complex that deposits H2A.Z.

  4. Discovery of common human genetic variants of GTP cyclohydrolase 1 (GCH1) governing nitric oxide, autonomic activity, and cardiovascular risk

    PubMed Central

    Zhang, Lian; Rao, Fangwen; Zhang, Kuixing; Khandrika, Srikrishna; Das, Madhusudan; Vaingankar, Sucheta M.; Bao, Xuping; Rana, Brinda K.; Smith, Douglas W.; Wessel, Jennifer; Salem, Rany M.; Rodriguez-Flores, Juan L.; Mahata, Sushil K.; Schork, Nicholas J.; Ziegler, Michael G.; O’Connor, Daniel T.

    2007-01-01

    GTP cyclohydrolase 1 (GCH1) is rate limiting in the provision of the cofactor tetrahydrobiopterin for biosynthesis of catecholamines and NO. We asked whether common genetic variation at GCH1 alters transmitter synthesis and predisposes to disease. Here we undertook a systematic search for polymorphisms in GCH1, then tested variants’ contributions to NO and catecholamine release as well as autonomic function in twin pairs. Renal NO and neopterin excretions were significantly heritable, as were baroreceptor coupling (heart rate response to BP fluctuation) and pulse interval (1/heart rate). Common GCH1 variant C+243T in the 3′-untranslated region (3′-UTRs) predicted NO excretion, as well as autonomic traits: baroreceptor coupling, maximum pulse interval, and pulse interval variability, though not catecholamine secretion. In individuals with the most extreme BP values in the population, C+243T affected both diastolic and systolic BP, principally in females. In functional studies, C+243T decreased reporter expression in transfected 3′-UTRs plasmids. We conclude that human NO secretion traits are heritable, displaying joint genetic determination with autonomic activity by functional polymorphism at GCH1. Our results document novel pathophysiological links between a key biosynthetic locus and NO metabolism and suggest new strategies for approaching the mechanism, diagnosis, and treatment of risk predictors for cardiovascular diseases such as hypertension. PMID:17717598

  5. Arsenic resistance strategy in Pantoea sp. IMH: Organization, function and evolution of ars genes

    PubMed Central

    Wang, Liying; Zhuang, Xuliang; Zhuang, Guoqiang; Jing, Chuanyong

    2016-01-01

    Pantoea sp. IMH is the only bacterium found in genus Pantoea with a high As resistance capacity, but its molecular mechanism is unknown. Herein, the organization, function, and evolution of ars genes in IMH are studied starting with analysis of the whole genome. Two ars systems - ars1 (arsR1B1C1H1) and ars2 (arsR2B2C2H2) - with low sequence homology and two arsC-like genes, were found in the IMH genome. Both ars1 and ars2 are involved in the As resistance, where ars1 is the major contributor at 15 °C and ars2 at 30 °C. The difference in the behavior of these two ars systems is attributed to the disparate activities of their arsR promoters at different temperatures. Sequence analysis based on concatenated ArsRBC indicates that ars1 and ars2 clusters may be acquired from Franconibacter helveticus LMG23732 and Serratia marcescens (plasmid R478), respectively, by horizontal gene transfer (HGT). Nevertheless, two arsC-like genes, probably arising from the duplication of arsC, do not contribute to the As resistance. Our results indicate that Pantoea sp. IMH acquired two different As resistance genetic systems by HGT, allowing the colonization of changing ecosystems, and highlighting the flexible adaptation of microorganisms to resist As. PMID:27966630

  6. Ar/Ar Dating Independent of Monitor Standard Ages

    NASA Astr