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Sample records for activated mammalian target

  1. Mammalian target of rapamycin activation underlies HSC defects in autoimmune disease and inflammation in mice.

    PubMed

    Chen, Chong; Liu, Yu; Liu, Yang; Zheng, Pan

    2010-11-01

    The mammalian target of rapamycin (mTOR) is a signaling molecule that senses environmental cues, such as nutrient status and oxygen supply, to regulate cell growth, proliferation, and other functions. Unchecked, sustained mTOR activity results in defects in HSC function. Inflammatory conditions, such as autoimmune disease, are often associated with defective hematopoiesis. Here, we investigated whether hyperactivation of mTOR in HSCs contributes to hematopoietic defects in autoimmunity and inflammation. We found that in mice deficient in Foxp3 (scurfy mice), a model of autoimmunity, the development of autoimmune disease correlated with progressive bone marrow loss and impaired regenerative capacity of HSCs in competitive bone marrow transplantation. Similarly, LPS-mediated inflammation in C57BL/6 mice led to massive bone marrow cell death and impaired HSC function. Importantly, treatment with rapamycin in both models corrected bone marrow hypocellularity and partially restored hematopoietic activity. In cultured mouse bone marrow cells, treatment with either of the inflammatory cytokines IL-6 or TNF-α was sufficient to activate mTOR, while preventing mTOR activation in vivo required simultaneous inhibition of CCL2, IL-6, and TNF-α. These data strongly suggest that mTOR activation in HSCs by inflammatory cytokines underlies defective hematopoiesis in autoimmune disease and inflammation.

  2. Activation of Mammalian target of rapamycin in canine mammary carcinomas: an immunohistochemical study.

    PubMed

    Delgado, L; Gärtner, F; Dias Pereira, P

    2015-01-01

    Mammalian target of rapamycin (mTOR) is a serine-threonine kinase involved in cell growth, proliferation and survival. Activation of mTOR has been reported in various tumour types, including human breast cancer; however, the expression of mTOR in canine mammary tumours has not been examined. In the present study, expression of the activated form of mTOR (phospho-mTOR [p-mTOR]) was examined immunohistochemically in five normal canine mammary glands, 45 canine mammary carcinomas and their corresponding metastatic lesions (n = 15). Phospho-mTOR was not expressed in normal canine mammary tissue, but cytoplasmic labelling was observed in 78% of canine mammary carcinomas. Two carcinomas had both cytoplasmic and nuclear labelling. No significant relationship was found between p-mTOR cytoplasmic expression and histological type or grading of carcinomas, degree of tubular formation, anisokaryosis, mitotic activity or lymph node metastasis. In all except one case, the expression pattern of p-mTOR in lymph node metastases was similar or decreased when compared with the primary lesion. The findings suggest that p-mTOR is involved in mammary carcinogenesis in dogs. However, p-mTOR cytoplasmic expression does not appear to be a prognostic indicator in canine mammary carcinomas, which may be related to its subcellular location in the neoplastic cells. Canine mammary tumours may provide a model for the development of innovative medical strategies involving mTOR inhibitors in human breast cancer. PMID:25670666

  3. Mammalian target of rapamycin is activated in association with myometrial proliferation during pregnancy.

    PubMed

    Jaffer, Shabana; Shynlova, Oksana; Lye, Stephen

    2009-10-01

    The adaptive growth of the uterus during gestation involves gradual changes in cellular phenotypes from the early proliferative to the intermediate synthetic phase of cellular hypertrophy, ending in the final contractile/labour phenotype. The mammalian target of rapamycin (mTOR) signaling pathway regulates cell growth and proliferation in many tissues. We hypothesized that mTOR was a mediator of hormone-initiated myometrial hyperplasia during gestation. The protein expression and phosphorylation levels of mTOR, its upstream regulators [insulin receptor substrate-1, phosphoinositide-3-kinase (PI3K), Akt], and downstream effectors [S6-kinase-1 (S6K1) and eI4FE-binding protein 1 (4EBP1)] were analyzed throughout normal pregnancy in rats. In addition, we used an ovariectomized (OVX) rat model to analyze the modulation of the mTOR pathway and proliferative activity of the uterine myocytes by estradiol alone and in combination with the mTOR-specific inhibitor rapamycin. Our results demonstrate that insulin receptor substrate-1 protein levels and the phosphorylated (activated) forms of PI3K, mTOR, and S6K1 were significantly up-regulated in the rat myometrium during the proliferative phase of pregnancy. Treatment of the OVX rats with estradiol caused a transient increase in IGF-I followed by an up-regulation of the PI3K/mTOR pathway, which became apparent by a cascade of phosphorylation reactions (P-P85, P-Akt, P-mTOR, P-S6K1, and P-4EBP1). Rapamycin blocked activation of P-mTOR, P-S6K1, and P-4EBP1 proteins and significantly reduced the number of proliferating cells in the myometrium of OVX rats. Our in vivo data demonstrate that estradiol was able to activate the PI3K/mTOR signaling pathway in uterine myocytes and suggest that this activation is responsible for the induction of myometrial hyperplasia during early gestation.

  4. Mechanical stretch activates mammalian target of rapamycin and AMP-activated protein kinase pathways in skeletal muscle cells.

    PubMed

    Nakai, Naoya; Kawano, Fuminori; Nakata, Ken

    2015-08-01

    Cellular protein synthesis is believed to be antagonistically regulated by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) signaling pathways. In the present study, we examined the relationship between mTOR/p70 S6 kinase (p70S6K) and AMPK in response to mechanical stretch. C2C12 myoblasts were grown on a silicone elastomer chamber to confluence and further cultured in differentiation medium for 4 days to form multinucleated myotubes. Cells were subjected to 15% cyclic uniaxial stretch for 4 h at a frequency of 1 Hz. Phosphorylation of p70S6K at threonine 389 and AMPK at threonine 172 of the catalytic α subunit were concomitantly increased by mechanical stretch. Stimulation of the mTOR pathway by adding leucine and insulin increased the phosphorylation of p70S6K without inactivation of AMPK. In contrast, addition of compound C, a pharmacological inhibitor of AMPK, increased the phosphorylation of p70S6K in stretched cells. Activation of AMPK by the addition of 5-amino-4-imidazolecarboxamide ribonucleoside reduced the phosphorylation of p70S6K in response to mechanical stretch. In conclusion, crosstalk between mTOR and AMPK signaling was not tightly regulated in response to physiological stimuli, such as mechanical stress and/or nutrients. However, pharmacological modulation of AMPK influenced the mTOR/p70S6K signaling pathway. PMID:25971373

  5. Clustering of mammalian Hox genes with other H3K27me3 targets within an active nuclear domain.

    PubMed

    Vieux-Rochas, Maxence; Fabre, Pierre J; Leleu, Marion; Duboule, Denis; Noordermeer, Daan

    2015-04-14

    Embryogenesis requires the precise activation and repression of many transcriptional regulators. The Polycomb group proteins and the associated H3K27me3 histone mark are essential to maintain the inactive state of many of these genes. Mammalian Hox genes are targets of Polycomb proteins and form local 3D clusters centered on the H3K27me3 mark. More distal contacts have also been described, yet their selectivity, dynamics, and relation to other layers of chromatin organization remained elusive. We report that repressed Hox genes form mutual intra- and interchromosomal interactions with other genes located in strong domains labeled by H3K27me3. These interactions occur in a central and active nuclear environment that consists of the HiC compartment A, away from peripheral lamina-associated domains. Interactions are independent of nearby H3K27me3-marked loci and determined by chromosomal distance and cell-type-specific scaling factors, thus inducing a moderate reorganization during embryogenesis. These results provide a simplified view of nuclear organization whereby Polycomb proteins may have evolved to repress genes located in gene-dense regions whose position is restricted to central, active, nuclear environments.

  6. Clustering of mammalian Hox genes with other H3K27me3 targets within an active nuclear domain

    PubMed Central

    Vieux-Rochas, Maxence; Fabre, Pierre J.; Leleu, Marion; Duboule, Denis; Noordermeer, Daan

    2015-01-01

    Embryogenesis requires the precise activation and repression of many transcriptional regulators. The Polycomb group proteins and the associated H3K27me3 histone mark are essential to maintain the inactive state of many of these genes. Mammalian Hox genes are targets of Polycomb proteins and form local 3D clusters centered on the H3K27me3 mark. More distal contacts have also been described, yet their selectivity, dynamics, and relation to other layers of chromatin organization remained elusive. We report that repressed Hox genes form mutual intra- and interchromosomal interactions with other genes located in strong domains labeled by H3K27me3. These interactions occur in a central and active nuclear environment that consists of the HiC compartment A, away from peripheral lamina-associated domains. Interactions are independent of nearby H3K27me3-marked loci and determined by chromosomal distance and cell-type–specific scaling factors, thus inducing a moderate reorganization during embryogenesis. These results provide a simplified view of nuclear organization whereby Polycomb proteins may have evolved to repress genes located in gene-dense regions whose position is restricted to central, active, nuclear environments. PMID:25825760

  7. Mammalian apoptotic signalling pathways: multiple targets of protozoan parasites to activate or deactivate host cell death.

    PubMed

    Graumann, Kristin; Hippe, Diana; Gross, Uwe; Lüder, Carsten G K

    2009-11-01

    Programmed cell death is an essential mechanism of the host to combat infectious agents and to regulate immunity during infection. Consequently, activation and deactivation of the hosts' cell death pathways by protozoan parasites play critical roles in parasite control, pathogenesis, immune evasion and parasite dissemination within the host. Here, we discuss advances in the understanding of these fascinating host-parasite interactions with special emphasis on how protozoa can modulate the cell death apparatus of its host.

  8. Biochemical, cellular, and in vivo activity of novel ATP-competitive and selective inhibitors of the mammalian target of rapamycin.

    PubMed

    Yu, Ker; Toral-Barza, Lourdes; Shi, Celine; Zhang, Wei-Guo; Lucas, Judy; Shor, Boris; Kim, Jamie; Verheijen, Jeroen; Curran, Kevin; Malwitz, David J; Cole, Derek C; Ellingboe, John; Ayral-Kaloustian, Semiramis; Mansour, Tarek S; Gibbons, James J; Abraham, Robert T; Nowak, Pawel; Zask, Arie

    2009-08-01

    The mammalian target of rapamycin (mTOR) is centrally involved in cell growth, metabolism, and angiogenesis. While showing clinical efficacy in a subset of tumors, rapamycin and rapalogs are specific and allosteric inhibitors of mTOR complex 1 (mTORC1), but they do not directly inhibit mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report chemical structure and biological characterization of three pyrazolopyrimidine ATP-competitive mTOR inhibitors, WAY-600, WYE-687, and WYE-354 (IC(50), 5-9 nmol/L), with significant selectivity over phosphatidylinositol 3-kinase (PI3K) isofoms (>100-fold). Unlike the rapalogs, these inhibitors acutely blocked substrate phosphorylation by mTORC1 and mTORC2 in vitro and in cells in response to growth factor, amino acids, and hyperactive PI3K/AKT. Unlike the inhibitors of PI3K or dual-pan PI3K/mTOR, cellular inhibition of P-S6K1(T389) and P-AKT(S473) by the pyrazolopyrimidines occurred at significantly lower inhibitor concentrations than those of P-AKT(T308) (PI3K-PDK1 readout), showing mTOR selectivity in cellular setting. mTOR kinase inhibitors reduced AKT downstream function and inhibited proliferation of diverse cancer cell lines. These effects correlated with a strong G(1) cell cycle arrest in both the rapamycin-sensitive and rapamycin-resistant cells, selective induction of apoptosis, repression of global protein synthesis, and down-regulation of angiogenic factors. When injected into tumor-bearing mice, WYE-354 inhibited mTORC1 and mTORC2 and displayed robust antitumor activity in PTEN-null tumors. Together, our results highlight mechanistic differentiation between rapalogs and mTOR kinase inhibitors in targeting cancer cell growth and survival and provide support for clinical development of mTOR kinase inhibitors as new cancer therapy.

  9. cAMP-dependent activation of mammalian target of rapamycin (mTOR) in thyroid cells. Implication in mitogenesis and activation of CDK4.

    PubMed

    Blancquaert, Sara; Wang, Lifu; Paternot, Sabine; Coulonval, Katia; Dumont, Jacques E; Harris, Thurl E; Roger, Pierre P

    2010-07-01

    How cAMP-dependent protein kinases [protein kinase A (PKA)] transduce the mitogenic stimulus elicited by TSH in thyroid cells to late activation of cyclin D3-cyclin-dependent kinase 4 (CDK4) remains enigmatic. Here we show in PC Cl3 rat thyroid cells that TSH/cAMP, like insulin, activates the mammalian target of rapamycin (mTOR)-raptor complex (mTORC1) leading to phosphorylation of S6K1 and 4E-BP1. mTORC1-dependent S6K1 phosphorylation in response to both insulin and cAMP required amino acids, whereas inhibition of AMP-activated protein kinase and glycogen synthase kinase 3 enhanced insulin but not cAMP effects. Unlike insulin, TSH/cAMP did not activate protein kinase B or induce tuberous sclerosis complex 2 phosphorylation at T1462 and Y1571. However, like insulin, TSH/cAMP produced a stable increase in mTORC1 kinase activity that was associated with augmented 4E-BP1 binding to raptor. This could be caused in part by T246 phosphorylation of PRAS40, which was found as an in vitro substrate of PKA. Both in PC Cl3 cells and primary dog thyrocytes, rapamycin inhibited DNA synthesis and retinoblastoma protein phosphorylation induced by TSH and insulin. Although rapamycin reduced cyclin D3 accumulation, the abundance of cyclin D3-CDK4 complexes was not affected. However, rapamycin inhibited the activity of these complexes by decreasing the TSH and insulin-mediated stimulation of activating T172 phosphorylation of CDK4. We propose that mTORC1 activation by TSH, at least in part through PKA-dependent phosphorylation of PRAS40, crucially contributes to mediate cAMP-dependent mitogenesis by regulating CDK4 T172-phosphorylation. PMID:20484410

  10. Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway.

    PubMed

    Varghese, Binny V; Koohestani, Faezeh; McWilliams, Michelle; Colvin, Arlene; Gunewardena, Sumedha; Kinsey, William H; Nowak, Romana A; Nothnick, Warren B; Chennathukuzhi, Vargheese M

    2013-02-01

    Uterine fibroids (leiomyomas) are the most common tumors of the female reproductive tract, occurring in up to 77% of reproductive-aged women, yet molecular pathogenesis remains poorly understood. A role for atypically activated mammalian target of rapamycin (mTOR) pathway in the pathogenesis of uterine fibroids has been suggested in several studies. We identified that G protein-coupled receptor 10 [GPR10, a putative signaling protein upstream of the phosphoinositide 3-kinase-protein kinase B/AKT-mammalian target of rapamycin (PI3K/AKT-mTOR) pathway] is aberrantly expressed in uterine fibroids. The activation of GPR10 by its cognate ligand, prolactin releasing peptide, promotes PI3K-AKT-mTOR pathways and cell proliferation specifically in cultured primary leiomyoma cells. Additionally, we report that RE1 suppressing transcription factor/neuron-restrictive silencing factor (REST/NRSF), a known tumor suppressor, transcriptionally represses GPR10 in the normal myometrium, and that the loss of REST in fibroids permits GPR10 expression. Importantly, mice overexpressing human GPR10 in the myometrium develop myometrial hyperplasia with excessive extracellular matrix deposition, a hallmark of uterine fibroids. We demonstrate previously unrecognized roles for GPR10 and its upstream regulator REST in the pathogenesis of uterine fibroids. Importantly, we report a unique genetically modified mouse model for a gene that is misexpressed in uterine fibroids. PMID:23284171

  11. Protein kinase C and P2Y12 take center stage in thrombin-mediated activation of mammalian target of rapamycin complex 1 in human platelets

    PubMed Central

    Moore, S F; Hunter, R W; Hers, I

    2014-01-01

    Background Rapamycin, an inhibitor of mammalian target of rapamycin complex-1 (mTORC1), reduces platelet spreading, thrombus stability, and clot retraction. Despite an important role of mTORC1 in platelet function, little is known about how it is regulated. The objective of this study was to determine the signaling pathways that regulate mTORC1 in human platelets. Methods Mammalian target of rapamycin complex-1 activation was assessed by measuring the phosphorylation of its downstream substrate ribosomal S6 kinase 1 (p70S6K). Results Thrombin or the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate stimulated activation of mTORC1 in a PKC-dependent, Akt-independent manner that correlated with phosphorylation of tuberin/tuberous sclerosis 2 (TSC2) (Ser939 and Thr1462). In contrast, insulin-like growth factor 1 (IGF-1)–stimulated TSC2 phosphorylation was completely dependent on phosphoinositide 3 kinase (PI3 kinase)/Akt but did not result in any detectable mTORC1 activation. Early (Ser939 and Thr1462) and late (Thr1462) TSC2 phosphorylation in response to thrombin were directly PKC dependent, whereas later TSC2 (Ser939) and p70S6K phosphorylation were largely dependent on paracrine signaling through P2Y12. PKC-mediated adenosine diphosphate (ADP) secretion was essential for thrombin-stimulated mTORC1 activation, as (i) ADP rescued p70S6K phosphorylation in the presence of a PKC inhibitor and (ii) P2Y12 antagonism prevented thrombin-mediated mTORC1 activation. Rescue of mTORC1 activation with exogenous ADP was completely dependent on the Src family kinases but independent of PI3 kinase/Akt. Interestingly, although inhibition of Src blocked the ADP rescue, it had little effect on thrombin-stimulated p70S6K phosphorylation under conditions where PKC was not inhibited. Conclusion These results demonstrate that thrombin activates the mTORC1 pathway in human platelets through PKC-mediated ADP secretion and subsequent activation of P2Y12, in a manner

  12. Novel role of the small GTPase Rheb: its implication in endocytic pathway independent of the activation of mammalian target of rapamycin.

    PubMed

    Saito, Kota; Araki, Yasuhiro; Kontani, Kenji; Nishina, Hiroshi; Katada, Toshiaki

    2005-03-01

    The Ras-homologous GTPase Rheb that is conserved from yeast to human appears to be involved not only in cell growth but also in nutrient uptake. Recent biochemical analysis revealed that tuberous sclerosis complex (TSC), a GTPase-activating protein (GAP), deactivates Rheb and that phosphatidylinositol 3'-kinase (PI3k)-Akt/PKB kinase pathway activates Rheb through inhibition of the GAP-mediated deactivation. Although mammalian target of rapamycin (mTOR) kinase is implicated in the downstream target of Rheb, the direct effector(s) and exact functions of Rheb have not been fully elucidated. Here we identified that Rheb expression in cultured cells induces the formation of large cytoplasmic vacuoles, which are characterized as late endocytic (late endosome- and lysosome-like) components. The vacuole formation required the GTP form of Rheb, but not the activation of the downstream mTOR kinase. These results suggest that Rheb regulates endocytic trafficking pathway independent of the previously identified mTOR pathway. The physiological roles of the two Rheb-dependent signaling pathways are discussed in terms of nutrient uptake and cell growth or cell cycle progression. PMID:15809346

  13. Palmitate Induces mRNA Translation and Increases ER Protein Load in Islet β-Cells via Activation of the Mammalian Target of Rapamycin Pathway

    PubMed Central

    Hatanaka, Masayuki; Maier, Bernhard; Sims, Emily K.; Templin, Andrew T.; Kulkarni, Rohit N.; Evans-Molina, Carmella

    2014-01-01

    Saturated free fatty acids (FFAs) have complex effects on the islet β-cell, acutely promoting adaptive hyperplasia but chronically impairing insulin release. The acute effects of FFAs remain incompletely defined. To elucidate these early molecular events, we incubated mouse β-cells and islets with palmitate and then studied mRNA translation by polyribosomal profiling and analyzed signaling pathways by immunoblot analysis. We found that palmitate acutely increases polyribosome occupancy of total RNA, consistent with an increase in mRNA translation. This effect on translation was attributable to activation of mammalian target of rapamycin (mTOR) pathways via L-type Ca2+ channels but was independent of insulin signaling. Longer incubations led to depletion of polyribosome-associated RNA, consistent with activation of the unfolded protein response (UPR). Pharmacologic inhibition of mTOR suppressed both the acute effects of palmitate on mRNA translation and the chronic effects on the UPR. Islets from mice fed a high-fat diet for 7 days showed increases in polyribosome-associated RNA and phosphorylation of S6K, both consistent with activation of mTOR. Our results suggest that palmitate acutely activates mRNA translation and that this increase in protein load contributes to the later UPR. PMID:24834975

  14. The role of diacylglycerol kinase ζ and phosphatidic acid in the mechanical activation of mammalian target of rapamycin (mTOR) signaling and skeletal muscle hypertrophy.

    PubMed

    You, Jae-Sung; Lincoln, Hannah C; Kim, Chan-Ran; Frey, John W; Goodman, Craig A; Zhong, Xiao-Ping; Hornberger, Troy A

    2014-01-17

    The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass. PMID:24302719

  15. The role of diacylglycerol kinase ζ and phosphatidic acid in the mechanical activation of mammalian target of rapamycin (mTOR) signaling and skeletal muscle hypertrophy.

    PubMed

    You, Jae-Sung; Lincoln, Hannah C; Kim, Chan-Ran; Frey, John W; Goodman, Craig A; Zhong, Xiao-Ping; Hornberger, Troy A

    2014-01-17

    The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass.

  16. Rapamycin does not improve insulin sensitivity despite elevated mammalian target of rapamycin complex 1 activity in muscles of ob/ob mice

    PubMed Central

    Miller, Andrew M.; Brestoff, Jonathan R.; Phelps, Charles B.; Berk, E. Zachary; Reynolds, Thomas H.

    2008-01-01

    Studies of cultured cells have indicated that the mammalian target of rapamycin complex 1 (mTORC1) mediates the development of insulin resistance. Because a role for mTORC1 in the development of skeletal muscle insulin resistance has not been established, we studied mTORC1 activity in skeletal muscles of ob/ob (OB) mice and wild-type (WT) mice. In vivo insulin action was assessed in muscles of mice 15 min following an intraperitoneal injection of insulin or an equivalent volume of saline. In the basal state, the phosphorylation of S6K on Thr389, mTOR on Ser2448, and PRAS40 on Thr246 were increased significantly in muscles from OB mice compared with WT mice. The increase in basal mTORC1 signaling was associated with an increase in basal PKB phosphorylation on Thr308 and Ser473. In the insulin-stimulated state, no differences existed in the phosphorylation of S6K on Thr389, but PKB phosphorylation on Thr308 and Ser473 was significantly reduced in muscles of OB compared with WT mice. Despite elevated mTORC1 activity in OB mice, rapamycin treatment did not improve either glucose tolerance or insulin tolerance. These results indicate that the insulin resistance of OB mice is mediated, in part, by factors other than mTORC1. PMID:18768766

  17. Activation of mammalian target of rapamycin contributes to pain nociception induced in rats by BmK I, a sodium channel-specific modulator.

    PubMed

    Jiang, Feng; Hua, Li-Ming; Jiao, Yun-Lu; Ye, Pin; Fu, Jin; Cheng, Zhi-Jun; Ding, Gang; Ji, Yong-Hua

    2014-02-01

    The mammalian target of rapamycin (mTOR) pathway is essential for maintenance of the sensitivity of certain adult sensory neurons. Here, we investigated whether the mTOR cascade is involved in scorpion envenomation-induced pain hypersensitivity in rats. The results showed that intraplantar injection of a neurotoxin from Buthus martensii Karsch, BmK I (10 μg), induced the activation of mTOR, as well as its downstream molecules p70 ribosomal S6 protein kinase (p70 S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), in lumbar 5-6 dorsal root ganglia neurons on both sides in rats. The activation peaked at 2 h and recovered 1 day after injection. Compared with the control group, the ratios of p-mTOR/p-p70 S6K/p-4EBP1 in three types of neurons changed significantly. The cell typology of p-mTOR/p-p70 S6K/p-4E-BP1 immuno-reactive neurons also changed. Intrathecal administration of deforolimus, a specific inhibitor of mTOR, attenuated BmK I-induced pain responses (spontaneous flinching, paroxysmal pain-like behavior, and mechanical hypersensitivity). Together, these results imply that the mTOR signaling pathway is mobilized by and contributes to experimental scorpion sting-induced pain. PMID:24132796

  18. Stromal liver kinase B1 [STK11] signaling loss induces oviductal adenomas and endometrial cancer by activating mammalian Target of Rapamycin Complex 1.

    PubMed

    Tanwar, Pradeep S; Kaneko-Tarui, Tomoko; Zhang, Lihua; Tanaka, Yoshihiro; Crum, Christopher P; Teixeira, Jose M

    2012-01-01

    Germline mutations of the Liver Kinase b1 (LKB1/STK11) tumor suppressor gene have been linked to Peutz-Jeghers Syndrome (PJS), an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. We have conditionally deleted Lkb1 in Müllerian duct mesenchyme-derived cells of the female reproductive tract and observed expansion of the stromal compartment and hyperplasia and/or neoplasia of adjacent epithelial cells throughout the reproductive tract with paratubal cysts and adenomyomas in oviducts and, eventually, endometrial cancer. Examination of the proliferation marker phospho-histone H3 and mammalian Target Of Rapamycin Complex 1 (mTORC1) pathway members revealed increased proliferation and mTORC1 activation in stromal cells of both the oviduct and uterus. Treatment with rapamycin, an inhibitor of mTORC1 activity, decreased tumor burden in adult Lkb1 mutant mice. Deletion of the genes for Tuberous Sclerosis 1 (Tsc1) or Tsc2, regulators of mTORC1 that are downstream of LKB1 signaling, in the oviductal and uterine stroma phenocopies some of the defects observed in Lkb1 mutant mice, confirming that dysregulated mTORC1 activation in the Lkb1-deleted stroma contributes to the phenotype. Loss of PTEN, an upstream regulator of mTORC1 signaling, along with Lkb1 deletion significantly increased tumor burden in uteri and induced tumorigenesis in the cervix and vagina. These studies show that LKB1/TSC1/TSC2/mTORC1 signaling in mesenchymal cells is important for the maintenance of epithelial integrity and suppression of carcinogenesis in adjacent epithelial cells. Because similar changes in the stromal population are also observed in human oviductal/ovarian adenoma and endometrial adenocarcinoma patients, we predict that dysregulated mTORC1 activity by upstream mechanisms similar to those described in these model systems contributes to the pathogenesis of these human diseases.

  19. Mammalian Target of Rapamycin Inhibitors and Nephrotoxicity: Fact or Fiction.

    PubMed

    Barbari, Antoine; Maawad, Maria; Kfoury Kassouf, Hala; Kamel, Gaby

    2015-10-01

    Mammalian target of rapamycin inhibitors, such as rapamycin and more recently everolimus, have substituted calcineurin inhibitors in many minimization strategies. Despite their acclaimed renal safety profile, several lines of evidence are emerging on their potential nephrotoxic effect. Predisposing conditions for nephrotoxicity involve a complex interplay between several environmental and genetic factors in the donor-recipient pair. Renal injury may be enhanced by pharmacodynamic interactions when combined with other drugs such as calcineurin inhibitors or nutrients that are predominantly related to an increase in local tissue exposure. These toxic interactions may occur within adequate doses and therapeutic blood levels. This explains the occurrence of nephrotoxicity in some but not all cases. Here, we postulated that activity of a low permeability glycoprotein efflux pump related to low protein expression and/or inhibition enhanced immunosuppressive drug entry in different cells. A rise in intracellular drug concentration increases bioactivity, leading to greater immunosuppression and more immune-related, nonrenal adverse events in the recipient and increased nephrotoxicity in the kidney graft. Under specific isolated or combined environmental and/or genetic conditions in both the recipient and donor affecting the glycoprotein efflux pump and/or the mammalian target of rapamycin pathway, these renal injuries may be aggravated by heightened drug tissue concentrations despite adherence to therapeutic drug and blood levels. Mammalian target of rapamycin inhibitors may induce predominantly a dose-dependent renal epithelial cell injury affecting either the glomerular or the renal tubular epithelial cells, leading to cell death and apoptosis. Epithelial mesenchymal transition mediated interstitial fibrosis and tubular atrophy observed with these drugs may be the result of a cumulative toxic renal tubular injury induced by the direct insult of the drug itself and

  20. Ciliary transport regulates PDGF-AA/αα signaling via elevated mammalian target of rapamycin signaling and diminished PP2A activity.

    PubMed

    Umberger, Nicole L; Caspary, Tamara

    2015-01-15

    Primary cilia are built and maintained by intraflagellar transport (IFT), whereby the two IFT complexes, IFTA and IFTB, carry cargo via kinesin and dynein motors for anterograde and retrograde transport, respectively. Many signaling pathways, including platelet- derived growth factor (PDGF)-AA/αα, are linked to primary cilia. Active PDGF-AA/αα signaling results in phosphorylation of Akt at two residues: P-Akt(T308) and P-Akt(S473), and previous work showed decreased P-Akt(S473) in response to PDGF-AA upon anterograde transport disruption. In this study, we investigated PDGF-AA/αα signaling via P-Akt(T308) and P-Akt(S473) in distinct ciliary transport mutants. We found increased Akt phosphorylation in the absence of PDGF-AA stimulation, which we show is due to impaired dephosphorylation resulting from diminished PP2A activity toward P-Akt(T308). Anterograde transport mutants display low platelet-derived growth factor receptor (PDGFR)α levels, whereas retrograde mutants exhibit normal PDGFRα levels. Despite this, neither shows an increase in P-Akt(S473) or P-Akt(T308) upon PDGF-AA stimulation. Because mammalian target of rapamycin complex 1 (mTORC1) signaling is increased in ciliary transport mutant cells and mTOR signaling inhibits PDGFRα levels, we demonstrate that inhibition of mTORC1 rescues PDGFRα levels as well as PDGF-AA-dependent phosphorylation of Akt(S473) and Akt(T308) in ciliary transport mutant MEFs. Taken together, our data indicate that the regulation of mTORC1 signaling and PP2A activity by ciliary transport plays key roles in PDGF-AA/αα signaling.

  1. Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways*

    PubMed Central

    Agarwal, Swati; Tiwari, Shashi Kant; Seth, Brashket; Yadav, Anuradha; Singh, Anshuman; Mudawal, Anubha; Chauhan, Lalit Kumar Singh; Gupta, Shailendra Kumar; Choubey, Vinay; Tripathi, Anurag; Kumar, Amit; Ray, Ratan Singh; Shukla, Shubha; Parmar, Devendra; Chaturvedi, Rajnish Kumar

    2015-01-01

    The human health hazards related to persisting use of bisphenol-A (BPA) are well documented. BPA-induced neurotoxicity occurs with the generation of oxidative stress, neurodegeneration, and cognitive dysfunctions. However, the cellular and molecular mechanism(s) of the effects of BPA on autophagy and association with oxidative stress and apoptosis are still elusive. We observed that BPA exposure during the early postnatal period enhanced the expression and the levels of autophagy genes/proteins. BPA treatment in the presence of bafilomycin A1 increased the levels of LC3-II and SQSTM1 and also potentiated GFP-LC3 puncta index in GFP-LC3-transfected hippocampal neural stem cell-derived neurons. BPA-induced generation of reactive oxygen species and apoptosis were mitigated by a pharmacological activator of autophagy (rapamycin). Pharmacological (wortmannin and bafilomycin A1) and genetic (beclin siRNA) inhibition of autophagy aggravated BPA neurotoxicity. Activation of autophagy against BPA resulted in intracellular energy sensor AMP kinase (AMPK) activation, increased phosphorylation of raptor and acetyl-CoA carboxylase, and decreased phosphorylation of ULK1 (Ser-757), and silencing of AMPK exacerbated BPA neurotoxicity. Conversely, BPA exposure down-regulated the mammalian target of rapamycin (mTOR) pathway by phosphorylation of raptor as a transient cell's compensatory mechanism to preserve cellular energy pool. Moreover, silencing of mTOR enhanced autophagy, which further alleviated BPA-induced reactive oxygen species generation and apoptosis. BPA-mediated neurotoxicity also resulted in mitochondrial loss, bioenergetic deficits, and increased PARKIN mitochondrial translocation, suggesting enhanced mitophagy. These results suggest implication of autophagy against BPA-mediated neurodegeneration through involvement of AMPK and mTOR pathways. Hence, autophagy, which arbitrates cell survival and demise during stress conditions, requires further assessment to be

  2. Mammalian Tribbles Homologs at the Crossroads of Endoplasmic Reticulum Stress and Mammalian Target of Rapamycin Pathways

    PubMed Central

    Cunard, Robyn

    2013-01-01

    In 2000, investigators discovered Tribbles, a Drosophila protein that coordinates morphogenesis by inhibiting mitosis. Further work has delineated Xenopus (Xtrb2), Nematode (Nipi-3), and mammalian homologs of Drosophila tribbles, which include TRB1, TRB2, and TRB3. The sequences of tribbles homologs are highly conserved, and despite their protein kinase structure, to date they have not been shown to have kinase activity. TRB family members play a role in the differentiation of macrophages, lymphocytes, muscle cells, adipocytes, and osteoblasts. TRB isoforms also coordinate a number of critical cellular processes including glucose and lipid metabolism, inflammation, cellular stress, survival, apoptosis, and tumorigenesis. TRB family members modulate multiple complex signaling networks including mitogen activated protein kinase cascades, protein kinase B/AKT signaling, mammalian target of rapamycin, and inflammatory pathways. The following review will discuss metazoan homologs of Drosophila tribbles, their structure, expression patterns, and functions. In particular, we will focus on TRB3 function in the kidney in podocytes. This review will also discuss the key signaling pathways with which tribbles proteins interact and provide a rationale for developing novel therapeutics that exploit these interactions to provide better treatment options for both acute and chronic kidney disease. PMID:24490110

  3. Activation of the Mammalian Target of Rapamycin in the Rostral Ventromedial Medulla Contributes to the Maintenance of Nerve Injury-Induced Neuropathic Pain in Rat.

    PubMed

    Wang, Jian; Feng, Da-Yun; Li, Zhi-Hua; Feng, Ban; Zhang, Han; Zhang, Ting; Chen, Tao; Li, Yun-Qing

    2015-01-01

    The mammalian target of rapamycin (mTOR), a serine-threonine protein kinase, integrates extracellular signals, thereby modulating several physiological and pathological processes, including pain. Previous studies have suggested that rapamycin (an mTOR inhibitor) can attenuate nociceptive behaviors in many pain models, most likely at the spinal cord level. However, the mechanisms of mTOR at the supraspinal level, particularly at the level of the rostral ventromedial medulla (RVM), remain unclear. Thus, the aim of this study was to elucidate the role of mTOR in the RVM, a key relay region for the descending pain control pathway, under neuropathic pain conditions. Phosphorylated mTOR was mainly expressed in serotonergic spinally projecting neurons and was significantly increased in the RVM after spared nerve injury- (SNI-) induced neuropathic pain. Moreover, in SNI rat brain slices, rapamycin infusion both decreased the amplitude instead of the frequency of spontaneous excitatory postsynaptic currents and reduced the numbers of action potentials in serotonergic neurons. Finally, intra-RVM microinjection of rapamycin effectively alleviated established mechanical allodynia but failed to affect the development of neuropathic pain. In conclusion, our data provide strong evidence for the role of mTOR in the RVM in nerve injury-induced neuropathic pain, indicating a novel mechanism of mTOR inhibitor-induced analgesia. PMID:26770837

  4. Activation of the Mammalian Target of Rapamycin in the Rostral Ventromedial Medulla Contributes to the Maintenance of Nerve Injury-Induced Neuropathic Pain in Rat

    PubMed Central

    Wang, Jian; Feng, Da-Yun; Li, Zhi-Hua; Feng, Ban; Zhang, Han; Zhang, Ting; Chen, Tao; Li, Yun-Qing

    2015-01-01

    The mammalian target of rapamycin (mTOR), a serine-threonine protein kinase, integrates extracellular signals, thereby modulating several physiological and pathological processes, including pain. Previous studies have suggested that rapamycin (an mTOR inhibitor) can attenuate nociceptive behaviors in many pain models, most likely at the spinal cord level. However, the mechanisms of mTOR at the supraspinal level, particularly at the level of the rostral ventromedial medulla (RVM), remain unclear. Thus, the aim of this study was to elucidate the role of mTOR in the RVM, a key relay region for the descending pain control pathway, under neuropathic pain conditions. Phosphorylated mTOR was mainly expressed in serotonergic spinally projecting neurons and was significantly increased in the RVM after spared nerve injury- (SNI-) induced neuropathic pain. Moreover, in SNI rat brain slices, rapamycin infusion both decreased the amplitude instead of the frequency of spontaneous excitatory postsynaptic currents and reduced the numbers of action potentials in serotonergic neurons. Finally, intra-RVM microinjection of rapamycin effectively alleviated established mechanical allodynia but failed to affect the development of neuropathic pain. In conclusion, our data provide strong evidence for the role of mTOR in the RVM in nerve injury-induced neuropathic pain, indicating a novel mechanism of mTOR inhibitor-induced analgesia. PMID:26770837

  5. Phospshoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitors: discovery and structure-activity relationships of a series of quinoline and quinoxaline derivatives.

    PubMed

    Nishimura, Nobuko; Siegmund, Aaron; Liu, Longbin; Yang, Kevin; Bryan, Marian C; Andrews, Kristin L; Bo, Yunxin; Booker, Shon K; Caenepeel, Sean; Freeman, Daniel; Liao, Hongyu; McCarter, John; Mullady, Erin L; San Miguel, Tisha; Subramanian, Raju; Tamayo, Nuria; Wang, Ling; Whittington, Douglas A; Zalameda, Leeanne; Zhang, Nancy; Hughes, Paul E; Norman, Mark H

    2011-07-14

    The phosphoinositide 3-kinase (PI3K) family catalyzes the ATP-dependent phosphorylation of the 3'-hydroxyl group of phosphatidylinositols and plays an important role in cell growth and survival. There is abundant evidence demonstrating that PI3K signaling is dysregulated in many human cancers, suggesting that therapeutics targeting the PI3K pathway may have utility for the treatment of cancer. Our efforts to identify potent, efficacious, and orally available PI3K/mammalian target of rapamycin (mTOR) dual inhibitors resulted in the discovery of a series of substituted quinolines and quinoxalines derivatives. In this report, we describe the structure-activity relationships, selectivity, and pharmacokinetic data of this series and illustrate the in vivo pharmacodynamic and efficacy data for a representative compound.

  6. CRISPR Technology for Genome Activation and Repression in Mammalian Cells.

    PubMed

    Du, Dan; Qi, Lei S

    2016-01-01

    Targeted modulation of transcription is necessary for understanding complex gene networks and has great potential for medical and industrial applications. CRISPR is emerging as a powerful system for targeted genome activation and repression, in addition to its use in genome editing. This protocol describes how to design, construct, and experimentally validate the function of sequence-specific single guide RNAs (sgRNAs) for sequence-specific repression (CRISPRi) or activation (CRISPRa) of transcription in mammalian cells. In this technology, the CRISPR-associated protein Cas9 is catalytically deactivated (dCas9) to provide a general platform for RNA-guided DNA targeting of any locus in the genome. Fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in mammalian cells. Delivery of multiple sgRNAs further enables activation or repression of multiple genes. By using scaffold RNAs (scRNAs), different effectors can be recruited to different genes for simultaneous activation of some and repression of others. The CRISPRi and CRISPRa methods provide powerful tools for sequence-specific control of gene expression on a genome-wide scale to aid understanding gene functions and for engineering genetic regulatory systems. PMID:26729910

  7. CRISPR Technology for Genome Activation and Repression in Mammalian Cells.

    PubMed

    Du, Dan; Qi, Lei S

    2016-01-04

    Targeted modulation of transcription is necessary for understanding complex gene networks and has great potential for medical and industrial applications. CRISPR is emerging as a powerful system for targeted genome activation and repression, in addition to its use in genome editing. This protocol describes how to design, construct, and experimentally validate the function of sequence-specific single guide RNAs (sgRNAs) for sequence-specific repression (CRISPRi) or activation (CRISPRa) of transcription in mammalian cells. In this technology, the CRISPR-associated protein Cas9 is catalytically deactivated (dCas9) to provide a general platform for RNA-guided DNA targeting of any locus in the genome. Fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in mammalian cells. Delivery of multiple sgRNAs further enables activation or repression of multiple genes. By using scaffold RNAs (scRNAs), different effectors can be recruited to different genes for simultaneous activation of some and repression of others. The CRISPRi and CRISPRa methods provide powerful tools for sequence-specific control of gene expression on a genome-wide scale to aid understanding gene functions and for engineering genetic regulatory systems.

  8. Mammalian target of rapamycin signaling in diabetic cardiovascular disease.

    PubMed

    Chong, Zhao Zhong; Maiese, Kenneth

    2012-07-16

    Diabetes mellitus currently affects more than 170 million individuals worldwide and is expected to afflict another 200 million individuals in the next 30 years. Complications of diabetes as a result of oxidant stress affect multiple systems throughout the body, but involvement of the cardiovascular system may be one of the most severe in light of the impact upon cardiac and vascular function that can result in rapid morbidity and mortality for individuals. Given these concerns, the signaling pathways of the mammalian target of rapamycin (mTOR) offer exciting prospects for the development of novel therapies for the cardiovascular complications of diabetes. In the cardiovascular and metabolic systems, mTOR and its multi-protein complexes of TORC1 and TORC2 regulate insulin release and signaling, endothelial cell survival and growth, cardiomyocyte proliferation, resistance to β-cell injury, and cell longevity. Yet, mTOR can, at times, alter insulin signaling and lead to insulin resistance in the cardiovascular system during diabetes mellitus. It is therefore vital to understand the complex relationship mTOR and its downstream pathways hold during metabolic disease in order to develop novel strategies for the complications of diabetes mellitus in the cardiovascular system.

  9. Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

    PubMed

    Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

    2016-07-01

    Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application. PMID:27408334

  10. Autophagy regulates the apoptosis of bone marrow-derived mesenchymal stem cells under hypoxic condition via AMP-activated protein kinase/mammalian target of rapamycin pathway.

    PubMed

    Zhang, Zheng; Yang, Ming; Wang, Yabin; Wang, Le; Jin, Zhitao; Ding, Liping; Zhang, Lijuan; Zhang, Lina; Jiang, Wei; Gao, Guojie; Yang, Junke; Lu, Bingwei; Cao, Feng; Hu, Taohong

    2016-06-01

    Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been demonstrated as an ideal autologous stem cells source for cell-based therapy for myocardial infarction (MI). However, poor viability of donor stem cells after transplantation limits their therapeutic efficiency, whereas the underlying mechanism is still poorly understood. Autophagy, a highly conserved process of cellular degradation, is required for maintaining homeostasis and normal function. Here, we investigated the potential role of autophagy on apoptosis in BM-MSCs induced by hypoxic injury. BM-MSCs, isolated from male C57BL/6 mice, were subjected to hypoxia and serum deprivation (H/SD) injury for 6, 12, and 24 h, respectively. The autophagy state was regulated by 3-methyladenine (3MA) and rapamycin administration. Furthermore, compound C was administrated to inhibit AMPK. The apoptosis induced by H/SD was determined by TUNEL assays. Meanwhile, autophagy was measured by GFP-LC3 plasmids transfection and transmission electron microscope. Moreover, protein expressions were evaluated by Western blot assay. In the present study, we found that hypoxic stress increased autophagy and apoptosis in BM-MSCs time dependently. Meanwhile, hypoxia increased the activity of AMPK/mTOR signal pathway. Moreover, increased apoptosis in BM-MSCs under hypoxia was abolished by 3-MA, whereas was aggravated by rapamycin. Furthermore, the increased autophagy and apoptosis in BM-MSCs induced by hypoxia were abolished by AMPK inhibitor compound C. These data provide evidence that hypoxia induced AMPK/mTOR signal pathway activation which regulated the apoptosis and autophagy in BM-MSCs. Furthermore, the apoptosis of BM-MSCs under hypoxic condition was regulated by autophagy via AMPK/mTOR pathway. PMID:27005844

  11. Differences in Mammalian Target of Rapamycin Gene Expression in the Peripheral Blood and Articular Cartilages of Osteoarthritic Patients and Disease Activity

    PubMed Central

    Tchetina, Elena V.; Poole, A. Robin; Zaitseva, Elena M.; Sharapova, Eugeniya P.; Kashevarova, Natalya G.; Taskina, Elena A.; Alekseeva, Liudmila I.; Semyonova, Liudmila A.; Glukhova, Svetlana I.; Kuzin, Alexandr N.; Makarov, Maxim A.; Makarov, Sergey A.

    2013-01-01

    The gene expression of mTOR, autophagy-related ULK1, caspase 3, CDK-inhibitor p21, and TNFα was measured in the peripheral blood of osteoarthritic (OA) patients at different stages of the disease aiming to establish a gene expression profile that might indicate the activity of the disease and joint destruction. Whole blood of 65 OA outpatients, 27 end-stage OA patients, 27 healthy volunteers, and knee articular cartilages of 28 end-stage OA patients and 26 healthy subjects were examined. OA outpatients were subjected to clinical testing, ultrasonography, and radiographic and WOMAC scoring. Protein levels of p70-S6K, p21, and caspase 3 were quantified by ELISA. Gene expression was measured using real-time RT-PCR. Upregulation of mTOR gene expression was observed in PBMCs of 42 OA outpatients (“High mTOR expression subset”) and in PBMCs and articular cartilages of all end-stage OA patients. A positive correlation between mTOR gene expression in PBMCs and cartilage was observed in the end-stage OA patients. 23 OA outpatients in the “Low mTOR expression subset” exhibited significantly lower mTOR gene expression in PBMCs compared to healthy controls. These “Low mTOR” subset subjects experienced significantly more pain upon walking, and standing and increased total joint stiffness versus “High mTOR” subset, while the latter more often exhibited synovitis. The protein concentrations of p70-S6K, p21, and caspase 3 in PBMCs were significantly lower in the “Low” subset versus “High” subset and end-stage subjects. Increases in the expression of mTOR in PBMCs of OA patients are related to disease activity, being associated with synovitis more than with pain. PMID:23864948

  12. A hexane fraction of guava Leaves (Psidium guajava L.) induces anticancer activity by suppressing AKT/mammalian target of rapamycin/ribosomal p70 S6 kinase in human prostate cancer cells.

    PubMed

    Ryu, Nae Hyung; Park, Kyung-Ran; Kim, Sung-Moo; Yun, Hyung-Mun; Nam, Dongwoo; Lee, Seok-Geun; Jang, Hyeung-Jin; Ahn, Kyoo Seok; Kim, Sung-Hoon; Shim, Bum Sang; Choi, Seung-Hoon; Mosaddik, Ashik; Cho, Somi K; Ahn, Kwang Seok

    2012-03-01

    This study was carried out to evaluate the anticancer effects of guava leaf extracts and its fractions. The chemical compositions of the active extracts were also determined. In the present study, we set out to determine whether the anticancer effects of guava leaves are linked with their ability to suppress constitutive AKT/mammalian target of rapamycin (mTOR)/ribosomal p70 S6 kinase (S6K1) and mitogen-activated protein kinase (MAPK) activation pathways in human prostate cancer cells. We found that guava leaf hexane fraction (GHF) was the most potent inducer of cytotoxic and apoptotic effects in PC-3 cells. The molecular mechanism or mechanisms of GHF apoptotic potential were correlated with the suppression of AKT/mTOR/S6K1 and MAPK signaling pathways. This effect of GHF correlated with down-regulation of various proteins that mediate cell proliferation, cell survival, metastasis, and angiogenesis. Analysis of GHF by gas chromatography and gas chromatography-mass spectrometry tentatively identified 60 compounds, including β-eudesmol (11.98%), α-copaene (7.97%), phytol (7.95%), α-patchoulene (3.76%), β-caryophyllene oxide (CPO) (3.63%), caryophylla-3(15),7(14)-dien-6-ol (2.68%), (E)-methyl isoeugenol (1.90%), α-terpineol (1.76%), and octadecane (1.23%). Besides GHF, CPO, but not phytol, also inhibited the AKT/mTOR/S6K1 signaling pathway and induced apoptosis in prostate cancer cells. Overall, these findings suggest that guava leaves can interfere with multiple signaling cascades linked with tumorigenesis and provide a source of potential therapeutic compounds for both the prevention and treatment of cancer.

  13. A Hexane Fraction of Guava Leaves (Psidium guajava L.) Induces Anticancer Activity by Suppressing AKT/Mammalian Target of Rapamycin/Ribosomal p70 S6 Kinase in Human Prostate Cancer Cells

    PubMed Central

    Ryu, Nae Hyung; Park, Kyung-Ran; Kim, Sung-Moo; Yun, Hyung-Mun; Nam, Dongwoo; Lee, Seok-Geun; Jang, Hyeung-Jin; Ahn, Kyoo Seok; Kim, Sung-Hoon; Shim, Bum Sang; Choi, Seung-Hoon; Mosaddik, Ashik

    2012-01-01

    Abstract This study was carried out to evaluate the anticancer effects of guava leaf extracts and its fractions. The chemical compositions of the active extracts were also determined. In the present study, we set out to determine whether the anticancer effects of guava leaves are linked with their ability to suppress constitutive AKT/mammalian target of rapamycin (mTOR)/ribosomal p70 S6 kinase (S6K1) and mitogen-activated protein kinase (MAPK) activation pathways in human prostate cancer cells. We found that guava leaf hexane fraction (GHF) was the most potent inducer of cytotoxic and apoptotic effects in PC-3 cells. The molecular mechanism or mechanisms of GHF apoptotic potential were correlated with the suppression of AKT/mTOR/S6K1 and MAPK signaling pathways. This effect of GHF correlated with down-regulation of various proteins that mediate cell proliferation, cell survival, metastasis, and angiogenesis. Analysis of GHF by gas chromatography and gas chromatography–mass spectrometry tentatively identified 60 compounds, including β-eudesmol (11.98%), α-copaene (7.97%), phytol (7.95%), α-patchoulene (3.76%), β-caryophyllene oxide (CPO) (3.63%), caryophylla-3(15),7(14)-dien-6-ol (2.68%), (E)-methyl isoeugenol (1.90%), α-terpineol (1.76%), and octadecane (1.23%). Besides GHF, CPO, but not phytol, also inhibited the AKT/mTOR/S6K1 signaling pathway and induced apoptosis in prostate cancer cells. Overall, these findings suggest that guava leaves can interfere with multiple signaling cascades linked with tumorigenesis and provide a source of potential therapeutic compounds for both the prevention and treatment of cancer. PMID:22280146

  14. Recent advances in developing molecular tools for targeted genome engineering of mammalian cells.

    PubMed

    Lim, Kwang-il

    2015-01-01

    Various biological molecules naturally existing in diversified species including fungi, bacteria, and bacteriophage have functionalities for DNA binding and processing. The biological molecules have been recently actively engineered for use in customized genome editing of mammalian cells as the molecule-encoding DNA sequence information and the underlying mechanisms how the molecules work are unveiled. Excitingly, multiple novel methods based on the newly constructed artificial molecular tools have enabled modifications of specific endogenous genetic elements in the genome context at efficiencies that are much higher than that of the conventional homologous recombination based methods. This minireview introduces the most recently spotlighted molecular genome engineering tools with their key features and ongoing modifications for better performance. Such ongoing efforts have mainly focused on the removal of the inherent DNA sequence recognition rigidity from the original molecular platforms, the addition of newly tailored targeting functions into the engineered molecules, and the enhancement of their targeting specificity. Effective targeted genome engineering of mammalian cells will enable not only sophisticated genetic studies in the context of the genome, but also widely-applicable universal therapeutics based on the pinpointing and correction of the disease-causing genetic elements within the genome in the near future.

  15. Recent advances in developing molecular tools for targeted genome engineering of mammalian cells.

    PubMed

    Lim, Kwang-il

    2015-01-01

    Various biological molecules naturally existing in diversified species including fungi, bacteria, and bacteriophage have functionalities for DNA binding and processing. The biological molecules have been recently actively engineered for use in customized genome editing of mammalian cells as the molecule-encoding DNA sequence information and the underlying mechanisms how the molecules work are unveiled. Excitingly, multiple novel methods based on the newly constructed artificial molecular tools have enabled modifications of specific endogenous genetic elements in the genome context at efficiencies that are much higher than that of the conventional homologous recombination based methods. This minireview introduces the most recently spotlighted molecular genome engineering tools with their key features and ongoing modifications for better performance. Such ongoing efforts have mainly focused on the removal of the inherent DNA sequence recognition rigidity from the original molecular platforms, the addition of newly tailored targeting functions into the engineered molecules, and the enhancement of their targeting specificity. Effective targeted genome engineering of mammalian cells will enable not only sophisticated genetic studies in the context of the genome, but also widely-applicable universal therapeutics based on the pinpointing and correction of the disease-causing genetic elements within the genome in the near future. PMID:25104401

  16. Pharmacological Targeting of the Mammalian Clock Regulates Sleep Architecture and Emotional Behavior

    PubMed Central

    Banerjee, Subhashis; Wang, Yongjun; Solt, Laura A.; Griffett, Kristine; Kazantzis, Melissa; Amador, Ariadna; El-Gendy, Bahaa M.; Huitron-Resendiz, Salvador; Roberts, Amanda J.; Shin, Youseung; Kamenecka, Theodore M.; Burris, Thomas P.

    2014-01-01

    Synthetic drug-like molecules that directly modulate the activity of key clock proteins offer the potential to directly modulate the endogenous circadian rhythm and treat diseases associated with clock dysfunction. Here, we demonstrate that synthetic ligands targeting a key component of the mammalian clock, the nuclear receptors REV-ERBα and β, regulate sleep architecture and emotional behavior in mice. REV-ERB agonists induce wakefulness and reduce REM and slow-wave sleep. Interestingly, REV-ERB agonists also reduce anxiety-like behavior. These data are consistent with increased anxiety-like behavior of REV-ERBβ null mice, in which REV-ERB agonists have no effect Also consistent with these effects being mediated by REV-ERB, the effect of the agonist on sleep and anxiety was suppressed by lithium treatment. These results indicate that pharmacological targeting of REVERB may lead to the development of novel therapeutics to treat sleep disorders and anxiety. PMID:25536025

  17. Pharmacological targeting of the mammalian clock regulates sleep architecture and emotional behaviour.

    PubMed

    Banerjee, Subhashis; Wang, Yongjun; Solt, Laura A; Griffett, Kristine; Kazantzis, Melissa; Amador, Ariadna; El-Gendy, Bahaa M; Huitron-Resendiz, Salvador; Roberts, Amanda J; Shin, Youseung; Kamenecka, Theodore M; Burris, Thomas P

    2014-12-23

    Synthetic drug-like molecules that directly modulate the activity of key clock proteins offer the potential to directly modulate the endogenous circadian rhythm and treat diseases associated with clock dysfunction. Here we demonstrate that synthetic ligands targeting a key component of the mammalian clock, the nuclear receptors REV-ERBα and β, regulate sleep architecture and emotional behaviour in mice. REV-ERB agonists induce wakefulness and reduce REM and slow-wave sleep. Interestingly, REV-ERB agonists also reduce anxiety-like behaviour. These data are consistent with increased anxiety-like behaviour of REV-ERBβ-null mice, in which REV-ERB agonists have no effect. These results indicate that pharmacological targeting of REV-ERB may lead to the development of novel therapeutics to treat sleep disorders and anxiety.

  18. Transcriptional regulation of gene expression during osmotic stress responses by the mammalian target of rapamycin.

    PubMed

    Ortells, M Carmen; Morancho, Beatriz; Drews-Elger, Katherine; Viollet, Benoit; Laderoute, Keith R; López-Rodríguez, Cristina; Aramburu, Jose

    2012-05-01

    Although stress can suppress growth and proliferation, cells can induce adaptive responses that allow them to maintain these functions under stress. While numerous studies have focused on the inhibitory effects of stress on cell growth, less is known on how growth-promoting pathways influence stress responses. We have approached this question by analyzing the effect of mammalian target of rapamycin (mTOR), a central growth controller, on the osmotic stress response. Our results showed that mammalian cells exposed to moderate hypertonicity maintained active mTOR, which was required to sustain their cell size and proliferative capacity. Moreover, mTOR regulated the induction of diverse osmostress response genes, including targets of the tonicity-responsive transcription factor NFAT5 as well as NFAT5-independent genes. Genes sensitive to mTOR-included regulators of stress responses, growth and proliferation. Among them, we identified REDD1 and REDD2, which had been previously characterized as mTOR inhibitors in other stress contexts. We observed that mTOR facilitated transcription-permissive conditions for several osmoresponsive genes by enhancing histone H4 acetylation and the recruitment of RNA polymerase II. Altogether, these results reveal a previously unappreciated role of mTOR in regulating transcriptional mechanisms that control gene expression during cellular stress responses. PMID:22287635

  19. The cytotoxic T cell proteome and its shaping by mammalian Target of Rapamycin

    PubMed Central

    Hukelmann, Jens L.; Anderson, Karen E.; Sinclair, Linda V.; Grzes, Katarzyna M.; Murillo, Alejandro Brenes; Hawkins, Phillip T.; Stephens, Len R.; Lamond, Angus I.; Cantrell, Doreen A.

    2015-01-01

    High-resolution mass spectrometry maps the cytotoxic T lymphocyte (CTL) proteome and the impact of mammalian target of rapamycin complex 1 (mTORC1) on CTLs. The CTL proteome was dominated by metabolic regulators and granzymes and mTORC1 selectively repressed and promoted expression of subset of CTL proteins (~10%). These included key CTL effector molecules, signaling proteins and a subset of metabolic enzymes. Proteomic data highlighted the potential for mTORC1 negative control of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) production in CTL. mTORC1 was shown to repress PtdIns(3,4,5)P3 production and to determine the mTORC2 requirement for activation of the kinase Akt. Unbiased proteomic analysis thus provides a comprehensive understanding of CTL identity and mTORC1 control of CTL function. PMID:26551880

  20. EPR characterization of molecular targets for NO · in mammalian cells

    NASA Astrophysics Data System (ADS)

    Guissani, A.; Henry, Y. A.

    1997-02-01

    First some elementary properties of nitric oxide NO · are presented: NO · is synthesized in mammalian cells from L-arginine, the reaction being catalyzed by the enzyme NO-synthase. The EPR spectroscopy of NO-complexes is shown; the formation of paramagnetic complexes with some metalloproteins including haemoglobin enables the EPR detection of such complexes. EPR-detectable targets for NO · in mammalian cells are then (rapidly) described, including first their detection (after induction of NO-synthases) through interaction of NO · with specific metalloenzymes, then NO · localization, the fact that the induction of NO-synthases in a generator cell such as a macrophage gives the same metabolic effects in target cells, and finally the implication of NO · in pathological states, where the inducible L-arginine-NO pathway plays an important role. It is concluded that EPR spectroscopy enables the unambiguous detection of such specific molecular targets for NO ·; in mammalian cells.

  1. Mammalian plasma membrane proteins as potential biomarkers and drug targets.

    PubMed

    Rucevic, Marijana; Hixson, Douglas; Josic, Djuro

    2011-06-01

    Defining the plasma membrane proteome is crucial to understand the role of plasma membrane in fundamental biological processes. Change in membrane proteins is one of the first events that take place under pathological conditions, making plasma membrane proteins a likely source of potential disease biomarkers with prognostic or diagnostic potential. Membrane proteins are also potential targets for monoclonal antibodies and other drugs that block receptors or inhibit enzymes essential to the disease progress. Despite several advanced methods recently developed for the analysis of hydrophobic proteins and proteins with posttranslational modifications, integral membrane proteins are still under-represented in plasma membrane proteome. Recent advances in proteomic investigation of plasma membrane proteins, defining their roles as diagnostic and prognostic disease biomarkers and as target molecules in disease treatment, are presented.

  2. Strategies to Detect Endogenous Ubiquitination of a Target Mammalian Protein.

    PubMed

    Sigismund, Sara; Polo, Simona

    2016-01-01

    Different biochemical techniques are well established to investigate target's ubiquitination in mammals without overexpressing a tagged version of ubiquitin (Ub). The simplest and more direct approach is to immunoprecipitate (IP) your target protein from cell lysate (stimulated and/or properly treated), followed by western blot analysis utilizing specific antibodies against Ub (see Subheading 3.1). This approach requires a good antibody against the target working in IP; alternatively, one could express a tagged version of the protein, possibly at the endogenous level. Another approach consists in IP ubiquitinated proteins from total cell lysate followed by detection with the antibody against the protein of interest. This second method relies on the availability of specific and very efficient antibodies against Ub (see Subheading 3.2). A more quantitative approach is the DELFIA assay (Perkin Elmer), an ELISA-based assay, which allows comparing more samples and conditions (see Subheading 3.3). Cross-validation with more than one approach is usually recommended in order to prove that your protein is modified by ubiquitin.Here we will use the EGFR as model system but protocols can be easily modified according to the protein of interest. PMID:27613032

  3. The mammalian target of rapamycin modulates the immunoproteasome system in the heart.

    PubMed

    Zhang, Hong-Mei; Fu, Jianliang; Hamilton, Ryan; Diaz, Vivian; Zhang, Yiqiang

    2015-09-01

    The mammalian target of rapamycin (mTOR) plays an important role in cardiac development and function. Inhibition of mTOR by rapamycin has been shown to attenuate pathological cardiac hypertrophy and improve the function of aging heart, accompanied by an inhibition of the cardiac proteasome activity. The current study aimed to determine the potential mechanism(s) by which mTOR inhibition modulates cardiac proteasome. Inhibition of mTOR by rapamycin was found to reduce primarily the immunoproteasome in both H9c2 cells in vitro and mouse heart in vivo, without significant effect on the constitutive proteasome and protein ubiquitination. Concurrent with the reduction of the immunoproteasome, rapamycin reduced two important inflammatory response pathways, the NF-κB and Stat3 signaling. In addition, rapamycin attenuated the induction of the immunoproteasome in H9c2 cells by inflammatory cytokines, including INFγ and TNFα, by suppressing NF-κB signaling. These data indicate that rapamycin indirectly modulated immunoproteasome through the suppression of inflammatory response pathways. Lastly, the role of the immunoproteasome during the development of cardiac hypertrophy was investigated. Administration of a specific inhibitor of the immunoproteasome ONX 0914 attenuated isoproterenol-induced cardiac hypertrophy, suggesting that the immunoproteasome may be involved in the development of cardiac hypertrophy and therefore could be a therapeutic target. In conclusion, rapamycin inhibits the immunoproteasome through its effect on the inflammatory signaling pathways and the immunoproteasome could be a potential therapeutic target for pathological cardiac hypertrophy. PMID:26239133

  4. Passive versus active local microrheology in mammalian cells and amoebae

    NASA Astrophysics Data System (ADS)

    Riviere, C.; Gazeau, F.; Marion, S.; Bacri, J.-C.; Wilhelm, C.

    2004-12-01

    We compare in this paper the rotational magnetic microrheology detailed by Marion et al [18] and Wilhelm et al [19] to the passive tracking microrheology. The rotational microrheology has been designed to explore, using magnetic rotating probes, the local intracellular microenvironment of living cells in terms of viscoelasticity. Passive microrheology techniques is based on the analysis of spontaneous diffusive motions of Brownian probes. The dependence of mean square displacement (MSD) with the time then directly reflects the type of movement (sub-, hyper- or diffusive motions). Using the same intracellular probes, we performed two types of measurements (active and passive). Based on the fluctuation-dissipation theorem, one should obtain the same information from the both techniques in a thermally equilibrium system. Interestingly, our measurements differ, and the discordances directly inform on active biological processes, which add to thermally activated fluctuations in our out-of equilibrium systems. In both cell models used, mammalian Hela cells and amoebae Entamoeba Histolytica, a hyper-diffusive regime at a short time is observed, which highlights the presence of an active non-thermal driving force, acting on the probe. However, the nature of this active force in mammalian cells and amoebae is different, according to their different phenotypes. In mammalian cells active processes are governed by the transport, via molecular motors, on the microtubule network. In amoebae, which are highly motile cells free of microtubule network, the active processes are dominated by strong fluxes of cytoplasm driven by extension of pseudopodia, in random directions, leading to an amplitude of motion one order of magnitude higher than for mammalian cells. Figs 7, Refs 32.

  5. Mammalian target of rapamycin signaling modulates photic entrainment of the suprachiasmatic circadian clock

    PubMed Central

    Cao, Ruifeng; Li, Aiqing; Cho, Hee-yeon; Lee, Boyoung; Obrietan, Karl

    2010-01-01

    Inducible gene expression appears to be an essential event that couples light to entrainment of the master mammalian circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Recently, we reported that light triggers phase-dependent activation of the mammalian target of rapamycin (mTOR) signaling pathway, a major regulator of protein synthesis, in the SCN, thus raising the possibility that mTOR-evoked mRNA translation contributes to clock entrainment. Here, we employed a combination of cellular, molecular and behavioral assays to address this question. To this end, we show that the in vivo infusion of the mTOR inhibitor rapamycin led to a significant attenuation of the phase-delaying effect of early night light. Conversely, disruption of mTOR during the late night augmented the phase-advancing effect of light. To assess the role of mTOR signaling within the context of molecular entrainment, the effects of rapamycin on light-induced expression of PERIOD1 and PERIOD2 were examined. At both the early and late night time points, abrogation of mTOR signaling led to a significant attenuation of light-evoked PERIOD protein expression. Our results also reveal that light-induced mTOR activation leads to translation of mRNAs with a 5′-terminal oligopyrimidine tract such as eukaryotic elongation factor 1 A (eEF1A) and the immediate early gene JunB. Together, these data indicate that the mTOR pathway functions as potent and selective regulator of light-evoked protein translation and SCN clock entrainment. PMID:20445056

  6. Superoxide radical and iron modulate aconitase activity in mammalian cells.

    PubMed

    Gardner, P R; Raineri, I; Epstein, L B; White, C W

    1995-06-01

    Aconitase is a member of a family of iron-sulfur-containing (de)hydratases whose activities are modulated in bacteria by superoxide radical (O2-.)-mediated inactivation and iron-dependent reactivation. The inactivation-reactivation of aconitase(s) in cultured mammalian cells was explored since these reactions may impact important and diverse aconitase functions in the cytoplasm and mitochondria. Conditions which increase O2-. production including exposure to the redox-cycling agent phenazine methosulfate (PMS), inhibitors of mitochondrial ubiquinol-cytochrome c oxidoreductase, or hyperoxia inactivated aconitase in mammalian cells. Overproduction of mitochondrial Mn-superoxide dismutase protected aconitase from inactivation by PMS or inhibitors of ubiquinol-cytochrome c oxidoreductase, but not from normobaric hyperoxia. Aconitase activity was reactivated (t1/2 of 12 +/- 3 min) upon removal of PMS. The iron chelator deferoxamine impaired reactivation and increased net inactivation of aconitase by O2-.. The ability of ubiquinol-cytochrome c oxidoreductase-generated O2-. to inactivate aconitase in several cell types correlated with the fraction of the aconitase activity localized in mitochondria. Extracellular O2-. generated with xanthine oxidase did not affect aconitase activity nor did exogenous superoxide dismutase decrease aconitase inactivation by PMS. The results demonstrate a dynamic and cyclical O2-.-mediated inactivation and iron-dependent reactivation of the mammalian [4Fe-4S] aconitases under normal and stress conditions and provide further evidence for the membrane compartmentalization of O2-.. PMID:7768942

  7. Deoxyribophosphate lyase activity of mammalian endonuclease VIII-like proteins.

    PubMed

    Grin, Inga R; Khodyreva, Svetlana N; Nevinsky, Georgy A; Zharkov, Dmitry O

    2006-09-01

    Base excision repair (BER) protects cells from nucleobase DNA damage. In eukaryotic BER, DNA glycosylases generate abasic sites, which are then converted to deoxyribo-5'-phosphate (dRP) and excised by a dRP lyase (dRPase) activity of DNA polymerase beta (Polbeta). Here, we demonstrate that NEIL1 and NEIL2, mammalian homologs of bacterial endonuclease VIII, excise dRP by beta-elimination with the efficiency similar to Polbeta. DNA duplexes imitating BER intermediates after insertion of a single nucleotide were better substrates. NEIL1 and NEIL2 supplied dRPase activity in BER reconstituted with dRPase-null Polbeta. Our results suggest a role for NEILs as backup dRPases in mammalian cells.

  8. Targeting the mammalian target of Rapamycin to inhibit VEGF and cytokines for the treatment of primary effusion lymphoma

    PubMed Central

    Gasperini, Paola; Tosato, Giovanna

    2009-01-01

    Primary effusion lymphoma (PEL) is a fatal malignancy, which typically presents as a lymphomatous effusion that later disseminates. Rapamycin (Rapa), which targets mTOR (mammalian target of Rapa), is currently evaluated as a treatment for PEL, but the recent development of PEL in Rapa-treated post-transplant recipients questions the drug's use in PEL. Here, we used a murine model of PEL effusion that mimics the human disease to investigate the anti-PEL activity of Rapa. We found that Rapa reduces ascites accumulation and extends mouse survival. Initially, Rapa reduced PEL load compared to control mice, but most mice rapidly showed PEL progression. Levels of VEGF, which promotes vascular permeability contributing to effusion formation, were significantly reduced in ascites of Rapa-treated mice compared to controls. Expression of IL-10, the principal autocrine growth factor for PEL, was initially reduced in PEL from Rapa-treated mice but rapidly increased despite treatment. We found that the hypoxic environment of ascites and Rapa cooperate in stimulating IL-10 expression in PEL, which likely contributes to the emergence of drug resistance. These results identify Rapa an effective drug to reduce PEL effusions but illustrate the rapid development of drug resistance, which likely limits the efficacy of Rapa in PEL. PMID:19554030

  9. Double-strand gap repair in a mammalian gene targeting reaction.

    PubMed Central

    Valancius, V; Smithies, O

    1991-01-01

    To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells. A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium. The structures of the recombinant loci were then determined by genomic Southern blot hybridizations. We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction. Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms. Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost. These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments. Images PMID:1875928

  10. Clinical significance of aberrant mammalian target of rapamycin expression in stage IIIB colon cancer

    PubMed Central

    WEN, MEILING; LI, BAOXIU; CAO, XIAOFEI; WENG, CHENGYIN; WU, YONG; FANG, XISHENG; ZHANG, XIAOSHI; LIU, GUOLONG

    2014-01-01

    The aim of the present study was to investigate the significance of aberrant expression of mammalian target of rapamycin (mTOR) and the activated form of mTOR kinase, phosphorylated mTOR (pmTOR), in human stage IIIB colon cancer. The expression of mTOR and pmTOR was detected by immunohistochemistry in the tumor tissue of stage IIIB colon cancer patients. The association between the expression of mTOR, pmTOR and clinicopathological parameters of patients was analyzed. The positive expression of mTOR and pmTOR was observed to be higher in 75.5% (80/106) and 76.4% (81/106) of the 106 colon cancer specimens, compared with the adjacent normal tissues. The high level of pmTOR expression was found to be significantly higher in the invasive tumor front cells and resulted in a higher risk of mortality. The results suggested that mTOR and pmTOR may be promising clinical markers and present novel molecular targets for designing novel therapeutic strategies to treat this malignancy. PMID:25120661

  11. Targeting of a histone acetyltransferase domain to a promoter enhances protein expression levels in mammalian cells.

    PubMed

    Kwaks, T H J; Sewalt, R G A B; van Blokland, R; Siersma, T J; Kasiem, M; Kelder, A; Otte, A P

    2005-01-12

    Silencing of transfected genes in mammalian cells is a fundamental problem that probably involves the (in)accessibility status of chromatin. A potential solution to this problem is to provide a cell with protein factors that make the chromatin of a promoter more open or accessible for transcription. We tested this by targeting such proteins to different promoters. We found that targeting the p300 histone acetyltransferase (HAT) domain to strong viral or cellular promoters is sufficient to result in higher expression levels of a reporter protein. In contrast, targeting the chromatin-remodeling factor Brahma does not result in stable, higher protein expression levels. The long-term effects of the targeted p300HAT domain on protein expression levels are positively reinforced, when also anti-repressor elements are applied to flank the reporter construct. These elements were previously shown to be potent blockers of chromatin-associated repressors. The simultaneous application of the targeted p300HAT domain and anti-repressor elements conveys long-term stability to protein expression. Whereas no copy number dependency is achieved by targeting of the p300HAT domain alone, copy number dependency is improved when anti-repressor elements are included. We conclude that targeting of protein domains such as HAT domains helps to facilitate expression of transfected genes in mammalian cells. However, the simultaneous application of other genomic elements such as the anti-repressor elements prevents silencing more efficiently.

  12. Mammalian target of rapamycin: hitting the bull's-eye for neurological disorders.

    PubMed

    Chong, Zhao Zhong; Shang, Yan Chen; Zhang, Lijie; Wang, Shaohui; Maiese, Kenneth

    2010-01-01

    The mammalian target of rapamycin (mTOR) and its associated cell signaling pathways have garnered significant attention for their roles in cell biology and oncology. Interestingly, the explosion of information in this field has linked mTOR to neurological diseases with promising initial studies. mTOR, a 289 kDa serine/threonine protein kinase, plays an important role in cell growth and proliferation and is activated through phosphorylation in response to growth factors, mitogens, and hormones. Growth factors, amino acids, cellular nutrients, and oxygen deficiency can down-regulate mTOR activity. The function of mTOR signaling is mediated primarily through two mTOR complexes: mTORC1 and mTORC2. mTORC1 initiates cap-dependent protein translation, a rate-limiting step of protein synthesis, through the phosphorylation of the targets eukaryotic initiation factor 4E-binding protein 1 (4EBP1) and p70 ribosomal S6 kinase (p70S6K). In contrast, mTORC2 regulates development of the cytoskeleton and also controls cell survival. Although closely tied to tumorigenesis, mTOR and the downstream signaling pathways are significantly involved in the central nervous system (CNS) with synaptic plasticity, memory retention, neuroendocrine regulation associated with food intake and puberty, and modulation of neuronal repair following injury. The signaling pathways of mTOR also are believed to be a significant component in a number of neurological diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, tuberous sclerosis, neurofibromatosis, fragile X syndrome, epilepsy, traumatic brain injury, and ischemic stroke. Here we describe the role of mTOR in the CNS and illustrate the potential for new strategies directed against neurological disorders.

  13. Garcinielliptone FC: antiparasitic activity without cytotoxicity to mammalian cells.

    PubMed

    Silva, Ana P; Silva, Marcos P; Oliveira, Cristiano G; Monteiro, Daniela C; Pinto, Pedro L; Mendonça, Ronaldo Z; Costa Júnior, Joaquim S; Freitas, Rivelilson M; de Moraes, Josué

    2015-06-01

    Garcinielliptone FC (GFC) is a natural prenylated benzophenone found in the seeds of Platonia insignis Mart. (Clusiaceae), a native Brazilian plant. It has been chemically characterized and it is known that GFC has several biological activities such as antioxidant and vasorelaxant properties. In this study, we report the in vitro effect of GFC against the blood fluke Schistosoma mansoni, the parasite responsible for schistosomiasis mansoni. The anti-S. mansoni activity and cytotoxicity toward mammalian cells were determined for the compound. GFC⩾6.25 μM showed antischistosomal activity and confocal laser scanning microscopy analysis demonstrated several morphological alterations on the tegument of worms, and a correlation between viability and tegumental damage was observed. In addition, at sub-lethal concentrations of GFC (⩽3.125 μM), the number of S. mansoni eggs was reduced. More importantly, GFC exhibited no activity toward mammalian cells and, therefore, there is an appreciable selectivity of this compound against the helminths. In conclusion, these findings indicate the potential of GFC as an antiparasitic agent. PMID:25553916

  14. Mammalian target of rapamycin coordinates iron metabolism with iron-sulfur cluster assembly enzyme and tristetraprolin.

    PubMed

    Guan, Peng; Wang, Na

    2014-09-01

    Both iron deficiency and excess are relatively common health concerns. Maintaining the body's levels of iron within precise boundaries is critical for cell functions. However, the difference between iron deficiency and overload is often a question of a scant few milligrams of iron. The mammalian target of rapamycin (mTOR), an atypical Ser/Thr protein kinase, is attracting significant amounts of interest due to its recently described role in iron homeostasis. Despite extensive study, a complete understanding of mTOR function has remained elusive. mTOR can form two multiprotein complexes that consist of mTOR complex 1 (mTORC1) and mTOR complex 2. Recent advances clearly demonstrate that mTORC1 can phosphorylate iron-sulfur cluster assembly enzyme ISCU and affect iron-sulfur clusters assembly. Moreover, mTOR is reported to control iron metabolism through modulation of tristetraprolin expression. It is now well appreciated that the hormonal hepcidin-ferroportin system and the cellular iron-responsive element/iron-regulatory protein regulatory network play important regulatory roles for systemic iron metabolism. Sustained ISCU protein levels enhanced by mTORC1 can inhibit iron-responsive element and iron-regulatory protein binding activities. In this study, hepcidin gene and protein expression in the livers of tristetraprolin knockout mice were dramatically reduced. Here, we highlight and summarize the current understanding of how mTOR pathways serve to modulate iron metabolism and homeostasis as the third iron-regulatory system.

  15. Drosophila homolog of the mammalian jun oncogene is expressed during embryonic development and activates transcription in mammalian cells.

    PubMed Central

    Zhang, K; Chaillet, J R; Perkins, L A; Halazonetis, T D; Perrimon, N

    1990-01-01

    By means of low-stringency cross-species hybridization to Southern DNA blots, human c-jun sequences were used to identify a unique Drosophila melanogaster locus (Djun). The predicted DJun protein is highly homologous to members of the mammalian Jun family in both the DNA binding and leucine zipper regions. Djun was mapped by in situ hybridization to position 46E of the second chromosome. It encodes a 1.7-kilobase transcript constitutively expressed at all developmental stages. Functionally, Djun in cooperation with mouse c-fos can trans-activate activator protein 1 DNA binding site when introduced into mammalian cells. Taken together, these data suggest that Djun, much like its mammalian homolog, may activate transcription of genes involved in regulation of cell growth, differentiation, and development. Furthermore, the identification of Djun allows one to exploit the genetics of Drosophila to identify genes in signal transduction pathways involving Djun and thus c-jun. Images PMID:1696724

  16. Testosterone induces cardiomyocyte hypertrophy through mammalian target of rapamycin complex 1 pathway.

    PubMed

    Altamirano, Francisco; Oyarce, César; Silva, Patricio; Toyos, Marcela; Wilson, Carlos; Lavandero, Sergio; Uhlén, Per; Estrada, Manuel

    2009-08-01

    Elevated testosterone concentrations induce cardiac hypertrophy but the molecular mechanisms are poorly understood. Anabolic properties of testosterone involve an increase in protein synthesis. The mammalian target of rapamycin complex 1 (mTORC1) pathway is a major regulator of cell growth, but the relationship between testosterone action and mTORC1 in cardiac cells remains unknown. Here, we investigated whether the hypertrophic effects of testosterone are mediated by mTORC1 signaling in cultured cardiomyocytes. Testosterone increases the phosphorylation of mTOR and its downstream targets 40S ribosomal protein S6 kinase 1 (S6K1; also known as RPS6KB1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The S6K1 phosphorylation induced by testosterone was blocked by rapamycin and small interfering RNA to mTOR. Moreover, the hormone increased both extracellular-regulated kinase (ERK1/2) and protein kinase B (Akt) phosphorylation. ERK1/2 inhibitor PD98059 blocked the testosterone-induced S6K1 phosphorylation, whereas Akt inhibition (Akt-inhibitor-X) had no effect. Testosterone-induced ERK1/2 and S6K1 phosphorylation increases were blocked by either 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethylester or by inhibitors of inositol 1,4,5-trisphosphate (IP(3)) pathway: U-73122 and 2-aminoethyl diphenylborate. Finally, cardiomyocyte hypertrophy was evaluated by, the expression of beta-myosin heavy chain, alpha-skeletal actin, cell size, and amino acid incorporation. Testosterone increased all four parameters and the increase being blocked by mTOR inhibition. Our findings suggest that testosterone activates the mTORC1/S6K1 axis through IP(3)/Ca(2+) and MEK/ERK1/2 to induce cardiomyocyte hypertrophy. PMID:19474060

  17. An Introduction to CRISPR Technology for Genome Activation and Repression in Mammalian Cells.

    PubMed

    Du, Dan; Qi, Lei S

    2016-01-04

    CRISPR interference/activation (CRISPRi/a) technology provides a simple and efficient approach for targeted repression or activation of gene expression in the mammalian genome. It is highly flexible and programmable, using an RNA-guided nuclease-deficient Cas9 (dCas9) protein fused with transcriptional regulators for targeting specific genes to effect their regulation. Multiple studies have shown how this method is an effective way to achieve efficient and specific transcriptional repression or activation of single or multiple genes. Sustained transcriptional modulation can be obtained by stable expression of CRISPR components, which enables directed reprogramming of cell fate. Here, we introduce the basics of CRISPRi/a technology for genome repression or activation.

  18. Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

    SciTech Connect

    Zhang, Pengpeng; Shan, Tizhong; Liang, Xinrong; Deng, Changyan; Kuang, Shihuan

    2014-09-12

    Highlights: • mTOR is a critical regulator of many biological processes yet its function in heart is not well understood. • MCK-Cre/Mtor{sup flox/flox} mice were established to delete Mtor in cardiomyocytes. • The mTOR-mKO mice developed normally but die prematurely within 5 weeks after birth due to heart disease. • The mTOR-mKO mice had dilated myocardium and increased cell death. • mTOR-mKO hearts had reduced expression of metabolic genes and activation of mTOR target proteins. - Abstract: Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor{sup flox/flox} mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function.

  19. Active zones of mammalian neuromuscular junctions: formation, density, and aging

    PubMed Central

    Nishimune, Hiroshi

    2012-01-01

    Presynaptic active zones are synaptic vesicle release sites that playessential roles in the function and pathology of mammalian neuromuscular junctions (NMJs). The molecular mechanisms of active zone organization utilize presynaptic voltage-dependent calcium channels (VDCCs) in NMJs as scaffolding proteins. VDCCs interact extracellularly with the muscle-derived synapse organizer, laminin β2, and interact intracellularly with active zone-specific proteins, such as Bassoon, CAST/Erc2/ELKS2alpha, ELKS, Piccolo, and RIMs. These molecular mechanisms are supported by studies in P/Q- and N-type VDCCs double-knockout mice, and they are consistent with the pathological conditions of Lambert-Eaton myasthenic syndrome and Pierson syndrome, which are caused by autoantibodies against VDCCs or by a laminin β2 mutation. During normal postnatal maturation, NMJs maintain the density of active zones, while NMJs triple their size. However, active zones become impaired during aging. Propitiously, muscle exercise ameliorates the active zone impairment in aged NMJs, which suggests the potential for therapeutic strategies. PMID:23252894

  20. The Microprocessor controls the activity of mammalian retrotransposons.

    PubMed

    Heras, Sara R; Macias, Sara; Plass, Mireya; Fernandez, Noemí; Cano, David; Eyras, Eduardo; Garcia-Perez, José L; Cáceres, Javier F

    2013-10-01

    More than half of the human genome is made of transposable elements whose ongoing mobilization is a driving force in genetic diversity; however, little is known about how the host regulates their activity. Here, we show that the Microprocessor (Drosha-DGCR8), which is required for microRNA biogenesis, also recognizes and binds RNAs derived from human long interspersed element 1 (LINE-1), Alu and SVA retrotransposons. Expression analyses demonstrate that cells lacking a functional Microprocessor accumulate LINE-1 mRNA and encoded proteins. Furthermore, we show that structured regions of the LINE-1 mRNA can be cleaved in vitro by Drosha. Additionally, we used a cell culture-based assay to show that the Microprocessor negatively regulates LINE-1 and Alu retrotransposition in vivo. Altogether, these data reveal a new role for the Microprocessor as a post-transcriptional repressor of mammalian retrotransposons and a defender of human genome integrity.

  1. Prometaphase APCcdh1 activity prevents non-disjunction in mammalian oocytes.

    PubMed

    Reis, Alexandra; Madgwick, Suzanne; Chang, Heng-Yu; Nabti, Ibtissem; Levasseur, Mark; Jones, Keith T

    2007-10-01

    The first female meiotic division (meiosis I, MI) is uniquely prone to chromosome segregation errors through non-disjunction, resulting in trisomies and early pregnancy loss. Here, we show a fundamental difference in the control of mammalian meiosis that may underlie such susceptibility. It involves a reversal in the well-established timing of activation of the anaphase-promoting complex (APC) by its co-activators cdc20 and cdh1. APC(cdh1) was active first, during prometaphase I, and was needed in order to allow homologue congression, as loss of cdh1 speeded up MI, leading to premature chromosome segregation and a non-disjunction phenotype. APC(cdh1) targeted cdc20 for degradation, but did not target securin or cyclin B1. These were degraded later in MI through APC(cdc20), making cdc20 re-synthesis essential for successful meiotic progression. The switch from APC(cdh1) to APC(cdc20) activity was controlled by increasing CDK1 and cdh1 loss. These findings demonstrate a fundamentally different mechanism of control for the first meiotic division in mammalian oocytes that is not observed in meioses of other species. PMID:17891138

  2. Mechanisms of immune system activation in mammalians by small interfering RNA (siRNA).

    PubMed

    Mansoori, Behzad; Mohammadi, Ali; Shir Jang, Solmaz; Baradaran, Behzad

    2016-11-01

    RNA interference (RNAi) guided by small interfering RNAs (siRNA), because of its potential to target and silence the expression of specific genes is utilized as an effective tool in a variety of biological applications. RNAi guided by siRNAs is a powerful tool to attain gene silencing in mammalian cells. One of the features which make siRNA as an amazing biological tool is extremely specific knockdown of target genes by degradation of analogous mRNAs. However, various non-specific effects limit the use of RNAi including the activation of innate immunity and inhibition of inadvertent target genes. One of the most common non-specific effects is inducing the innate immune system including cytoplasmic and endosomal activation of innate immune system, potentially offending the single in mammals. This activation is mainly interceded by immune cells, regularly through a Toll-like receptor (TLR) pathway. The siRNA sequence association of these pathways changes with the sort and position of the TLR involved. In contrast, non-immune cell activation can also arise generally siRNAs which enter into cytoplasm interacting with cytoplasmic RNA sensors such as retinoic acid-inducible gene I. Here, we explain the off-target effects of siRNAs that activate innate immune system and methods to alleviate them, to help enable impressive application of this exciting technology, Also we bold the aspect of molecular strategies permitting the design of therapeutic siRNAs with minute off-target effects.

  3. Glutamate-dependent phosphorylation of the mammalian target of rapamycin (mTOR) in Bergmann glial cells.

    PubMed

    Zepeda, Rossana C; Barrera, Iliana; Castelán, Francisco; Suárez-Pozos, Edna; Melgarejo, Yaaziel; González-Mejia, Elba; Hernández-Kelly, Luisa C; López-Bayghen, Esther; Aguilera, José; Ortega, Arturo

    2009-09-01

    Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, plays an important role in neuronal development and synaptic plasticity. It activates a variety of signaling pathways that regulate gene expression at the transcriptional and translational levels. Within glial cells, besides transcription, glutamate also regulates translation initiation and elongation. The mammalian target of rapamycin (mTOR), a key participant in the translation process, represents an important regulatory locus for translational control. Therefore, in the present communication we sought to characterize the mTOR phosphorylation pattern after glutamate treatment in chick cerebellar Bergmann glia primary cultures. A time- and dose-dependent increase in mTOR Ser 2448 phosphorylation was found. Pharmacological tools established that the glutamate effect is mediated through ionotropic and metabotropic receptors and interestingly, the glutamate transporter system is also involved. The signaling cascade triggered by glutamate includes an increase in intracellular Ca2+ levels, and the activation of the p60(Src)/PI-3K/PKB pathway. These results suggest that glia cells participate in the activity-dependent change in the brain protein repertoire.

  4. microRNA target predictions across seven Drosophila species and comparison to mammalian targets.

    PubMed

    Grün, Dominic; Wang, Yi-Lu; Langenberger, David; Gunsalus, Kristin C; Rajewsky, Nikolaus

    2005-06-01

    microRNAs are small noncoding genes that regulate the protein production of genes by binding to partially complementary sites in the mRNAs of targeted genes. Here, using our algorithm PicTar, we exploit cross-species comparisons to predict, on average, 54 targeted genes per microRNA above noise in Drosophila melanogaster. Analysis of the functional annotation of target genes furthermore suggests specific biological functions for many microRNAs. We also predict combinatorial targets for clustered microRNAs and find that some clustered microRNAs are likely to coordinately regulate target genes. Furthermore, we compare microRNA regulation between insects and vertebrates. We find that the widespread extent of gene regulation by microRNAs is comparable between flies and mammals but that certain microRNAs may function in clade-specific modes of gene regulation. One of these microRNAs (miR-210) is predicted to contribute to the regulation of fly oogenesis. We also list specific regulatory relationships that appear to be conserved between flies and mammals. Our findings provide the most extensive microRNA target predictions in Drosophila to date, suggest specific functional roles for most microRNAs, indicate the existence of coordinate gene regulation executed by clustered microRNAs, and shed light on the evolution of microRNA function across large evolutionary distances. All predictions are freely accessible at our searchable Web site http://pictar.bio.nyu.edu.

  5. Activation of mammalian Chk1 during DNA replication arrest

    PubMed Central

    Feijoo, Carmen; Hall-Jackson, Clare; Wu, Rong; Jenkins, David; Leitch, Jane; Gilbert, David M.; Smythe, Carl

    2001-01-01

    Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block–induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited. PMID:11535615

  6. Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases.

    PubMed

    Sugden, Mary C; Holness, Mark J

    2006-07-01

    The mechanisms that control mammalian pyruvate dehydrogenase complex (PDC) activity include its phosphorylation (inactivation) by a family of pyruvate dehydrogenase kinases (PDKs 1 - 4). Here we review new developments in the regulation of the activities and expression of the PDKs, in particular PDK2 and PDK4, in relation to glucose and lipid homeostasis. This review describes recent advances relating to the acute and long-term modes of regulation of the PDKs, with particular emphasis on the regulatory roles of nuclear receptors including peroxisome proliferator-activated receptor (PPAR) alpha and Liver X receptor (LXR), PPAR gamma coactivator alpha (PGC-1alpha) and insulin, and the impact of changes in PDK activity and expression in glucose and lipid homeostasis. Since PDK4 may assist in lipid clearance when there is an imbalance between lipid delivery and oxidation, it may represent an attractive target for interventions aimed at rectifying abnormal lipid as well as glucose homeostasis in disease states. PMID:17132539

  7. Mammalian Dopa decarboxylase: structure, catalytic activity and inhibition.

    PubMed

    Bertoldi, Mariarita

    2014-03-15

    Mammalian Dopa decarboxylase catalyzes the conversion of L-Dopa and L-5-hydroxytryptophan to dopamine and serotonin, respectively. Both of them are biologically active neurotransmitters whose levels should be finely tuned. In fact, an altered concentration of dopamine is the cause of neurodegenerative diseases, such as Parkinson's disease. The chemistry of the enzyme is based on the features of its coenzyme pyridoxal 5'-phosphate (PLP). The cofactor is highly reactive and able to perform multiple reactions, besides decarboxylation, such as oxidative deamination, half-transamination and Pictet-Spengler cyclization. The structure resolution shows that the enzyme has a dimeric arrangement and provides a molecular basis to identify the residues involved in each catalytic activity. This information has been combined with kinetic studies under steady-state and pre-steady-state conditions as a function of pH to shed light on residues important for catalysis. A great effort in DDC research is devoted to design efficient and specific inhibitors in addition to those already used in therapy that are not highly specific and are responsible for the side effects exerted by clinical approach to either Parkinson's disease or aromatic amino acid decarboxylase deficiency. PMID:24407024

  8. Mammalian Rest/Activity Patterns Explained by Physiologically Based Modeling

    PubMed Central

    Phillips, A. J. K.; Fulcher, B. D.; Robinson, P. A.; Klerman, E. B.

    2013-01-01

    Circadian rhythms are fundamental to life. In mammals, these rhythms are generated by pacemaker neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN is remarkably consistent in structure and function between species, yet mammalian rest/activity patterns are extremely diverse, including diurnal, nocturnal, and crepuscular behaviors. Two mechanisms have been proposed to account for this diversity: (i) modulation of SCN output by downstream nuclei, and (ii) direct effects of light on activity. These two mechanisms are difficult to disentangle experimentally and their respective roles remain unknown. To address this, we developed a computational model to simulate the two mechanisms and their influence on temporal niche. In our model, SCN output is relayed via the subparaventricular zone (SPZ) to the dorsomedial hypothalamus (DMH), and thence to ventrolateral preoptic nuclei (VLPO) and lateral hypothalamus (LHA). Using this model, we generated rich phenotypes that closely resemble experimental data. Modulation of SCN output at the SPZ was found to generate a full spectrum of diurnal-to-nocturnal phenotypes. Intriguingly, we also uncovered a novel mechanism for crepuscular behavior: if DMH/VLPO and DMH/LHA projections act cooperatively, daily activity is unimodal, but if they act competitively, activity can become bimodal. In addition, we successfully reproduced diurnal/nocturnal switching in the rodent Octodon degu using coordinated inversions in both masking and circadian modulation. Finally, the model correctly predicted the SCN lesion phenotype in squirrel monkeys: loss of circadian rhythmicity and emergence of ∼4-h sleep/wake cycles. In capturing these diverse phenotypes, the model provides a powerful new framework for understanding rest/activity patterns and relating them to underlying physiology. Given the ubiquitous effects of temporal organization on all aspects of animal behavior and physiology, this study sheds light on the physiological

  9. Prometaphase APCcdh1 activity prevents non-disjunction in mammalian oocytes

    PubMed Central

    Reis, Alexandra; Madgwick, Suzanne; Chang, Heng-Yu; Nabti, Ibtissem; Levasseur, Mark; Jones, Keith T

    2008-01-01

    Summary The first female meiotic division (MI) is uniquely prone to chromosome segregation errors through non-disjunction, resulting in trisomies and early pregnancy loss1. Here, we show a fundamental difference in the control of mammalian meiosis which may underlie such susceptibility. It involved a reversal in the well-established timing of activation of the Anaphase-Promoting Complex (APC)2, 3 by its co-activators cdc20 and cdh1. APCcdh1 was active first, during prometaphase I, and was needed in order to allow homologue congression, since loss of cdh1 speeded up MI, leading to premature chromosome segregation and a non-disjunction phenotype. APCcdh1 targeted cdc20 for degradation but not securin and cyclin B1. These were degraded later in MI through APCcdc20, making cdc20 re-synthesis essential for successful meiotic progression. The switch from APCcdh1 to APCcdc20 activity was controlled by increasing CDK1 and cdh1 loss. These findings demonstrate a fundamentally different mechanism of control for the first meiotic division in mammalian oocytes not observed in meioses of other species. PMID:17891138

  10. Targeted recombination with single-stranded DNA vectors in mammalian cells.

    PubMed Central

    Fujioka, K; Aratani, Y; Kusano, K; Koyama, H

    1993-01-01

    We studied the ability of single-stranded DNA (ssDNA) to participate in targeted recombination in mammalian cells. A 5' end-deleted adenine phosphoribosyltransferase (aprt) gene was subcloned into M13 vector, and the resulting ssDNA and its double-stranded DNA (dsDNA) were transfected to APRT-Chinese hamster ovary cells with a deleted aprt gene. APRT+ recombinants with the ssDNA was obtained at a frequency of 3 x 10(-7) per survivor, which was almost equal to that with the double-stranded equivalent. Analysis of the genome in recombinant clones produced by ssDNA revealed that 12 of 14 clones resulted from correction of the deletion in the aprt locus. On the other hand, the locus of the remaining 2 was not corrected; instead, the 5' deletion of the vector was corrected by end extension, followed by integration into random sites of the genome. To exclude the possibility that input ssDNA was converted into its duplex form before participating in a recombination reaction, we compared the frequency of extrachromosomal recombination between noncomplementary ssDNAs, and between one ssDNA and one dsDNA, of two phage vectors. The frequency with the ssDNAs was 0.4 x 10(-5), being 10-fold lower than that observed with the ssDNA and the dsDNA, suggesting that as little as 10% of the transfected ssDNA was converted into duplex forms before the recombination event, hence 90% remained unchanged as single-stranded molecules. Nevertheless, the above finding that ssDNA was as efficient as dsDNA in targeted recombination suggests that ssDNA itself is able to participate directly in targeted recombination reactions in mammalian cells. Images PMID:8441653

  11. N-Acetylcysteine and mucociliary activity in mammalian airways.

    PubMed

    Iravani, J; Melville, G N; Horstmann, G

    1978-01-01

    The effect of N-acetylcysteine on mucus trasnport velocity (MV), ciliary beat frequency (CBF), mucus production (MP), mucus lysis and on the micro-morphology of the secretory cells was studied in mammalian airways. The results showed that: 1. MV increased in healthy rats and rabbits, as well as in bronchitic rats, after concentrations as low as 10(-14) g/ml. Depression of MV occurred first at 10(-6) and 10(-5) g/ml in healthy and bronchitic animals, respectively. 2. CBF was stimulated at concentrations between 10(-12) and 10(-10) g/ml and decreased at concentrations above 10(-8) g/ml. 3. MP increased by approximately 100% over control values. 4. Lysis of stagnant mucus was evident first at a concentration of 10(-11) g/ml after 15 min incubation. 5. TEM confirmed the increased activity of the mucus secreting cells and showed that no pathological changes occurred within the cell following incubation at 10(-7) g/ml for up to 150 min. The importance of these findings on the overall mucociliary function is discussed.

  12. Larger Mammalian Body Size Leads to Lower Retroviral Activity

    PubMed Central

    Katzourakis, Aris; Magiorkinis, Gkikas; Lim, Aaron G.; Gupta, Sunetra; Belshaw, Robert; Gifford, Robert

    2014-01-01

    Retroviruses have been infecting mammals for at least 100 million years, leaving descendants in host genomes known as endogenous retroviruses (ERVs). The abundance of ERVs is partly determined by their mode of replication, but it has also been suggested that host life history traits could enhance or suppress their activity. We show that larger bodied species have lower levels of ERV activity by reconstructing the rate of ERV integration across 38 mammalian species. Body size explains 37% of the variance in ERV integration rate over the last 10 million years, controlling for the effect of confounding due to other life history traits. Furthermore, 68% of the variance in the mean age of ERVs per genome can also be explained by body size. These results indicate that body size limits the number of recently replicating ERVs due to their detrimental effects on their host. To comprehend the possible mechanistic links between body size and ERV integration we built a mathematical model, which shows that ERV abundance is favored by lower body size and higher horizontal transmission rates. We argue that because retroviral integration is tumorigenic, the negative correlation between body size and ERV numbers results from the necessity to reduce the risk of cancer, under the assumption that this risk scales positively with body size. Our model also fits the empirical observation that the lifetime risk of cancer is relatively invariant among mammals regardless of their body size, known as Peto's paradox, and indicates that larger bodied mammals may have evolved mechanisms to limit ERV activity. PMID:25033295

  13. CX-5461 induces autophagy and inhibits tumor growth via mammalian target of rapamycin-related signaling pathways in osteosarcoma

    PubMed Central

    Li, Leiming; Li, Yan; Zhao, Jiansong; Fan, Shuli; Wang, Liguo; Li, Xu

    2016-01-01

    Osteosarcoma (OS) is the most common primary bone tumor, but molecular mechanisms of the disease have not been well understood, and treatment of metastatic OS remains a challenge. Rapid ribosomal RNA synthesis in cancer is transcribed by RNA polymerase I, which results in unbridled cell growth. The recent discovery of CX-5461, a selective RNA polymerase I inhibitor, exerted its inhibitory effect of ribosomal RNA synthesis and antiproliferative potency. Here, we demonstrate that CX-5461 induces G2 arrest in the cell cycle and expression of microtubule-associated protein 1 light chain 3 II isoform in OS cell lines. Autophagic vacuoles could be observed in electron microscopy and 3-methyladenine prevented cell death mediated by CX-5461. Moreover, it significantly augmented phosphorylated AMP-Activated Protein Kinases α (p-AMPK α). (Thr172) expression in U2-OS cells and decreased p-Akt (Ser473) expression in MNNG cells, respectively, which repressed their downstream effector, mammalian target of rapamycin. On the other hand, CX-5461 increased p53 accumulation and messenger RNA level of its target genes, p21, MDM2, and Sestrin1/2 in U2-OS cells. Knockdown of p53 expression markedly impaired cell death as well as the expression of light chain 3-II and p21 induced by CX-5461. It also significantly enhanced doxorubicin-mediated cytotoxic effect in vitro and in vivo together with additive expression of p53, p21, and light chain 3-II in U2-OS cells. Our data indicate that CX-5461 might induce autophagy via mammalian target of rapamycin-associated signaling pathways dependent on p53 status and exert p53-dependent synergistic antitumor effect combined with doxorubicin in OS. These results suggest that CX-5461 might be promising in clinical therapy for OS, especially cases harboring wild-type p53. PMID:27729807

  14. Discovery of GSK2126458, a Highly Potent Inhibitor of PI3K and the Mammalian Target of Rapamycin

    SciTech Connect

    Knight, Steven D.; Adams, Nicholas D.; Burgess, Joelle L.; Chaudhari, Amita M.; Darcy, Michael G.; Donatelli, Carla A.; Luengo, Juan I.; Newlander, Ken A.; Parrish, Cynthia A.; Ridgers, Lance H.; Sarpong, Martha A.; Schmidt, Stanley J.; Aller, Glenn S.Van; Carson, Jeffrey D.; Diamond, Melody A.; Elkins, Patricia A.; Gardiner, Christine M.; Garver, Eric; Gilbert, Seth A.; Gontarek, Richard R.; Jackson, Jeffrey R.; Kershner, Kevin L.; Luo, Lusong; Raha, Kaushik; Sherk, Christian S.; Sung, Chiu-Mei; Sutton, David; Tummino, Peter J.; Wegrzyn, Ronald J.; Auger, Kurt R.; Dhanak, Dashyant

    2010-09-30

    Phosphoinositide 3-kinase {alpha} (PI3K{alpha}) is a critical regulator of cell growth and transformation, and its signaling pathway is the most commonly mutated pathway in human cancers. The mammalian target of rapamycin (mTOR), a class IV PI3K protein kinase, is also a central regulator of cell growth, and mTOR inhibitors are believed to augment the antiproliferative efficacy of PI3K/AKT pathway inhibition. 2,4-Difluoro-N-{l_brace}2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl{r_brace}benzenesulfonamide (GSK2126458, 1) has been identified as a highly potent, orally bioavailable inhibitor of PI3K{alpha} and mTOR with in vivo activity in both pharmacodynamic and tumor growth efficacy models. Compound 1 is currently being evaluated in human clinical trials for the treatment of cancer.

  15. Coupling nutrient sensing to metabolic homoeostasis: the role of the mammalian target of rapamycin complex 1 pathway.

    PubMed

    André, Caroline; Cota, Daniela

    2012-11-01

    The mammalian target of rapamycin complex 1 (mTORC1) pathway is known to couple different environmental cues to the regulation of several energy-demanding functions within the cell, spanning from protein translation to mitochondrial activity. As a result, at the organism level, mTORC1 activity affects energy balance and general metabolic homoeostasis by modulating both the activity of neuronal populations that play key roles in the control of food intake and body weight, as well as by determining storage and use of fuel substrates in peripheral tissues. This review focuses on recent advances made in understanding the role of the mTORC1 pathway in the regulation of energy balance. More particularly, it aims at providing an overview of the status of knowledge regarding the mechanisms underlying the ability of certain amino acids, glucose and fatty acids, to affect mTORC1 activity and in turn illustrates how the mTORC1 pathway couples nutrient sensing to the hypothalamic regulation of the organisms' energy homoeostasis and to the control of intracellular metabolic processes, such as glucose uptake, protein and lipid biosynthesis. The evidence reviewed pinpoints the mTORC1 pathway as an integrator of the actions of nutrients on metabolic health and provides insight into the relevance of this intracellular pathway as a potential target for the therapy of metabolic diseases such as obesity and type-2 diabetes.

  16. Construction and development of a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma.

    PubMed

    Li, Feng; Liu, Yan-Hong; Li, Yan-Wen; Li, Yue-Hui; Xie, Ping-Li; Ju, Qiang; Chen, Lin; Li, Guan-Cheng

    2012-12-01

    We present a detailed method for constructing a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma. Two novel mammalian library vectors pcDNA3-CHm and pcDNA3-CLm were constructed that contained restriction enzyme sites NheI, ClaI and antibody constant domain. Mammalian expression vector pcDNA3-CHm contains IgG heavy-chain (HC) constant region and glycosylphosphatidylinositol anchor (GPI) that could be anchored full-length antibodies on the surface of mammalian cells. GOLPH2 prokaryotic expression vector was carried out in Escherichia coli and purified by immobilized metal affinity chromatography. Variable domain of heavy-chain and variable domain of light-chain genes were respectively inserted into the vector pcDNA3-CHm and pcDNA3-CLm by ligation, and antibody libraries are displayed as whole IgG molecules on the cell surface by co-transfecting this HC-GPI with a light chain. By screening the cell library using magnetic beads and cell ELISA, the cell clone that displayed GOLPH2-specific antibodies on cell surfaces was identified. The mammalian cell-based antibody display library is a great potential application for displaying full-length functional antibodies of targeting hepatocellular carcinoma on the surface of mammalian cells. Anti-GOLPH2 display antibody was successfully isolated from the library.

  17. Live imaging and modeling of inner nuclear membrane targeting reveals its molecular requirements in mammalian cells

    PubMed Central

    Boni, Andrea; Politi, Antonio Z.; Strnad, Petr; Xiang, Wanqing; Hossain, M. Julius

    2015-01-01

    Targeting of inner nuclear membrane (INM) proteins is essential for nuclear architecture and function, yet its mechanism remains poorly understood. Here, we established a new reporter that allows real-time imaging of membrane protein transport from the ER to the INM using Lamin B receptor and Lap2β as model INM proteins. These reporters allowed us to characterize the kinetics of INM targeting and establish a mathematical model of this process and enabled us to probe its molecular requirements in an RNA interference screen of 96 candidate genes. Modeling of the phenotypes of genes involved in transport of these INM proteins predicted that it critically depended on the number and permeability of nuclear pores and the availability of nuclear binding sites, but was unaffected by depletion of most transport receptors. These predictions were confirmed with targeted validation experiments on the functional requirements of nucleoporins and nuclear lamins. Collectively, our data support a diffusion retention model of INM protein transport in mammalian cells. PMID:26056140

  18. Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells.

    PubMed

    Trinkle-Mulcahy, L; Sleeman, J E; Lamond, A I

    2001-12-01

    Protein phosphatase 1 (PP1) is expressed in mammalian cells as three closely related isoforms, alpha, beta/delta and gamma1, which are encoded by separate genes. It has yet to be determined whether the separate isoforms behave in a similar fashion or play distinct roles in vivo. We report here on analyses by fluorescence microscopy of functional and fluorescently tagged PP1 isoforms in live cells. PP1alpha and PP1gamma fluorescent protein fusions show largely complimentary localization patterns, particularly within the nucleus where tagged PP1gamma accumulates in the nucleolus, whereas tagged PP1alpha is primarily found in the nucleoplasm. Overexpression of NIPP1 (nuclear inhibitor of PP1), a PP1 targeting subunit that accumulates at interchromatin granule clusters in the nucleoplasm, results in a retargeting of both isoforms to these structures, indicating that steady-state localization is based, at least in part, on relative affinities for various targeting subunits. Photobleaching analyses show that PP1gamma is rapidly exchanging between the nucleolar, nucleoplasmic and cytoplasmic compartments. Fluorescence resonance energy transfer (FRET) analyses indicate that the direct interaction of the two proteins predominantly occurs at or near interchromatin granule clusters. These data indicate that PP1 isoforms are highly mobile in cells and can be dynamically (re)localized through direct interaction with targeting subunits. PMID:11739654

  19. Rho-associated kinase connects a cell cycle-controlling anchorage signal to the mammalian target of rapamycin pathway.

    PubMed

    Park, Jung-ha; Arakawa-Takeuchi, Shiho; Jinno, Shigeki; Okayama, Hiroto

    2011-07-01

    When deprived of anchorage to the extracellular matrix, fibroblasts arrest in G(1) phase at least in part due to inactivation of G(1) cyclin-dependent kinases. Despite great effort, how anchorage signals control the G(1)-S transition of fibroblasts remains highly elusive. We recently found that the mammalian target of rapamycin (mTOR) cascade might convey an anchorage signal that regulates S phase entry. Here, we show that Rho-associated kinase connects this signal to the TSC1/TSC2-RHEB-mTOR pathway. Expression of a constitutively active form of ROCK1 suppressed all of the anchorage deprivation effects suppressible by tsc2 mutation in rat embryonic fibroblasts. TSC2 contains one evolutionarily conserved ROCK target-like sequence, and an alanine substitution for Thr(1203) in this sequence severely impaired the ability of ROCK1 to counteract the anchorage loss-imposed down-regulation of both G(1) cell cycle factors and mTORC1 activity. Moreover, TSC2 Thr(1203) underwent ROCK-dependent phosphorylation in vivo and could be phosphorylated by bacterially expressed active ROCK1 in vitro, providing biochemical evidence for a direct physical interaction between ROCK and TSC2.

  20. Mis-regulation of Mammalian Target of Rapamycin (mTOR) Complexes Induced by Albuminuria in Proximal Tubules*

    PubMed Central

    Peruchetti, Diogo B.; Cheng, Jie; Caruso-Neves, Celso; Guggino, William B.

    2014-01-01

    High albumin concentrations in the proximal tubule of the kidney causes tubulointerstitial injury, but how this process occurs is not completely known. To address the signal transduction pathways mis-regulated in renal injury, we studied the modulation of mammalian target of rapamycin (mTOR) complexes by physiologic and pathophysiologic albumin concentrations in proximal tubule cells. Physiologic albumin concentrations activated the PI3K/mTORC2/PKB/mTORC1/S6 kinase (S6K) pathway, but pathophysiologically high albumin concentrations overactivated mTORC1 and inhibited mTORC2 activity. This control process involved the activation of ERK1/2, which promoted the inhibition of TSC2 and activation of S6K. Furthermore, S6K was crucial to promoting the over activation of mTORC1 and inhibition of mTORC2. Megalin expression at the luminal membrane is reduced by high concentrations of albumin. In addition, knockdown of megalin mimicked all the effects of pathophysiologic albumin concentrations, which disrupt normal signal transduction pathways and lead to an overactivation of mTORC1 and inhibition of mTORC2. These data provide new perspectives for understanding the molecular mechanisms behind the effects of albumin on the progression of renal disease. PMID:24790108

  1. Dazl is a target RNA suppressed by mammalian NANOS2 in sexually differentiating male germ cells

    PubMed Central

    Kato, Yuzuru; Katsuki, Takeo; Kokubo, Hiroki; Masuda, Aki; Saga, Yumiko

    2016-01-01

    Evolutionally conserved Nanos RNA-binding proteins play crucial roles in germ cell development. While a mammalian Nanos family protein, NANOS2, is required for sexual differentiation of male (XY) germ cells in mice, the underlying mechanisms and the identities of its target RNAs in vivo remain elusive. Using comprehensive microarray analysis and a bacterial artificial chromosome transgenic system, here we identify Dazl, a germ cell-specific gene encoding an RNA-binding protein implicated in translation, as a crucial target of NANOS2. Importantly, removal of the Dazl 3′-untranslated region in XY germ cells stabilizes the Dazl mRNA, resulting in elevated meiotic gene expression, abnormal resumption of the cell cycle and impaired processing-body formation, reminiscent of Nanos2-knockout phenotypes. Furthermore, our data suggest that NANOS2 acts as an antagonist of the DAZL protein. We propose a dual system of NANOS2-mediated suppression of Dazl expression as a pivotal molecular mechanism promoting sexual differentiation of XY germ cells. PMID:27072294

  2. Dazl is a target RNA suppressed by mammalian NANOS2 in sexually differentiating male germ cells.

    PubMed

    Kato, Yuzuru; Katsuki, Takeo; Kokubo, Hiroki; Masuda, Aki; Saga, Yumiko

    2016-01-01

    Evolutionally conserved Nanos RNA-binding proteins play crucial roles in germ cell development. While a mammalian Nanos family protein, NANOS2, is required for sexual differentiation of male (XY) germ cells in mice, the underlying mechanisms and the identities of its target RNAs in vivo remain elusive. Using comprehensive microarray analysis and a bacterial artificial chromosome transgenic system, here we identify Dazl, a germ cell-specific gene encoding an RNA-binding protein implicated in translation, as a crucial target of NANOS2. Importantly, removal of the Dazl 3'-untranslated region in XY germ cells stabilizes the Dazl mRNA, resulting in elevated meiotic gene expression, abnormal resumption of the cell cycle and impaired processing-body formation, reminiscent of Nanos2-knockout phenotypes. Furthermore, our data suggest that NANOS2 acts as an antagonist of the DAZL protein. We propose a dual system of NANOS2-mediated suppression of Dazl expression as a pivotal molecular mechanism promoting sexual differentiation of XY germ cells. PMID:27072294

  3. Blocking mammalian target of rapamycin alleviates bone cancer pain and morphine tolerance via µ-opioid receptor.

    PubMed

    Jiang, Zongming; Wu, Shaoyong; Wu, Xiujuan; Zhong, Junfeng; Lv, Anqing; Jiao, Jing; Chen, Zhonghua

    2016-04-15

    The current study was to examine the underlying mechanisms responsible for the role of mammalian target of rapamycin (mTOR) in regulating bone cancer-evoked pain and the tolerance of systemic morphine. Breast sarcocarcinoma Walker 256 cells were implanted into the tibia bone cavity of rats and this evoked significant mechanical and thermal hyperalgesia. Our results showed that the protein expression of p-mTOR, mTOR-mediated phosphorylation of 4E-binding protein 4 (4E-BP1), p70 ribosomal S6 protein kinase 1 (S6K1) as well as phosphatidylinositide 3-kinase (p-PI3K) pathways were amplified in the superficial dorsal horn of the spinal cord of bone cancer rats compared with control rats. Blocking spinal mTOR by using rapamycin significantly attenuated activities of PI3K signaling pathways as well as mechanical and thermal hyperalgesia. Additionally, rapamycin enhanced attenuations of protein kinase Cɛ (PKCɛ)/protein kinase A (PKA) induced by morphine and further extended analgesia of morphine via µ-opioid receptor (MOR). Our data for the first time revealed specific signaling pathways leading to bone cancer pain, including the activation of mTOR and PI3K and downstream PKCɛ/PKA, and resultant sensitization of MOR. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of bone cancer pain often observed in clinics. PMID:26566757

  4. Engineering Escherichia coli for light-activated cytolysis of mammalian cells.

    PubMed

    Magaraci, Michael S; Veerakumar, Avin; Qiao, Peter; Amurthur, Ashwin; Lee, Justin Y; Miller, Jordan S; Goulian, Mark; Sarkar, Casim A

    2014-12-19

    By delivering payloads in response to specific exogenous stimuli, smart bacterial therapeutics have the potential to overcome many limitations of conventional therapies, including poor targeting specificity and dosage control in current cancer treatments. Although not yet explored as a trigger for bacterial drug delivery, light is an ideal induction mechanism because it offers fine spatiotemporal control and is easily and safely administered. Using recent advances in optogenetics, we have engineered two strains of Escherichia coli to secrete a potent mammalian cytotoxin in response to blue or red light. The tools in this study demonstrate the initial feasibility of light-activated bacterial therapeutics for applications such as tumor cytolysis, and their modular nature should enable simple substitution of other payloads of interest.

  5. ATP-Competitive Inhibitors of the Mammalian Target of Rapamycin: Design and Synthesis of Highly Potent and Selective Pyrazolopyrimidines

    SciTech Connect

    Zask, Arie; Verheijen, Jeroen C.; Curran, Kevin; Kaplan, Joshua; Richard, David J.; Nowak, Pawel; Malwitz, David J.; Brooijmans, Natasja; Bard, Joel; Svenson, Kristine; Lucas, Judy; Toral-Barza, Lourdes; Zhang, Wei-Guo; Hollander, Irwin; Gibbons, James J.; Abraham, Robert T.; Ayral-Kaloustian, Semiramis; Mansour, Tarek S.; Yu, Ker

    2009-09-18

    The mammalian target of rapamycin (mTOR), a central regulator of growth, survival, and metabolism, is a validated target for cancer therapy. Rapamycin and its analogues, allosteric inhibitors of mTOR, only partially inhibit one mTOR protein complex. ATP-competitive, global inhibitors of mTOR that have the potential for enhanced anticancer efficacy are described. Structural features leading to potency and selectivity were identified and refined leading to compounds with in vivo efficacy in tumor xenograft models.

  6. The role of phosphoinositide 3-kinase and phosphatidic acid in the regulation of mammalian target of rapamycin following eccentric contractions.

    PubMed

    O'Neil, T K; Duffy, L R; Frey, J W; Hornberger, T A

    2009-07-15

    Resistance exercise induces a hypertrophic response in skeletal muscle and recent studies have begun to shed light on the molecular mechanisms involved in this process. For example, several studies indicate that signalling by the mammalian target of rapamycin (mTOR) is necessary for a hypertrophic response. Furthermore, resistance exercise has been proposed to activate mTOR signalling through an upstream pathway involving the phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB); however, this hypothesis has not been thoroughly tested. To test this hypothesis, we first evaluated the temporal pattern of signalling through PI3K-PKB and mTOR following a bout of resistance exercise with eccentric contractions (EC). Our results indicated that the activation of signalling through PI3K-PKB is a transient event (<15 min), while the activation of mTOR is sustained for a long duration (>12 h). Furthermore, inhibition of PI3K-PKB activity did not prevent the activation of mTOR signalling by ECs, indicating that PI3K-PKB is not part of the upstream regulatory pathway. These observations led us to investigate an alternative pathway for the activation of mTOR signalling involving the synthesis of phosphatidic acid (PA) by phospholipase D (PLD). Our results demonstrate that ECs induce a sustained elevation in [PA] and inhibiting the synthesis of PA by PLD prevented the activation of mTOR. Furthermore, we determined that similar to ECs, PA activates mTOR signalling through a PI3K-PKB-independent mechanism. Combined, the results of this study indicate that the activation of mTOR following eccentric contractions occurs through a PI3K-PKB-independent mechanism that requires PLD and PA. PMID:19470781

  7. The role of phosphoinositide 3-kinase and phosphatidic acid in the regulation of mammalian target of rapamycin following eccentric contractions.

    PubMed

    O'Neil, T K; Duffy, L R; Frey, J W; Hornberger, T A

    2009-07-15

    Resistance exercise induces a hypertrophic response in skeletal muscle and recent studies have begun to shed light on the molecular mechanisms involved in this process. For example, several studies indicate that signalling by the mammalian target of rapamycin (mTOR) is necessary for a hypertrophic response. Furthermore, resistance exercise has been proposed to activate mTOR signalling through an upstream pathway involving the phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB); however, this hypothesis has not been thoroughly tested. To test this hypothesis, we first evaluated the temporal pattern of signalling through PI3K-PKB and mTOR following a bout of resistance exercise with eccentric contractions (EC). Our results indicated that the activation of signalling through PI3K-PKB is a transient event (<15 min), while the activation of mTOR is sustained for a long duration (>12 h). Furthermore, inhibition of PI3K-PKB activity did not prevent the activation of mTOR signalling by ECs, indicating that PI3K-PKB is not part of the upstream regulatory pathway. These observations led us to investigate an alternative pathway for the activation of mTOR signalling involving the synthesis of phosphatidic acid (PA) by phospholipase D (PLD). Our results demonstrate that ECs induce a sustained elevation in [PA] and inhibiting the synthesis of PA by PLD prevented the activation of mTOR. Furthermore, we determined that similar to ECs, PA activates mTOR signalling through a PI3K-PKB-independent mechanism. Combined, the results of this study indicate that the activation of mTOR following eccentric contractions occurs through a PI3K-PKB-independent mechanism that requires PLD and PA.

  8. Dysregulation of Mammalian Target of Rapamycin Signaling in Mouse Models of Autism

    PubMed Central

    Huber, Kimberly M.; Klann, Eric; Costa-Mattioli, Mauro

    2015-01-01

    The mammalian target of rapamycin (mTOR) is a central regulator of a diverse array of cellular processes, including cell growth, proliferation, autophagy, translation, and actin polymerization. Components of the mTOR cascade are present at synapses and influence synaptic plasticity and spine morphogenesis. A prevailing view is that the study of mTOR and its role in autism spectrum disorders (ASDs) will elucidate the molecular mechanisms by which mTOR regulates neuronal function under physiological and pathological conditions. Although many ASDs arise as a result of mutations in genes with multiple molecular functions, they appear to converge on common biological pathways that give rise to autism-relevant behaviors. Dysregulation of mTOR signaling has been identified as a phenotypic feature common to fragile X syndrome, tuberous sclerosis complex 1 and 2, neurofibromatosis 1, phosphatase and tensin homolog, and potentially Rett syndrome. Below are a summary of topics covered in a symposium that presents dysregulation of mTOR as a unifying theme in a subset of ASDs. PMID:26468183

  9. Advances in the therapeutic use of mammalian target of rapamycin (mTOR) inhibitors in dermatology.

    PubMed

    Fogel, Alexander L; Hill, Sharleen; Teng, Joyce M C

    2015-05-01

    Significant developments in the use of mammalian target of rapamycin (mTOR) inhibitors (mTORIs) as immunosuppressant and antiproliferative agents have been made. Recent advances in the understanding of the mTOR signaling pathway and its downstream effects on tumorigenesis and vascular proliferation have broadened the clinical applications of mTORIs in many challenging disorders such as tuberous sclerosis complex, pachyonychia congenita, complex vascular anomalies, and inflammatory dermatoses. Systemic mTORI therapy has shown benefits in these areas, but is associated with significant side effects that sometimes necessitate drug holidays. To mitigate the side effects of systemic mTORIs for dermatologic applications, preliminary work to assess the potential of percutaneous therapy has been performed, and the evidence suggests that percutaneous delivery of mTORIs may allow for effective long-term therapy while avoiding systemic toxicities. Additional large placebo-controlled, double-blinded, randomized studies are needed to assess the efficacy, safety, duration, and tolerability of topical treatments. The objective of this review is to provide updated information on the novel use of mTORIs in the management of many cutaneous disorders. PMID:25769191

  10. The Rapamycin-Sensitive Complex of Mammalian Target of Rapamycin Is Essential to Maintain Male Fertility.

    PubMed

    Schell, Christoph; Kretz, Oliver; Liang, Wei; Kiefer, Betina; Schneider, Simon; Sellung, Dominik; Bork, Tillmann; Leiber, Christian; Rüegg, Markus A; Mallidis, Con; Schlatt, Stefan; Mayerhofer, Artur; Huber, Tobias B; Grahammer, Florian

    2016-02-01

    The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin and its analogs are being increasingly used in solid-organ transplantation. A commonly reported side effect is male subfertility to infertility, yet the precise mechanisms of mTOR interference with male fertility remain obscure. With the use of a conditional mouse genetic approach we demonstrate that deficiency of mTORC1 in the epithelial derivatives of the Wolffian duct is sufficient to cause male infertility. Analysis of spermatozoa from Raptor fl/fl*KspCre mice revealed an overall decreased motility pattern. Both epididymis and seminal vesicles displayed extensive organ regression with increasing age. Histologic and ultrastructural analyses demonstrated increased amounts of destroyed and absorbed spermatozoa in different segments of the epididymis. Mechanistically, genetic and pharmacologic mTORC1 inhibition was associated with an impaired cellular metabolism and a disturbed protein secretion of epididymal epithelial cells. Collectively, our data highlight the role of mTORC1 to preserve the function of the epididymis, ductus deferens, and the seminal vesicles. We thus reveal unexpected new insights into the frequently observed mTORC1 inhibitor side effect of male infertility in transplant recipients. PMID:26683665

  11. Dysregulation of Mammalian Target of Rapamycin Signaling in Mouse Models of Autism.

    PubMed

    Huber, Kimberly M; Klann, Eric; Costa-Mattioli, Mauro; Zukin, R Suzanne

    2015-10-14

    The mammalian target of rapamycin (mTOR) is a central regulator of a diverse array of cellular processes, including cell growth, proliferation, autophagy, translation, and actin polymerization. Components of the mTOR cascade are present at synapses and influence synaptic plasticity and spine morphogenesis. A prevailing view is that the study of mTOR and its role in autism spectrum disorders (ASDs) will elucidate the molecular mechanisms by which mTOR regulates neuronal function under physiological and pathological conditions. Although many ASDs arise as a result of mutations in genes with multiple molecular functions, they appear to converge on common biological pathways that give rise to autism-relevant behaviors. Dysregulation of mTOR signaling has been identified as a phenotypic feature common to fragile X syndrome, tuberous sclerosis complex 1 and 2, neurofibromatosis 1, phosphatase and tensin homolog, and potentially Rett syndrome. Below are a summary of topics covered in a symposium that presents dysregulation of mTOR as a unifying theme in a subset of ASDs.

  12. Taking aim at Alzheimer’s disease through the mammalian target of rapamycin

    PubMed Central

    Maiese, Kenneth

    2014-01-01

    A significant portion of the world’s population suffers from sporadic Alzheimer’s disease (AD) with available present therapies limited to symptomatic care that does not alter disease progression. Over the next decade, advancing age of the global population will dramatically increase the incidence of AD and severely impact health care resources, necessitating novel, safe, and efficacious strategies for AD. The mammalian target of rapamycin (mTOR) and its protein complexes mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2) offer exciting and unique avenues of intervention for AD through the oversight of programmed cell death pathways of apoptosis, autophagy, and necroptosis. mTOR modulates multi-faceted signal transduction pathways that involve phosphoinositide 3-kinase (PI 3-K), protein kinase B (Akt), hamartin (tuberous sclerosis 1)/tuberin (tuberous sclerosis 2) (TSC1/TSC2) complex, proline-rich Akt substrate 40 kDa (PRAS40), and p70 ribosomal S6 kinase (p70S6K) and can interface with the neuroprotective pathways of growth factors, sirtuins, wingless, fork-head transcription factors, and glycogen synthase kinase-3β. With the ability of mTOR to broadly impact cellular function, clinical strategies for AD that implement mTOR must achieve parallel objectives of protecting neuronal, vascular, and immune cell survival in conjunction with preserving networks that determine memory and cognitive function. PMID:25105207

  13. A central role for the mammalian target of rapamycin in LPS-induced anorexia in mice.

    PubMed

    Yue, Yunshuang; Wang, Yi; Li, Dan; Song, Zhigang; Jiao, Hongchao; Lin, Hai

    2015-01-01

    Bacterial lipopolysaccharide (LPS), also known as endotoxin, induces profound anorexia. However, the LPS-provoked pro-inflammatory signaling cascades and the neural mechanisms underlying the development of anorexia are not clear. Mammalian target of rapamycin (mTOR) is a key regulator of metabolism, cell growth, and protein synthesis. This study aimed to determine whether the mTOR pathway is involved in LPS-induced anorexia. Effects of LPS on hypothalamic gene/protein expression in mice were measured by RT-PCR or western blotting analysis. To determine whether inhibition of mTOR signaling could attenuate LPS-induced anorexia, we administered an i.c.v. injection of rapamycin, an mTOR inhibitor, on LPS-treated male mice. In this study, we showed that LPS stimulates the mTOR signaling pathway through the enhanced phosphorylation of mTOR(Ser2448) and p70S6K(Thr389). We also showed that LPS administration increased the phosphorylation of FOXO1(Ser256), the p65 subunit of nuclear factor kappa B (P<0.05), and FOXO1/3a(Thr) (24) (/) (32) (P<0.01). Blocking the mTOR pathway significantly attenuated the LPS-induced anorexia by decreasing the phosphorylation of p70S6K(Thr389), FOXO1(Ser256), and FOXO1/3a(Thr) (24) (/) (32). These results suggest promising approaches for the prevention and treatment of LPS-induced anorexia.

  14. Targeting the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway: an emerging treatment strategy for squamous cell lung carcinoma.

    PubMed

    Beck, Joseph Thaddeus; Ismail, Amen; Tolomeo, Christina

    2014-09-01

    Squamous cell lung carcinoma accounts for approximately 30% of all non-small cell lung cancers (NSCLCs). Despite progress in the understanding of the biology of cancer, cytotoxic chemotherapy remains the standard of care for patients with squamous cell lung carcinoma, but the prognosis is generally poor. The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is one of the most commonly activated signaling pathways in cancer, leading to cell proliferation, survival, and differentiation. It has therefore become a major focus of clinical research. Various alterations in the PI3K/AKT/mTOR pathway have been identified in squamous cell lung carcinoma and a number of agents targeting these alterations are in clinical development for use as single agents and in combination with other targeted and conventional treatments. These include pan-PI3K inhibitors, isoform-specific PI3K inhibitors, AKT inhibitors, mTOR inhibitors, and dual PI3K/mTOR inhibitors. These agents have demonstrated antitumor activity in preclinical models of NSCLC and preliminary clinical evidence is also available for some agents. This review will discuss the role of the PI3K/AKT/mTOR pathway in cancer and how the discovery of genetic alterations in this pathway in patients with squamous cell lung carcinoma can inform the development of targeted therapies for this disease. An overview of ongoing clinical trials investigating PI3K/AKT/mTOR pathway inhibitors in squamous cell lung carcinoma will also be included.

  15. Inhibition of mammalian target of rapamycin protects against reperfusion injury in diabetic heart through STAT3 signaling.

    PubMed

    Das, Anindita; Salloum, Fadi N; Filippone, Scott M; Durrant, David E; Rokosh, Gregg; Bolli, Roberto; Kukreja, Rakesh C

    2015-05-01

    Diabetic patients suffer augmented severity of myocardial infarction. Excessive activation of the mammalian target of rapamycin (mTOR) and decreased activation of STAT3 are implicated in diabetic complications. Considering the potent cardioprotective effect of mTOR inhibitor, rapamycin, we hypothesized that reperfusion therapy with rapamycin would reduce infarct size in the diabetic hearts through STAT3 signaling. Hearts from adult male db/db or wild type (WT) C57 mice were isolated and subjected to 30 min of normothermic global ischemia and 60 min of reperfusion in Langendorff mode. Rapamycin (100 nM) was infused at the onset of reperfusion. Myocardial infarct size (IS) was significantly reduced in rapamycin-treated mice (13.3 ± 2.4 %) compared to DMSO vehicle control (35.9 ± 0.9 %) or WT mice (27.7 ± 1.1 %). Rapamycin treatment restored phosphorylation of STAT3 and enhanced AKT phosphorylation (target of mTORC2), but significantly reduced ribosomal protein S6 phosphorylation (target of mTORC1) in the diabetic heart. To determine the cause and effect relationship of STAT3 in cardioprotection, inducible cardiac-specific STAT3-deficient (MCM TG:STAT3(flox/flox)) and WT mice (MCM TG:STAT3(flox/flox)) were made diabetic by feeding high fat diet (HFD). Rapamycin given at reperfusion reduced IS in WT mice but not in STAT3-deficient mice following I/R. Moreover, cardiomyocytes isolated from HFD-fed WT mice showed resistance against necrosis (trypan blue staining) and apoptosis (TUNEL assay) when treated with rapamycin during reoxygenation following simulated ischemia. Such protection was absent in cardiomyocytes from HFD-fed STAT3-deficient mice. STAT3 signaling plays critical role in reducing IS and attenuates cardiomyocyte death following reperfusion therapy with rapamycin in diabetic heart.

  16. Myeloproliferative defects following targeting of the Drf1 gene encoding the mammalian diaphanous related formin mDia1.

    PubMed

    Peng, Jun; Kitchen, Susan M; West, Richard A; Sigler, Robert; Eisenmann, Kathryn M; Alberts, Arthur S

    2007-08-15

    Rho GTPase-effector mammalian diaphanous (mDia)-related formins assemble nonbranched actin filaments as part of cellular processes, including cell division, filopodia assembly, and intracellular trafficking. Whereas recent efforts have led to thorough characterization of formins in cytoskeletal remodeling and actin assembly in vitro, little is known about the role of mDia proteins in vivo. To fill this knowledge gap, the Drf1 gene, which encodes the canonical formin mDia1, was targeted by homologous recombination. Upon birth, Drf1+/- and Drf1-/- mice were developmentally and morphologically indistinguishable from their wild-type littermates. However, both Drf1+/- and Drf1-/- developed age-dependent myeloproliferative defects. The phenotype included splenomegaly, fibrotic and hypercellular bone marrow, extramedullary hematopoiesis in both spleen and liver, and the presence of immature myeloid progenitor cells with high nucleus-to-cytoplasm ratios. Analysis of cell surface markers showed an age-dependent increase in the percentage of CD11b+-activated and CD14+-activated monocytes/macrophages in both spleen and bone marrow in Drf1+/- and Drf1-/- animals. Analysis of the erythroid compartment showed a significant increase in the proportion of splenic cells in S phase and an expansion of erythroid precursors (TER-119+ and CD71+) in Drf1-targeted mice. Overall, knocking out mDia1 expression in mice leads to a phenotype similar to human myeloproliferative syndrome (MPS) and myelodysplastic syndromes (MDS). These observations suggest that defective DRF1 expression or mDia1 function may contribute to myeloid malignancies and point to mDia1 as an attractive therapeutic target in MDS and MPS.

  17. Role of the mammalian target of rapamycin pathway and rapamycin in lentiviral vector gene transduction of hematopoietic stem cells

    PubMed Central

    Wang, Cathy X.; Torbett, Bruce E.

    2015-01-01

    Purpose of review A major goal in repopulating hematopoietic stem cell (HSC) gene therapies is achieving high-efficacy gene transfer, while maintaining robust HSC engraftment and differentiation in vivo. Recent studies have documented that rapamycin treatment of HSC during lentiviral vector transduction enhances gene transfer to human and mouse HSCs and maintains engraftment capacity. In this review, we place into context the role of mammalian target of rapamycin (mTOR) pathways in HSC quiescence and function, endocytic regulation, and lentiviral gene delivery. Recent findings Lentiviral vector transduction of human and mouse HSCs is considerably enhanced by rapamycin treatment. Furthermore, rapamycin preserves long-term engraftment of human and mouse HSCs. Investigations of cellular mechanisms that contribute to increased transduction in HSCs uncovered a role for mTOR inhibition-dependent activation of endocytosis. Summary Rapamycin enhances lentiviral vector transduction of HSCs through regulation of endocytic activity via mTOR inhibition. An important attribute of rapamycin treatment during transduction is the preservation of HSC function, allowing reconstitution of long-term hematopoiesis in vivo in murine models. PMID:26049750

  18. Mammalian target of rapamycin hyperactivity mediates the detrimental effects of a high sucrose diet on Alzheimer's disease pathology.

    PubMed

    Orr, Miranda E; Salinas, Angelica; Buffenstein, Rochelle; Oddo, Salvatore

    2014-06-01

    High sugar consumption and diabetes increase the risk of developing Alzheimer's disease (AD) by unknown mechanisms. Using an animal model of AD, here we show that high sucrose intake induces obesity with changes in central and peripheral insulin signaling. These pre-diabetic changes are associated with an increase in amyloid-β production and deposition. Moreover, high sucrose ingestion exacerbates tau phosphorylation by increasing Cdk5 activity. Mechanistically, the sucrose-mediated increase in AD-like pathology results from hyperactive mammalian target of rapamycin (mTOR), a key nutrient sensor important in regulating energy homeostasis. Specifically, we show that rapamycin, an mTOR inhibitor, prevents the detrimental effects of sucrose in the brain without altering changes in peripheral insulin resistance. Overall, our data suggest that high sucrose intake and dysregulated insulin signaling, which are known to contribute to the occurrence of diabetes, increase the risk of developing AD by upregulating brain mTOR signaling. Therefore, early interventions to modulate mTOR activity in individuals at high risk of developing diabetes may decrease their AD susceptibility. PMID:24411482

  19. Characterization of the RNA Silencing Suppression Activity of the Ebola Virus VP35 Protein in Plants and Mammalian Cells

    PubMed Central

    Zhu, Yali; Cherukuri, Nil Celebi; Jackel, Jamie N.; Wu, Zetang; Crary, Monica; Buckley, Kenneth J.; Bisaro, David M.

    2012-01-01

    Ebola virus (EBOV) causes a lethal hemorrhagic fever for which there is no approved effective treatment or prevention strategy. EBOV VP35 is a virulence factor that blocks innate antiviral host responses, including the induction of and response to alpha/beta interferon. VP35 is also an RNA silencing suppressor (RSS). By inhibiting microRNA-directed silencing, mammalian virus RSSs have the capacity to alter the cellular environment to benefit replication. A reporter gene containing specific microRNA target sequences was used to demonstrate that prior expression of wild-type VP35 was able to block establishment of microRNA silencing in mammalian cells. In addition, wild-type VP35 C-terminal domain (CTD) protein fusions were shown to bind small interfering RNA (siRNA). Analysis of mutant proteins demonstrated that reporter activity in RSS assays did not correlate with their ability to antagonize double-stranded RNA (dsRNA)-activated protein kinase R (PKR) or bind siRNA. The results suggest that enhanced reporter activity in the presence of VP35 is a composite of nonspecific translational enhancement and silencing suppression. Moreover, most of the specific RSS activity in mammalian cells is RNA binding independent, consistent with VP35's proposed role in sequestering one or more silencing complex proteins. To examine RSS activity in a system without interferon, VP35 was tested in well-characterized plant silencing suppression assays. VP35 was shown to possess potent plant RSS activity, and the activities of mutant proteins correlated strongly, but not exclusively, with RNA binding ability. The results suggest the importance of VP35-protein interactions in blocking silencing in a system (mammalian) that cannot amplify dsRNA. PMID:22238300

  20. Mammalian target of rapamycin is required for phrenic long-term facilitation following severe but not moderate acute intermittent hypoxia

    PubMed Central

    Dougherty, Brendan J.; Fields, Daryl P.

    2015-01-01

    Phrenic long-term facilitation (pLTF) is a persistent increase in phrenic nerve activity after acute intermittent hypoxia (AIH). Distinct cell-signaling cascades give rise to pLTF depending on the severity of hypoxemia within hypoxic episodes. Moderate AIH (mAIH; three 5-min episodes, PaO2 ∼35–55 mmHG) elicits pLTF by a serotonin (5-HT)-dependent mechanism that requires new synthesis of brain-derived neurotrophic factor (BDNF), activation of its high-affinity receptor (TrkB), and ERK MAPK signaling. In contrast, severe AIH (sAIH; three 5-min episodes, PaO2 ∼25–30 mmHG) elicits pLTF by an adenosine-dependent mechanism that requires new TrkB synthesis and Akt signaling. Although both mechanisms require spinal protein synthesis, the newly synthesized proteins are distinct, as are the neurochemicals inducing plasticity (serotonin vs. adenosine). In many forms of neuroplasticity, new protein synthesis requires translational regulation via mammalian target of rapamycin (mTOR) signaling. Since Akt regulates mTOR activity, we hypothesized that mTOR activity is necessary for sAIH- but not mAIH-induced pLTF. Phrenic nerve activity in anesthetized, paralyzed, and ventilated rats was recorded before, during, and 60 min after mAIH or sAIH. Rats were pretreated with intrathecal injections of 20% DMSO (vehicle controls) or rapamycin (0.1 mM, 12 μl), a selective mTOR complex 1 inhibitor. Consistent with our hypothesis, rapamycin blocked sAIH- but not mAIH-induced pLTF. Thus spinal mTOR activity is required for adenosine-dependent (sAIH) but not serotonin-dependent (mAIH) pLTF, suggesting that distinct mechanisms regulate new protein synthesis in these forms of spinal neuroplasticity. PMID:26224775

  1. Contribution of mammalian target of rapamycin in the pathophysiology of cirrhotic cardiomyopathy

    PubMed Central

    Saeedi Saravi, Seyed Soheil; Ghazi-Khansari, Mahmoud; Ejtemaei Mehr, Shahram; Nobakht, Maliheh; Mousavi, Seyyedeh Elaheh; Dehpour, Ahmad Reza

    2016-01-01

    AIM: To explore the role of mammalian target of rapamycin (mTOR) in the pathogenesis of cirrhotic cardiomyopathy and the potential of rapamycin to improve this pathologic condition. METHODS: Male albino Wistar rats weighing 100-120 g were treated with tetrachloride carbon (CCl4) for 8 wk to induce cirrhosis. Subsequently, animals were administered rapamycin (2 mg/kg per day). The QTc intervals were calculated in a 5-min electrocardiogram. Then, the left ventricular papillary muscles were isolated to examine inotropic responsiveness to β-adrenergic stimulation using a standard organ bath equipped by Powerlab system. Phosphorylated-mTOR localization in left ventricles was immunohistochemically assessed, and ventricular tumor necrosis factor (TNF)-α was measured. Western blot was used to measure levels of ventricular phosphorylated-mTOR protein. RESULTS: Cirrhosis was confirmed by hematoxylin and eosin staining of liver tissues, visual observation of lethargy, weight loss, jaundice, brown urine, ascites, liver stiffness, and a significant increase of spleen weight (P < 0.001). A significant prolongation in QTc intervals occurred in cirrhotic rats exposed to CCl4 (P < 0.001), while this prolongation was decreased with rapamycin treatment (P < 0.01). CCl4-induced cirrhosis caused a significant decrease of contractile responsiveness to isoproterenol stimulation and a significant increase in cardiac TNF-α. These findings were correlated with data from western blot and immunohistochemical studies on phosphorylated-mTOR expression in left ventricles. Phosphorylated-mTOR was significantly enhanced in cirrhotic rats, especially in the endothelium, compared to controls. Rapamycin treatment significantly increased contractile force and myocardial localization of phosphorylated-mTOR and decreased cardiac TNF-α concentration compared to cirrhotic rats with no treatment. CONCLUSION: In this study, we demonstrated a potential role for cardiac mTOR in the pathophysiology of

  2. Olive Oil-derived Oleocanthal as Potent Inhibitor of Mammalian Target of Rapamycin: Biological Evaluation and Molecular Modeling Studies

    PubMed Central

    Khanfar, Mohammad A.; Bardaweel, Sanaa K.; Akl, Mohamed R.; El Sayed, Khalid A.

    2016-01-01

    The established anticancer and neuroprotective properties of oleocanthal combined with the reported role of mammalian target of rapamycin (mTOR) in cancer and Alzheimer’s disease development encouraged us to examine the possibility that oleocanthal inhibits mTOR. To validate this hypothesis, we docked oleocanthal into the adenosine triphosphate binding pocket of a close mTOR protein homologue, namely, PI3K-γ. Apparently, oleocanthal shared nine out of ten critical binding interactions with a potent dual PIK3-γ/mTOR natural inhibitor. Subsequent experimental validation indicated that oleocanthal indeed inhibited the enzymatic activity of mTOR with an IC50 value of 708 nM. Oleocanthal inhibits the growth of several breast cancer cell lines at low micromolar concentration in a dose-dependent manner. Oleocanthal treatment caused a marked downregulation of phosphorylated mTOR in metastatic breast cancer cell line (MDA-MB-231). These results strongly indicate that mTOR inhibition is at least one of the factors of the reported anticancer and neuroprotective properties of oleocanthal. PMID:26248874

  3. Olive Oil-derived Oleocanthal as Potent Inhibitor of Mammalian Target of Rapamycin: Biological Evaluation and Molecular Modeling Studies.

    PubMed

    Khanfar, Mohammad A; Bardaweel, Sanaa K; Akl, Mohamed R; El Sayed, Khalid A

    2015-11-01

    The established anticancer and neuroprotective properties of oleocanthal combined with the reported role of mammalian target of rapamycin (mTOR) in cancer and Alzheimer's disease development encouraged us to examine the possibility that oleocanthal inhibits mTOR. To validate this hypothesis, we docked oleocanthal into the adenosine triphosphate binding pocket of a close mTOR protein homologue, namely, PI3K-γ. Apparently, oleocanthal shared nine out of ten critical binding interactions with a potent dual PIK3-γ/mTOR natural inhibitor. Subsequent experimental validation indicated that oleocanthal indeed inhibited the enzymatic activity of mTOR with an IC50 value of 708 nM. Oleocanthal inhibits the growth of several breast cancer cell lines at low micromolar concentration in a dose-dependent manner. Oleocanthal treatment caused a marked downregulation of phosphorylated mTOR in metastatic breast cancer cell line (MDA-MB-231). These results strongly indicate that mTOR inhibition is at least one of the factors of the reported anticancer and neuroprotective properties of oleocanthal. PMID:26248874

  4. Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA

    PubMed Central

    Zhang, Lin; Hou, Dongxia; Chen, Xi; Li, Donghai; Zhu, Lingyun; Zhang, Yujing; Li, Jing; Bian, Zhen; Liang, Xiangying; Cai, Xing; Yin, Yuan; Wang, Cheng; Zhang, Tianfu; Zhu, Dihan; Zhang, Dianmu; Xu, Jie; Chen, Qun; Ba, Yi; Liu, Jing; Wang, Qiang; Chen, Jianqun; Wang, Jin; Wang, Meng; Zhang, Qipeng; Zhang, Junfeng; Zen, Ke; Zhang, Chen-Yu

    2012-01-01

    Our previous studies have demonstrated that stable microRNAs (miRNAs) in mammalian serum and plasma are actively secreted from tissues and cells and can serve as a novel class of biomarkers for diseases, and act as signaling molecules in intercellular communication. Here, we report the surprising finding that exogenous plant miRNAs are present in the sera and tissues of various animals and that these exogenous plant miRNAs are primarily acquired orally, through food intake. MIR168a is abundant in rice and is one of the most highly enriched exogenous plant miRNAs in the sera of Chinese subjects. Functional studies in vitro and in vivo demonstrated that MIR168a could bind to the human/mouse low-density lipoprotein receptor adapter protein 1 (LDLRAP1) mRNA, inhibit LDLRAP1 expression in liver, and consequently decrease LDL removal from mouse plasma. These findings demonstrate that exogenous plant miRNAs in food can regulate the expression of target genes in mammals. PMID:21931358

  5. Induction of Direct Antimicrobial Activity Through Mammalian Toll-Like Receptors

    NASA Astrophysics Data System (ADS)

    Thoma-Uszynski, Sybille; Stenger, Steffen; Takeuchi, Osamu; Ochoa, Maria Teresa; Engele, Matthias; Sieling, Peter A.; Barnes, Peter F.; Röllinghoff, Martin; Bölcskei, Pal L.; Wagner, Manfred; Akira, Shizuo; Norgard, Michael V.; Belisle, John T.; Godowski, Paul J.; Bloom, Barry R.; Modlin, Robert L.

    2001-02-01

    The mammalian innate immune system retains from Drosophila a family of homologous Toll-like receptors (TLRs) that mediate responses to microbial ligands. Here, we show that TLR2 activation leads to killing of intracellular Mycobacterium tuberculosis in both mouse and human macrophages, through distinct mechanisms. In mouse macrophages, bacterial lipoprotein activation of TLR2 leads to a nitric oxide-dependent killing of intracellular tubercle bacilli, but in human monocytes and alveolar macrophages, this pathway was nitric oxide-independent. Thus, mammalian TLRs respond (as Drosophila Toll receptors do) to microbial ligands and also have the ability to activate antimicrobial effector pathways at the site of infection.

  6. Stress-responsive sestrins link p53 with redox regulation and mammalian target of rapamycin signaling.

    PubMed

    Budanov, Andrei V

    2011-09-15

    The tumor suppressor p53 protects organisms from most types of cancer through multiple mechanisms. The p53 gene encodes a stress-activated transcriptional factor that transcriptionally regulates a large set of genes with versatile functions. These p53-activated genes mitigate consequences of stress regulating cell viability, growth, proliferation, repair, and metabolism. Recently, we described a novel antioxidant function of p53, which is important for its tumor suppressor activity. Among the many antioxidant genes activated by p53, Sestrins (Sesns) are critical for suppression of reactive oxygen species (ROS) and protection from oxidative stress, transformation, and genomic instability. Sestrins can regulate ROS through their direct effect on antioxidant peroxiredoxin proteins and through the AMP-activated protein kinase-target of rapamycin signaling pathway. The AMP-activated protein kinase-target of rapamycin axis is critical for regulation of metabolism and autophagy, two processes associated with ROS production, and deregulation of this pathway increases vulnerability of the organism to stress, aging, and age-related diseases, including cancer. Recently, we have shown that inactivation of Sestrin in fly causes accumulation of age-associated damage. Hence, Sestrins can link p53 with aging and age-related diseases. PMID:20712410

  7. The role of amino acid-induced mammalian target of rapamycin complex 1(mTORC1) signaling in insulin resistance

    PubMed Central

    Yoon, Mee-Sup; Choi, Cheol Soo

    2016-01-01

    Mammalian target of rapamycin (mTOR) controls cell growth and metabolism in response to nutrients, energy, and growth factors. Recent findings have placed the lysosome at the core of mTOR complex 1 (mTORC1) regulation by amino acids. Two parallel pathways, Rag GTPase-Ragulator and Vps34-phospholipase D1 (PLD1), regulate mTOR activation on the lysosome. This review describes the recent advances in understanding amino acid-induced mTOR signaling with a particular focus on the role of mTOR in insulin resistance. PMID:27534530

  8. TALE activators regulate gene expression in a position- and strand-dependent manner in mammalian cells.

    PubMed

    Uhde-Stone, Claudia; Cheung, Edna; Lu, Biao

    2014-01-24

    Transcription activator-like effectors (TALEs) are a class of transcription factors that are readily programmable to regulate gene expression. Despite their growing popularity, little is known about binding site parameters that influence TALE-mediated gene activation in mammalian cells. We demonstrate that TALE activators modulate gene expression in mammalian cells in a position- and strand-dependent manner. To study the effects of binding site location, we engineered TALEs customized to recognize specific DNA sequences located in either the promoter or the transcribed region of reporter genes. We found that TALE activators robustly activated reporter genes when their binding sites were located within the promoter region. In contrast, TALE activators inhibited the expression of reporter genes when their binding sites were located on the sense strand of the transcribed region. Notably, this repression was independent of the effector domain utilized, suggesting a simple blockage mechanism. We conclude that TALE activators in mammalian cells regulate genes in a position- and strand-dependent manner that is substantially different from gene activation by native TALEs in plants. These findings have implications for optimizing the design of custom TALEs for genetic manipulation in mammalian cells.

  9. Leptin induces macrophage lipid body formation by a phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent mechanism.

    PubMed

    Maya-Monteiro, Clarissa M; Almeida, Patricia E; D'Avila, Heloisa; Martins, Aline S; Rezende, Ana Paula; Castro-Faria-Neto, Hugo; Bozza, Patricia T

    2008-01-25

    Leptin is an adipocyte-derived hormone/cytokine that links nutritional status with neuroendocrine and immune functions. Lipid bodies (lipid droplets) are emerging as dynamic organelles with roles in lipid metabolism and inflammation. Here we investigated the roles of leptin in signaling pathways involved in cytoplasmic lipid body biogenesis and leukotriene B(4) synthesis in macrophages. Our results demonstrated that leptin directly activated macrophages and induced the formation of adipose differentiation-related protein-enriched lipid bodies. Newly formed lipid bodies were sites of 5-lipoxygenase localization and correlated with an enhanced capacity of leukotriene B(4) production. We demonstrated that leptin-induced macrophage activation was dependent on phosphatidylinositol 3-kinase (PI3K) activity, since the lipid body formation was inhibited by LY294002 and was absent in the PI3K knock-out mice. Leptin induces phosphorylation of p70(S6K) and 4EBP1 key downstream signaling intermediates of the mammalian target of rapamycin (mTOR) pathway in a rapamycin-sensitive mechanism. The mTOR inhibitor, rapamycin, inhibited leptin-induced lipid body formation, both in vivo and in vitro. In addition, rapamycin inhibited leptin-induced adipose differentiation-related protein accumulation in macrophages and lipid body-dependent leukotriene synthesis, demonstrating a key role for mTOR in lipid body biogenesis and function. Our results establish PI3K/mTOR as an important signaling pathway for leptin-induced cytoplasmic lipid body biogenesis and adipose differentiation-related protein accumulation. Furthermore, we demonstrate a previously unrecognized link between intracellular (mTOR) and systemic (leptin) nutrient sensors in macrophage lipid metabolism. Leptin-induced increased formation of cytoplasmic lipid bodies and enhanced inflammatory mediator production in macrophages may have implications for obesity-related cardiovascular diseases. PMID:18039669

  10. Mammalian target of rapamycin signalling modulates amino acid uptake by regulating transporter cell surface abundance in primary human trophoblast cells.

    PubMed

    Rosario, Fredrick J; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

    2013-02-01

    Abnormal fetal growth increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Emerging evidence suggests that changes in placental amino acid transport directly contribute to altered fetal growth. However, the molecular mechanisms regulating placental amino acid transport are largely unknown. Here we combined small interfering (si) RNA-mediated silencing approaches with protein expression/localization and functional studies in cultured primary human trophoblast cells to test the hypothesis that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) regulate amino acid transporters by post-translational mechanisms. Silencing raptor (inhibits mTORC1) or rictor (inhibits mTORC2) markedly decreased basal System A and System L amino acid transport activity but had no effect on growth factor-stimulated amino acid uptake. Simultaneous inhibition of mTORC1 and 2 completely inhibited both basal and growth factor-stimulated amino acid transport activity. In contrast, mTOR inhibition had no effect on serotonin transport. mTORC1 or mTORC2 silencing markedly decreased the plasma membrane expression of specific System A (SNAT2, SLC38A2) and System L (LAT1, SLC7A5) transporter isoforms without affecting global protein expression. In conclusion, mTORC1 and mTORC2 regulate human trophoblast amino acid transporters by modulating the cell surface abundance of specific transporter isoforms. This is the first report showing regulation of amino acid transport by mTORC2. Because placental mTOR activity and amino acid transport are decreased in human intrauterine growth restriction our data are consistent with the possibility that dysregulation of placental mTOR plays an important role in the development of abnormal fetal growth.

  11. Activation Domain-Mediated Enhancement of Activator Binding to Chromatin in Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Bunker, Christopher A.; Kingston, Robert E.

    1996-10-01

    DNA binding by transcriptional activators is typically an obligatory step in the activation of gene expression. Activator binding and subsequent steps in transcription are repressed by genomic chromatin. Studies in vitro have suggested that overcoming this repression is an important function of some activation domains. Here we provide quantitative in vivo evidence that the activation domain of GAL4-VP16 can increase the affinity of GAL4 for its binding site on genomic DNA in mammalian cells. Moreover, the VP16 activation domain has a much greater stimulatory effect on expression from a genomic reporter gene than on a transiently transfected reporter gene, where factor binding is more permissive. We found that not all activation domains showed a greater activation potential in a genomic context, suggesting that only some activation domains can function in vivo to alleviate the repressive effects of chromatin. These data demonstrate the importance of activation domains in relieving chromatin-mediated repression in vivo and suggest that one way they function is to increase binding of the activator itself.

  12. Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain.

    PubMed

    Tsunekawa, Yuji; Terhune, Raymond Kunikane; Fujita, Ikumi; Shitamukai, Atsunori; Suetsugu, Taeko; Matsuzaki, Fumio

    2016-09-01

    Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues. PMID:27578183

  13. Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain

    PubMed Central

    Tsunekawa, Yuji; Terhune, Raymond Kunikane; Fujita, Ikumi; Shitamukai, Atsunori; Suetsugu, Taeko

    2016-01-01

    Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues. PMID:27578183

  14. Mitochondrial Nitroreductase Activity Enables Selective Imaging and Therapeutic Targeting.

    PubMed

    Chevalier, Arnaud; Zhang, Yanmin; Khdour, Omar M; Kaye, Justin B; Hecht, Sidney M

    2016-09-21

    Nitroreductase (NTR) activities have been known for decades, studied extensively in bacteria and also in systems as diverse as yeast, trypanosomes, and hypoxic tumors. The putative bacterial origin of mitochondria prompted us to explore the possible existence of NTR activity within this organelle and to probe its behavior in a cellular context. Presently, by using a profluorescent near-infrared (NIR) dye, we characterize the nature of NTR activity localized in mammalian cell mitochondria. Further, we demonstrate that this mitochondrially localized enzymatic activity can be exploited both for selective NIR imaging of mitochondria and for mitochondrial targeting by activating a mitochondrial poison specifically within that organelle. This constitutes a new mechanism for mitochondrial imaging and targeting. These findings represent the first use of mitochondrial enzyme activity to unmask agents for mitochondrial fluorescent imaging and therapy, which may prove to be more broadly applicable.

  15. A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells

    PubMed Central

    Weng, Kuo-Feng; Hung, Chuan-Tien; Hsieh, Po-Ting; Li, Mei-Ling; Chen, Guang-Wu; Kung, Yu-An; Huang, Peng-Nien; Kuo, Rei-Lin; Chen, Li-Lien; Lin, Jing-Yi; Wang, Robert Yung-Liang; Chen, Shu-Jen; Tang, Petrus; Horng, Jim-Tong; Huang, Hsing-I; Wang, Jen-Ren; Ojcius, David M.; Brewer, Gary; Shih, Shin-Ru

    2014-01-01

    The roles of virus-derived small RNAs (vsRNAs) have been studied in plants and insects. However, the generation and function of small RNAs from cytoplasmic RNA viruses in mammalian cells remain unexplored. This study describes four vsRNAs that were detected in enterovirus 71-infected cells using next-generation sequencing and northern blots. Viral infection produced substantial levels (>105 copy numbers per cell) of vsRNA1, one of the four vsRNAs. We also demonstrated that Dicer is involved in vsRNA1 generation in infected cells. vsRNA1 overexpression inhibited viral translation and internal ribosomal entry site (IRES) activity in infected cells. Conversely, blocking vsRNA1 enhanced viral yield and viral protein synthesis. We also present evidence that vsRNA1 targets stem-loop II of the viral 5′ untranslated region and inhibits the activity of the IRES through this sequence-specific targeting. Our study demonstrates the ability of a cytoplasmic RNA virus to generate functional vsRNA in mammalian cells. In addition, we also demonstrate a potential novel mechanism for a positive-stranded RNA virus to regulate viral translation: generating a vsRNA that targets the IRES. PMID:25352551

  16. Distinct roles for mammalian target of rapamycin complexes in the fibroblast response to transforming growth factor-beta.

    PubMed

    Rahimi, Rod A; Andrianifahanana, Mahefatiana; Wilkes, Mark C; Edens, Maryanne; Kottom, Theodore J; Blenis, John; Leof, Edward B

    2009-01-01

    Transforming growth factor-beta (TGF-beta) promotes a multitude of diverse biological processes, including growth arrest of epithelial cells and proliferation of fibroblasts. Although the TGF-beta signaling pathways that promote inhibition of epithelial cell growth are well characterized, less is known about the mechanisms mediating the positive response to this growth factor. Given that TGF-beta has been shown to promote fibrotic diseases and desmoplasia, identifying the fibroblast-specific TGF-beta signaling pathways is critical. Here, we investigate the role of mammalian target of rapamycin (mTOR), a known effector of phosphatidylinositol 3-kinase (PI3K) and promoter of cell growth, in the fibroblast response to TGF-beta. We show that TGF-beta activates mTOR complex 1 (mTORC1) in fibroblasts but not epithelial cells via a PI3K-Akt-TSC2-dependent pathway. Rapamycin, the pharmacologic inhibitor of mTOR, prevents TGF-beta-mediated anchorage-independent growth without affecting TGF-beta transcriptional responses or extracellular matrix protein induction. In addition to mTORC1, we also examined the role of mTORC2 in TGF-beta action. mTORC2 promotes TGF-beta-induced morphologic transformation and is required for TGF-beta-induced Akt S473 phosphorylation but not mTORC1 activation. Interestingly, both mTOR complexes are necessary for TGF-beta-mediated growth in soft agar. These results define distinct and overlapping roles for mTORC1 and mTORC2 in the fibroblast response to TGF-beta and suggest that inhibitors of mTOR signaling may be useful in treating fibrotic processes, such as desmoplasia. PMID:19117990

  17. Profiling the role of mammalian target of rapamycin in the vascular smooth muscle metabolome in pulmonary arterial hypertension

    PubMed Central

    Kudryashova, Tatiana V.; Goncharov, Dmitry A.; Pena, Andressa; Ihida-Stansbury, Kaori; DeLisser, Horace; Kawut, Steven M.

    2015-01-01

    Abstract Increased proliferation and resistance to apoptosis of pulmonary arterial vascular smooth muscle cells (PAVSMCs), coupled with metabolic reprogramming, are key components of pulmonary vascular remodeling, a major and currently irreversible pathophysiological feature of pulmonary arterial hypertension (PAH). We recently reported that activation of mammalian target of rapamycin (mTOR) plays a key role in increased energy generation and maintenance of the proliferative, apoptosis-resistant PAVSMC phenotype in human PAH, but the downstream effects of mTOR activation on PAH PAVSMC metabolism are not clear. Using liquid and gas chromatography–based mass spectrometry, we performed pilot metabolomic profiling of human microvascular PAVSMCs from idiopathic-PAH subjects before and after treatment with the selective adenosine triphosphate–competitive mTOR inhibitor PP242 and from nondiseased lungs. We have shown that PAH PAVSMCs have a distinct metabolomic signature of altered metabolites—components of fatty acid synthesis, deficiency of sugars, amino sugars, and nucleotide sugars—intermediates of protein and lipid glycosylation, and downregulation of key biochemicals involved in glutathione and nicotinamide adenine dinucleotide (NAD) metabolism. We also report that mTOR inhibition attenuated or reversed the majority of the PAH-specific abnormalities in lipogenesis, glycosylation, glutathione, and NAD metabolism without affecting altered polyunsaturated fatty acid metabolism. Collectively, our data demonstrate a critical role of mTOR in major PAH PAVSMC metabolic abnormalities and suggest the existence of de novo lipid synthesis in PAVSMCs in human PAH that may represent a new, important component of disease pathogenesis worthy of future investigation. PMID:26697174

  18. Targeted Gene Activation Using RNA-Guided Nucleases.

    PubMed

    Brown, Alexander; Woods, Wendy S; Perez-Pinera, Pablo

    2017-01-01

    The discovery of the prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) system and its adaptation for targeted manipulation of DNA in diverse species has revolutionized the field of genome engineering. In particular, the fusion of catalytically inactive Cas9 to any number of transcriptional activator domains has resulted in an array of easily customizable synthetic transcription factors that are capable of achieving robust, specific, and tunable activation of target gene expression within a wide variety of tissues and cells. This chapter describes key experimental design considerations, methods for plasmid construction, gene delivery protocols, and procedures for analysis of targeted gene activation in mammalian cell lines using CRISPR-Cas transcription factors. PMID:27662880

  19. Diversity between mammalian tolloid proteinases: Oligomerisation and non-catalytic domains influence activity and specificity

    PubMed Central

    Bayley, Christopher P.; Ruiz Nivia, Hilda D.; Dajani, Rana; Jowitt, Thomas A.; Collins, Richard F.; Rada, Heather; Bird, Louise E.; Baldock, Clair

    2016-01-01

    The mammalian tolloid family of metalloproteinases is essential for tissue patterning and extracellular matrix assembly. The four members of the family: bone morphogenetic protein-1 (BMP-1), mammalian tolloid (mTLD), tolloid-like (TLL)-1 and TLL-2 differ in their substrate specificity and activity levels, despite sharing similar domain organization. We have previously described a model of substrate exclusion by dimerisation to explain differences in the activities of monomeric BMP-1 and dimers of mTLD and TLL-1. Here we show that TLL-2, the least active member of the tolloid family, is predominantly monomeric in solution, therefore it appears unlikely that substrate exclusion via dimerisation is a mechanism for regulating TLL-2 activity. X-ray scattering and electron microscopy structural and biophysical analyses reveal an elongated shape for the monomer and flexibility in the absence of calcium. Furthermore, we show that TLL-2 can cleave chordin in vitro, similar to other mammalian tolloids, but truncated forms of TLL-2 mimicking BMP-1 are unable to cleave chordin. However, both the N- and C-terminal non-catalytic domains from all mammalian tolloids bind chordin with high affinity. The mechanisms underlying substrate specificity and activity in the tolloid family are complex with variation between family members and depend on both multimerisation and substrate interaction. PMID:26902455

  20. Motor unit regulation of mammalian pharyngeal dilator muscle activity.

    PubMed Central

    van Lunteren, E; Dick, T E

    1989-01-01

    The present study examined the cellular regulation of one of the pharyngeal dilator muscles, the geniohyoid, by assessing its motor unit (MU) behavior in anesthetized cats. During spontaneous breathing, MU that (a) were active during inspiration only (I-MU) and (b) were active during both inspiration and expiration (I/E-MU) were identified. I-MU had a later inspiratory onset time and a shorter duration of inspiratory firing than did I/E-MU (P less than 0.002 and P less than 0.0001, respectively). I-MU were usually quiescent whereas I/E-MU were usually active during the last 20% of inspiration. I/E-MU fired more rapidly (P less than 0.00001) and for relatively longer periods of time (P less than 0.00001) during inspiration than during expiration. End-expiratory airway occlusion (preventing lung expansion during inspiration) augmented the inspiratory activity of both I-MU and I/E-MU. Conversely, end-expiratory airway occlusion reduced the absolute and relative firing durations (P less than 0.002 and P less than 0.00002, respectively) and the firing frequency (P less than 0.001) of I/E-MU activity during expiration. These results indicate that (a) the complex pattern of pharyngeal dilator muscle activity is due to the integrated activity of a heterogeneous group of MU, (b) changes in the degree to which pharyngeal dilator muscles are active result from combinations of MU recruitment/decruitment and modulations of the frequency and duration of MU firing, and (c) gating of lung-volume afferent information occurs during the respiratory cycle. PMID:2760202

  1. Platelet-derived growth factor regulates vascular smooth muscle phenotype via mammalian target of rapamycin complex 1

    SciTech Connect

    Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan; Jin, Seo Yeon; Lee, Hye Sun; Song, Sang Heon; Shin, Hwa Kyoung; Bae, Sun Sik

    2015-08-14

    Mammalian target of rapamycin complex (mTORC) regulates various cellular processes including proliferation, growth, migration and differentiation. In this study, we showed that mTORC1 regulates platelet-derived growth factor (PDGF)-induced phenotypic conversion of vascular smooth muscle cells (VSMCs). Stimulation of contractile VSMCs with PDGF significantly reduced the expression of contractile marker proteins in a time- and dose-dependent manner. In addition, angiotensin II (AngII)-induced contraction of VSMCs was completely blocked by the stimulation of VSMCs with PDGF. PDGF-dependent suppression of VSMC marker gene expression was significantly blocked by inhibition of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and mTOR whereas inhibition of p38 MAPK had no effect. In particular, inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked the PDGF-dependent phenotypic change of VSMCs whereas silencing of Rictor had no effect. In addition, loss of AngII-dependent contraction by PDGF was significantly retained by silencing of Raptor. Inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked PDGF-induced proliferation of VSMCs. Taken together, we suggest that mTORC1 plays an essential role in PDGF-dependent phenotypic changes of VSMCs. - Graphical abstract: Regulation of VSMC phenotype by PDGF-dependent activation of mTORC1. - Highlights: • The expression of contractile marker proteins was reduced by PDGF stimulation. • PDGF-dependent phenotypic conversion of VSMCs was blocked by inhibition of mTOR. • PDGF-induced proliferation of VSMCs was attenuated by inhibition of mTORC1. • mTORC1 plays a critical role in PDGF-dependent phenotypic conversion of VSMCs.

  2. Investigating Deformylase and Deacylase Activity of Mammalian and Bacterial Sirtuins.

    PubMed

    Seidel, Julian; Klockenbusch, Cordula; Schwarzer, Dirk

    2016-03-01

    Lysine acylation constitutes a major group of post-translational modifications of proteins, and is found in the proteomes of organisms from all kingdoms of life. Sirtuins are considered the main erasers of these modification marks, and thus contribute to acylation-dependent regulation of enzyme activity, and potentially of protein quality control. We have established a substrate scaffold to enable the analysis of sirtuin activity with a broad range of acyl-lysine modifications, including hydrophobic fatty acids. Characterization of the deacylase activity of the bacterial SrtN, which is encoded by the yhdZ gene of Bacillus subtilis, showed that this enzyme is capable of removing a broad range of acyl groups. These investigations further showed that SrtN and human SIRT1 are efficient lysine-deformylases, thereby providing a first clue as to how this nonenzymatic modification might be removed from affected proteins.

  3. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002.

    PubMed Central

    Brunn, G J; Williams, J; Sabers, C; Wiederrecht, G; Lawrence, J C; Abraham, R T

    1996-01-01

    The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases. This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein, PHAS-1, in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation. A mutant YAC-1 T lymphoma cell line, which was selected for resistance to the growth-inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation. In contrast, the PI 3-kinase inhibitor, wortmannin, reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells. At similar drug concentrations (0.1-1 microM), wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor, LY294002, at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer. These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3-kinase homologs, are directly affected by cellular treatment with wortmannin or LY294002. Images PMID:8895571

  4. Mechanism of arsenate activation of mammalian phosphoglycerate mutase

    SciTech Connect

    Rea, D.W.; McWilliams, A.D.; Hass, L.F.

    1987-05-01

    Towne demonstrated that arsenate (As/sub i/) can replace D-glycerate-2,3-P/sub 2/ (2,3-DPG) as an activator for cofactor-dependent phosphoglycerate mutase (PGM). Arsenate activation was found to be accompanied by a lag phase which, over a period of several minutes, gradually evolved into a region of steady-state kinetics. The authors have verified and expanded Towne's findings through isotope exchange studies and kinetic analysis. In the absence of 2,3-DPG, reciprocal plots of PGM-catalyzed steady-state velocities versus As/sub i/ concentrations at different D-glycerate-3-P (3-PGA) levels yield a family of curves which suggest a ping-pong mechanism accompanied by double competitive substrate inhibition. Other experiments show that incubation of doubly-labelled 0.25 mM (U-/sup 14/C, /sup 32/P)-3-PGA with 20 mM As/sub i/ and PGM for several hrs. promotes the release of P/sub i/ with the concomitant formation of D-glycerate. Addition of 0.2 mM glycolate-2-P to the reaction medium accelerates the process. P/sub i/, but not vanadate, also promotes hydrolysis of 3-PGA, but to a much lesser extent than As/sub i/, even in the presence of glycolate-2-P. The pH optimum for 3-PGA phosphatase activity is 6.0-6.2. These and other findings suggest that As/sub i/ accelerates PGM catalysis by first forming 2-As-3-PGA which in turn forms phosphoenzyme (EP). Previous studies have shown that EP is the active form of PGM.

  5. Multiple RNase H activities in mammalian type C retravirus lysates.

    PubMed Central

    Gerard, G F

    1978-01-01

    Lysates of Moloney murine sarcoma-leukemia virus [M-MSV(MLV)], a virus complex grown in the rat cell line 78A-1, were found to contain three RNase H species separable by polycytidylic acid[poly(C)]-agarose chromatography. RNase H activity (RNase H I) associated with RNA-directed DNA polymerase eluted at 0.23 M KCI from poly(C)-agarose. RNase H II, which eluted from poly(C)-agarose at 0.12 M KCI and was not associated with DNA polymerase activity, was shown to be identical to an RNase H species (designated RNase H II) previously isolated from M-MSV(MLV) by a different procedure (G. F. Gerard and D. P. Grandgenett, J. Virol. 15:785-797, 1975). M-MSV(MLV) RNase H II was established to be a random exohybridase that requires free-chain termini in its hybrid substrate for activity. Lysates of Rickard feline leukemia virus also contained RNase H activity not associated with DNA polymerase activity that eluted from poly(C)-agarose at 0.12 M KCl. A third species of enzyme from M-MSV(MLV) lysates, called RNase H III, did not bind to poly(C)-agarose in 0.06 M KCl. RNase H III was purified from lysates of M-MSV(MLV) and M-MLV (grown in mouse cells) by sequential chromatography on poly(C)-agarose, DEAE-cellulose, phosphocellulose, and polyuridylic acid-Sepharose. Purified RNase H III (i) was free of any associated DNA polymerase activity, (ii) had an apparent molecular weight of 30,000 determined by Sephadex G-100 gel filtration, (iii) had an absolute requirement for Mn2+ (1 mM optimum) for the degradation of [3H](A)n.(dT)n, (iv) was inhibited by the presence of any salt in reaction mixtures, and (v) was endoribonucleolytic in its mode of action as indicated by the size distribution of limited degradation products of [3H](A)n.(dT)n. RNase H III was inhibited by antisera prepared against Rauscher MLV and simian sarcoma virus reverse transcriptase, and the quantity of RNase H III and RNase H I present in lysates of M-MLV were reduced and increased proportionately if virus was

  6. Rapid Feedforward Inhibition and Asynchronous Excitation Regulate Granule Cell Activity in the Mammalian Main Olfactory Bulb

    PubMed Central

    Burton, Shawn D.

    2015-01-01

    Granule cell-mediated inhibition is critical to patterning principal neuron activity in the olfactory bulb, and perturbation of synaptic input to granule cells significantly alters olfactory-guided behavior. Despite the critical role of granule cells in olfaction, little is known about how sensory input recruits granule cells. Here, we combined whole-cell patch-clamp electrophysiology in acute mouse olfactory bulb slices with biophysical multicompartmental modeling to investigate the synaptic basis of granule cell recruitment. Physiological activation of sensory afferents within single glomeruli evoked diverse modes of granule cell activity, including subthreshold depolarization, spikelets, and suprathreshold responses with widely distributed spike latencies. The generation of these diverse activity modes depended, in part, on the asynchronous time course of synaptic excitation onto granule cells, which lasted several hundred milliseconds. In addition to asynchronous excitation, each granule cell also received synchronous feedforward inhibition. This inhibition targeted both proximal somatodendritic and distal apical dendritic domains of granule cells, was reliably recruited across sniff rhythms, and scaled in strength with excitation as more glomeruli were activated. Feedforward inhibition onto granule cells originated from deep short-axon cells, which responded to glomerular activation with highly reliable, short-latency firing consistent with tufted cell-mediated excitation. Simulations showed that feedforward inhibition interacts with asynchronous excitation to broaden granule cell spike latency distributions and significantly attenuates granule cell depolarization within local subcellular compartments. Collectively, our results thus identify feedforward inhibition onto granule cells as a core feature of olfactory bulb circuitry and establish asynchronous excitation and feedforward inhibition as critical regulators of granule cell activity. SIGNIFICANCE

  7. Novel pentapeptide activators of mammalian and mushroom tyrosinase.

    PubMed

    Ubeid, Anan Abu; Hantash, Basil M

    2014-01-01

    Melanoma incidence continues to rise due to intentional exposure to ultraviolet radiation (UVR) from sunlight and indoor tanning beds. Eumelanin exhibits photoprotective effects; thus, agents that induce its synthesis offer a means for sunless tanning without UVR damage. Herein, we report the development of two pentapeptides, P9 and P10, capable of enhancing melanin synthesis in B16 melanoma cells by activating mushroom and mouse tyrosinases without any effect on cell viability or proliferation. P9 and P10 significantly increased melanin content in a dose-dependent manner comparable to the positive controls, IBMX, scoparone, and α-MSH. However, unlike IBMX and scoparone, but similar to α-MSH, P9 and P10 were able to reverse 6BH4-dependent tyrosinase inhibition. We hypothesize that P9 and P10 allosterically activate tyrosinase and consequently enhance epidermal melanin synthesis. P9 and P10 may offer an alternative to tanning bed use and non-photoprotective tanning products. Moreover, sustained increase of melanin content in skin has the potential to reduce symptoms of photosensitivity disorders such as erythropoietic protoporphyria (EPP), solar urticaria (SU) and polymorphic light eruption (PLE), which lack fully effective treatments and result in significant morbidity.

  8. 4-Alkynylphenylsilatranes: Insecticidal activity, mammalian toxicity, and mode of action

    SciTech Connect

    Horsham, M.A.; Palmer, C.J.; Cole, L.M.; Casida, J.E. )

    1990-08-01

    4-Ethynyl- and 4-(prop-1-ynyl)phenylsilatranes (N(CH{sub 2}CH{sub 2}O){sub 3}SiR, R = C{sub 6}H{sub 4}-4-C{triple bond}CH or C{sub 6}H{sub 4}-4-C{triple bond}CCH{sub 3}) are highly toxic to houseflies (pretreated with piperonyl butoxide) and milkweed bugs (topical LD{sub 50}s 3-14 {mu}g/g) and to mice (intraperitoneal LD{sub 50}s 0.4-0.9 mg/kg), and they are moderately potent inhibitors of the ({sup 35}S)-tert-butylbicyclophosphorothionate or TBPS binding site (GABA-gated chloride channel) of mouse brain membranes. Scatchard analysis indicates noncompetitive interaction of 4-ethynylphenylsilatrane with the TBPS binding site. Phenylsilatrane analogues with 4-substituents of H, CH{sub 3}, Cl, Br, and C{triple bond}CSi(CH{sub 3}){sub 3} are highly toxic to mice but have little or no activity in the insect and receptor assays. Radioligand binding studies with (4-{sup 3}H)phenylsilatrane failed to reveal a specific binding site in mouse brain. Silatranes with R = H, CH{sub 3}, CH{sub 2}Cl, CH{double bond}CH{sub 2}, OCH{sub 2}CH{sub 3}, and C{sub 6}H{sub 4}-4-CH{sub 2}CH{sub 3} are of little or no activity in the insect and mouse toxicity and TBPS binding site assays as are the trithia and monocyclic analogues of phenylsilatrane. 4-Alkynylphenylsilatranes are new probes to examine the GABA receptor-ionophore complex of insects and mammals.

  9. Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher.

    PubMed

    Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

    2015-01-01

    Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein-DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells. PMID:26151127

  10. Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

    NASA Astrophysics Data System (ADS)

    Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

    2015-07-01

    Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein-DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

  11. Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher.

    PubMed

    Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

    2015-07-07

    Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein-DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

  12. Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

    PubMed Central

    Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

    2015-01-01

    Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein–DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells. PMID:26151127

  13. Discovery of a novel class of highly potent, selective, ATP-competitive, and orally bioavailable inhibitors of the mammalian target of rapamycin (mTOR).

    PubMed

    Takeuchi, Craig S; Kim, Byung Gyu; Blazey, Charles M; Ma, Sunghoon; Johnson, Henry W B; Anand, Neel K; Arcalas, Arlyn; Baik, Tae Gon; Buhr, Chris A; Cannoy, Jonah; Epshteyn, Sergey; Joshi, Anagha; Lara, Katherine; Lee, Matthew S; Wang, Longcheng; Leahy, James W; Nuss, John M; Aay, Naing; Aoyama, Ron; Foster, Paul; Lee, Jae; Lehoux, Isabelle; Munagala, Narsimha; Plonowski, Arthur; Rajan, Sharmila; Woolfrey, John; Yamaguchi, Kyoko; Lamb, Peter; Miller, Nicole

    2013-03-28

    A series of novel, highly potent, selective, and ATP-competitive mammalian target of rapamycin (mTOR) inhibitors based on a benzoxazepine scaffold have been identified. Lead optimization resulted in the discovery of inhibitors with low nanomolar activity and greater than 1000-fold selectivity over the closely related PI3K kinases. Compound 28 (XL388) inhibited cellular phosphorylation of mTOR complex 1 (p-p70S6K, pS6, and p-4E-BP1) and mTOR complex 2 (pAKT (S473)) substrates. Furthermore, this compound displayed good pharmacokinetics and oral exposure in multiple species with moderate bioavailability. Oral administration of compound 28 to athymic nude mice implanted with human tumor xenografts afforded significant and dose-dependent antitumor activity. PMID:23394126

  14. Discovery of a novel class of highly potent, selective, ATP-competitive, and orally bioavailable inhibitors of the mammalian target of rapamycin (mTOR).

    PubMed

    Takeuchi, Craig S; Kim, Byung Gyu; Blazey, Charles M; Ma, Sunghoon; Johnson, Henry W B; Anand, Neel K; Arcalas, Arlyn; Baik, Tae Gon; Buhr, Chris A; Cannoy, Jonah; Epshteyn, Sergey; Joshi, Anagha; Lara, Katherine; Lee, Matthew S; Wang, Longcheng; Leahy, James W; Nuss, John M; Aay, Naing; Aoyama, Ron; Foster, Paul; Lee, Jae; Lehoux, Isabelle; Munagala, Narsimha; Plonowski, Arthur; Rajan, Sharmila; Woolfrey, John; Yamaguchi, Kyoko; Lamb, Peter; Miller, Nicole

    2013-03-28

    A series of novel, highly potent, selective, and ATP-competitive mammalian target of rapamycin (mTOR) inhibitors based on a benzoxazepine scaffold have been identified. Lead optimization resulted in the discovery of inhibitors with low nanomolar activity and greater than 1000-fold selectivity over the closely related PI3K kinases. Compound 28 (XL388) inhibited cellular phosphorylation of mTOR complex 1 (p-p70S6K, pS6, and p-4E-BP1) and mTOR complex 2 (pAKT (S473)) substrates. Furthermore, this compound displayed good pharmacokinetics and oral exposure in multiple species with moderate bioavailability. Oral administration of compound 28 to athymic nude mice implanted with human tumor xenografts afforded significant and dose-dependent antitumor activity.

  15. Mammalian target of rapamycin (mTOR) inhibition reduces cerebral vasospasm following a subarachnoid hemorrhage injury in canines.

    PubMed

    Zhang, Weiguang; Khatibi, Nikan H; Yamaguchi-Okada, Mitsuo; Yan, Junhao; Chen, Chunhua; Hu, Qin; Meng, Haiwei; Han, Hongbin; Liu, Shuwei; Zhou, Changman

    2012-02-01

    Mammalian target of rapamycin (mTOR) pathway is a serine/threonine protein kinase that plays a vital role in regulating growth, proliferation, survival, and protein synthesis among cells. In the present study, we investigated the role of the mTOR pathway following subarachnoid hemorrhage brain injury--specifically investigating its ability to mediate the activation of cerebral vasospasm. Additionally, we investigated whether key signaling pathway molecules such as the mTOR, P70S6K1, and 4E-BP1 play a role in the process. Thirty dogs were randomly divided into 5 groups: sham, SAH (subarachnoid hemorrhage), SAH+DMSO (dimethyl sulfoxide), SAH+Rapamycin and SAH+AZD8055. An established canine double-hemorrhage model of SAH was used by injecting autologous arterial blood into the cisterna magna on days 0 and 2. Angiography was performed at days 0 and 7. Clinical behavior, histology, immunohistochemistry, and Western blot of mTOR, P70S6K1, 4E-BP1 and PCNA (proliferating cell nuclear antigen) in the basilar arteries were examined. In the SAH and SAH+DMSO groups, severe angiographic vasospasm was obtained (34.3±19.8%, 38.4±10.3) compared with that in Sham (93.9±5.0%) respectively. mTOR, P70S6K1, 4E-BP1 and PCNA increased in the sample of spastic basilar arteries (p<0.05). In the SAH+RAPA and SAH+AZD8055 groups, Rapamycin and AZD8055 attenuated angiographic vasospasm (62.3±15.9% and 65.2±10.3%) while improving appetite and activity scores (p<0.05) on days 5 through 7. Rapamycin and AZD8055 significantly reduced the level and expression of mTOR, P70S6K1, 4E-BP1 and PCNA (p<0.05). In conclusion, our study suggests that the mTOR molecular signaling pathway plays a significant role in cerebral vasospasm following SAH, and that inhibition of the mTOR pathway has the potential to become an attractive strategy to treat vasospasm following SAH. PMID:22177999

  16. The endosomal sorting complex required for transport pathway mediates chemokine receptor CXCR4-promoted lysosomal degradation of the mammalian target of rapamycin antagonist DEPTOR.

    PubMed

    Verma, Rita; Marchese, Adriano

    2015-03-13

    G protein-coupled receptor (GPCR) signaling mediates many cellular functions, including cell survival, proliferation, and cell motility. Many of these processes are mediated by GPCR-promoted activation of Akt signaling by mammalian target of rapamycin complex 2 (mTORC2) and the phosphatidylinositol 3-kinase (PI3K)/phosphoinositide-dependent kinase 1 (PDK1) pathway. However, the molecular mechanisms by which GPCRs govern Akt activation by these kinases remain poorly understood. Here, we show that the endosomal sorting complex required for transport (ESCRT) pathway mediates Akt signaling promoted by the chemokine receptor CXCR4. Pharmacological inhibition of heterotrimeric G protein Gαi or PI3K signaling and siRNA targeting ESCRTs blocks CXCR4-promoted degradation of DEPTOR, an endogenous antagonist of mTORC2 activity. Depletion of ESCRTs by siRNA leads to increased levels of DEPTOR and attenuated CXCR4-promoted Akt activation and signaling, consistent with decreased mTORC2 activity. In addition, ESCRTs likely have a broad role in Akt signaling because ESCRT depletion also attenuates receptor tyrosine kinase-promoted Akt activation and signaling. Our data reveal a novel role for the ESCRT pathway in promoting intracellular signaling, which may begin to identify the signal transduction pathways that are important in the physiological roles of ESCRTs and Akt.

  17. Identification of Adeno-Associated Viral Vectors That Target Neonatal and Adult Mammalian Inner Ear Cell Subtypes.

    PubMed

    Shu, Yilai; Tao, Yong; Wang, Zhengmin; Tang, Yong; Li, Huawei; Dai, Pu; Gao, Guangping; Chen, Zheng-Yi

    2016-09-01

    The mammalian inner ear consists of diverse cell types with important functions. Gene mutations in these diverse cell types have been found to underlie different forms of genetic hearing loss. Targeting these mutations for gene therapy development represents a future therapeutic strategy to treat hearing loss. Adeno-associated viral (AAV) vectors have become the vector of choice for gene delivery in animal models in vivo. To identify AAV vectors that target inner ear cell subtypes, we systemically screened 12 AAV vectors with different serotypes (AAV1, 2, 5, 6, 6.2, 7, 8, 9, rh.8, rh.10, rh.39, and rh.43) that carry a reporter gene GFP in neonatal and adult mice by microinjection in vivo. We found that most AAVs infect both neonatal and adult inner ear, with different specificities and expression levels. The inner ear cochlear sensory epithelial region, which includes auditory hair cells and supporting cells, is most frequently targeted for gene delivery. Expression of the transgene is sustained, and neonatal inner ear delivery does not adversely affect hearing. Adult inner ear injection of AAV has a similar infection pattern as the younger inner ear, with the exception that outer hair cell death caused by the injection procedure can lead to hearing loss. In the adult, more so than in the neonatal mice, cell types infected and efficiency of infection are correlated with the site of injection. Most infected cells survive in neonatal and adult inner ears. The study adds to the list of AAV vectors that transduce the mammalian inner ear efficiently, providing the tools that are important to study inner ear gene function and for the development of gene therapy to treat hearing loss. PMID:27342665

  18. Otx2 is a target of N-myc and acts as a suppressor of sensory development in the mammalian cochlea.

    PubMed

    Vendrell, Victor; López-Hernández, Iris; Durán Alonso, María Beatriz; Feijoo-Redondo, Ana; Abello, Gina; Gálvez, Héctor; Giráldez, Fernando; Lamonerie, Thomas; Schimmang, Thomas

    2015-08-15

    Transcriptional regulatory networks are essential during the formation and differentiation of organs. The transcription factor N-myc is required for proper morphogenesis of the cochlea and to control correct patterning of the organ of Corti. We show here that the Otx2 gene, a mammalian ortholog of the Drosophila orthodenticle homeobox gene, is a crucial target of N-myc during inner ear development. Otx2 expression is lost in N-myc mouse mutants, and N-myc misexpression in the chick inner ear leads to ectopic expression of Otx2. Furthermore, Otx2 enhancer activity is increased by N-myc misexpression, indicating that N-myc may directly regulate Otx2. Inactivation of Otx2 in the mouse inner ear leads to ectopic expression of prosensory markers in non-sensory regions of the cochlear duct. Upon further differentiation, these domains give rise to an ectopic organ of Corti, together with the re-specification of non-sensory areas into sensory epithelia, and the loss of Reissner's membrane. Therefore, the Otx2-positive domain of the cochlear duct shows a striking competence to develop into a mirror-image copy of the organ of Corti. Taken together, these data show that Otx2 acts downstream of N-myc and is essential for patterning and spatial restriction of the sensory domain of the mammalian cochlea. PMID:26160903

  19. Controlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection.

    PubMed

    Cottle, Renee N; Lee, Ciaran M; Archer, David; Bao, Gang

    2015-11-12

    Tal-effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins are genome editing tools with unprecedented potential. However, the ability to deliver optimal amounts of these nucleases into mammalian cells with minimal toxicity poses a major challenge. Common delivery approaches are transfection- and viral-based methods; each associated with significant drawbacks. An alternative method for directly delivering genome-editing reagents into single living cells with high efficiency and controlled volume is microinjection. Here, we characterize a glass microcapillary-based injection system and demonstrate controlled co-injection of TALENs or CRISPR/Cas9 together with donor template into single K562 cells for targeting the human β-globin gene. We quantified nuclease induced insertions and deletions (indels) and found that, with β-globin-targeting TALENs, similar levels of on- and off-target activity in cells could be achieved by microinjection compared with nucleofection. Furthermore, we observed 11% and 2% homology directed repair in single K562 cells co-injected with a donor template along with CRISPR/Cas9 and TALENs respectively. These results demonstrate that a high level of targeted gene modification can be achieved in human cells using glass-needle microinjection of genome editing reagents.

  20. Modulating Antimicrobial Activity and Mammalian Cell Biocompatibility with Glucosamine-Functionalized Star Polymers.

    PubMed

    Wong, Edgar H H; Khin, Mya Mya; Ravikumar, Vikashini; Si, Zhangyong; Rice, Scott A; Chan-Park, Mary B

    2016-03-14

    The development of novel reagents and antibiotics for combating multidrug resistance bacteria has received significant attention in recent years. In this study, new antimicrobial star polymers (14-26 nm in diameter) that consist of mixtures of polylysine and glycopolymer arms were developed and were shown to possess antimicrobial efficacy toward Gram positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) (with MIC values as low as 16 μg mL(-1)) while being non-hemolytic (HC50 > 10,000 μg mL(-1)) and exhibit excellent mammalian cell biocompatibility. Structure function analysis indicated that the antimicrobial activity and mammalian cell biocompatibility of the star nanoparticles could be optimized by modifying the molar ratio of polylysine to glycopolymers arms. The technology described herein thus represents an innovative approach that could be used to fight deadly infectious diseases.

  1. Predicting effective microRNA target sites in mammalian mRNAs

    PubMed Central

    Agarwal, Vikram; Bell, George W; Nam, Jin-Wu; Bartel, David P

    2015-01-01

    MicroRNA targets are often recognized through pairing between the miRNA seed region and complementary sites within target mRNAs, but not all of these canonical sites are equally effective, and both computational and in vivo UV-crosslinking approaches suggest that many mRNAs are targeted through non-canonical interactions. Here, we show that recently reported non-canonical sites do not mediate repression despite binding the miRNA, which indicates that the vast majority of functional sites are canonical. Accordingly, we developed an improved quantitative model of canonical targeting, using a compendium of experimental datasets that we pre-processed to minimize confounding biases. This model, which considers site type and another 14 features to predict the most effectively targeted mRNAs, performed significantly better than existing models and was as informative as the best high-throughput in vivo crosslinking approaches. It drives the latest version of TargetScan (v7.0; targetscan.org), thereby providing a valuable resource for placing miRNAs into gene-regulatory networks. DOI: http://dx.doi.org/10.7554/eLife.05005.001 PMID:26267216

  2. Targeted toxin-based selectable drug-free enrichment of Mammalian cells with high transgene expression.

    PubMed

    Sato, Masahiro; Akasaka, Eri; Saitoh, Issei; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

    2013-01-01

    Almost all transfection protocols for mammalian cells use a drug resistance gene for the selection of transfected cells. However, it always requires the characterization of each isolated clone regarding transgene expression, which is time-consuming and labor-intensive. In the current study, we developed a novel method to selectively isolate clones with high transgene expression without drug selection. Porcine embryonic fibroblasts were transfected with pCEIEnd, an expression vector that simultaneously expresses enhanced green fluorescent protein (EGFP) and endo-b-galactosidase C(EndoGalC; an enzyme capable of digesting cell surface a-Gal epitope) upon transfection. After transfection, the surviving cells were briefly treated with IB4SAP (a-Gal epitope-specific BS-I-B4 lectin conjugated with a toxin saporin). The treated cells were then allowed to grow in normal medium, during which only cells strongly expressing EndoGalC and EGFP would survive because of the absence of a-Gal epitopes on their cell surface. Almost all the surviving colonies after IB4SAP treatment were in fact negative for BS-I-B4 staining, and also strongly expressed EGFP. This system would be particularly valuable for researchers who wish to perform large-scale production of therapeutically important recombinant proteins. PMID:24832665

  3. Mammalian Target of Rapamycin: Its Role in Early Neural Development and in Adult and Aged Brain Function.

    PubMed

    Garza-Lombó, Carla; Gonsebatt, María E

    2016-01-01

    The kinase mammalian target of rapamycin (mTOR) integrates signals triggered by energy, stress, oxygen levels, and growth factors. It regulates ribosome biogenesis, mRNA translation, nutrient metabolism, and autophagy. mTOR participates in various functions of the brain, such as synaptic plasticity, adult neurogenesis, memory, and learning. mTOR is present during early neural development and participates in axon and dendrite development, neuron differentiation, and gliogenesis, among other processes. Furthermore, mTOR has been shown to modulate lifespan in multiple organisms. This protein is an important energy sensor that is present throughout our lifetime its role must be precisely described in order to develop therapeutic strategies and prevent diseases of the central nervous system. The aim of this review is to present our current understanding of the functions of mTOR in neural development, the adult brain and aging. PMID:27378854

  4. Mammalian Target of Rapamycin: Its Role in Early Neural Development and in Adult and Aged Brain Function

    PubMed Central

    Garza-Lombó, Carla; Gonsebatt, María E.

    2016-01-01

    The kinase mammalian target of rapamycin (mTOR) integrates signals triggered by energy, stress, oxygen levels, and growth factors. It regulates ribosome biogenesis, mRNA translation, nutrient metabolism, and autophagy. mTOR participates in various functions of the brain, such as synaptic plasticity, adult neurogenesis, memory, and learning. mTOR is present during early neural development and participates in axon and dendrite development, neuron differentiation, and gliogenesis, among other processes. Furthermore, mTOR has been shown to modulate lifespan in multiple organisms. This protein is an important energy sensor that is present throughout our lifetime its role must be precisely described in order to develop therapeutic strategies and prevent diseases of the central nervous system. The aim of this review is to present our current understanding of the functions of mTOR in neural development, the adult brain and aging. PMID:27378854

  5. Transcriptional activation of muscle atrophy promotes cardiac muscle remodeling during mammalian hibernation.

    PubMed

    Zhang, Yichi; Aguilar, Oscar A; Storey, Kenneth B

    2016-01-01

    Background. Mammalian hibernation in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) is characterized by dramatic changes on a physiological and molecular level. During hibernation, mammalian hearts show a propensity to hypertrophy due to the need for increasing contractility to pump colder and more viscous blood. While cardiac hypertrophy is quite often a process characterized by decompensation, the ground squirrel studied is an excellent model of cardiac plasticity and cardioprotection under conditions of hypothermia and ischemia. The forkhead box O (Foxo) family of proteins and myogenin (MyoG) are transcription factors that control protein degradation and muscle atrophy by regulating the expression of the E3 ubiquitin ligases, MAFbx and MuRF1. These ligases are part of the ubiquitin proteasome system by transferring ubiquitin to proteins and targeting these proteins for degradation. Regulation of Foxo1 and 3a occurs through phosphorylation at different residues. The threonine-24 (Thr-24) and serine-319 (Ser-319) residues on Foxo1, and the Thr-32 residue on Foxo3a are phosphorylated by Akt, leading to cytoplasmic localization of Foxo. We propose that the described mechanism contributes to the changes taking place in cardiac muscle throughout hibernation. Methods. Total and phosphorylated protein levels of Foxo1 and Foxo3a, as well as total protein levels of MyoG, MAFbx, and MuRF1, were studied using immunoblotting. Results. Immunoblotting results demonstrated upregulations in Foxo1 and Foxo3a total protein levels (1.3- and 4.5-fold increases relative to euthermic control, for Foxo1 and 3a respectively) during late torpor, and protein levels remained elevated throughout the rest of torpor and at interbout arousal. We also observed decreases in inactive, phosphorylated Foxo1 and 3a proteins during throughout torpor, where levels of p-Foxo1 Ser(319) and Thr(24), as well as p-Foxo3a Thr(32) decreased by at least 45% throughout torpor. MyoG was

  6. Transcriptional activation of muscle atrophy promotes cardiac muscle remodeling during mammalian hibernation

    PubMed Central

    Zhang, Yichi; Aguilar, Oscar A.

    2016-01-01

    Background. Mammalian hibernation in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) is characterized by dramatic changes on a physiological and molecular level. During hibernation, mammalian hearts show a propensity to hypertrophy due to the need for increasing contractility to pump colder and more viscous blood. While cardiac hypertrophy is quite often a process characterized by decompensation, the ground squirrel studied is an excellent model of cardiac plasticity and cardioprotection under conditions of hypothermia and ischemia. The forkhead box O (Foxo) family of proteins and myogenin (MyoG) are transcription factors that control protein degradation and muscle atrophy by regulating the expression of the E3 ubiquitin ligases, MAFbx and MuRF1. These ligases are part of the ubiquitin proteasome system by transferring ubiquitin to proteins and targeting these proteins for degradation. Regulation of Foxo1 and 3a occurs through phosphorylation at different residues. The threonine-24 (Thr-24) and serine-319 (Ser-319) residues on Foxo1, and the Thr-32 residue on Foxo3a are phosphorylated by Akt, leading to cytoplasmic localization of Foxo. We propose that the described mechanism contributes to the changes taking place in cardiac muscle throughout hibernation. Methods. Total and phosphorylated protein levels of Foxo1 and Foxo3a, as well as total protein levels of MyoG, MAFbx, and MuRF1, were studied using immunoblotting. Results. Immunoblotting results demonstrated upregulations in Foxo1 and Foxo3a total protein levels (1.3- and 4.5-fold increases relative to euthermic control, for Foxo1 and 3a respectively) during late torpor, and protein levels remained elevated throughout the rest of torpor and at interbout arousal. We also observed decreases in inactive, phosphorylated Foxo1 and 3a proteins during throughout torpor, where levels of p-Foxo1 Ser319 and Thr24, as well as p-Foxo3a Thr32 decreased by at least 45% throughout torpor. MyoG was upregulated only

  7. Transcriptional activation of muscle atrophy promotes cardiac muscle remodeling during mammalian hibernation

    PubMed Central

    Zhang, Yichi; Aguilar, Oscar A.

    2016-01-01

    Background. Mammalian hibernation in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) is characterized by dramatic changes on a physiological and molecular level. During hibernation, mammalian hearts show a propensity to hypertrophy due to the need for increasing contractility to pump colder and more viscous blood. While cardiac hypertrophy is quite often a process characterized by decompensation, the ground squirrel studied is an excellent model of cardiac plasticity and cardioprotection under conditions of hypothermia and ischemia. The forkhead box O (Foxo) family of proteins and myogenin (MyoG) are transcription factors that control protein degradation and muscle atrophy by regulating the expression of the E3 ubiquitin ligases, MAFbx and MuRF1. These ligases are part of the ubiquitin proteasome system by transferring ubiquitin to proteins and targeting these proteins for degradation. Regulation of Foxo1 and 3a occurs through phosphorylation at different residues. The threonine-24 (Thr-24) and serine-319 (Ser-319) residues on Foxo1, and the Thr-32 residue on Foxo3a are phosphorylated by Akt, leading to cytoplasmic localization of Foxo. We propose that the described mechanism contributes to the changes taking place in cardiac muscle throughout hibernation. Methods. Total and phosphorylated protein levels of Foxo1 and Foxo3a, as well as total protein levels of MyoG, MAFbx, and MuRF1, were studied using immunoblotting. Results. Immunoblotting results demonstrated upregulations in Foxo1 and Foxo3a total protein levels (1.3- and 4.5-fold increases relative to euthermic control, for Foxo1 and 3a respectively) during late torpor, and protein levels remained elevated throughout the rest of torpor and at interbout arousal. We also observed decreases in inactive, phosphorylated Foxo1 and 3a proteins during throughout torpor, where levels of p-Foxo1 Ser319 and Thr24, as well as p-Foxo3a Thr32 decreased by at least 45% throughout torpor. MyoG was upregulated only

  8. Transcriptional activation of muscle atrophy promotes cardiac muscle remodeling during mammalian hibernation.

    PubMed

    Zhang, Yichi; Aguilar, Oscar A; Storey, Kenneth B

    2016-01-01

    Background. Mammalian hibernation in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) is characterized by dramatic changes on a physiological and molecular level. During hibernation, mammalian hearts show a propensity to hypertrophy due to the need for increasing contractility to pump colder and more viscous blood. While cardiac hypertrophy is quite often a process characterized by decompensation, the ground squirrel studied is an excellent model of cardiac plasticity and cardioprotection under conditions of hypothermia and ischemia. The forkhead box O (Foxo) family of proteins and myogenin (MyoG) are transcription factors that control protein degradation and muscle atrophy by regulating the expression of the E3 ubiquitin ligases, MAFbx and MuRF1. These ligases are part of the ubiquitin proteasome system by transferring ubiquitin to proteins and targeting these proteins for degradation. Regulation of Foxo1 and 3a occurs through phosphorylation at different residues. The threonine-24 (Thr-24) and serine-319 (Ser-319) residues on Foxo1, and the Thr-32 residue on Foxo3a are phosphorylated by Akt, leading to cytoplasmic localization of Foxo. We propose that the described mechanism contributes to the changes taking place in cardiac muscle throughout hibernation. Methods. Total and phosphorylated protein levels of Foxo1 and Foxo3a, as well as total protein levels of MyoG, MAFbx, and MuRF1, were studied using immunoblotting. Results. Immunoblotting results demonstrated upregulations in Foxo1 and Foxo3a total protein levels (1.3- and 4.5-fold increases relative to euthermic control, for Foxo1 and 3a respectively) during late torpor, and protein levels remained elevated throughout the rest of torpor and at interbout arousal. We also observed decreases in inactive, phosphorylated Foxo1 and 3a proteins during throughout torpor, where levels of p-Foxo1 Ser(319) and Thr(24), as well as p-Foxo3a Thr(32) decreased by at least 45% throughout torpor. MyoG was

  9. Exchange protein directly activated by cAMP encoded by the mammalian rapgef3 gene: Structure, function and therapeutics.

    PubMed

    Banerjee, Upasana; Cheng, Xiaodong

    2015-10-10

    Mammalian exchange protein directly activated by cAMP isoform 1 (EPAC1), encoded by the RAPGEF3 gene, is one of the two-membered family of cAMP sensors that mediate the intracellular functions of cAMP by acting as guanine nucleotide exchange factors for the Ras-like Rap small GTPases. Extensive studies have revealed that EPAC1-mediated cAMP signaling is highly coordinated spatiotemporally through the formation of dynamic signalosomes by interacting with a diverse array of cellular partners. Recent functional analyses of genetically engineered mouse models further suggest that EPAC1 functions as an important stress response switch and is involved in pathophysiological conditions of cardiac stresses, chronic pain, cancer and infectious diseases. These findings, coupled with the development of EPAC specific small molecule modulators, validate EPAC1 as a promising target for therapeutic interventions.

  10. Exchange protein directly activated by cAMP encoded by the mammalian rapgef3 gene: Structure, function and therapeutics

    PubMed Central

    Banerjee, Upasana; Cheng, Xiaodong

    2015-01-01

    Mammalian exchange protein directly activated by cAMP isoform 1 (EPAC1), encoded by the RAPGEF3 gene, is one of the two-membered family of cAMP sensors that mediate the intracellular functions of cAMP by acting as guanine nucleotide exchange factors for the Ras-like Rap small GTPases. Extensive studies have revealed that EPAC1-mediated cAMP signaling is highly coordinated spatiotemporally through the formation of dynamic signalosomes by interacting with a diverse array of cellular partners. Recent functional analyses of genetically engineered mouse models further suggest that EPAC1 functions as an important stress response switch and is involved in pathophysiological conditions of cardiac stresses, chronic pain, cancer and infectious diseases. These findings, coupled with the development of EPAC specific small molecule modulators, validate EPAC1 as a promising target for therapeutic interventions. PMID:26119090

  11. Spatiotemporal regulation of GLI target genes in the mammalian limb bud

    PubMed Central

    Lewandowski, Jordan P.; Du, Fang; Zhang, Shilu; Powell, Marian B.; Falkenstein, Kristin N.; Ji, Hongkai; Vokes, Steven A.

    2015-01-01

    GLI proteins convert Sonic hedgehog (Shh) signaling into a transcriptional output in a tissue-specific fashion. The Shh pathway has been extensively studied in the limb bud, where it helps regulate growth through a SHH-FGF feedback loop. However, the transcriptional response is still poorly understood. We addressed this by determining the gene expression patterns of approximately 200 candidate GLI-target genes, and identified three discrete SHH-responsive expression domains. GLI-target genes expressed in the three domains are predominately regulated by derepression of GLI3 but have different temporal requirements for SHH. The GLI binding regions associated with these genes harbor both distinct and common DNA motifs. Given the potential for interaction between the SHH and FGF pathways, we also measured the response of GLI-target genes to inhibition of FGF signaling and found the majority were either unaffected or upregulated. These results provide the first characterization of the spatiotemporal response of a large group of GLI-target genes and lay the foundation for a systems-level understanding of the gene regulatory networks underlying SHH-mediated limb patterning. PMID:26238476

  12. Fluorescent protein-based detection of φC31 integrase activity in mammalian cells.

    PubMed

    Liu, Taian; Fang, Yongxiang; Jia, Huaijie; Chen, Guohua; Guan, Qisai; He, Xiaobing; Yao, Wenjuan; Zeng, Shuang; Jing, Zhizhong

    2013-10-15

    The enzyme φC31 integrase from Streptomyces phage has been documented as functional in mammalian cells and, therefore, has the potential to be a powerful gene manipulation tool. However, the activity of this enzyme is cell-type dependent. The more active mutant forms of φC31 integrase are required. Therefore, a rapid and effective method should be developed to detect the intracellular activity of φC31 integrase. We devised in this study an integrase-inversion cassette that contains the enhanced green fluorescent protein (EGFP) gene and the reverse complementary DsRed gene, which are flanked by attB and reverse complementary attP. This cassette can be inverted by φC31 integrase, thereby altering the fluorescent protein expression. Thus, φC31 integrase activity can be qualitatively or quantitatively evaluated based on the detected fluorescence. Furthermore, this cassette-based method was applied to several cell types, demonstrating that it is an efficient and reliable tool for measuring φC31 integrase activity in mammalian cells.

  13. Targeted Cytoplasmic Irradiation with Alpha Particles Induces Mutations in Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Wu, Li-Jun; Randers-Pehrson, Gerhard; Xu, An; Waldren, Charles A.; Geard, Charles R.; Yu, Zengliang; Hei, Tom K.

    1999-04-01

    Ever since x-rays were shown to induce mutation in Drosophila more than 70 years ago, prevailing dogma considered the genotoxic effects of ionizing radiation, such as mutations and carcinogenesis, as being due mostly to direct damage to the nucleus. Although there was indication that alpha particle traversal through cellular cytoplasm was innocuous, the full impact remained unknown. The availability of the microbeam at the Radiological Research Accelerator Facility of Columbia University made it possible to target and irradiate the cytoplasm of individual cells in a highly localized spatial region. By using dual fluorochrome dyes (Hoechst and Nile Red) to locate nucleus and cellular cytoplasm, respectively, thereby avoiding inadvertent traversal of nuclei, we show here that cytoplasmic irradiation is mutagenic at the CD59 (S1) locus of human-hamster hybrid (AL) cells, while inflicting minimal cytotoxicity. The principal class of mutations induced are similar to those of spontaneous origin and are entirely different from those of nuclear irradiation. Furthermore, experiments with radical scavenger and inhibitor of intracellular glutathione indicated that the mutagenicity of cytoplasmic irradiation depends on generation of reactive oxygen species. These findings suggest that cytoplasm is an important target for genotoxic effects of ionizing radiation, particularly radon, the second leading cause of lung cancer in the United States. In addition, cytoplasmic traversal by alpha particles may be more dangerous than nuclear traversal, because the mutagenicity is accomplished by little or no killing of the target cells.

  14. Target-specific variants of Flp recombinase mediate genome engineering reactions in mammalian cells.

    PubMed

    Shah, Riddhi; Li, Feng; Voziyanova, Eugenia; Voziyanov, Yuri

    2015-09-01

    Genome engineering relies on DNA-modifying enzymes that are able to locate a DNA sequence of interest and initiate a desired genome rearrangement. Currently, the field predominantly utilizes site-specific DNA nucleases that depend on the host DNA repair machinery to complete a genome modification task. We show here that genome engineering approaches that employ target-specific variants of the self-sufficient, versatile site-specific DNA recombinase Flp can be developed into promising alternatives. We demonstrate that the Flp variant evolved to recombine an FRT-like sequence, FL-IL10A, which is located upstream of the human interleukin-10 gene, and can target this sequence in the model setting of Chinese hamster ovary and human embryonic kidney 293 cells. This target-specific Flp variant is able to perform the integration reaction and, when paired with another recombinase, the dual recombinase-mediated cassette exchange reaction. The efficiency of the integration reaction in human cells can be enhanced by 'humanizing' the Flp variant gene and by adding the nuclear localization sequence to the recombinase.

  15. End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter

    PubMed Central

    Ide, Satoru; Dejardin, Jerome

    2015-01-01

    The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes. The proteomics of isolated chromatin fragment (PICh) method has previously been developed to purify telomeres and identify associated proteins. This approach is based on the affinity capture of endogenous chromatin segments by hybridization with oligonucleotide containing locked nucleic acids. However, PICh is only efficient with highly abundant genomic targets, limiting its applicability. Here we develop an approach for identifying factors bound to the promoter region of the ribosomal RNA genes that we call end-targeting PICh (ePICh). Using ePICh, we could specifically enrich the RNA polymerase I pre-initiation complex, including the selectivity factor 1. The high purity of the ePICh material allowed the identification of ZFP106, a novel factor regulating transcription initiation by targeting RNA polymerase I to the promoter. Our results demonstrate that ePICh can uncover novel proteins controlling endogenous regulatory elements in mammals. PMID:25812914

  16. End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter.

    PubMed

    Ide, Satoru; Dejardin, Jerome

    2015-01-01

    The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes. The proteomics of isolated chromatin fragment (PICh) method has previously been developed to purify telomeres and identify associated proteins. This approach is based on the affinity capture of endogenous chromatin segments by hybridization with oligonucleotide containing locked nucleic acids. However, PICh is only efficient with highly abundant genomic targets, limiting its applicability. Here we develop an approach for identifying factors bound to the promoter region of the ribosomal RNA genes that we call end-targeting PICh (ePICh). Using ePICh, we could specifically enrich the RNA polymerase I pre-initiation complex, including the selectivity factor 1. The high purity of the ePICh material allowed the identification of ZFP106, a novel factor regulating transcription initiation by targeting RNA polymerase I to the promoter. Our results demonstrate that ePICh can uncover novel proteins controlling endogenous regulatory elements in mammals. PMID:25812914

  17. Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate*

    PubMed Central

    Schwarz, Jennifer Jasmin; Wiese, Heike; Tölle, Regine Charlotte; Zarei, Mostafa; Dengjel, Jörn; Warscheid, Bettina; Thedieck, Kathrin

    2015-01-01

    The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and investigated in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation. PMID:25907765

  18. Phophatidylinositol-3 kinase/mammalian target of rapamycin/p70S6K regulates contractile protein accumulation in airway myocyte differentiation.

    PubMed

    Halayko, Andrew J; Kartha, Sreedharan; Stelmack, Gerald L; McConville, John; Tam, John; Camoretti-Mercado, Blanca; Forsythe, Sean M; Hershenson, Marc B; Solway, Julian

    2004-09-01

    Increased airway smooth muscle in airway remodeling results from myocyte proliferation and hypertrophy. Skeletal and vascular smooth muscle hypertrophy is induced by phosphatidylinositide-3 kinase (PI(3) kinase) via mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K). We tested the hypothesis that this pathway regulates contractile protein accumulation in cultured canine airway myocytes acquiring an elongated contractile phenotype in serum-free culture. In vitro assays revealed a sustained activation of PI(3) kinase and p70S6K during serum deprivation up to 12 d, with concomitant accumulation of SM22 and smooth muscle myosin heavy chain (smMHC) proteins. Immunocytochemistry revealed that activation of PI3K/mTOR/p70S6K occurred almost exclusively in myocytes that acquire the contractile phenotype. Inhibition of PI(3) kinase or mTOR with LY294002 or rapamycin blocked p70S6K activation, prevented formation of large elongated contractile phenotype myocytes, and blocked accumulation of SM22 and smMHC. Inhibition of MEK had no effect. Steady-state mRNA abundance for SM22 and smMHC was unaffected by blocking p70S6K activation. These studies provide primary evidence that PI(3) kinase and mTOR activate p70S6K in airway myocytes leading to the accumulation of contractile apparatus proteins, differentiation, and growth of large, elongated contractile phenotype airway smooth muscle cells. PMID:15105162

  19. Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets.

    PubMed

    Turanov, Anton A; Kehr, Sebastian; Marino, Stefano M; Yoo, Min-Hyuk; Carlson, Bradley A; Hatfield, Dolph L; Gladyshev, Vadim N

    2010-09-01

    The classical Trx (thioredoxin) system, composed of TR (Trx reductase), Trx and NADPH, defines a major pathway of cellular thiol-based redox regulation. Three TRs have been identified in mammals: (i) cytosolic TR1, (ii) mitochondrial TR3 and (iii) testes-specific TGR (Trx-glutathione reductase). All three are selenocysteine-containing enzymes with broad substrate specificity in in vitro assays, but which protein substrates are targeted by TRs in vivo is not well understood. In the present study, we used a mechanism-based approach to characterize the molecular targets of TR1. Cytosolic Trx1 was the major target identified in rat and mouse liver, as well as in rat brain and mouse serum. The results suggest that the main function of TR1 is to reduce Trx1. We also found that TR1-based affinity resins provide a convenient tool for specific isolation of Trxs from a variety of biological samples. To better assess the role of TRs in redox homoeostasis, we comparatively analysed TR1- and TR3-knockdown cells. Although cells deficient in TR1 were particularly sensitive to diamide, TR3-knockdown cells were more sensitive to hydrogen peroxide. To further examine the TR1-Trx1 redox pair, we used mice with a liver-specific knockout of selenocysteine tRNA. In this model, selenocysteine insertion into TR1 was blocked, but the truncated form of this protein was not detected. Instead, TR1 and TR3 levels were decreased in the knockout samples. Diminished hepatic TR1 function was associated with elevated Trx1 levels, but this protein was mostly in the oxidized state. Overall, this study provides evidence for the key role of the TR1-Trx1 pair in redox homoeostasis.

  20. The interaction between early life epilepsy and autistic-like behavioral consequences: a role for the mammalian target of rapamycin (mTOR) pathway.

    PubMed

    Talos, Delia M; Sun, Hongyu; Zhou, Xiangping; Fitzgerald, Erin C; Jackson, Michele C; Klein, Peter M; Lan, Victor J; Joseph, Annelise; Jensen, Frances E

    2012-01-01

    Early life seizures can result in chronic epilepsy, cognitive deficits and behavioral changes such as autism, and conversely epilepsy is common in autistic children. We hypothesized that during early brain development, seizures could alter regulators of synaptic development and underlie the interaction between epilepsy and autism. The mammalian Target of Rapamycin (mTOR) modulates protein translation and is dysregulated in Tuberous Sclerosis Complex, a disorder characterized by epilepsy and autism. We used a rodent model of acute hypoxia-induced neonatal seizures that results in long term increases in neuronal excitability, seizure susceptibility, and spontaneous seizures, to determine how seizures alter mTOR Complex 1 (mTORC1) signaling. We hypothesized that seizures occurring at a developmental stage coinciding with a critical period of synaptogenesis will activate mTORC1, contributing to epileptic networks and autistic-like behavior in later life. Here we show that in the rat, baseline mTORC1 activation peaks during the first three postnatal weeks, and induction of seizures at postnatal day 10 results in further transient activation of its downstream targets phospho-4E-BP1 (Thr37/46), phospho-p70S6K (Thr389) and phospho-S6 (Ser235/236), as well as rapid induction of activity-dependent upstream signaling molecules, including BDNF, phospho-Akt (Thr308) and phospho-ERK (Thr202/Tyr204). Furthermore, treatment with the mTORC1 inhibitor rapamycin immediately before and after seizures reversed early increases in glutamatergic neurotransmission and seizure susceptibility and attenuated later life epilepsy and autistic-like behavior. Together, these findings suggest that in the developing brain the mTORC1 signaling pathway is involved in epileptogenesis and altered social behavior, and that it may be a target for development of novel therapies that eliminate the progressive effects of neonatal seizures.

  1. The effects of selenium on glutathione peroxidase activity and radioprotection in mammalian cells

    SciTech Connect

    Diamond, A.M.; Murray, J.L.; Dale, P.; Tritz, R.; Grdina, D.J.

    1995-09-05

    The media of representative mammalian cell lines were supplemented with low levels of selenium in the form of sodium selenite in order to investigate the effects of selenium on mammalian cells. Following incubation in 30 nM sodium selenite, these cells were assayed for changes in glutathione peroxidase (GPx) activity. The cells examined included NIH 3T3 mouse fibroblasts, PC12 rat sympathetic precursor cells, SupT-1 human lymphocytes, MCF-7{sup adr} human breast carcinoma cells and AA8 Chinese hamster ovary cells. Selenium supplementation resulted in a marginal increase in GPx activity for the NIH 3T3, MCF-7{sup adr} and Supt-1 cells but stimulated GPx activity approximately 5-fold in PC12 and AA8 cells. AA8 cells were selected to evaluate whether selenium supplementation was radioprotective against {sup 60}cobalt gamma irradiation. Protection against radiation-induced mutation was measured by evaluating mutation frequency at the hprt locus. In this assay, preincubation of AA8 CHO cells significantly protected these cells from exposure to 8 Gy.

  2. Molecular targets, DNA breakage, DNA repair: Their roles in mutation induction in mammalian germ cells

    SciTech Connect

    Sega, G.A.

    1989-01-01

    Variability in genetic sensitivity among different germ-cell stages in the mammal to various mutagens could be the result of how much chemical reaches the different stages, what molecular targets may be affected in the different stages and whether or not repair of lesions occurs. Several chemicals have been found to bind very strongly to protamine in late-spermatid and early-spermatozoa stages in the mouse. The chemicals also produce their greatest genetic damage in these same germ-cell stages. While chemical binding to DNA has not been correlated with the level of induced genetic damage, DNA breakage in the sensitive stages has been shown to increase. This DNA breakage is believed to indirectly result from chemical binding to sulfhydryl groups in protamine which prevents normal chromatin condensation within the sperm nucleus. 22 refs., 5 figs.

  3. Mitochondria are targets for peroxisome-derived oxidative stress in cultured mammalian cells.

    PubMed

    Wang, Bo; Van Veldhoven, Paul P; Brees, Chantal; Rubio, Noemí; Nordgren, Marcus; Apanasets, Oksana; Kunze, Markus; Baes, Myriam; Agostinis, Patrizia; Fransen, Marc

    2013-12-01

    Many cellular processes are driven by spatially and temporally regulated redox-dependent signaling events. Although mounting evidence indicates that organelles such as the endoplasmic reticulum and mitochondria can function as signaling platforms for oxidative stress-regulated pathways, little is known about the role of peroxisomes in these processes. In this study, we employ targeted variants of the genetically encoded photosensitizer KillerRed to gain a better insight into the interplay between peroxisomes and cellular oxidative stress. We show that the phototoxic effects of peroxisomal KillerRed induce mitochondria-mediated cell death and that this process can be counteracted by targeted overexpression of a select set of antioxidant enzymes, including peroxisomal glutathione S-transferase kappa 1, superoxide dismutase 1, and mitochondrial catalase. We also present evidence that peroxisomal disease cell lines deficient in plasmalogen biosynthesis or peroxisome assembly are more sensitive to KillerRed-induced oxidative stress than control cells. Collectively, these findings confirm and extend previous observations suggesting that disturbances in peroxisomal redox control and metabolism can sensitize cells to oxidative stress. In addition, they lend strong support to the ideas that peroxisomes and mitochondria share a redox-sensitive relationship and that the redox communication between these organelles is not only mediated by diffusion of reactive oxygen species from one compartment to the other. Finally, these findings indicate that mitochondria may act as dynamic receivers, integrators, and transmitters of peroxisome-derived mediators of oxidative stress, and this may have profound implications for our views on cellular aging and age-related diseases.

  4. Cellular senescence or EGFR signaling induces Interleukin 6 (IL-6) receptor expression controlled by mammalian target of rapamycin (mTOR)

    PubMed Central

    Garbers, Christoph; Kuck, Fabian; Aparicio-Siegmund, Samadhi; Konzak, Kirstin; Kessenbrock, Mareike; Sommerfeld, Annika; Häussinger, Dieter; Lang, Philipp A; Brenner, Dirk; Mak, Tak W.; Rose-John, Stefan; Essmann, Frank; Schulze-Osthoff, Klaus; Piekorz, Roland P; Scheller, Jürgen

    2013-01-01

    Interleukin 6 (IL-6) signaling plays a role in inflammation, cancer, and senescence. Here, we identified soluble IL-6 receptor (sIL-6R) as a member of the senescence-associated secretory phenotype (SASP). Senescence-associated sIL-6R upregulation was mediated by mammalian target of rapamycin (mTOR). sIL-6R was mainly generated by a disintegrin and metalloprotease 10 (ADAM10)-dependent ectodomain shedding to enable IL-6 trans-signaling. In vivo, heterozygous PTEN-knockout mice exhibited higher mTOR activity and increased sIL-6R levels. Moreover, aberrant EGF receptor (EGFR) activation triggered IL-6 synthesis. In analogy to senescence, EGFR-induced activation of mTOR also induced IL-6R expression and sIL-6R generation. Hence, mTOR activation reprograms IL-6 non-responder cells into IL-6 responder cells. Our data suggest that mTOR serves as a central molecular switch to facilitate cellular IL-6 classic and trans-signaling via IL-6R upregulation with direct implications for cellular senescence and tumor development. PMID:24047696

  5. Acetylcholine activates an inward current in single mammalian smooth muscle cells.

    PubMed

    Benham, C D; Bolton, T B; Lang, R J

    Acetylcholine, the major excitatory neurotransmitter to the smooth muscle of mammalian intestine, is known to depolarize smooth muscle cells with an apparent increase in membrane conductance. However, the ionic mechanisms that are triggered by muscarinic receptor activation and underlie this response are poorly understood, due in part to the technical problems associated with the electrophysiological study of smooth muscle. The muscarinic action of acetylcholine in certain neurones has been shown to involve the switching off of a resting K+ current (M-current) and a similar mechanism has recently also been identified in smooth muscle of amphibian stomach. We have now applied the patch-clamp technique to single smooth muscle cells of rabbit jejunum and find that muscarinic receptor activation switches on a nonselective, voltage-sensitive inward current. In addition, acetylcholine activates and then suppresses spontaneous K+ current transients, which are probably triggered by rises in intracellular Ca2+ in these cells.

  6. Quantitative Persulfide Site Identification (qPerS-SID) Reveals Protein Targets of H2S Releasing Donors in Mammalian Cells.

    PubMed

    Longen, Sebastian; Richter, Florian; Köhler, Yvette; Wittig, Ilka; Beck, Karl-Friedrich; Pfeilschifter, Josef

    2016-07-14

    H2S is an important signalling molecule involved in diverse biological processes. It mediates the formation of cysteine persulfides (R-S-SH), which affect the activity of target proteins. Like thiols, persulfides show reactivity towards electrophiles and behave similarly to other cysteine modifications in a biotin switch assay. In this manuscript, we report on qPerS-SID a mass spectrometry-based method allowing the isolation of persulfide containing peptides in the mammalian proteome. With this method, we demonstrated that H2S donors differ in their efficacy to induce persulfides in HEK293 cells. Furthermore, data analysis revealed that persulfide formation affects all subcellular compartments and various cellular processes. Negatively charged amino acids appeared more frequently adjacent to cysteines forming persulfides. We confirmed our proteomic data using pyruvate kinase M2 as a model protein and showed that several cysteine residues are prone to persulfide formation finally leading to its inactivation. Taken together, the site-specific identification of persulfides on a proteome scale can help to identify target proteins involved in H2S signalling and enlightens the biology of H2S and its releasing agents.

  7. Quantitative Persulfide Site Identification (qPerS-SID) Reveals Protein Targets of H2S Releasing Donors in Mammalian Cells.

    PubMed

    Longen, Sebastian; Richter, Florian; Köhler, Yvette; Wittig, Ilka; Beck, Karl-Friedrich; Pfeilschifter, Josef

    2016-01-01

    H2S is an important signalling molecule involved in diverse biological processes. It mediates the formation of cysteine persulfides (R-S-SH), which affect the activity of target proteins. Like thiols, persulfides show reactivity towards electrophiles and behave similarly to other cysteine modifications in a biotin switch assay. In this manuscript, we report on qPerS-SID a mass spectrometry-based method allowing the isolation of persulfide containing peptides in the mammalian proteome. With this method, we demonstrated that H2S donors differ in their efficacy to induce persulfides in HEK293 cells. Furthermore, data analysis revealed that persulfide formation affects all subcellular compartments and various cellular processes. Negatively charged amino acids appeared more frequently adjacent to cysteines forming persulfides. We confirmed our proteomic data using pyruvate kinase M2 as a model protein and showed that several cysteine residues are prone to persulfide formation finally leading to its inactivation. Taken together, the site-specific identification of persulfides on a proteome scale can help to identify target proteins involved in H2S signalling and enlightens the biology of H2S and its releasing agents. PMID:27411966

  8. Quantitative Persulfide Site Identification (qPerS-SID) Reveals Protein Targets of H2S Releasing Donors in Mammalian Cells

    PubMed Central

    Longen, Sebastian; Richter, Florian; Köhler, Yvette; Wittig, Ilka; Beck, Karl-Friedrich; Pfeilschifter, Josef

    2016-01-01

    H2S is an important signalling molecule involved in diverse biological processes. It mediates the formation of cysteine persulfides (R-S-SH), which affect the activity of target proteins. Like thiols, persulfides show reactivity towards electrophiles and behave similarly to other cysteine modifications in a biotin switch assay. In this manuscript, we report on qPerS-SID a mass spectrometry-based method allowing the isolation of persulfide containing peptides in the mammalian proteome. With this method, we demonstrated that H2S donors differ in their efficacy to induce persulfides in HEK293 cells. Furthermore, data analysis revealed that persulfide formation affects all subcellular compartments and various cellular processes. Negatively charged amino acids appeared more frequently adjacent to cysteines forming persulfides. We confirmed our proteomic data using pyruvate kinase M2 as a model protein and showed that several cysteine residues are prone to persulfide formation finally leading to its inactivation. Taken together, the site-specific identification of persulfides on a proteome scale can help to identify target proteins involved in H2S signalling and enlightens the biology of H2S and its releasing agents. PMID:27411966

  9. Screening of mammalian target of rapamycin inhibitors in natural product extracts by capillary electrophoresis in combination with high performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Zhang, Yanmei; Li, Feng; Li, Mingxia; Kang, Jingwu

    2015-04-01

    In this study, capillary electrophoresis (CE) combined with HPLC-MS/MS were used as a powerful platform for screening of inhibitors of mammalian target of rapamycin (mTOR) in natural product extracts. The screening system has been established by using 5-carboxyfluorescein labeled substrate peptide F-4EBP1, a known mTOR inhibitor AZD8055, and a small chemical library consisted of 18 natural product extracts. Biochemical screening of natural product extracts was performed by CE with laser induced fluorescence detection. The CE separation allowed a quantitative measurement of the phosphorylated product, hence the quantitation of enzymatic inhibition as well as inhibition kinetics. The hits are readily identified as long as the peak area of the phosphorylated product is reduced in comparison with the negative control. Subsequent assay-guided isolation of the active natural product extract was performed with HPLC-MS/MS to track the particular active components. The structures of the identified active components were elucidated by the molecular ions and fragmentation information provided by MS/MS analysis. The CE-based assay method only requires minute pure compounds, which can be readily purified by HPLC. Therefore, the combination of CE and HPLC-MS/MS provides a high-throughput platform for screening bioactive compounds from the crude nature extracts. By taking the advantage of the screening system, salvianolic acid A and C in extract of Salvia miltiorrhiza were discovered as the new mTOR inhibitors.

  10. Oral mucosal injury caused by mammalian target of rapamycin inhibitors: emerging perspectives on pathobiology and impact on clinical practice.

    PubMed

    Peterson, Douglas E; O'Shaughnessy, Joyce A; Rugo, Hope S; Elad, Sharon; Schubert, Mark M; Viet, Chi T; Campbell-Baird, Cynthia; Hronek, Jan; Seery, Virginia; Divers, Josephine; Glaspy, John; Schmidt, Brian L; Meiller, Timothy F

    2016-08-01

    In recent years oral mucosal injury has been increasingly recognized as an important toxicity associated with mammalian target of rapamycin (mTOR) inhibitors, including in patients with breast cancer who are receiving everolimus. This review addresses the state-of-the-science regarding mTOR inhibitor-associated stomatitis (mIAS), and delineates its clinical characteristics and management. Given the clinically impactful pain associated with mIAS, this review also specifically highlights new research focusing on the study of the molecular basis of pain. The incidence of mIAS varies widely (2-78%). As reported across multiple mTOR inhibitor clinical trials, grade 3/4 toxicity occurs in up to 9% of patients. Managing mTOR-associated oral lesions with topical oral, intralesional, and/or systemic steroids can be beneficial, in contrast to the lack of evidence supporting steroid treatment of oral mucositis caused by high-dose chemotherapy or radiation. However, steroid management is not uniformly efficacious in all patients receiving mTOR inhibitors. Furthermore, technology does not presently exist to permit clinicians to predict a priori which of their patients will develop these lesions. There thus remains a strategic need to define the pathobiology of mIAS, the molecular basis of pain, and risk prediction relative to development of the clinical lesion. This knowledge could lead to novel future interventions designed to more effectively prevent mIAS and improve pain management if clinically significant mIAS lesions develop. PMID:27334013

  11. Antisense Oligonucleotides Targeting Parasite Inositol 1,4,5-Trisphosphate Receptor Inhibits Mammalian Host Cell Invasion by Trypanosoma cruzi

    NASA Astrophysics Data System (ADS)

    Hashimoto, Muneaki; Nara, Takeshi; Hirawake, Hiroko; Morales, Jorge; Enomoto, Masahiro; Mikoshiba, Katsuhiko

    2014-02-01

    Chagas disease is caused by an intracellular parasitic protist, Trypanosoma cruzi. As there are no highly effective drugs against this agent that also demonstrate low toxicity, there is an urgent need for development of new drugs to treat Chagas disease. We have previously demonstrated that the parasite inositol 1,4,5-trisphosphate receptor (TcIP3R) is crucial for invasion of the mammalian host cell by T. cruzi. Here, we report that TcIP3R is a short-lived protein and that its expression is significantly suppressed in trypomastigotes. Treatment of trypomastigotes, an infective stage of T. cruzi, with antisense oligonucleotides specific to TcIP3R deceased TcIP3R protein levels and impaired trypomastigote invasion of host cells. Due to the resulting instability and very low expression level of TcIP3R in trypomastigotes indicates that TcIP3R is a promising target for antisense therapy in Chagas disease.

  12. Identification of secondary targets of N-containing bisphosphonates in mammalian cells via parallel competition analysis of the barcoded yeast deletion collection

    PubMed Central

    Bivi, Nicoletta; Romanello, Milena; Harrison, Richard; Clarke, Ian; Hoyle, David C; Moro, Luigi; Ortolani, Fulvia; Bonetti, Antonella; Quadrifoglio, Franco; Tell, Gianluca; Delneri, Daniela

    2009-01-01

    Background Nitrogen-containing bisphosphonates are the elected drugs for the treatment of diseases in which excessive bone resorption occurs, for example, osteoporosis and cancer-induced bone diseases. The only known target of nitrogen-containing bisphosphonates is farnesyl pyrophosphate synthase, which ensures prenylation of prosurvival proteins, such as Ras. However, it is likely that the action of nitrogen-containing bisphosphonates involves additional unknown mechanisms. To identify novel targets of nitrogen-containing bisphosphonates, we used a genome-wide high-throughput screening in which 5,936 Saccharomyces cerevisiae heterozygote barcoded mutants were grown competitively in the presence of sub-lethal doses of three nitrogen-containing bisphosphonates (risedronate, alendronate and ibandronate). Strains carrying deletions in genes encoding potential drug targets show a variation of the intensity of their corresponding barcodes on the hybridization array over the time. Results With this approach, we identified novel targets of nitrogen-containing bisphosphonates, such as tubulin cofactor B and ASK/DBF4 (Activator of S-phase kinase). The up-regulation of tubulin cofactor B may explain some previously unknown effects of nitrogen-containing bisphosphonates on microtubule dynamics and organization. As nitrogen-containing bisphosphonates induce extensive DNA damage, we also document the role of DBF4 as a key player in nitrogen-containing bisphosphonate-induced cytotoxicity, thus explaining the effects on the cell-cycle. Conclusions The dataset obtained from the yeast screen was validated in a mammalian system, allowing the discovery of new biological processes involved in the cellular response to nitrogen-containing bisphosphonates and opening up opportunities for development of new anticancer drugs. PMID:19744312

  13. Role of inhibitors of mammalian target of rapamycin in the treatment of luminal breast cancer.

    PubMed

    Ciruelos, Eva; Cortes-Funes, Hernán; Ghanem, Ismael; Manso, Luis; Arteaga, Carlos

    2013-09-01

    Approximately 75% of patients with breast cancer present hormone receptor-positive tumors. This subtype of breast cancer initially shows a high overall response rate to hormonal treatments. However, resistance eventually develops, resulting in tumor progression. The PI3K/Akt/mTOR pathway regulates several cellular functions in cancer such as cell growth, survival, and proliferation. In addition, a high activation level of the PI3K/Akt/mTOR pathway is related to resistance to conventional chemotherapy and hormone therapy. The mTOR inhibitor everolimus, in combination with hormonal treatments, has led to excellent results in progression-free survival in patients with metastatic breast cancer resistant to hormone therapies. Therefore, everolimus has entered the National Comprehensive Cancer Network (NCCN) guidelines 2012 and its combination with exemestane was approved recently by the US Food and Drug Administration and the European Medicines Agency. This is the first time that a drug will have been approved for the restoration of hormone sensitivity in breast cancer.

  14. The mechanism of mTOR (mammalian target of rapamycin) in a mouse model of polycystic ovary syndrome (PCOS)

    PubMed Central

    2012-01-01

    Polycystic ovary syndrome (PCOS) is a common and complex endocrine disorder affecting 5-10% of women in reproductive age that is characterized by hyperandrogenism, oligo- or anovulation and infertility. However the pathophysiology of PCOS still remains unknown. The mammalian target of rapamycin (mTOR) is a central component that regulates various processes including cell growth, proliferation, metabolism, and angiogenesis. mTOR signaling cascade has recently been examined in ovarian follicles where it regulates granulosa cell proliferation and differentiation. mTOR functions as two complexes, mTOR complex 1 and 2. Therefore, we hypothesized that mTORC1 and/or 2 may have important role in proliferation of theca and granulosa cells in PCOS. In the present study, we sought to determine the mTOR signaling pathway in PCOS mouse ovary. We designed 3 groups: Control (C, no treatment), PCOS (P, The injection of DHEA (6 mg/100 g BW in 0.1 ml of sesame oil) (s.c) for 20 consecutive days), Vehicle (V, daily (s.c) sesame oil alone injection). Our results showed that mTORC1 and mTORC2-mediated signaling may play a role in PCOS mouse ovary. These findings provide evidence that mTORC1 and mTORC2 may have responsibility in increased ovarian follicular cell proliferation and growth in PCOS. Consequently, these results suggest that the mTOR signaling pathways (mTORC1 and mTORC 2) may create new clinical strategies to optimize developmental competence of PCOS should target correction of the entire follicle growth, oocyte development process and anovulatory infertility in PCOS. PMID:23185989

  15. Mammalian Antimicrobial Peptides: Promising Therapeutic Targets Against Infection and Chronic Inflammation.

    PubMed

    Dutta, Pujarini; Das, Santasabuj

    2016-01-01

    Antimicrobial peptides (AMPs) are integral components of the host innate immune system and functional throughout the plant and animal kingdoms. AMPs are short cationic molecules and lethal against a wide range of bacteria, viruses, fungi, yeast and protozoa due to their membranolytic effects on the negatively-charged microbial membranes. In addition, they exert multiple immunomodulatory roles like chemotaxis, modulation of cytokine and chemokine expression, leukocyte activation etc. Since AMPs suffer loss of microbicidal properties under serum and tissue environments, their capacity to modulate the immune system may predominates under the physiological conditions. Discovery of new antibiotics is lagging far behind the rapidly spreading drug resistance among the microorganisms. Both natural and synthetic AMPs have shown promise as 'next generation antibiotics' due to their unique mode of action, which minimises the chance of development of microbial resistance. In addition, they have therapeutic potential against non-infectious diseases like chronic inflammation and cancer. Many of the synthetic AMPs are currently undergoing clinical trials for the treatment of debilitating diseases, such as catheter-related infections, diabetic foot ulcers, chemotherapy-associated infections etc. Some of them have already entered the market as topical preparations. In this review, we synopsise the current literature of natural and synthetic AMPs in different infectious and inflammatory diseases of human microfloral habitats, especially the gastrointestinal, respiratory and genitourinary tracts and the skin. We also discuss the classification of AMPs, their mode action and antimicrobial spectrum, including the pathogen evasion mechanisms. In short, we tried to present the locus standi of AMPs in relation to human diseases and highlight the most promising synthetic peptides emerging from the clinical trials. Finally, we focused on the limitations and hurdles in terms of cost of

  16. Mammalian TBX1 preferentially binds and regulates downstream targets via a tandem T-site repeat.

    PubMed

    Castellanos, Raquel; Xie, Qing; Zheng, Deyou; Cvekl, Ales; Morrow, Bernice E

    2014-01-01

    Haploinsufficiency or mutation of TBX1 is largely responsible for the etiology of physical malformations in individuals with velo-cardio-facial/DiGeorge syndrome (VCFS/DGS/22q11.2 deletion syndrome). TBX1 encodes a transcription factor protein that contains an evolutionarily conserved DNA binding domain termed the T-box that is shared with other family members. All T-box proteins, examined so far, bind to similar but not identical consensus DNA sequences, indicating that they have specific binding preferences. To identify the TBX1 specific consensus sequence, Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was performed. In contrast to other TBX family members recognizing palindrome sequences, we found that TBX1 preferentially binds to a tandem repeat of 5'-AGGTGTGAAGGTGTGA-3'. We also identified a second consensus sequence comprised of a tandem repeat with a degenerated downstream site. We show that three known human disease-causing TBX1 missense mutations (F148Y, H194Q and G310S) do not alter nuclear localization, or disrupt binding to the tandem repeat consensus sequences, but they reduce transcriptional activity in cell culture reporter assays. To identify Tbx1-downstream genes, we performed an in silico genome wide analysis of potential cis-acting elements in DNA and found strong enrichment of genes required for developmental processes and transcriptional regulation. We found that TBX1 binds to 19 different loci in vitro, which may correspond to putative cis-acting binding sites. In situ hybridization coupled with luciferase gene reporter assays on three gene loci, Fgf8, Bmper, Otog-MyoD, show that these motifs are directly regulated by TBX1 in vitro. Collectively, the present studies establish new insights into molecular aspects of TBX1 binding to DNA. This work lays the groundwork for future in vivo studies, including chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) to further elucidate the molecular

  17. Opposing motor activities are required for the organization of the mammalian mitotic spindle pole

    PubMed Central

    1996-01-01

    We use both in vitro and in vivo approaches to examine the roles of Eg5 (kinesin-related protein), cytoplasmic dynein, and dynactin in the organization of the microtubules and the localization of NuMA (Nu-clear protein that associates with the Mitotic Apparatus) at the polar ends of the mammalian mitotic spindle. Perturbation of the function of Eg5 through either immunodepletion from a cell free system for assembly of mitotic asters or antibody microinjection into cultured cells leads to organized astral microtubule arrays with expanded polar regions in which the minus ends of the microtubules emanate from a ring-like structure that contains NuMA. Conversely, perturbation of the function of cytoplasmic dynein or dynactin through either specific immunodepletition from the cell free system or expression of a dominant negative subunit of dynactin in cultured cells results in the complete lack of organization of microtubules and the failure to efficiently concentrate the NuMA protein despite its association with the microtubules. Simultaneous immunodepletion of these proteins from the cell free system for mitotic aster assembly indicates that the plus end- directed activity of Eg5 antagonizes the minus end-directed activity of cytoplasmic dynein and a minus end-directed activity associated with NuMA during the organization of the microtubules into a morphologic pole. Taken together, these results demonstrate that the unique organization of the minus ends of microtubules and the localization of NuMA at the polar ends of the mammalian mitotic spindle can be accomplished in a centrosome-independent manner by the opposing activities of plus end- and minus end-directed motors. PMID:8896597

  18. Target activation by regulatory RNAs in bacteria

    PubMed Central

    Papenfort, Kai; Vanderpool, Carin K.

    2015-01-01

    Bacterial small regulatory RNAs (sRNAs) are commonly known to repress gene expression by base pairing to target mRNAs. In many cases, sRNAs base pair with and sequester mRNA ribosome-binding sites, resulting in translational repression and accelerated transcript decay. In contrast, a growing number of examples of translational activation and mRNA stabilization by sRNAs have now been documented. A given sRNA often employs a conserved region to interact with and regulate both repressed and activated targets. However, the mechanisms underlying activation differ substantially from repression. Base pairing resulting in target activation can involve sRNA interactions with the 5′ untranslated region (UTR), the coding sequence or the 3′ UTR of the target mRNAs. Frequently, the activities of protein factors such as cellular ribonucleases and the RNA chaperone Hfq are required for activation. Bacterial sRNAs, including those that function as activators, frequently control stress response pathways or virulence-associated functions required for immediate responses to changing environments. This review aims to summarize recent advances in knowledge regarding target mRNA activation by bacterial sRNAs, highlighting the molecular mechanisms and biological relevance of regulation. PMID:25934124

  19. The Mammalian "Obesogen" Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish.

    PubMed

    Lyssimachou, Angeliki; Santos, Joana G; André, Ana; Soares, Joana; Lima, Daniela; Guimarães, Laura; Almeida, C Marisa R; Teixeira, Catarina; Castro, L Filipe C; Santos, Miguel M

    2015-01-01

    Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an "obesogenic" phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound.

  20. The Mammalian “Obesogen” Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish

    PubMed Central

    Lyssimachou, Angeliki; Santos, Joana G.; André, Ana; Soares, Joana; Lima, Daniela; Guimarães, Laura; Almeida, C. Marisa R.; Teixeira, Catarina; Castro, L. Filipe C.; Santos, Miguel M.

    2015-01-01

    Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an “obesogenic” phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound. PMID:26633012

  1. Dihydroartemisinin ameliorates inflammatory disease by its reciprocal effects on Th and regulatory T cell function via modulating the mammalian target of rapamycin pathway.

    PubMed

    Zhao, Yan G; Wang, Yunqi; Guo, Zengli; Gu, Ai-di; Dan, Han C; Baldwin, Albert S; Hao, Weidong; Wan, Yisong Y

    2012-11-01

    Dihydroartemisinin (DHA) is an important derivative of the herb medicine Artemisia annua L., used in ancient China. DHA is currently used worldwide to treat malaria by killing malaria-causing parasites. In addition to this prominent effect, DHA is thought to regulate cellular functions, such as angiogenesis, tumor cell growth, and immunity. Nonetheless, how DHA affects T cell function remains poorly understood. We found that DHA potently suppressed Th cell differentiation in vitro. Unexpectedly, however, DHA greatly promoted regulatory T cell (Treg) generation in a manner dependent on the TGF-βR:Smad signal. In addition, DHA treatment effectively reduced onset of experimental autoimmune encephalomyelitis (EAE) and ameliorated ongoing EAE in mice. Administration of DHA significantly decreased Th but increased Tregs in EAE-inflicted mice, without apparent global immune suppression. Moreover, DHA modulated the mammalian target of rapamycin (mTOR) pathway, because mTOR signal was attenuated in T cells upon DHA treatment. Importantly, enhanced Akt activity neutralized DHA-mediated effects on T cells in an mTOR-dependent fashion. This study therefore reveals a novel immune regulatory function of DHA in reciprocally regulating Th and Treg cell generation through the modulating mTOR pathway. It addresses how DHA regulates immune function and suggests a new type of drug for treating diseases in which mTOR activity is to be tempered. PMID:22993204

  2. Noncoding flavivirus RNA displays RNA interference suppressor activity in insect and Mammalian cells.

    PubMed

    Schnettler, Esther; Sterken, Mark G; Leung, Jason Y; Metz, Stefan W; Geertsema, Corinne; Goldbach, Rob W; Vlak, Just M; Kohl, Alain; Khromykh, Alexander A; Pijlman, Gorben P

    2012-12-01

    West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3'-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses. PMID:23035235

  3. Bothriurus bonariensis scorpion venom activates voltage-dependent sodium channels in insect and mammalian nervous systems.

    PubMed

    Dos Santos, Douglas Silva; Carvalho, Evelise Leis; de Lima, Jeferson Camargo; Breda, Ricardo Vaz; Oliveira, Raquel Soares; de Freitas, Thiago Carrazoni; Salamoni, Simone Denise; Domingues, Michelle Flores; Piovesan, Angela Regina; Boldo, Juliano Tomazzoni; de Assis, Dênis Reis; da Costa, Jaderson Costa; Dal Belo, Cháriston André; Pinto, Paulo Marcos

    2016-10-25

    Animal venoms have been widely recognized as a major source of biologically active molecules. Bothriurus bonariensis, popularly known as black scorpion, is the arthropod responsible for the highest number of accidents involving scorpion sting in Southern Brazil. Here we reported the first attempt to investigate the neurobiology of B. bonariensis venom (BBV) in the insect and mammalian nervous system. BBV (32 μg/g) induced a slow neuromuscular blockade in the in vivo cockroach nerve-muscle preparations (70 ± 4%, n = 6, p < 0.001), provoking repetitive twitches and significantly decreasing the frequency of spontaneous leg action potentials (SNCAPs) from 82 ± 3 min(-1) to 36 ± 1.3 min(-1) (n = 6, p < 0.05), without affecting the amplitude. When tested in primary cultures of rat hippocampal cells, BBV induced a massive increase of Ca(2+) influx (250 ± 1% peak increase, n = 3, p < 0.0001). The disturbance of calcium homeostasis induced by BBV on the mammalian central nervous system was not accompanied by cellular death and was prevented by the co-treatment of the hippocampal cells with tetrodotoxin, a selective sodium channel blocker. The results suggest that the biological activity of BBV is mostly related to a modulation of sodium channels function. Our biological activity survey suggests that BBV may have a promising insecticidal and therapeutic potential. PMID:27544632

  4. Noncoding Flavivirus RNA Displays RNA Interference Suppressor Activity in Insect and Mammalian Cells

    PubMed Central

    Schnettler, Esther; Sterken, Mark G.; Leung, Jason Y.; Metz, Stefan W.; Geertsema, Corinne; Goldbach, Rob W.; Vlak, Just M.; Kohl, Alain

    2012-01-01

    West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3′-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses. PMID:23035235

  5. Mammalian Pheromones

    PubMed Central

    Liberles, Stephen D.

    2015-01-01

    Mammalian pheromones control a myriad of innate social behaviors and acutely regulate hormone levels. Responses to pheromones are highly robust, reproducible, and stereotyped and likely involve developmentally predetermined neural circuits. Here, I review several facets of pheromone transduction in mammals, including (a) chemosensory receptors and signaling components of the main olfactory epithelium and vomeronasal organ involved in pheromone detection; (b) pheromone-activated neural circuits subject to sex-specific and state-dependent modulation; and (c) the striking chemical diversity of mammalian pheromones, which range from small, volatile molecules and sulfated steroids to large families of proteins. Finally, I review (d ) molecular mechanisms underlying various behavioral and endocrine responses, including modulation of puberty and estrous; control of reproduction, aggression, suckling, and parental behaviors; individual recognition; and distinguishing of own species from predators, competitors, and prey. Deconstruction of pheromone transduction mechanisms provides a critical foundation for understanding how odor response pathways generate instinctive behaviors. PMID:23988175

  6. The nematode Caenorhabditis elegans as a tool to predict chemical activity on mammalian development and identify mechanisms influencing toxicological outcome.

    PubMed

    Harlow, Philippa H; Perry, Simon J; Widdison, Stephanie; Daniels, Shannon; Bondo, Eddie; Lamberth, Clemens; Currie, Richard A; Flemming, Anthony J

    2016-01-01

    To determine whether a C. elegans bioassay could predict mammalian developmental activity, we selected diverse compounds known and known not to elicit such activity and measured their effect on C. elegans egg viability. 89% of compounds that reduced C. elegans egg viability also had mammalian developmental activity. Conversely only 25% of compounds found not to reduce egg viability in C. elegans were also inactive in mammals. We conclude that the C. elegans egg viability assay is an accurate positive predictor, but an inaccurate negative predictor, of mammalian developmental activity. We then evaluated C. elegans as a tool to identify mechanisms affecting toxicological outcomes among related compounds. The difference in developmental activity of structurally related fungicides in C. elegans correlated with their rate of metabolism. Knockdown of the cytochrome P450s cyp-35A3 and cyp-35A4 increased the toxicity to C. elegans of the least developmentally active compounds to the level of the most developmentally active. This indicated that these P450s were involved in the greater rate of metabolism of the less toxic of these compounds. We conclude that C. elegans based approaches can predict mammalian developmental activity and can yield plausible hypotheses for factors affecting the biological potency of compounds in mammals.

  7. The nematode Caenorhabditis elegans as a tool to predict chemical activity on mammalian development and identify mechanisms influencing toxicological outcome

    PubMed Central

    Harlow, Philippa H.; Perry, Simon J.; Widdison, Stephanie; Daniels, Shannon; Bondo, Eddie; Lamberth, Clemens; Currie, Richard A.; Flemming, Anthony J.

    2016-01-01

    To determine whether a C. elegans bioassay could predict mammalian developmental activity, we selected diverse compounds known and known not to elicit such activity and measured their effect on C. elegans egg viability. 89% of compounds that reduced C. elegans egg viability also had mammalian developmental activity. Conversely only 25% of compounds found not to reduce egg viability in C. elegans were also inactive in mammals. We conclude that the C. elegans egg viability assay is an accurate positive predictor, but an inaccurate negative predictor, of mammalian developmental activity. We then evaluated C. elegans as a tool to identify mechanisms affecting toxicological outcomes among related compounds. The difference in developmental activity of structurally related fungicides in C. elegans correlated with their rate of metabolism. Knockdown of the cytochrome P450s cyp-35A3 and cyp-35A4 increased the toxicity to C. elegans of the least developmentally active compounds to the level of the most developmentally active. This indicated that these P450s were involved in the greater rate of metabolism of the less toxic of these compounds. We conclude that C. elegans based approaches can predict mammalian developmental activity and can yield plausible hypotheses for factors affecting the biological potency of compounds in mammals. PMID:26987796

  8. Autophagy inhibition enhances colorectal cancer apoptosis induced by dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235

    PubMed Central

    YANG, XIAOYU; NIU, BINGXUAN; WANG, LIBO; CHEN, MEILING; KANG, XIAOCHUN; WANG, LUONAN; JI, YINGHUA; ZHONG, JIATENG

    2016-01-01

    Phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway performs a central role in tumorigenesis and is constitutively activated in many malignancies. As a novel dual PI3K/mTOR inhibitor currently undergoing evaluation in a phase I/II clinical trial, NVP-BEZ235 indicates a significant antitumor efficacy in diverse solid tumors, including colorectal cancer (CRC). Autophagy is a catabolic process that maintains cellular homeostasis and reduces diverse stresses through lysosomal recycling of the unnecessary and damaged cell components. This process is also observed to antagonize the antitumor efficacy of PI3K/mTOR inhibitor agents such as NVP-BEZ235, via apoptosis inhibition. In the present study, we investigated anti-proliferative and apoptosis-inducing ability of NVP-BEZ235 in SW480 cells and the crosstalk between autophagy and apoptosis in SW480 cells treated with NVP-BEZ235 in combination with an autophagy inhibitor. The results revealed that, NVP-BEZ235 effectively inhibit the growth of SW480 cells by targeting the PI3K/mTOR signaling pathway and induced apoptosis. The inhibition of autophagy with 3-methyladenine or chloroquine inhibitors in combination with NVP-BEZ235 in SW480 cells enhanced the apoptotic rate as componets to NVP-BEZ235 alone. In conclusion, the findings provide a rationale for chemotherapy targeting the PI3K/mTOR signaling pathway presenting a potential therapeutic strategy to enhance the efficacy of dual PI3K/mTOR inhibitor NVP-BEZ235 in combination with an autophagy inhibitor in CRC treatment and treatment of other tumors. PMID:27347108

  9. BAP, a mammalian BiP-associated protein, is a nucleotide exchange factor that regulates the ATPase activity of BiP.

    PubMed

    Chung, Kyung Tae; Shen, Ying; Hendershot, Linda M

    2002-12-01

    We identified a mammalian BiP-associated protein, BAP, using a yeast two-hybrid screen that shared low homology with yeast Sls1p/Sil1p and mammalian HspBP1, both of which regulate the ATPase activity of their Hsp70 partner. BAP encoded an approximately 54-kDa protein with an N-terminal endoplasmic reticulum (ER) targeting sequence, two sites of N-linked glycosylation, and a C-terminal ER retention sequence. Immunofluorescence staining demonstrated that BAP co-localized with GRP94 in the endoplasmic reticulum. BAP was ubiquitously expressed but showed the highest levels of expression in secretory organ tissues, a pattern similar to that observed with BiP. BAP binding was affected by the conformation of the ATPase domain of BiP based on in vivo binding studies with BiP mutants. BAP stimulated the ATPase activity of BiP when added alone or together with the ER DnaJ protein, ERdj4, by promoting the release of ADP from BiP. Together, these data demonstrate that BAP serves as a nucleotide exchange factor for BiP and provide insights into the mechanisms that control protein folding in the mammalian ER.

  10. High Inorganic Triphosphatase Activities in Bacteria and Mammalian Cells: Identification of the Enzymes Involved

    PubMed Central

    Lakaye, Bernard; Servais, Anne-Catherine; Scholer, Georges; Fillet, Marianne; Elias, Benjamin; Derochette, Jean-Michel; Crommen, Jacques; Wins, Pierre; Bettendorff, Lucien

    2012-01-01

    Background We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. Methodology/Principal Findings Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPPi) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPPi but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. Conclusions and General Significance We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPPi in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPPi, which could be cytotoxic because of its high affinity for Ca2+, thereby interfering with Ca2+ signaling. PMID:22984449

  11. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor.

    PubMed

    Ukhanov, K; Bobkov, Y; Corey, E A; Ache, B W

    2014-10-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca(2+) release and/or Ca(2+) influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. PMID:25149566

  12. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor

    PubMed Central

    Ukhanov, K.; Bobkov, Y.; Corey, E.A.; Ache, B.W.

    2014-01-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca2+ release and/or Ca2+ influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5 mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. PMID:25149566

  13. Mammalian Glucose Transporter Activity Is Dependent upon Anionic and Conical Phospholipids*

    PubMed Central

    Hresko, Richard C.; Kraft, Thomas E.; Quigley, Andrew; Carpenter, Elisabeth P.; Hruz, Paul W.

    2016-01-01

    The regulated movement of glucose across mammalian cell membranes is mediated by facilitative glucose transporters (GLUTs) embedded in lipid bilayers. Despite the known importance of phospholipids in regulating protein structure and activity, the lipid-induced effects on the GLUTs remain poorly understood. We systematically examined the effects of physiologically relevant phospholipids on glucose transport in liposomes containing purified GLUT4 and GLUT3. The anionic phospholipids, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, were found to be essential for transporter function by activating it and stabilizing its structure. Conical lipids, phosphatidylethanolamine and diacylglycerol, enhanced transporter activity up to 3-fold in the presence of anionic phospholipids but did not stabilize protein structure. Kinetic analyses revealed that both lipids increase the kcat of transport without changing the Km values. These results allowed us to elucidate the activation of GLUT by plasma membrane phospholipids and to extend the field of membrane protein-lipid interactions to the family of structurally and functionally related human solute carriers. PMID:27302065

  14. Mammalian Glucose Transporter Activity Is Dependent upon Anionic and Conical Phospholipids.

    PubMed

    Hresko, Richard C; Kraft, Thomas E; Quigley, Andrew; Carpenter, Elisabeth P; Hruz, Paul W

    2016-08-12

    The regulated movement of glucose across mammalian cell membranes is mediated by facilitative glucose transporters (GLUTs) embedded in lipid bilayers. Despite the known importance of phospholipids in regulating protein structure and activity, the lipid-induced effects on the GLUTs remain poorly understood. We systematically examined the effects of physiologically relevant phospholipids on glucose transport in liposomes containing purified GLUT4 and GLUT3. The anionic phospholipids, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, were found to be essential for transporter function by activating it and stabilizing its structure. Conical lipids, phosphatidylethanolamine and diacylglycerol, enhanced transporter activity up to 3-fold in the presence of anionic phospholipids but did not stabilize protein structure. Kinetic analyses revealed that both lipids increase the kcat of transport without changing the Km values. These results allowed us to elucidate the activation of GLUT by plasma membrane phospholipids and to extend the field of membrane protein-lipid interactions to the family of structurally and functionally related human solute carriers. PMID:27302065

  15. Rapamycin-mediated inhibition of mammalian target of rapamycin in skeletal muscle cells reduces glucose utilization and increases fatty acid oxidation.

    PubMed

    Sipula, Ian J; Brown, Nicholas F; Perdomo, German

    2006-12-01

    The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays an important role in cell growth and metabolism. mTOR has been postulated as a nutrient sensor, but its role in the regulation of fatty acid and glucose metabolism is poorly understood. For the first time, we show that mTOR inhibition in skeletal muscle cells has pronounced effects on intermediary metabolism. Rapamycin, a uniquely specific mTOR inhibitor with clinical applications, increased fatty acid oxidation by 60% accompanied by increased activities of carnitine palmitoyltransferases I and II, the former believed to be the primary intracellular regulatory enzyme of the fatty acid oxidation pathway. Furthermore, glucose transport capacity, glycogen synthesis, and glycolysis were reduced by approximately 40% under the same conditions. In addition, in the presence of rapamycin, hyperinsulinemic conditions (100 nmol/L insulin, 24 hours) were unable to suppress fatty acid oxidation in L6 myotubes. Rapamycin treatment also decreased baseline phosphorylation of mTOR residues S2448 and S2481 by 30% and almost completely abolished p70 S6 kinase phosphorylation. These results show that rapamycin causes a metabolic shift from glucose utilization to fatty acid oxidation in model muscle cells in the presence of nutrient abundance and underline the importance of mTOR as a key regulator in glucose and lipid metabolism. PMID:17142137

  16. Video Guidance Sensors Using Remotely Activated Targets

    NASA Technical Reports Server (NTRS)

    Bryan, Thomas C.; Howard, Richard T.; Book, Michael L.

    2004-01-01

    Four updated video guidance sensor (VGS) systems have been proposed. As described in a previous NASA Tech Briefs article, a VGS system is an optoelectronic system that provides guidance for automated docking of two vehicles. The VGS provides relative position and attitude (6-DOF) information between the VGS and its target. In the original intended application, the two vehicles would be spacecraft, but the basic principles of design and operation of the system are applicable to aircraft, robots, objects maneuvered by cranes, or other objects that may be required to be aligned and brought together automatically or under remote control. In the first two of the four VGS systems as now proposed, the tracked vehicle would include active targets that would light up on command from the tracking vehicle, and a video camera on the tracking vehicle would be synchronized with, and would acquire images of, the active targets. The video camera would also acquire background images during the periods between target illuminations. The images would be digitized and the background images would be subtracted from the illuminated-target images. Then the position and orientation of the tracked vehicle relative to the tracking vehicle would be computed from the known geometric relationships among the positions of the targets in the image, the positions of the targets relative to each other and to the rest of the tracked vehicle, and the position and orientation of the video camera relative to the rest of the tracking vehicle. The major difference between the first two proposed systems and prior active-target VGS systems lies in the techniques for synchronizing the flashing of the active targets with the digitization and processing of image data. In the prior active-target VGS systems, synchronization was effected, variously, by use of either a wire connection or the Global Positioning System (GPS). In three of the proposed VGS systems, the synchronizing signal would be generated on, and

  17. Activity of creatine kinase in a contracting mammalian muscle of uniform fiber type.

    PubMed Central

    McFarland, E W; Kushmerick, M J; Moerland, T S

    1994-01-01

    We investigated whether the creatine kinase-catalyzed phosphate exchange between PCr and gamma ATP in vivo equilibrated with cellular substrates and products as predicted by in vitro kinetic properties of the enzyme, or was a function of ATPase activity as predicted by obligatory "creatine phosphate shuttle" concepts. A transient NMR spin-transfer method was developed, tested, and applied to resting and stimulated ex vivo muscle, the soleus, which is a cellularly homogeneous slow-twitch mammalian muscle, to measure creatine kinase kinetics. The forward and reverse unidirectional CK fluxes were equal, being 1.6 mM.s-1 in unstimulated muscle at 22 degrees C, and 2.7 mM.s-1 at 30 degrees C. The CK fluxes did not differ during steady-state stimulation conditions giving a 10-fold range of ATPase rates in which the ATP/PCr ratio increased from approximately 0.3 to 1.6. The observed kinetic behavior of CK activity in the muscle was that expected from the enzyme in vitro in a homogeneous solution only if account was taken of inhibition by an anion-stabilized quaternary dead-end enzyme complex: E.Cr.MgADP.anion. The CK fluxes in soleus were not a function of ATPase activity as predicted by obligatory phosphocreatine shuttle models for cellular energetics. PMID:7858128

  18. 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.

  19. Isolation of the protein and RNA content of active sites of transcription from mammalian cells.

    PubMed

    Melnik, Svitlana; Caudron-Herger, Maïwen; Brant, Lilija; Carr, Ian M; Rippe, Karsten; Cook, Peter R; Papantonis, Argyris

    2016-03-01

    Mammalian cell nuclei contain three RNA polymerases (RNAP I, RNAP II and RNAP III), which transcribe different gene subsets, and whose active forms are contained in supramolecular complexes known as 'transcription factories.' These complexes are difficult to isolate because they are embedded in the 3D structure of the nucleus. Factories exchange components with the soluble nucleoplasmic pool over time as gene expression programs change during development or disease. Analysis of their content can provide information on the nascent transcriptome and its regulators. Here we describe a protocol for the isolation of large factory fragments under isotonic salt concentrations in <72 h. It relies on DNase I-mediated detachment of chromatin from the nuclear substructure of freshly isolated, unfixed cells, followed by caspase treatment to release multi-megadalton factory complexes. These complexes retain transcriptional activity, and isolation of their contents is compatible with downstream analyses by mass spectrometry (MS) or RNA-sequencing (RNA-seq) to catalog the proteins and RNA associated with sites of active transcription. PMID:26914315

  20. Dual inhibition of phosphatidylinositol 3-kinase/Akt and mammalian target of rapamycin signaling in human nonsmall cell lung cancer cells by a dietary flavonoid fisetin.

    PubMed

    Khan, Naghma; Afaq, Farrukh; Khusro, Fatima H; Mustafa Adhami, Vaqar; Suh, Yewseok; Mukhtar, Hasan

    2012-04-01

    Lung cancer is one of the most commonly occurring malignancies. It has been reported that mammalian target of rapamycin (mTOR) is phosphorylated in lung cancer and its activation was more frequent in tumors with overexpression of phosphatidylinositol 3-kinase (PI3K)/Akt. Therefore, dual inhibitors of PI3K/Akt and mTOR signaling could be valuable agents for treating lung cancer. In the present study, we show that fisetin, a dietary tetrahydroxyflavone inhibits cell growth with the concomitant suppression of PI3K/Akt and mTOR signaling in human nonsmall cell lung cancer (NSCLC) cells. Using autodock 4, we found that fisetin physically interacts with the mTOR complex at two sites. Fisetin treatment was also found to reduce the formation of A549 cell colonies in a dose-dependent manner. Treatment of cells with fisetin caused decrease in the protein expression of PI3K (p85 and p110), inhibition of phosphorylation of Akt, mTOR, p70S6K1, eIF-4E and 4E-BP1. Fisetin-treated cells also exhibited dose-dependent inhibition of the constituents of mTOR signaling complex such as Rictor, Raptor, GβL and PRAS40. There was an increase in the phosphorylation of AMPKα and a decrease in the phosphorylation of TSC2 on treatment of cells with fisetin. We also found that treatment of cells with mTOR inhibitor rapamycin and mTOR-siRNA caused decrease in phosphorylation of mTOR and its target proteins which were further downregulated on treatment with fisetin, suggesting that these effects are mediated in part, through mTOR signaling. Our results show that fisetin suppressed PI3K/Akt and mTOR signaling in NSCLC cells and thus, could be developed as a chemotherapeutic agent against human lung cancer.

  1. The novel orally bioavailable inhibitor of phosphoinositol-3-kinase and mammalian target of rapamycin, NVP-BEZ235, inhibits growth and proliferation in multiple myeloma

    SciTech Connect

    Baumann, Philipp Mandl-Weber, Sonja; Oduncu, Fuat; Schmidmaier, Ralf

    2009-02-01

    NVP-BEZ235 is a new inhibitor of phosphoinositol-3-kinase (PI3 kinase) and mammalian target of rapamycin (mTOR) whose efficacy in advanced solid tumours is currently being evaluated in a phase I/II clinical trial. Here we show that NVP-BEZ235 inhibits growth in common myeloma cell lines as well as primary myeloma cells at nanomolar concentrations in a time and dose dependent fashion. Further experiments revealed induction of apoptosis in three of four cell lines. Inhibition of cell growth was mainly due to inhibition of myeloma cell proliferation, as shown by the BrdU assay. Cell cycle analysis revealed induction of cell cycle arrest in the G1 phase, which was due to downregulation of cyclin D1, pRb and cdc25a. NVP-BEZ235 inhibited phosphorylation of protein kinase B (Akt), P70S6k and 4E-BP-1. Furthermore we show that the stimulatory effect of CD40-ligand (CD40L), insulin-like growth factor 1 (IGF-1), interleukin-6 (IL-6) and conditioned medium of HS-5 stromal cells on myeloma cell growth is completely abrogated by NVP-BEZ235. In addition, synergism studies revealed synergistic and additive activity of NVP-BEZ235 together with melphalan, doxorubicin and bortezomib. Taken together, inhibition of PI3 kinase/mTOR by NVP-BEZ235 is highly effective and NVP-BEZ235 represents a potential new candidate for targeted therapy in multiple myeloma.

  2. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    PubMed

    Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

    2007-10-01

    Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase. PMID:17850513

  3. Anti-Breast Cancer Potential of Quercetin via the Akt/AMPK/Mammalian Target of Rapamycin (mTOR) Signaling Cascade

    PubMed Central

    Rivera Rivera, Amilcar; Castillo-Pichardo, Linette; Gerena, Yamil; Dharmawardhane, Suranganie

    2016-01-01

    The Akt/adenosine monophosphate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has emerged as a critical signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth. Thus, dysregulation of this pathway contributes to the development of metabolic disorders such as obesity, type 2diabetes, and cancer. We previously reported that a combination of grape polyphenols (resveratrol, quercetin and catechin: RQC), at equimolar concentrations, reduces breast cancer (BC) growth and metastasis in nude mice, and inhibits Akt and mTOR activities and activates AMPK, an endogenous inhibitor of mTOR, in metastatic BC cells. The objective of the present study was to determine the contribution of individual polyphenols to the effect of combined RQC on mTOR signaling. Metastatic BC cells were treated with RQC individually or in combination, at various concentrations, and the activities (phosphorylation) of AMPK, Akt, and the mTOR downstream effectors, p70S6 kinase (p70S6K) and 4E binding protein (4EBP1), were determined by Western blot. Results show that quercetin was the most effective compound for Akt/mTOR inhibition. Treatment with quercetin at 15μM had a similar effect as the RQC combination in the inhibition of BC cell proliferation, apoptosis, and migration. However, cell cycle analysis showed that the RQC treatment arrested BC cells in the G1 phase, while quercetin arrested the cell cycle in G2/M. In vivo experiments, using SCID mice with implanted tumors from metastatic BC cells, demonstrated that administration of quercetin at 15mg/kg body weight resulted in a ~70% reduction in tumor growth. In conclusion, quercetin appears to be a viable grape polyphenol for future development as an anti BC therapeutic. PMID:27285995

  4. Anti-Breast Cancer Potential of Quercetin via the Akt/AMPK/Mammalian Target of Rapamycin (mTOR) Signaling Cascade.

    PubMed

    Rivera Rivera, Amilcar; Castillo-Pichardo, Linette; Gerena, Yamil; Dharmawardhane, Suranganie

    2016-01-01

    The Akt/adenosine monophosphate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has emerged as a critical signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth. Thus, dysregulation of this pathway contributes to the development of metabolic disorders such as obesity, type 2diabetes, and cancer. We previously reported that a combination of grape polyphenols (resveratrol, quercetin and catechin: RQC), at equimolar concentrations, reduces breast cancer (BC) growth and metastasis in nude mice, and inhibits Akt and mTOR activities and activates AMPK, an endogenous inhibitor of mTOR, in metastatic BC cells. The objective of the present study was to determine the contribution of individual polyphenols to the effect of combined RQC on mTOR signaling. Metastatic BC cells were treated with RQC individually or in combination, at various concentrations, and the activities (phosphorylation) of AMPK, Akt, and the mTOR downstream effectors, p70S6 kinase (p70S6K) and 4E binding protein (4EBP1), were determined by Western blot. Results show that quercetin was the most effective compound for Akt/mTOR inhibition. Treatment with quercetin at 15μM had a similar effect as the RQC combination in the inhibition of BC cell proliferation, apoptosis, and migration. However, cell cycle analysis showed that the RQC treatment arrested BC cells in the G1 phase, while quercetin arrested the cell cycle in G2/M. In vivo experiments, using SCID mice with implanted tumors from metastatic BC cells, demonstrated that administration of quercetin at 15mg/kg body weight resulted in a ~70% reduction in tumor growth. In conclusion, quercetin appears to be a viable grape polyphenol for future development as an anti BC therapeutic. PMID:27285995

  5. N-nitroso-N-ethylurea activates DNA damage surveillance pathways and induces transformation in mammalian cells

    PubMed Central

    2014-01-01

    treatment indicating NEU to have the potential to cause early transformation in the cells. Conclusion NEU causes damage in mammalian cells in the form of double strand and single strand breaks that temporally activate the major checkpoint signalling kinases without the occurrence of cross-talk between the pathways. NEU also appear to cause transformation in three-dimensional spheroid cultures. PMID:24758542

  6. Trans-activity of Plasma Membrane-associated Ganglioside Sialyltransferase in Mammalian Cells*

    PubMed Central

    Vilcaes, Aldo A.; Demichelis, Vanina Torres; Daniotti, Jose L.

    2011-01-01

    Gangliosides are acidic glycosphingolipids that contain sialic acid residues and are expressed in nearly all vertebrate cells. They are synthesized at the Golgi complex by a combination of glycosyltransferase activities followed by vesicular delivery to the plasma membrane, where they participate in a variety of physiological as well as pathological processes. Recently, a number of enzymes of ganglioside anabolism and catabolism have been shown to be associated with the plasma membrane. In particular, it was observed that CMP-NeuAc:GM3 sialyltransferase (Sial-T2) is able to sialylate GM3 at the plasma membrane (cis-catalytic activity). In this work, we demonstrated that plasma membrane-integrated ecto-Sial-T2 also displays a trans-catalytic activity at the cell surface of epithelial and melanoma cells. By using a highly sensitive enzyme-linked immunosorbent assay combined with confocal fluorescence microscopy, we observed that ecto-Sial-T2 was able to sialylate hydrophobically or covalently immobilized GM3 onto a solid surface. More interestingly, we observed that ecto-Sial-T2 was able to sialylate GM3 exposed on the membrane of neighboring cells by using both the exogenous and endogenous donor substrate (CMP-N-acetylneuraminic acid) available at the extracellular milieu. In addition, the trans-activity of ecto-Sial-T2 was considerably reduced when the expression of the acceptor substrate was inhibited by using a specific inhibitor of biosynthesis of glycolipids, indicating the lipidic nature of the acceptor. Our findings provide the first direct evidence that an ecto-sialyltransferase is able to trans-sialylate substrates exposed in the plasma membrane from mammalian cells, which represents a novel insight into the molecular events that regulate the local glycosphingolipid composition. PMID:21768099

  7. Unrestrained mammalian target of rapamycin complexes 1 and 2 increase expression of phosphatase and tensin homolog deleted on chromosome 10 to regulate phosphorylation of Akt kinase.

    PubMed

    Das, Falguni; Ghosh-Choudhury, Nandini; Dey, Nirmalya; Mandal, Chandi Charan; Mahimainathan, Lenin; Kasinath, Balakuntalam S; Abboud, Hanna E; Choudhury, Goutam Ghosh

    2012-02-01

    Tuberous sclerosis complex 2 (TSC2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) function to block growth factor-induced mammalian target of rapamycin (mTOR) signaling and are mutated in autosomal dominant hamartoma syndromes. mTOR binds to a spectrum of common and different proteins to form TOR complex 1 (TORC1) and TORC2, which regulate cell growth, division, and metabolism. TSC2 deficiency induces constitutive activation of mTOR, leading to a state of insulin resistance due to a negative feedback regulation, resulting in reduced Akt phosphorylation. We have recently described an alternative mechanism showing that in TSC2 deficiency, enhanced PTEN expression contributes to reduced Akt phosphorylation. To explore the mechanism of PTEN regulation, we used rapamycin and constitutively active mTOR to show that TORC1 increases the expression of PTEN mRNA and protein. We found that in TSC2(-/-) mouse embryonic fibroblasts expression of a kinase-dead mutant of mTOR, which inhibits both TORC1 and TORC2, decreases the expression of PTEN via transcriptional mechanism. Furthermore, kinase-dead mTOR increased and decreased phosphorylation of Akt at catalytic loop site Thr-308 and hydrophobic motif site Ser-473, respectively. Moreover, inhibition of deregulated TORC1 in TSC2-null mouse embryonic fibroblasts or in 293 cells by down-regulation of raptor decreased the levels of the transcription factor Hif1α and blocked PTEN expression, resulting in enhanced phosphorylation of Akt at Thr-308 and Ser-473. Finally, knockdown of rictor or mSin1 attenuated the expression of Hif1α, which decreased transcription of PTEN. These results unravel a previously unrecognized cell-autonomous function of TORC1 and TORC2 in the up-regulation of PTEN, which prevents phosphorylation of Akt and may shield against the development of malignancy in TSC patients. PMID:22184110

  8. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth muscle cell survival patterns to promote pulmonary arterial hypertension.

    PubMed

    Aghamohammadzadeh, Reza; Zhang, Ying-Yi; Stephens, Thomas E; Arons, Elena; Zaman, Paula; Polach, Kevin J; Matar, Majed; Yung, Lai-Ming; Yu, Paul B; Bowman, Frederick P; Opotowsky, Alexander R; Waxman, Aaron B; Loscalzo, Joseph; Leopold, Jane A; Maron, Bradley A

    2016-07-01

    Activation of the mammalian target of rapamycin complex 1 (mTORC1) subunit Raptor induces cell growth and is a downstream target of Akt. Elevated levels of aldosterone activate Akt, and, in pulmonary arterial hypertension (PAH), correlate with pulmonary arteriole thickening, which suggests that mTORC1 regulation by aldosterone may mediate adverse pulmonary vascular remodeling. We hypothesized that aldosterone-Raptor signaling induces abnormal pulmonary artery smooth muscle cell (PASMC) survival patterns to promote PAH. Remodeled pulmonary arterioles from SU-5416/hypoxia-PAH rats and monocrotaline-PAH rats with hyperaldosteronism expressed increased levels of the Raptor target, p70S6K, which provided a basis for investigating aldosterone-Raptor signaling in human PASMCs. Aldosterone (10(-9) to 10(-7) M) increased Akt/mTOR/Raptor to activate p70S6K and increase proliferation, viability, and apoptosis resistance in PASMCs. In PASMCs transfected with Raptor-small interfering RNA or treated with spironolactone/eplerenone, aldosterone or pulmonary arterial plasma from patients with PAH failed to increase p70S6K activation or to induce cell survival in vitro Optimal inhibition of pulmonary arteriole Raptor was achieved by treatment with Staramine-monomethoxy polyethylene glycol that was formulated with Raptor-small interfering RNA plus spironolactone in vivo, which decreased arteriole muscularization and pulmonary hypertension in 2 experimental animal models of PAH in vivo Up-regulation of mTORC1 by aldosterone is a critical pathobiologic mechanism that controls PASMC survival to promote hypertrophic vascular remodeling and PAH.-Aghamohammadzadeh, R., Zhang, Y.-Y., Stephens, T. E., Arons, E., Zaman, P., Polach, K. J., Matar, M., Yung, L.-M., Yu, P. B., Bowman, F. P., Opotowsky, A. R., Waxman, A. B., Loscalzo, J., Leopold, J. A., Maron, B. A. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth

  9. Oxidative DNA damage contributes to the toxic activity of propylparaben in mammalian cells.

    PubMed

    Pérez Martín, José Manuel; Peropadre, Ana; Herrero, Oscar; Fernández Freire, Paloma; Labrador, Verónica; Hazen, María José

    2010-09-30

    Propyl p-hydroxybenzoate, commonly referred to as propylparaben, is the most frequently used preservative to inhibit microbial growth and extend shelf life of a range of consumer products. The objective of this study was to provide further insight into the toxicological profile of this compound, because of the current discrepancy in the literature with regard to the safety of parabens. The Vero cell line, derived from the kidney of the green monkey, was selected to evaluate the adverse effects of propylparaben by use of a set of mechanistically relevant endpoints for detecting cytotoxicity and genotoxic activities. Our results demonstrate that exposure to the compound for 24h causes changes in cell-proliferation rates rather than in cell viability. A significant and dose-dependent decline in the percentage of mitotic cells was observed at the lowest concentration tested, mainly due to cell-cycle arrest at the G0/G1 phase. Immunodetection techniques revealed that induction of DNA double-strand breaks and oxidative damage underlies the cytostatic effect observed in treated Vero cells. Additional studies are in progress to extend these findings, which define a novel mode of action of propylparaben in cultured mammalian cells.

  10. Transcription is Associated with Z-DNA Formation in Metabolically Active Permeabilized Mammalian Cell Nuclei

    NASA Astrophysics Data System (ADS)

    Wittig, Burghardt; Dorbic, Tomislav; Rich, Alexander

    1991-03-01

    Mammalian cells have been encapsulated in agarose microbeads, and from these cells metabolically active permeabilized nuclei were prepared. Previously, we showed that biotin-labeled monoclonal antibodies against Z-DNA can be diffused into the nuclei and, over a specific concentration range, they will bind to Z-DNA within the nucleus in a concentration-independent manner. By using radiolabeled streptavidin, we showed that the amount of Z-DNA antibody bound is related to the torsional strain of the DNA in the nucleus. Relaxation of the DNA results in a decrease of Z-DNA formation, whereas increasing torsional strain through inhibiting topoisomerase I results in increased Z-DNA formation. Here we measure the influence of RNA transcription and DNA replication. Transcription is associated with a substantial increase in the binding of anti-Z-DNA antibodies, paralleling the increased level of RNA synthesized as the level of ribonucleoside triphosphate in the medium is increased. DNA replication yields smaller increases in the binding of Z-DNA antibodies. Stopping RNA transcription with inhibitors results in a large loss of Z-DNA antibody binding, whereas only a small decrease is associated with inhibition of DNA replication.

  11. Characterization of orderly spatiotemporal patterns of clock gene activation in mammalian suprachiasmatic nucleus

    PubMed Central

    Foley, Nicholas C.; Tong, Tina Y.; Foley, Duncan; LeSauter, Joseph; Welsh, David K.

    2012-01-01

    Because we can observe oscillation within individual cells and in the tissue as a whole, the suprachiasmatic nucleus (SCN) presents a unique system in the mammalian brain for the analysis of individual cells and the networks of which they are a part. While dispersed cells of the SCN sustain circadian oscillations in isolation, they are unstable oscillators that require network interactions for robust cycling. Using cluster analysis to assess bioluminescence in acute brain slices from PERIOD2∷Luciferase (PER2∷LUC) knockin mice, and immunochemistry of SCN from animals harvested at various circadian times, we assessed the spatiotemporal activation patterns of PER2 to explore the emergence of a coherent oscillation at the tissue level. The results indicate that circadian oscillation is characterized by a stable daily cycle of PER2 expression involving orderly serial activation of specific SCN subregions, followed by a silent interval, with substantial symmetry between the left and right side of the SCN. The biological significance of the clusters identified in living slices was confirmed by co-expression of LUC and PER2 in fixed, immunochemically stained brain sections, with the spatiotemporal pattern of LUC expression resembling that revealed in the cluster analysis of bioluminescent slices. We conclude that the precise timing of PER2 expression within individual neurons is dependent on their location within the nucleus, and that small groups of neurons within the SCN give rise to distinctive and identifiable subregions. We propose that serial activation of these subregions is the basis of robustness and resilience of the daily rhythm of the SCN. PMID:21488990

  12. Characterization of orderly spatiotemporal patterns of clock gene activation in mammalian suprachiasmatic nucleus.

    PubMed

    Foley, Nicholas C; Tong, Tina Y; Foley, Duncan; Lesauter, Joseph; Welsh, David K; Silver, Rae

    2011-05-01

    Because we can observe oscillation within individual cells and in the tissue as a whole, the suprachiasmatic nucleus (SCN) presents a unique system in the mammalian brain for the analysis of individual cells and the networks of which they are a part. While dispersed cells of the SCN sustain circadian oscillations in isolation, they are unstable oscillators that require network interactions for robust cycling. Using cluster analysis to assess bioluminescence in acute brain slices from PERIOD2::Luciferase (PER2::LUC) knockin mice, and immunochemistry of SCN from animals harvested at various circadian times, we assessed the spatiotemporal activation patterns of PER2 to explore the emergence of a coherent oscillation at the tissue level. The results indicate that circadian oscillation is characterized by a stable daily cycle of PER2 expression involving orderly serial activation of specific SCN subregions, followed by a silent interval, with substantial symmetry between the left and right side of the SCN. The biological significance of the clusters identified in living slices was confirmed by co-expression of LUC and PER2 in fixed, immunochemically stained brain sections, with the spatiotemporal pattern of LUC expression resembling that revealed in the cluster analysis of bioluminescent slices. We conclude that the precise timing of PER2 expression within individual neurons is dependent on their location within the nucleus, and that small groups of neurons within the SCN give rise to distinctive and identifiable subregions. We propose that serial activation of these subregions is the basis of robustness and resilience of the daily rhythm of the SCN.

  13. Inhibition of insulin-like growth factor receptor/AKT/mammalian target of rapamycin axis targets colorectal cancer stem cells by attenuating mevalonate-isoprenoid pathway in vitro and in vivo.

    PubMed

    Sharon, Chetna; Baranwal, Somesh; Patel, Nirmita J; Rodriguez-Agudo, Daniel; Pandak, William M; Majumdar, Adhip P N; Krystal, Geoffrey; Patel, Bhaumik B

    2015-06-20

    We observed a co-upregulation of the insulin-like growth factor receptor (IGF-1R)/AKT/mammalian target of rapamycin (mTOR) [InAT] axis and the mevalonate-isoprenoid biosynthesis (MIB) pathways in colorectal cancer stem cells (CSCs) in an unbiased approach. Hence, we hypothesized that the InAT axis might regulate the MIB pathway to govern colorectal CSCs growth. Stimulation (IGF-1) or inhibition (IGF-1R depletion and pharmacological inhibition of IGF-1R/mTOR) of the InAT axis produced induction or attenuation of CSC growth as well as expression of CSC markers and self-renewal factors respectively. Intriguingly, activation of the InAT axis (IGF-1) caused significant upregulation of the MIB pathway genes (both mRNA and protein); while its inhibition produced the opposite effects in colonospheres. More importantly, supplementation with dimethylallyl- and farnesyl-PP, MIB metabolites downstream of isopentenyl-diphosphate delta isomerase (IDI), but not mevalonate and isopentenyl-pp that are upstream of IDI, resulted in a near-complete reversal of the suppressive effect of the InAT axis inhibitors on CSCs growth. The latter findings suggest a specific regulation of the MIB pathway by the InAT axis distal to the target of statins that inhibit 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Effects of IGF-1R inhibition on colonic CSCs proliferation and the MIB pathway were confirmed in an 'in vivo' HCT-116 xenograft model. These observations establish a novel mechanistic link between the InAT axis that is commonly deregulated in colorectal cancer and the MIB pathway in regulation of colonic CSCs growth. Hence, the InAT-MIB corridor is a novel target for developing paradigm shifting optimum anti-CSCs therapies for colorectal cancer. PMID:25895029

  14. Inhibition of insulin-like growth factor receptor/AKT/mammalian target of rapamycin axis targets colorectal cancer stem cells by attenuating mevalonate-isoprenoid pathway in vitro and in vivo

    PubMed Central

    Sharon, Chetna; Baranwal, Somesh; Patel, Nirmita J.; Rodriguez-Agudo, Daniel; Pandak, William M.; Majumdar, Adhip PN; Krystal, Geoffrey; Patel, Bhaumik B.

    2015-01-01

    We observed a co-upregulation of the insulin-like growth factor receptor (IGF-1R)/AKT/mammalian target of rapamycin (mTOR) [InAT] axis and the mevalonate-isoprenoid biosynthesis (MIB) pathways in colorectal cancer stem cells (CSCs) in an unbiased approach. Hence, we hypothesized that the InAT axis might regulate the MIB pathway to govern colorectal CSCs growth. Stimulation (IGF-1) or inhibition (IGF-1R depletion and pharmacological inhibition of IGF-1R/mTOR) of the InAT axis produced induction or attenuation of CSC growth as well as expression of CSC markers and self-renewal factors respectively. Intriguingly, activation of the InAT axis (IGF-1) caused significant upregulation of the MIB pathway genes (both mRNA and protein); while its inhibition produced the opposite effects in colonospheres. More importantly, supplementation with dimethylallyl- and farnesyl-PP, MIB metabolites downstream of isopentenyl-diphosphate delta isomerase (IDI), but not mevalonate and isopentenyl-pp that are upstream of IDI, resulted in a near-complete reversal of the suppressive effect of the InAT axis inhibitors on CSCs growth. The latter findings suggest a specific regulation of the MIB pathway by the InAT axis distal to the target of statins that inhibit 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Effects of IGF-1R inhibition on colonic CSCs proliferation and the MIB pathway were confirmed in an ‘in vivo’ HCT-116 xenograft model. These observations establish a novel mechanistic link between the InAT axis that is commonly deregulated in colorectal cancer and the MIB pathway in regulation of colonic CSCs growth. Hence, the InAT-MIB corridor is a novel target for developing paradigm shifting optimum anti-CSCs therapies for colorectal cancer. PMID:25895029

  15. Designing an Active Target Test Projection Chamber

    NASA Astrophysics Data System (ADS)

    Koci, James; Tan Ahn Collaboration, Dr.; Nicolas Dixneuf Collaboration

    2015-10-01

    The development of instrumentation in nuclear physics is crucial for advancing our ability to measure the properties of exotic nuclei. One limitation of the use of exotic nuclei in experiment is their very low production intensities. Recently, detectors, called active-target dectectors, have been developed to address this issue. Active-target detectors use a gas medium to image charged-particle tracks that are emitted in nuclear reactions. Last semester, I designed a vacuum chamber to be used in developing Micro-Pattern Gas detectors that will upgrade the capabilities of an active-target detector called the Prototype AT-TPC. With the exterior of the chamber complete, I have now been using an electric field modeling program, Garfield, developed by CERN to design a field cage to be placed within the vacuum chamber. The field cage will be a box-like apparatus consisting of two parallel metal plates connected with a resistor chain and attached to wires wrapped between them. The cage will provide a uniform electric field within the chamber to drift electrons from nuclear reactions down to the detector in the bottom of the chamber. These signals are then amplified by a proportional counter, and the data is sent to a computer. For the long term, we would like to incorporate a Micro-Pattern Gas Detectors in the interior of the chamber and eventually use the AT-TPC to examine various nuclei. Dr. Ahn is my advising professor.

  16. Bile acid exposure up-regulates tuberous sclerosis complex 1/mammalian target of rapamycin pathway in Barrett's-associated esophageal adenocarcinoma.

    PubMed

    Yen, Chia-Jui; Izzo, Julie G; Lee, Dung-Fang; Guha, Sushovan; Wei, Yongkun; Wu, Tsung-Teh; Chen, Chun-Te; Kuo, Hsu-Ping; Hsu, Jung-Mao; Sun, Hui-Lung; Chou, Chao-Kai; Buttar, Navtej S; Wang, Kenneth K; Huang, Peng; Ajani, Jaffer; Hung, Mien-Chie

    2008-04-15

    Barrett's esophagus, a columnar metaplasia of the lower esophagus epithelium related to gastroesophageal reflux disease, is the strongest known risk factor for the development of esophageal adenocarcinoma (EAC). Understanding the signal transduction events involved in esophageal epithelium carcinogenesis may provide insights into the origins of EAC and may suggest new therapies. To elucidate the molecular pathways of bile acid-induced tumorigenesis, the newly identified inflammation-associated signaling pathway involving I kappaB kinases beta (IKK beta), tuberous sclerosis complex 1 (TSC1), and mammalian target of rapamycin (mTOR) downstream effector S6 kinase (S6K1) was confirmed to be activated in immortalized Barrett's CPC-A and CPC-C cells and esophageal cancer SEG-1 and BE3 cells. Phosphorylation of TSC1 and S6K1 was induced in response to bile acid stimulation. Treatment of these cells with the mTOR inhibitor rapamycin or the IKK beta inhibitor Bay 11-7082 suppressed bile acid-induced cell proliferation and anchorage-independent growth. We next used an orthotopic rat model to evaluate the role of bile acid in the progression of Barrett's esophagus to EAC. Of interest, we found high expression of phosphorylated IKK beta (pIKK beta) and phosphorylated S6K1 (pS6K1) in tumor tissues and the Barrett's epithelium compared with normal epithelium. Furthermore, immunostaining of clinical EAC tissue specimens revealed that pIKK beta expression was strongly correlated with pS6K1 level. Together, these results show that bile acid can deregulate TSC1/mTOR through IKK beta signaling, which may play a critical role in EAC progression. In addition, Bay 11-7082 and rapamycin may potentially be chemopreventive drugs against Barrett's esophagus-associated EAC.

  17. The Transcription Factor p8 Regulates Autophagy in Response to Palmitic Acid Stress via a Mammalian Target of Rapamycin (mTOR)-independent Signaling Pathway.

    PubMed

    Jia, Sheng-Nan; Lin, Cheng; Chen, Dian-Fu; Li, An-Qi; Dai, Li; Zhang, Li; Zhao, Ling-Ling; Yang, Jin-Shu; Yang, Fan; Yang, Wei-Jun

    2016-02-26

    Autophagy is an evolutionarily conserved degradative process that allows cells to maintain homoeostasis in numerous physiological situations. This process also functions as an essential protective response to endoplasmic reticulum (ER) stress, which promotes the removal and degradation of unfolded proteins. However, little is known regarding the mechanism by which autophagy is initiated and regulated in response to ER stress. In this study, different types of autophagy were identified in human gastric cancer MKN45 cells in response to the stress induced by nutrient starvation or lipotoxicity in which the regulation of these pathways is mammalian target of rapamycin (mTOR)-dependent or -independent, respectively. Interestingly, we found that p8, a stress-inducible transcription factor, was enhanced in MKN45 cells treated with palmitic acid to induce lipotoxicity. Furthermore, an increase in autophagy was observed in MKN45 cells stably overexpressing p8 using a lentivirus system, and autophagy induced by palmitic acid was blocked by p8 RNAi compared with the control. Western blotting analyses showed that autophagy was regulated by p8 or mTOR in response to the protein kinase-like endoplasmic reticulum kinase/activating transcription factor 6-mediated ER stress of lipotoxicity or the parkin-mediated mitochondrial stress of nutrient starvation, respectively. Furthermore, our results indicated that autophagy induced by palmitic acid is mTOR-independent, but this autophagy pathway was regulated by p8 via p53- and PKCα-mediated signaling in MKN45 cells. Our findings provide insights into the role of p8 in regulating autophagy induced by the lipotoxic effects of excess fat accumulation in cells. PMID:26733200

  18. GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells.

    PubMed Central

    Koide, H; Satoh, T; Nakafuku, M; Kaziro, Y

    1993-01-01

    Ras is involved in signal transduction of various factors for growth, differentiation, and oncogenesis. Recent studies have revealed several proteins that function upstream and downstream of the Ras signaling pathway. However, its immediate downstream target molecular has not yet been identified. In an effort to identify the Ras-associated downstream proteins, we added recombinant Ha-Ras in a GTP-bound form to cell-free lysates and used several antibodies against Ras to immunoprecipitate Ras complexes. We found that a serine/threonine kinase, Raf-1, was coimmunoprecipitated with Ha-Ras by two anti-Ras antibodies (LA069 and Y13-238), whereas a neutralizing antibody against Ras (Y13-259) could not precipitate Raf-1. The coimmunoprecipitation was observed with a complex of Ras and guanosine 5'-[gamma- thio]triphosphate but not with a complex of Ras and guanosine 5'-[beta-thio]diphosphate. The GTP-dependent association of Ha-Ras with Raf-1 was observed with lysates of various types of cultured cells, including NIH 3T3, pheochromocytoma (PC) 12, Ba/F3, and Jurkat T cells, and also with crude extracts from rat brain. Furthermore, Raf-1 was precipitated with a transforming Ha-Ras mutant ([Val12]Ras) and wild-type Ha-Ras but not with an effector-region mutant ([Leu35,ARg37]Ras) that lacks transforming activity. These results indicate that Ras.GTP physically associates with Raf either directly or through other component(s) and strongly suggest that Raf functions in close downstream proximity to Ras in mammalian cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8378348

  19. The effect of space microgravity on the physiological activity of mammalian resident cardiac stem cells

    NASA Astrophysics Data System (ADS)

    Belostotskaya, Galina; Zakharov, Eugeny

    Prolonged exposure to weightlessness during space flights is known to cause depression of heart function in mammals. The decrease in heart weight and its remodeling under the influence of prolonged weightlessness (or space microgravity) is assumed to be due to both morphological changes of working cardiomyocytes and their progressive loss, as well as to possible depletion of resident cardiac stem cells (CSCs) population, or their inability to self-renewal and regeneration of muscle tissue under conditions of weightlessness. We have previously shown that the presence of different maturity clones formed by resident CSCs not only in culture but also in the mammalian myocardium can be used as an indicator of the regenerative activity of myocardial cells [Belostotskaya, et al., 2013: 2014]. In this study, we were interested to investigate whether the 30-day near-Earth space flight on the spacecraft BION-M1 affects the regenerative potential of resident CSCs. Immediately after landing of the spacecraft, we had examined the presence of resident c-kit+, Sca-1+ and Isl1+ CSCs and their development in suspension of freshly isolated myocardial cells of C57BL mice in comparison to controls. Cardiac cell suspension was obtained by enzymatic digestion of the heart [Belostotskaya and Golovanova, 2014]. Immunocytochemically stained preparations of fixed cells were analyzed with confocal microscope Leica TCS SP5 (Germany) in the Resource Center of St-Petersburg State University. CSCs were labeled with appropriate antibodies. CSCs differentiation into mature cardiomyocytes was verified using antibodies to Sarcomeric α-Actinin and Cardiac Troponin T. Antibodies to Connexin43 were used to detect cell-cell contacts. All antibodies were conjugated with Alexa fluorochromes (488, 532, 546, 568, 594 and/or 647 nm), according to Zenon-technology (Invitrogen). It has been shown that, under identical conditions of cell isolation, more complete digestion of heart muscle was observed in

  20. Targeting the Channel Activity of Viroporins.

    PubMed

    To, Janet; Surya, Wahyu; Torres, Jaume

    2016-01-01

    Since the discovery that certain small viral membrane proteins, collectively termed as viroporins, can permeabilize host cellular membranes and also behave as ion channels, attempts have been made to link this feature to specific biological roles. In parallel, most viroporins identified so far are virulence factors, and interest has focused toward the discovery of channel inhibitors that would have a therapeutic effect, or be used as research tools to understand the biological roles of viroporin ion channel activity. However, this paradigm is being shifted by the difficulties inherent to small viral membrane proteins, and by the realization that protein-protein interactions and other diverse roles in the virus life cycle may represent an equal, if not, more important target. Therefore, although targeting the channel activity of viroporins can probably be therapeutically useful in some cases, the focus may shift to their other functions in following years. Small-molecule inhibitors have been mostly developed against the influenza A M2 (IAV M2 or AM2). This is not surprising since AM2 is the best characterized viroporin to date, with a well-established biological role in viral pathogenesis combined the most extensive structural investigations conducted, and has emerged as a validated drug target. For other viroporins, these studies are still mostly in their infancy, and together with those for AM2, are the subject of the present review.

  1. Mammalian target of rapamycin inhibitors induce tumor cell apoptosis in vivo primarily by inhibiting VEGF expression and angiogenesis.

    PubMed

    Frost, Patrick; Berlanger, Eileen; Mysore, Veena; Hoang, Bao; Shi, Yijiang; Gera, Joseph; Lichtenstein, Alan

    2013-01-01

    We found that rapalog mTOR inhibitors induce G1 arrest in the PTEN-null HS Sultan B-cell lymphoma line in vitro, but that administration of rapalogs in a HS Sultan xenograft model resulted in significant apoptosis, and that this correlated with induction of hypoxia and inhibition of neoangiogenesis and VEGF expression. Mechanistically, rapalogs prevent cap-dependent translation, but studies have shown that cap-independent, internal ribosome entry site (IRES)-mediated translation of genes, such as c-myc and cyclin D, can provide a fail-safe mechanism that regulates tumor survival. Therefore, we tested if IRES-dependent expression of VEGF could likewise regulate sensitivity of tumor cells in vivo. To achieve this, we developed isogenic HS Sultan cell lines that ectopically express the VEGF ORF fused to the p27 IRES, an IRES sequence that is insensitive to AKT-mediated inhibition of IRES activity and effective in PTEN-null tumors. Mice challenged with p27-VEGF transfected tumor cells were more resistant to the antiangiogenic and apoptotic effects of the rapalog, temsirolimus, and active site mTOR inhibitor, pp242. Our results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and underscore the importance of IRES activity as a resistance mechanism to such targeted therapy.

  2. GSK621 Targets Glioma Cells via Activating AMP-Activated Protein Kinase Signalings

    PubMed Central

    Jiang, Hong; Liu, Wei; Zhan, Shi-Kun; Pan, Yi-Xin; Bian, Liu-Guan; Sun, Bomin; Sun, Qing-Fang; Pan, Si-Jian

    2016-01-01

    Here, we studied the anti-glioma cell activity by a novel AMP-activated protein kinase (AMPK) activator GSK621. We showed that GSK621 was cytotoxic to human glioma cells (U87MG and U251MG lines), possibly via provoking caspase-dependent apoptotic cell death. Its cytotoxicity was alleviated by caspase inhibitors. GSK621 activated AMPK to inhibit mammalian target of rapamycin (mTOR) and downregulate Tetraspanin 8 (Tspan8) in glioma cells. AMPK inhibition, through shRNA knockdown of AMPKα or introduction of a dominant negative (T172A) AMPKα, almost reversed GSK621-induced AMPK activation, mTOR inhibition and Tspan8 degradation. Consequently, GSK621’s cytotoxicity in glioma cells was also significantly attenuated by AMPKα knockdown or mutation. Further studies showed that GSK621, at a relatively low concentration, significantly potentiated temozolomide (TMZ)’s sensitivity and lethality against glioma cells. We summarized that GSK621 inhibits human glioma cells possibly via activating AMPK signaling. This novel AMPK activator could be a novel and promising anti-glioma cell agent. PMID:27532105

  3. Regulation of succinate dehydrogenase activity by SIRT3 in mammalian mitochondria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A member of the sirtuin family of NAD (+) dependent deacetylases, SIRT3, is identified as one of the major mitochondrial deacetylases, located in mammalian mitochondria responsible for deacetylation of several metabolic enzymes and components of oxidative phosphorylation. Regulation of protein deace...

  4. Genome-scale RNA interference screen identifies antizyme 1 (OAZ1) as a target for improvement of recombinant protein production in mammalian cells.

    PubMed

    Xiao, Su; Chen, Yu Chi; Buehler, Eugen; Mandal, Swati; Mandal, Ajeet; Betenbaugh, Michael; Park, Myung Hee; Martin, Scott; Shiloach, Joseph

    2016-11-01

    For the purpose of improving recombinant protein production from mammalian cells, an unbiased, high-throughput whole-genome RNA interference screen was conducted using human embryonic kidney 293 (HEK 293) cells expressing firefly luciferase. A 21,585 human genes were individually silenced with three different siRNAs for each gene. The screen identified 56 genes that led to the greatest improvement in luciferase expression. These genes were found to be included in several pathways involved in spliceosome formation and mRNA processing, transcription, metabolic processes, transport, and protein folding. The 10 genes that most enhanced protein expression when downregulated, were further confirmed by measuring the effect of their silencing on the expression of three additional recombinant proteins. Among the confirmed genes, OAZ1-the gene encoding the ornithine decarboxylase antizyme1-was selected for detailed investigation, since its silencing improved the reporter protein production without affecting cell viability. Silencing OAZ1 caused an increase of the ornithine decarboxylase enzyme and the cellular levels of putrescine and spermidine; an indication that increased cellular polyamines enhances luciferase expression without affecting its transcription. The study shows that OAZ1 is a novel target for improving expression of recombinant proteins. The genome-scale screening performed in this work can establish the foundation for targeted design of an efficient mammalian cell platform for various biotechnological applications. Biotechnol. Bioeng. 2016;113: 2403-2415. © 2016 Wiley Periodicals, Inc. PMID:27215166

  5. Efficacy and Toxicity of Mammalian Target Rapamycin Inhibitors in Patients with Metastatic Renal Cell Carcinoma with Renal Insufficiency: The Korean Cancer Study Group GU 14-08

    PubMed Central

    Kim, Ki Hyang; Kim, Joo Hoon; Lee, Ji Young; Kim, Hyo Song; Heo, Su Jin; Kim, Ji Hyung; Kim, Ho Young; Rha, Sun Young

    2016-01-01

    Purpose We evaluated the efficacy and toxicity of mammalian target rapamycin inhibitors in Korean patients with metastatic renal cell carcinoma (mRCC) with chronic renal insufficiency not requiring dialysis. Materials and Methods Korean patients with mRCC and chronic renal insufficiency not requiring dialysis treated with everolimus or temsirolimus between January 2008 and December 2014 were included. Patient characteristics, clinical outcomes, and toxicities were evaluated. Overall survival (OS) and progression-free survival (PFS) durations were evaluated according to the degree of renal impairment. Results Eighteen patients were considered eligible for the study (median age, 59 years). The median glomerular filtration rate was 51.5 mL/min/1.73 m2. The best response was partial response in six patients and stable disease in 11 patients. The median PFS and OS durations were 8 months (95% confidence interval [CI], 0 to 20.4) and 32 months (95% CI, 27.5 to 36.5), respectively. The most common non-hematologic and grade 3/4 adverse events included stomatitis, fatigue, flu-like symptoms, and anorexia as well as elevated creatinine level. Conclusion Mammalian target rapamycin inhibitors were efficacious and did not increase toxicity in Korean patients with mRCC and chronic renal insufficiency not requiring dialysis. PMID:26875195

  6. Active mammalian replication origins are associated with a high-density cluster of mCpG dinucleotides.

    PubMed Central

    Rein, T; Zorbas, H; DePamphilis, M L

    1997-01-01

    ori-beta is a well-characterized origin of bidirectional replication (OBR) located approximately 17 kb downstream of the dihydrofolate reductase gene in hamster cell chromosomes. The approximately 2-kb region of ori-beta that exhibits greatest replication initiation activity also contains 12 potential methylation sites in the form of CpG dinucleotides. To ascertain whether DNA methylation might play a role at mammalian replication origins, the methylation status of these sites was examined with bisulfite to chemically distinguish cytosine (C) from 5-methylcytosine (mC). All of the CpGs were methylated, and nine of them were located within 356 bp flanking the minimal OBR, creating a high-density cluster of mCpGs that was approximately 10 times greater than average for human DNA. However, the previously reported densely methylated island in which all cytosines were methylated regardless of their dinucleotide composition was not detected and appeared to be an experimental artifact. A second OBR, located at the 5' end of the RPS14 gene, exhibited a strikingly similar methylation pattern, and the organization of CpG dinucleotides at other mammalian origins revealed the potential for high-density CpG methylation. Moreover, analysis of bromodeoxyuridine-labeled nascent DNA confirmed that active replication origins were methylated. These results suggest that a high-density cluster of mCpG dinucleotides may play a role in either the establishment or the regulation of mammalian replication origins. PMID:8972222

  7. A plant scaffold attached region detected close to a T-DNA integration site is active in mammalian cells.

    PubMed Central

    Dietz, A; Kay, V; Schlake, T; Landsmann, J; Bode, J

    1994-01-01

    Integration of foreign genes into plant genomes by the Agrobacterium T-DNA transfer system has been considered to occur at random. It has been speculated that the chromosomal structure of the integration site might affect the expression pattern of the introduced genes. To gain insight into the molecular structure of T-DNA integration sites and its possible impact on gene expression, we have examined plant DNA sequences in the vicinity of T-DNA borders. Analysis of a transgenic petunia plant containing a chloramphenicol acetyltransferase (CAT) gene regulated by the hemoglobin promoter (PAR) from Parasponia andersonii revealed a scaffold attachment region (SAR) close to one T-DNA end. In addition to having strong binding affinities for both animal and plant nuclear scaffolds this petunia SAR element is as active in mammalian cells as the authentic elements from mammalian sources. Images PMID:8052530

  8. The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae.

    PubMed

    Ye, Tian; Bendrioua, Loubna; Carmena, David; García-Salcedo, Raúl; Dahl, Peter; Carling, David; Hohmann, Stefan

    2014-06-01

    The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomyces cerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase.

  9. The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae.

    PubMed

    Ye, Tian; Bendrioua, Loubna; Carmena, David; García-Salcedo, Raúl; Dahl, Peter; Carling, David; Hohmann, Stefan

    2014-06-01

    The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomyces cerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase. PMID:24815694

  10. Δ²,³-ivermectin ethyl secoester, a conjugated ivermectin derivative with leishmanicidal activity but without inhibitory effect on mammalian P-type ATPases.

    PubMed

    Noël, François; Pimenta, Paulo Henrique Cotrim; Dos Santos, Anderson Rouge; Tomaz, Erick Carlos Loureiro; Quintas, Luis Eduardo Menezes; Kaiser, Carlos Roland; Silva, Claudia Lucia Martins; Férézou, Jean-Pierre

    2011-01-01

    Looking at a new putative target for the large spectrum antiparasitic drug ivermectin, we recently showed that avermectin-derived drugs are active against promastigote and amastigote forms of Leishmania amazonensis at low micromolar concentrations. However, we then reported that at this concentration range ivermectin is also able to inhibit three important mammalian P-type ATPases so that unacceptable adverse effects could occur if this drug were used at such high doses therapeutically. The present work aimed to test the activity of ten ivermectin analogs on these rat ATPases in search of a compound with similar leishmanicidal activity but with no effect on the mammalian (host) ATPases at effective concentrations. We synthesized three new ivermectin analogs for testing on rat SERCA (1a and 1b), Na+, K+-ATPase (α₁ and α₂/α₃ isoforms) and H+/K+-ATPase activity, along with seven analogs already characterized for their leishmanicidal activity. Our main finding is that one of the prepared derivatives, Δ²,³-ivermectin ethyl secoester 8, is equipotent to ivermectin 1 for the in vitro leishmanicidal effects but is nearly without effect on the rat ATPases, indicating that it could have a better therapeutic index in vivo and could serve as a candidate for hit-to-lead progression. This conclusion is further supported by the fact that compound 8 produced only 6% (vs 77% for ivermectin) inhibition of the human kidney enzyme at 5 μM, a concentration corresponding to the IC₅₀ for the activity against L. amazonensis amastigotes. PMID:21088826

  11. Roles of 5'-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis.

    PubMed Central

    Rutter, Guy A; Da Silva Xavier, Gabriela; Leclerc, Isabelle

    2003-01-01

    AMPK (5'-AMP-activated protein kinase) is emerging as a metabolic master switch, by which cells in both mammals and lower organisms sense and decode changes in energy status. Changes in AMPK activity have been shown to regulate glucose transport in muscle and glucose production by the liver. Moreover, AMPK appears to be a key regulator of at least one transcription factor linked to a monogenic form of diabetes mellitus. As a result, considerable efforts are now under way to explore the usefulness of AMPK as a therapeutic target for other forms of this disease. Here we review this topic, and discuss new findings which suggest that AMPK may play roles in regulating insulin release and the survival of pancreatic islet beta-cells, and nutrient sensing by the brain. PMID:12839490

  12. An improved method of electroporation for introducing biologically active foreign genes into cultured mammalian cells

    SciTech Connect

    Tatsuka, Masaaki; Orita, Satoshi; Yagi, Takashi; Kakunaga, Takeo )

    1988-09-01

    The authors have developed a modified, reproducible, and efficient method for introducing cloned genes into mammalian cells by using an electric field followed by treatment with sodium butyrate. Transfection frequencies with plasmid pSV2-neo, consisting of an antibiotic (G418) resistance gene and simian virus 40 (SV40) early promoter, by electroporation were higher than those by calcium phosphate DNA precipitation. Treatment with sodium butyrate following electroporation significantly increased the frequency of transfection in various types of cell lines and primary cultured cells including human skin fibroblasts. Treatment with sodium butyrate also increased the transient expression of the gene for chloramphenicol acetyltransferase when the gene was introduced into BALB/c 3T3 cells by eletroporation. Electroporation combined with sodium butyrate treatment is an improved method for stable and transient biochemical transformation of foreign genes in cultured mammalian cells.

  13. Rhodopsin Kinase Activity in the Mammalian Pineal Gland and Other Tissues

    NASA Astrophysics Data System (ADS)

    Somers, Robert L.; Klein, David C.

    1984-10-01

    Rhodopsin kinase, an enzyme involved in photochemical transduction in the retina, has been found in the mammalian pineal gland in amounts equal to those in the retina; other tissues had 7 percent of this amount, or less. This finding suggests that, in mammals, rhodopsin kinase functions in the pineal gland and other tissues to phosphorylate rhodopsin-like integral membrane receptors and is thereby involved in signal transduction.

  14. Factors Influencing the Central Nervous System Distribution of a Novel Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitor GSK2126458: Implications for Overcoming Resistance with Combination Therapy for Melanoma Brain Metastases.

    PubMed

    Vaidhyanathan, Shruthi; Wilken-Resman, Brynna; Ma, Daniel J; Parrish, Karen E; Mittapalli, Rajendar K; Carlson, Brett L; Sarkaria, Jann N; Elmquist, William F

    2016-02-01

    Small molecule inhibitors targeting the mitogen-activated protein kinase pathway (Braf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase) have had success in extending survival for patients with metastatic melanoma. Unfortunately, resistance may occur via cross-activation of alternate signaling pathways. One approach to overcome resistance is to simultaneously target the phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathway. Recent reports have shown that GSK2126458 [2,4-difluoro-N-(2-methoxy-5-(4-(pyridazin-4-yl)quinolin-6-yl)pyridin-3-yl) benzenesulfonamide], a dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor, can overcome acquired resistance to Braf and mitogen-activated protein kinase kinase inhibitors in vitro. These resistance mechanisms may be especially important in melanoma brain metastases because of limited drug delivery across the blood-brain barrier. The purpose of this study was to investigate factors that influence the brain distribution of GSK2126458 and to examine the efficacy of GSK2126458 in a novel patient-derived melanoma xenograft (PDX) model. Both in vitro and in vivo studies indicate that GSK2126458 is a substrate for P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), two dominant active efflux transporters in the blood-brain barrier. The steady-state brain distribution of GSK2126458 was 8-fold higher in the P-gp/Bcrp knockout mice compared with the wild type. We also observed that when simultaneously infused to steady state, GSK212658, dabrafenib, and trametinib, a rational combination to overcome mitogen-activated protein kinase inhibitor resistance, all had limited brain distribution. Coadministration of elacridar, a P-gp/Bcrp inhibitor, increased the brain distribution of GSK2126458 by approximately 7-fold in wild-type mice. In the PDX model, GSK2126458 showed efficacy in flank tumors but was ineffective in intracranial melanoma. These results show that

  15. High-resolution genetic mapping of mammalian motor activity levels in mice.

    PubMed

    Kas, M J H; de Mooij-van Malsen, J G; de Krom, M; van Gassen, K L I; van Lith, H A; Olivier, B; Oppelaar, H; Hendriks, J; de Wit, M; Groot Koerkamp, M J A; Holstege, F C P; van Oost, B A; de Graan, P N E

    2009-02-01

    The generation of motor activity levels is under tight neural control to execute essential behaviors, such as movement toward food or for social interaction. To identify novel neurobiological mechanisms underlying motor activity levels, we studied a panel of chromosome substitution (CS) strains derived from mice with high (C57BL/6J strain) or low motor activity levels (A/J strain) using automated home cage behavioral registration. In this study, we genetically mapped the expression of baseline motor activity levels (horizontal distance moved) to mouse chromosome 1. Further genetic mapping of this trait revealed an 8.3-Mb quantitative trait locus (QTL) interval. This locus is distinct from the QTL interval for open-field anxiety-related motor behavior on this chromosome. By data mining, an existing phenotypic and genotypic data set of 2445 genetically heterogeneous mice (http://gscan.well.ox.ac.uk/), we confirmed linkage to the peak marker at 79 970 253 bp and refined the QTL to a 312-kb interval containing a single gene (A830043J08Rik). Sequence analysis showed a nucleotide deletion in the 3' untranslated region of the Riken gene. Genome-wide microarray gene expression profiling in brains of discordant F(2) individuals from CS strain 1 showed a significant upregulation of Epha4 in low-active F(2) individuals. Inclusion of a genetic marker for Epha4 confirmed that this gene is located outside of the QTL interval. Both Epha4 and A830043J08Rik are expressed in brain motor circuits, and similar to Epha4 mutants, we found linkage between reduced motor neurons number and A/J chromosome 1. Our findings provide a novel QTL and a potential downstream target underlying motor circuitry development and the expression of physical activity levels.

  16. Herpes simplex virus virion host shutoff protein requires a mammalian factor for efficient in vitro endoribonuclease activity.

    PubMed

    Lu, P; Jones, F E; Saffran, H A; Smiley, J R

    2001-02-01

    The virion host shutoff protein (vhs) of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated mRNA turnover during virus infection and induces endoribonucleolytic cleavage of exogenous RNA substrates when it is produced in a rabbit reticulocyte (RRL) in vitro translation system. Although vhs induces RNA turnover in the absence of other HSV gene products, it is not yet known whether cellular factors are required for its activity. As one approach to addressing this question, we expressed vhs in the budding yeast Saccharomyces cerevisiae. Expression of vhs inhibited colony formation, and the severity of this effect varied with the carbon source. The biological relevance of this effect was assessed by examining the activity of five mutant forms of vhs bearing previously characterized in-frame linker insertions. The results indicated a complete concordance between the growth inhibition phenotype in yeast and mammalian host cell shutoff. Despite these results, expression of vhs did not trigger global mRNA turnover in vivo, and cell extracts of yeast expressing vhs displayed little if any vhs-dependent endoribonuclease activity. However, activity was readily detected when such extracts were mixed with RRL. These data suggest that the vhs-dependent endoribonuclease requires one or more mammalian macromolecular factors for efficient activity.

  17. Activation and proliferation of lymphocytes and other mammalian cells in microgravity

    NASA Technical Reports Server (NTRS)

    Cogoli, A.; Cogoli-Greuter, M.

    1997-01-01

    The experimental findings reviewed in this chapter support the following conclusions: Proliferation. Human T-lymphocytes, associated with monocytes as accessory cells, show dramatic changes in the centrifuge, in the clinostat and in space. In free-floating cells the mitogenic response is depressed by 90% in microgravity, whereas in cells attached to a substratum activation is enhanced by 100% compared to 1-G ground and inflight controls. The duration of phase G1 of the mitotic cycle of HeLa cells is reduced in hypergravity, resulting in an increased proliferation rate. Other systems like Friend cells and WI38 human embryonic lung cells do not show significant changes. Genetic expression and signal transduction. T-lymphocytes and monocytes show important changes in the expression of cytokines like interleukin-1, interleukin-2, interferon-gamma and tumor necrosis factor. The data from space experiments in Spacelab, Space Shuttle mid-deck, and Biokosmos have helped to clarify certain aspects of the mechanism of T-cell activation. Epidermoid A431 cells show changes in the genetic expression of the proto-oncogenes c-fos and c-jun in the clinostat and in sounding rockets. Membrane function, in particular the binding of ligates as first messengers of a signal, is not changed in most of the cell systems in microgravity. Morphology and Mortility. Free cells, lymphocytes in particular, are able to move and form aggregates in microgravity, indicating that cell-cell contacts and cell communications do take place in microgravity. Dramatic morphological and ultrastructural changes are not detected in cells cultured in microgravity. Important experiments with single mammalian cells, including immune cells, were carried out recently in three Spacelab flights, (SL-J, D-2, and IML-2 in 1992, 1993, and 1994, respectively). The results of the D-2 mission have been published in ref. 75; those of the IML-2 mission in ref. 76. Finally, many cell biology experiments in space have suffered

  18. Antidepressant-like effect of ascorbic acid is associated with the modulation of mammalian target of rapamycin pathway.

    PubMed

    Moretti, Morgana; Budni, Josiane; Freitas, Andiara Espíndola; Rosa, Priscila Batista; Rodrigues, Ana Lúcia S

    2014-01-01

    The present study investigated the involvement of the PI3K, GSK-3β, heme oxygenase-1 (HO-1) and mTOR in the antidepressant-like effect of ascorbic acid in the tail suspension test (TST). Male Swiss mice were pretreated with ascorbic acid (1 mg/kg, p.o.) or vehicle and 45 min after, LY294002 (10 μg/site, i.c.v., reversible PI3K inhibitor), rapamycin (0.2 nmol/site, i.c.v., selective mTOR inhibitor), zinc protoporphyrin (ZnPP - 10 ng/site, i.c.v., HO-1 inhibitor) or vehicle was administered. We also investigated the synergistic effect of ascorbic acid (0.1 mg/kg, p.o., sub-effective dose in the TST) with lithium chloride (10 mg/kg, p.o., non-selective GSK-3β inhibitor), AR-A014418 (0.01 μg/site, i.c.v., selective GSK-3β inhibitor) or cobalt protoporphyrin (CoPP - 0.01 μg/site, i.c.v., HO-1 inducer) in the TST. The antidepressant-like effect of ascorbic acid (1 mg/kg, p.o.) was prevented by the treatment of mice with LY294002, rapamycin or ZnPP. In addition, sub-effective doses of lithium chloride, AR-A014418 or CoPP, combined with a sub-effective dose of ascorbic acid produced a synergistic antidepressant-like effect. We also demonstrated that 1 h after its administration, ascorbic acid increased the phosphorylation of p70S6K and the immunocontent of PSD-95 in the hippocampus of mice. These results indicate that the antidepressant-like effect of ascorbic acid in the TST might be dependent on the activation of PI3K and mTOR, inhibition of GSK-3β as well as induction of HO-1, reinforcing the notion that these are important targets for antidepressant activity and contributing to better elucidate the mechanisms underlying the antidepressant-like effect of ascorbic acid.

  19. Two small enzyme isoforms mediate mammalian mitochondrial poly(ADP-ribose) glycohydrolase (PARG) activity

    SciTech Connect

    Meyer, Ralph G. . E-mail: meyerg@vet.upenn.edu; Meyer-Ficca, Mirella L.; Whatcott, Clifford J.; Jacobson, Elaine L.; Jacobson, Myron K.

    2007-08-01

    Poly(ADP-ribose)glycohydrolase (PARG) is the major enzyme capable of rapidly hydrolyzing poly(ADP-ribose) (PAR) formed by the diverse members of the PARP enzyme family. This study presents an alternative splice mechanism by which two novel PARG protein isoforms of 60 kDa and 55 kDa are expressed from the human PARG gene, termed hPARG60 and hPARG55, respectively. Homologous forms were found in the mouse (mPARG63 and mPARG58) supporting the hypothesis that expression of small PARG isoforms is conserved among mammals. A PARG protein of {approx} 60 kDa has been described for decades but with its genetic basis unknown, it was hypothesized to be a product of posttranslational cleavage of larger PARG isoforms. While this is not excluded entirely, isolation and expression of cDNA clones from different sources of RNA indicate that alternative splicing leads to expression of a catalytically active hPARG60 in multiple cell compartments. A second enzyme, hPARG55, that can be expressed through alternative translation initiation from hPARG60 transcripts is strictly targeted to the mitochondria. Functional studies of a mitochondrial targeting signal (MTS) in PARG exon IV suggest that hPARG60 may be capable of shuttling between nucleus and mitochondria, which would be in line with a proposed function of PAR in genotoxic stress-dependent, nuclear-mitochondrial crosstalk.

  20. Localization and Expression of VMAT2 Aross Mammalian Species: A Translational Guide for Its Visualization and Targeting in Health and Disease

    PubMed Central

    Schafer, Martin K.-H.; Weihe, Eberhard; Eiden, Lee E.

    2014-01-01

    VMAT2 is the vesicular monoamine transporter that allows DA, NE, Epi, His, and 5-HT uptake into neurons and endocrine cells. A second isoform, VMAT1, has similar structure and function, but does not recognize histamine as a substrate. VMAT1 is absent from neurons, and its major function appears to be in endocrine cells, that is, enterochromaffin cells, which scavenge 5-HT, but not histamine, from dietary sources. This chapter provides an update on the neuroanatomical distribution of VMAT2 across various mammalian species, including human, primate, pig, rat, and mouse. When necessary, VMAT1 expression is provided as a contrast. The main purpose of this chapter is to allow clinicians, in particular endocrinologists and diagnosing neuroradiologists and neuropathologists, an acquaintanceship with the possibilities for VMAT2 as a target for in vivo imaging, and drug development, based on this updated information. PMID:24054151

  1. Construction of simple and efficient siRNA validation systems for screening and identification of effective RNAi-targeted sequences from mammalian genes.

    PubMed

    Tsai, Wen-Hui; Chang, Wen-Tsan

    2014-01-01

    RNA interference (RNAi) is an evolutionarily conserved mechanism of gene silencing induced by double-stranded RNAs (dsRNAs). Among the widely used dsRNAs, small interfering RNAs (siRNAs) and short hairpin RNAs have evolved as extremely powerful and the most popular gene silencing reagents. The key challenge to achieving efficient gene silencing especially for the purpose of therapeutics is mainly dependent on the effectiveness and specificity of the selected RNAi-targeted sequences. Practically, only a small number of dsRNAs are capable of inducing highly effective and sequence-specific gene silencing via RNAi mechanism. In addition, the efficiency of gene silencing induced by dsRNAs can only be experimentally examined based on inhibition of the target gene expression. Therefore, it is essential to develop a fully robust and comparative validation system for measuring the efficacy of designed dsRNAs. In this chapter, we focus our discussion on a reliable and quantitative reporter-based siRNA validation system that has been previously established in our laboratory. The system consists of a short synthetic DNA fragment containing an RNAi-targeted sequence of interest and two expression vectors for targeting reporter and triggering siRNA expressions. The efficiency of siRNAs is determined by their abilities to inhibit expression of the targeting reporters with easily quantified readouts including enhanced green fluorescence protein and firefly luciferase. Since only a readily available short synthetic DNA fragment is needed for constructing this reliable and efficient reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective RNAi-targeted sequences from mammalian genes but also implicates the use of RNAi-based dsRNA reagents for reverse functional genomics and molecular therapeutics.

  2. In vivo activation of a conserved microRNA program induces robust mammalian heart regeneration

    PubMed Central

    Aguirre, A.; Montserrat, N.; Zachiggna, S.; Nivet, E.; Hishida, T.; Krause, M. N.; Kurian, L.; Ocampo, A.; Vazquez-Ferrer, E.; Rodriguez-Esteban, C.; Kumar, S.; Moresco, J.J.; Yates, J.R.; Campistol, J. M.; Sancho-Martinez, I.; Giacca, M.; Belmonte, J.C. Izpisua

    2014-01-01

    SUMMARY Heart failure is a leading cause of mortality and morbidity in the developed world, partly because mammals lack the ability to regenerate heart tissue. Whether this is due to evolutionary loss of regenerative mechanisms present in other organisms or to an inability to activate such mechanisms is currently unclear. Here, we decipher mechanisms underlying heart regeneration in adult zebrafish and show that the molecular regulators of this response are conserved in mammals. We identified miR-99/100 and Let-7a/c, and their protein targets smarca5 and fntb, as critical regulators of cardiomyocyte dedifferentiation and heart regeneration in zebrafish. Although human and murine adult cardiomyocytes fail to elicit an endogenous regenerative response following myocardial infarction, we show that in vivo manipulation of this molecular machinery in mice results in cardiomyocyte dedifferentiation and improved heart functionality after injury. These data provide a proof-of-concept for identifying and activating conserved molecular programs to regenerate the damaged heart. PMID:25517466

  3. Specific and nonspecific membrane-binding determinants cooperate in targeting phosphatidylinositol transfer protein beta-isoform to the mammalian trans-Golgi network.

    PubMed

    Phillips, Scott E; Ile, Kristina E; Boukhelifa, Malika; Huijbregts, Richard P H; Bankaitis, Vytas A

    2006-06-01

    Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and specific steps in membrane trafficking through the secretory pathway in eukaryotes. Herein, we describe the cis-acting information that controls PITPbeta localization in mammalian cells. We demonstrate PITPbeta localizes predominantly to the trans-Golgi network (TGN) and that this localization is independent of the phospholipid-bound state of PITPbeta. Domain mapping analyses show the targeting information within PITPbeta consists of three short C-terminal specificity elements and a nonspecific membrane-binding element defined by a small motif consisting of adjacent tryptophan residues (the W(202)W(203) motif). Combination of the specificity elements with the W(202)W(203) motif is necessary and sufficient to generate an efficient TGN-targeting module. Finally, we demonstrate that PITPbeta association with the TGN is tolerant to a range of missense mutations at residue serine 262, we describe the TGN localization of a novel PITPbeta isoform with a naturally occurring S262Q polymorphism, and we find no other genetic or pharmacological evidence to support the concept that PITPbeta localization to the TGN is obligately regulated by conventional protein kinase C (PKC) or the Golgi-localized PKC isoforms delta or epsilon. These latter findings are at odds with a previous report that conventional PKC-mediated phosphorylation of residue Ser262 is required for PITPbeta targeting to Golgi membranes.

  4. Specific and Nonspecific Membrane-binding Determinants Cooperate in Targeting Phosphatidylinositol Transfer Protein β-Isoform to the Mammalian Trans-Golgi Network

    PubMed Central

    Phillips, Scott E.; Ile, Kristina E.; Boukhelifa, Malika; Huijbregts, Richard P.H.; Bankaitis, Vytas A.

    2006-01-01

    Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and specific steps in membrane trafficking through the secretory pathway in eukaryotes. Herein, we describe the cis-acting information that controls PITPβ localization in mammalian cells. We demonstrate PITPβ localizes predominantly to the trans-Golgi network (TGN) and that this localization is independent of the phospholipid-bound state of PITPβ. Domain mapping analyses show the targeting information within PITPβ consists of three short C-terminal specificity elements and a nonspecific membrane-binding element defined by a small motif consisting of adjacent tryptophan residues (the W202W203 motif). Combination of the specificity elements with the W202W203 motif is necessary and sufficient to generate an efficient TGN-targeting module. Finally, we demonstrate that PITPβ association with the TGN is tolerant to a range of missense mutations at residue serine 262, we describe the TGN localization of a novel PITPβ isoform with a naturally occurring S262Q polymorphism, and we find no other genetic or pharmacological evidence to support the concept that PITPβ localization to the TGN is obligately regulated by conventional protein kinase C (PKC) or the Golgi-localized PKC isoforms δ or ε. These latter findings are at odds with a previous report that conventional PKC-mediated phosphorylation of residue Ser262 is required for PITPβ targeting to Golgi membranes. PMID:16540520

  5. CUL4-DDB1-CDT2 E3 Ligase Regulates the Molecular Clock Activity by Promoting Ubiquitination-Dependent Degradation of the Mammalian CRY1.

    PubMed

    Tong, Xin; Zhang, Deqiang; Guha, Anirvan; Arthurs, Blake; Cazares, Victor; Gupta, Neil; Yin, Lei

    2015-01-01

    The CUL4-DDB1 E3 ligase complex serves as a critical regulator in various cellular processes, including cell proliferation, DNA damage repair, and cell cycle progression. However, whether this E3 ligase complex regulates clock protein turnover and the molecular clock activity in mammalian cells is unknown. Here we show that CUL4-DDB1-CDT2 E3 ligase ubiquitinates CRY1 and promotes its degradation both in vitro and in vivo. Depletion of the major components of this E3 ligase complex, including Ddb1, Cdt2, and Cdt2-cofactor Pcna, leads to CRY1 stabilization in cultured cells or in the mouse liver. CUL4A-DDB1-CDT2 E3 ligase targets lysine 585 within the C-terminal region of CRY1 protein, shown by the CRY1 585KA mutant's resistance to ubiquitination and degradation mediated by the CUL4A-DDB1 complex. Surprisingly, both depletion of Ddb1 and over-expression of Cry1-585KA mutant enhance the oscillatory amplitude of the Bmal1 promoter activity without altering its period length, suggesting that CUL4A-DDB1-CDT2 E3 targets CRY1 for degradation and reduces the circadian amplitude. All together, we uncovered a novel biological role for CUL4A-DDB1-CDT2 E3 ligase that regulates molecular circadian behaviors via promoting ubiquitination-dependent degradation of CRY1.

  6. Tunicamycins: translocase-I inhibitors that target bacterial cell wall and mammalian N-glycoproteins. The potential for selective inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tunicamycins are a heterologous family of nucleoside antibiotics that target the biosynthesis of bacterial peptidoglycan and eukaryotic N-glycoproteins. The mechanism of action is known, with the tunicamycin-Mg2+ complex established as a transition state analog for hexosamine-1-phosphate: prenol pho...

  7. Class IIa Histone Deacetylases are Hormone-activated regulators of FOXO and Mammalian Glucose Homeostasis

    PubMed Central

    Mihaylova, Maria M.; Vasquez, Debbie S.; Ravnskjaer, Kim; Denechaud, Pierre-Damien; Yu, Ruth T.; Alvarez, Jacqueline G.; Downes, Michael; Evans, Ronald M.; Montminy, Marc; Shaw, Reuben J.

    2011-01-01

    SUMMARY Class IIa histone deacetylases (HDACs) are signal-dependent modulators of transcription with established roles in muscle differentiation and neuronal survival. We show here that in liver, Class IIa HDACs (HDAC4, 5, and 7) are phosphorylated and excluded from the nucleus by AMPK family kinases. In response to the fasting hormone glucagon, Class IIa HDACs are rapidly dephosphorylated and translocated to the nucleus where they associate with the promoters of gluconeogenic enzymes such as G6Pase. In turn, HDAC4/5 recruit HDAC3, which results in the acute transcriptional induction of these genes via deacetylation and activation of Foxo family transcription factors. Loss of Class IIa HDACs in murine liver results in inhibition of FOXO target genes and lowers blood glucose, resulting in increased glycogen storage. Finally, suppression of Class IIa HDACs in mouse models of Type 2 Diabetes ameliorates hyperglycemia, suggesting that inhibitors of Class I/II HDACs may be potential therapeutics for metabolic syndrome. PMID:21565617

  8. Definition of two agonist types at the mammalian cold-activated channel TRPM8.

    PubMed

    Janssens, Annelies; Gees, Maarten; Toth, Balazs Istvan; Ghosh, Debapriya; Mulier, Marie; Vennekens, Rudi; Vriens, Joris; Talavera, Karel; Voets, Thomas

    2016-01-01

    Various TRP channels act as polymodal sensors of thermal and chemical stimuli, but the mechanisms whereby chemical ligands impact on TRP channel gating are poorly understood. Here we show that AITC (allyl isothiocyanate; mustard oil) and menthol represent two distinct types of ligands at the mammalian cold sensor TRPM8. Kinetic analysis of channel gating revealed that AITC acts by destabilizing the closed channel, whereas menthol stabilizes the open channel, relative to the transition state. Based on these differences, we classify agonists as either type I (menthol-like) or type II (AITC-like), and provide a kinetic model that faithfully reproduces their differential effects. We further demonstrate that type I and type II agonists have a distinct impact on TRPM8 currents and TRPM8-mediated calcium signals in excitable cells. These findings provide a theoretical framework for understanding the differential actions of TRP channel ligands, with important ramifications for TRP channel structure-function analysis and pharmacology. PMID:27449282

  9. Cosmogenic activation of a natural tellurium target

    NASA Astrophysics Data System (ADS)

    Lozza, V.; Petzoldt, J.

    2015-02-01

    130Te is one of the candidates for the search for neutrinoless double beta decay. It is currently planned to be used in two experiments: CUORE and SNO+. In the CUORE experiment TeO2 crystals cooled at cryogenic temperatures will be used. In the SNO+ experiment natTe will be deployed up to 0.3% loading in the liquid scintillator volume. A possible background for the signal searched for, are the high Q-value, long-lived isotopes, produced by cosmogenic neutron and proton spallation reaction on the target material. A total of 18 isotopes with Q-value larger than 2 MeV and T1/2 > 20 days have been identified as potential backgrounds. In addition low Q-value, high rate isotopes can be problematic due to pile-up effects, specially in liquid scintillator based detectors. Production rates have been calculated using the ACTIVIA program, the TENDL library, and the cosmogenic neutron and proton flux parametrization at sea level from Armstrong and Gehrels for both long and short lived isotopes. The obtained values for the cross sections are compared with the existing experimental data and calculations. Good agreement has been generally found. The results have been applied to the SNO+ experiment for one year of exposure at sea level. Two possible cases have been considered: a two years of cooling down period deep underground, or a first purification on surface and 6 months of cooling down deep underground. Deep underground activation at the SNOLAB location has been considered.

  10. Effects of Intermediates between Vitamins K2 and K3 on Mammalian DNA Polymerase Inhibition and Anti-Inflammatory Activity

    PubMed Central

    Mizushina, Yoshiyuki; Maeda, Jun; Irino, Yasuhiro; Nishida, Masayuki; Nishiumi, Shin; Kondo, Yasuyuki; Nishio, Kazuyuki; Kuramochi, Kouji; Tsubaki, Kazunori; Kuriyama, Isoko; Azuma, Takeshi; Yoshida, Hiromi; Yoshida, Masaru

    2011-01-01

    Previously, we reported that vitamin K3 (VK3), but not VK1 or VK2 (=MK-4), inhibits the activity of human DNA polymerase γ (pol γ). In this study, we chemically synthesized three intermediate compounds between VK2 and VK3, namely MK-3, MK-2 and MK-1, and investigated the inhibitory effects of all five compounds on the activity of mammalian pols. Among these compounds, MK-2 was the strongest inhibitor of mammalian pols α, κ and λ, which belong to the B, Y and X families of pols, respectively; whereas VK3 was the strongest inhibitor of human pol γ, an A-family pol. MK-2 potently inhibited the activity of all animal species of pol tested, and its inhibitory effect on pol λ activity was the strongest with an IC50 value of 24.6 μM. However, MK-2 did not affect the activity of plant or prokaryotic pols, or that of other DNA metabolic enzymes such as primase of pol α, RNA polymerase, polynucleotide kinase or deoxyribonuclease I. Because we previously found a positive relationship between pol λ inhibition and anti-inflammatory action, we examined whether these compounds could inhibit inflammatory responses. Among the five compounds tested, MK-2 caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in mouse ear. In addition, in a cell culture system using mouse macrophages, MK-2 displayed the strongest suppression of the production of tumor necrosis factor (TNF)-α induced by lipopolysaccharide (LPS). Moreover, MK-2 was found to inhibit the action of nuclear factor (NF)-κB. In an in vivo mouse model of LPS-evoked acute inflammation, intraperitoneal injection of MK-2 in mice led to suppression of TNF-α production in serum. In conclusion, this study has identified VK2 and VK3 intermediates, such as MK-2, that are promising anti-inflammatory candidates. PMID:21541047

  11. Mammalian DNA Repair. Final Report

    SciTech Connect

    2003-01-24

    The Gordon Research Conference (GRC) on Mammalian DNA Repair was held at Harbortown Resort, Ventura Beach, CA. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  12. How targets select activation or repression in response to Wnt.

    PubMed

    Murgan, Sabrina; Bertrand, Vincent

    2015-01-01

    In metazoans, the Wnt signaling pathway plays a key role in the regulation of binary decisions during development. During this process different sets of target genes are activated in cells where the Wnt pathway is active (classic target genes) versus cells where the pathway is inactive (opposite target genes). While the mechanism of transcriptional activation is well understood for classic target genes, how opposite target genes are activated in the absence of Wnt remains poorly characterized. Here we discuss how the key transcriptional mediator of the Wnt pathway, the TCF family member POP-1, regulates opposite target genes during C. elegans development. We examine recent findings suggesting that the direction of the transcriptional output (activation or repression) can be determined by the way TCF is recruited and physically interacts with its target gene. PMID:27123368

  13. Transforming Growth Factor-β Is an Upstream Regulator of Mammalian Target of Rapamycin Complex 2-Dependent Bladder Cancer Cell Migration and Invasion.

    PubMed

    Gupta, Sounak; Hau, Andrew M; Al-Ahmadie, Hikmat A; Harwalkar, Jyoti; Shoskes, Aaron C; Elson, Paul; Beach, Jordan R; Hussey, George S; Schiemann, William P; Egelhoff, Thomas T; Howe, Philip H; Hansel, Donna E

    2016-05-01

    Our prior work identified the mammalian target of rapamycin complex 2 (mTORC2) as a key regulator of bladder cancer cell migration and invasion, although upstream growth factor mediators of this pathway in bladder cancer have not been well delineated. We tested whether transforming growth factor (TGF)-β, which can function as a promotility factor in bladder cancer cells, could regulate mTORC2-dependent bladder cancer cell motility and invasion. In human bladder cancers, the highest levels of phosphorylated SMAD2, a TGF-β signaling intermediate, were present in high-grade invasive bladder cancers and associated with more frequent recurrence and decreased disease-specific survival. Increased expression of TGF-β isoforms, receptors, and signaling components was detected in invasive high-grade bladder cancer cells that expressed Vimentin and lacked E-cadherin. Application of TGF-β induced phosphorylation of the Ser473 residue of AKT, a selective target of mTORC2, in a SMAD2- and SMAD4-independent manner and increased bladder cancer cell migration in a modified scratch wound assay and invasion through Matrigel. Inhibition of TGF-β receptor I using SB431542 ablated TGF-β-induced migration and invasion. A similar effect was seen when Rictor, a key mTORC2 component, was selectively silenced. Our results suggest that TGF-β can induce bladder cancer cell invasion via mTORC2 signaling, which may be applicable in most bladder cancers.

  14. Pirarubicin induces an autophagic cytoprotective response through suppression of the mammalian target of rapamycin signaling pathway in human bladder cancer cells

    SciTech Connect

    Li, Kuiqing; Chen, Xu; Liu, Cheng; Gu, Peng; Li, Zhuohang; Wu, Shaoxu; Xu, Kewei; Lin, Tianxin; Huang, Jian

    2015-05-01

    Pirarubicin is widely used in intravesical chemotherapy for bladder cancer, but its efficacy is limited due to drug resistance; the mechanism has not been well studied. Emerging evidence shows that autophagy can be a novel target for cancer therapy. This study aimed to investigate the role of autophagy in pirarubicin-treated bladder cancer cells. Bladder cancer cells EJ and J82 were treated with pirarubicin, siRNA, 3-methyladenine or hydroxychloroquine. Cell proliferation and apoptosis were tested by cell survival assay and flow cytometric analysis, respectively. Autophagy was evaluated by immunoblotting before and after the treatments. The phosphorylated mammalian target of rapamycin, serine/threonine kinase p70 S6 kinase, and eukaryotic translation initiation factor 4E binding protein 1 were also investigated by immunoblotting. We found that pirarubicin could induce autophagy in bladder cancer cells. Inhibition of autophagy by 3-methyladenine, hydroxychloroquine or knockdown of autophagy related gene 3 significantly increased apoptosis in pirarubicin-treated bladder cancer cells. Pirarubicin-induced autophagy was mediated via the mTOR/p70S6K/4E-BP1 signaling pathway. In conclusion, autophagy induced by pirarubicin plays a cytoprotective role in bladder cancer cells, suggesting that inhibition of autophagy may improve efficacy over traditional pirarubicin chemotherapy in bladder cancer patients. - Highlights: • Pirarubicin induced autophagy in bladder cancer cells. • Inhibition of autophagy enhanced pirarubicin-induced apoptosis. • Pirarubicin induced autophagy through inhibition of mTOR signaling pathway.

  15. Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons

    PubMed Central

    Lazcano-Pérez, Fernando; Castro, Héctor; Arenas, Isabel; García, David E.; González-Muñoz, Ricardo; Arreguín-Espinosa, Roberto

    2016-01-01

    The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7), voltage-gated calcium channel (CaV2.2), the A-type transient outward (IA) and delayed rectifier (IDR) currents of KV channels of the superior cervical ganglion (SCG) neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels. PMID:27164140

  16. Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons.

    PubMed

    Lazcano-Pérez, Fernando; Castro, Héctor; Arenas, Isabel; García, David E; González-Muñoz, Ricardo; Arreguín-Espinosa, Roberto

    2016-01-01

    The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7), voltage-gated calcium channel (CaV2.2), the A-type transient outward (IA) and delayed rectifier (IDR) currents of KV channels of the superior cervical ganglion (SCG) neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels. PMID:27164140

  17. Mammalian development in a changing environment: exposure to endocrine disruptors reveals the developmental plasticity of steroid-hormone target organs.

    PubMed

    Markey, Caroline M; Coombs, Macall A; Sonnenschein, Carlos; Soto, Ana M

    2003-01-01

    Recent findings in the field of environmental endocrine disruption have revealed that developmental exposure to estrogenic chemicals induces morphological, functional, and behavioral anomalies associated with reproduction. The aim of the present study was to determine the effects of in utero exposure to low doses of the estrogenic chemical bisphenol A (BPA) on the development of the female reproductive tissues and mammary glands in CD-1 mice. Humans are exposed to BPA, which leaches from dental materials and plastic food and beverage containers. Here we report that prenatal exposure to BPA induces alterations in tissue organization within the ovaries and mammary glands and disrupts estrous cyclicity in adulthood. Because estrogen receptors are expressed developmentally in these estrogen-target organs, we propose that BPA may directly affect the expression of genes involved in their morphogenesis. In addition, alterations in the sexual differentiation of the brain, and thus the hypothalamic-pituitary-gonadal axis, may further contribute to the observed phenotype. The emerging field of endocrine disruptors promises to provide new insights into the mechanisms underlying the development of hormone-target organs and demonstrates that the environment plays important roles in the making of phenotypes.

  18. Sustained ERK activation underlies reprogramming in regeneration-competent salamander cells and distinguishes them from their mammalian counterparts.

    PubMed

    Yun, Maximina H; Gates, Phillip B; Brockes, Jeremy P

    2014-07-01

    In regeneration-competent vertebrates, such as salamanders, regeneration depends on the ability of various differentiated adult cell types to undergo natural reprogramming. This ability is rarely observed in regeneration-incompetent species such as mammals, providing an explanation for their poor regenerative potential. To date, little is known about the molecular mechanisms mediating natural reprogramming during regeneration. Here, we have identified the extent of extracellular signal-regulated kinase (ERK) activation as a key component of such mechanisms. We show that sustained ERK activation following serum induction is required for re-entry into the cell cycle of postmitotic salamander muscle cells, partially by promoting the downregulation of p53 activity. Moreover, ERK activation induces epigenetic modifications and downregulation of muscle-specific genes such as Sox6. Remarkably, while long-term ERK activation is found in salamander myotubes, only transient activation is seen in their mammalian counterparts, suggesting that the extent of ERK activation could underlie differences in regenerative competence between species.

  19. Short KR-12 analogs designed from human cathelicidin LL-37 possessing both antimicrobial and antiendotoxic activities without mammalian cell toxicity.

    PubMed

    Jacob, Binu; Park, Il-Seon; Bang, Jeong-Kyu; Shin, Song Yub

    2013-11-01

    KR-12 (residues 18-29 of LL-37) was known to be the smallest peptide of human cathelicidin LL-37 possessing antimicrobial activity. In order to optimize α-helical short antimicrobial peptides having both antimicrobial and antiendotoxic activities without mammalian cell toxicity, we designed and synthesized a series of KR-12 analogs. Highest hydrophobic analogs KR-12-a5 and KR-12-a6 displayed greater inhibition of lipopolysaccharide (LPS)-stimulated tumor necrosis factor-α production and higher LPS-binding activity. We have observed that antimicrobial activity is independent of charge, but LPS neutralization requires a balance of hydrophobicity and net positive charge. Among KR-12 analogs, KR-12-a2, KR-12-a3 and KR-12-a4 showed much higher cell specificity for bacteria over erythrocytes and retained antiendotoxic activity, relative to parental LL-37. KR-12-a5 displayed the strongest antiendotoxic activity but almost similar cell specificity as compared with LL-37. Also, these KR-12 analogs (KR-12-a2, KR-12-a3, KR-12-a4 and KR-12-a5) exhibited potent antimicrobial activity (minimal inhibitory concentration: 4 μM) against methicillin-resistant Staphylococcus aureus. Taken together, these KR-12 analogs have the potential for future development as a novel class of antimicrobial and anti-inflammatory therapeutic agents.

  20. Definition of two agonist types at the mammalian cold-activated channel TRPM8

    PubMed Central

    Janssens, Annelies; Gees, Maarten; Toth, Balazs Istvan; Ghosh, Debapriya; Mulier, Marie; Vennekens, Rudi; Vriens, Joris; Talavera, Karel; Voets, Thomas

    2016-01-01

    Various TRP channels act as polymodal sensors of thermal and chemical stimuli, but the mechanisms whereby chemical ligands impact on TRP channel gating are poorly understood. Here we show that AITC (allyl isothiocyanate; mustard oil) and menthol represent two distinct types of ligands at the mammalian cold sensor TRPM8. Kinetic analysis of channel gating revealed that AITC acts by destabilizing the closed channel, whereas menthol stabilizes the open channel, relative to the transition state. Based on these differences, we classify agonists as either type I (menthol-like) or type II (AITC-like), and provide a kinetic model that faithfully reproduces their differential effects. We further demonstrate that type I and type II agonists have a distinct impact on TRPM8 currents and TRPM8-mediated calcium signals in excitable cells. These findings provide a theoretical framework for understanding the differential actions of TRP channel ligands, with important ramifications for TRP channel structure-function analysis and pharmacology. DOI: http://dx.doi.org/10.7554/eLife.17240.001 PMID:27449282

  1. Activation of the protein deacetylase SIRT6 by long-chain fatty acids and widespread deacylation by mammalian sirtuins.

    PubMed

    Feldman, Jessica L; Baeza, Josue; Denu, John M

    2013-10-25

    Mammalian sirtuins (SIRT1 through SIRT7) are members of a highly conserved family of NAD(+)-dependent protein deacetylases that function in metabolism, genome maintenance, and stress responses. Emerging evidence suggests that some sirtuins display substrate specificity toward other acyl groups attached to the lysine ε-amine. SIRT6 was recently reported to preferentially hydrolyze long-chain fatty acyl groups over acetyl groups. Here we investigated the catalytic ability of all sirtuins to hydrolyze 13 different acyl groups from histone H3 peptides, ranging in carbon length, saturation, and chemical diversity. We find that long-chain deacylation is a general feature of mammalian sirtuins, that SIRT1 and SIRT2 act as efficient decrotonylases, and that SIRT1, SIRT2, SIRT3, and SIRT4 can remove lipoic acid. These results provide new insight into sirtuin function and a means for cellular removal of an expanding list of endogenous lysine modifications. Given that SIRT6 is a poor deacetylase in vitro, but binds and prefers to hydrolyze long-chain acylated peptides, we hypothesize that binding of certain free fatty acids (FFAs) could stimulate deacetylation activity. Indeed, we demonstrate that several biologically relevant FFAs (including myristic, oleic, and linoleic acids) at physiological concentrations induce up to a 35-fold increase in catalytic efficiency of SIRT6 but not SIRT1. The activation mechanism is consistent with fatty acid inducing a conformation that binds acetylated H3 with greater affinity. Binding of long-chain FFA and myristoylated H3 peptide is mutually exclusive. We discuss the implications of discovering endogenous, small-molecule activators of SIRT6.

  2. Role of the Phosphoinositide 3-Kinase-Akt-Mammalian Target of the Rapamycin Signaling Pathway in Long-Term Potentiation and Trace Fear Conditioning Memory in Rat Medial Prefrontal Cortex

    ERIC Educational Resources Information Center

    Sui, Li; Wang, Jing; Li, Bao-Ming

    2008-01-01

    Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt (also known as protein kinase B, PKB), mammalian target of rapamycin (mTOR), the 70-kDa ribosomal S6 kinase (p70S6k), and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), may play important roles in long-term synaptic plasticity and memory in many…

  3. Resveratrol inhibits cancer cell metabolism by down regulating pyruvate kinase M2 via inhibition of mammalian target of rapamycin.

    PubMed

    Iqbal, Mohd Askandar; Bamezai, Rameshwar N K

    2012-01-01

    Metabolism of cancer cells with pyruvate kinase M2 (PKM2) at its centre stage has assumed a prime significance in cancer research in recent times. Cancer cell metabolism, characterized by enhanced glucose uptake, production of lactate and anabolism is considered an ideal target for therapeutic interventions. Expression of PKM2 switches metabolism in favor of cancer cells, therefore, the present study was designed to investigate the hitherto unknown effect of resveratrol, a phytoalexin, on PKM2 expression and resultant implications on cancer metabolism. We observed that resveratrol down-regulated PKM2 expression by inhibiting mTOR signaling and suppressed cancer metabolism, adjudged by decreased glucose uptake, lactate production (aerobic glycolysis) and reduced anabolism (macromolecule synthesis) in various cancer cell lines. A contingent decrease in intracellular levels of ribose-5-phosphate (R5P), a critical intermediate of pentose phosphate pathway, accounted for a reduced anabolism. Consequently, the state of suppressed cancer metabolism resulted in decreased cellular proliferation. Interestingly, shRNA-mediated silencing of PKM2 inhibited glucose uptake and lactate production, providing evidence for the critical role of PKM2 and its mediation in the observed effects of resveratrol on cancer metabolism. Further, an over-expression of PKM2 abolished the observed effects of resveratrol, signifying the role of PKM2 downregulation as a critical function of resveratrol. The study reports a novel PKM2-mediated effect of resveratrol on cancer metabolism and provides a new dimension to its therapeutic potential.

  4. Mammalian target of rapamycin complex 1 (mTORC1) Is required for mouse spermatogonial differentiation in vivo.

    PubMed

    Busada, Jonathan T; Niedenberger, Bryan A; Velte, Ellen K; Keiper, Brett D; Geyer, Christopher B

    2015-11-01

    Spermatogonial stem cells (SSCs) must balance self-renewal with production of transit-amplifying progenitors that differentiate in response to retinoic acid (RA) before entering meiosis. This self-renewal vs. differentiation spermatogonial fate decision is critical for maintaining tissue homeostasis, as imbalances cause spermatogenesis defects that can lead to human testicular cancer or infertility. A great deal of effort has been exerted to understand how the SSC population is maintained. In contrast, little is known about the essential program of differentiation initiated by retinoic acid (RA) that precedes meiosis, and the pathways and proteins involved are poorly defined. We recently reported a novel role for RA in stimulating the PI3/AKT/mTOR kinase signaling pathway to activate translation of repressed mRNAs such as Kit. Here, we examined the requirement for mTOR complex 1 (mTORC1) in mediating the RA signal to direct spermatogonial differentiation in the neonatal testis. We found that in vivo inhibition of mTORC1 by rapamycin blocked spermatogonial differentiation, which led to an accumulation of undifferentiated spermatogonia. In addition, rapamycin also blocked the RA-induced translational activation of mRNAs encoding KIT, SOHLH1, and SOHLH2 without affecting expression of STRA8. These findings highlight dual roles for RA in germ cell development - transcriptional activation of genes, and kinase signaling to stimulate translation of repressed messages required for spermatogonial differentiation.

  5. Activation of Heat Shock and Antioxidant Responses by the Natural Product Celastrol: Transcriptional Signatures of a Thiol-targeted Molecule

    PubMed Central

    Trott, Amy; West, James D.; Klaić, Lada; Westerheide, Sandy D.; Silverman, Richard B.; Morimoto, Richard I.

    2008-01-01

    Stress response pathways allow cells to sense and respond to environmental changes and adverse pathophysiological states. Pharmacological modulation of cellular stress pathways has implications in the treatment of human diseases, including neurodegenerative disorders, cardiovascular disease, and cancer. The quinone methide triterpene celastrol, derived from a traditional Chinese medicinal herb, has numerous pharmacological properties, and it is a potent activator of the mammalian heat shock transcription factor HSF1. However, its mode of action and spectrum of cellular targets are poorly understood. We show here that celastrol activates Hsf1 in Saccharomyces cerevisiae at a similar effective concentration seen in mammalian cells. Transcriptional profiling revealed that celastrol treatment induces a battery of oxidant defense genes in addition to heat shock genes. Celastrol activated the yeast Yap1 oxidant defense transcription factor via the carboxy-terminal redox center that responds to electrophilic compounds. Antioxidant response genes were likewise induced in mammalian cells, demonstrating that the activation of two major cell stress pathways by celastrol is conserved. We report that celastrol's biological effects, including inhibition of glucocorticoid receptor activity, can be blocked by the addition of excess free thiol, suggesting a chemical mechanism for biological activity based on modification of key reactive thiols by this natural product. PMID:18199679

  6. Cionin, a protochordean hybrid of cholecystokinin and gastrin: biological activity in mammalian systems.

    PubMed

    Schjoldager, B; Park, J; Johnsen, A H; Yamada, T; Rehfeld, J F

    1991-06-01

    The protochordean octapeptide cionin is structurally a hybrid of mammalian cholecystokinin (CCK) and gastrin, and thus their possible common ancestor. To determine whether cionin behaves like CCK or gastrin, we examined its effect on canine fundic somatostatin cells and on porcine and bovine gallbladder muscles. Cionin released somatostatin with a potency (ED50 0.15 nM) and efficacy (14.8% of cell content) similar to that of CCK-8 (ED50 0.12 nM, efficacy 16.7%). The efficacies but not the potencies of CCK-8 and cionin differed from those of sulfated gastrin (0.12 nM, 9.7%), nonsulfated gastrin (0.20 nM, 9.4%), and nonsulfated CCK-8 (0.30 nM, 10.4%). CCK and gastrin stimulated contractions of porcine gallbladder muscle strips in a concentration-dependent manner with no differences in efficacy but with characteristic differences in potency. CCK-8 and cionin displayed similar potencies of ED50 2.0 and 2.6 nM; both were significantly different from the ED50 of 0.4 microM for sulfated gastrin and 2.3 microM for nonsulfated gastrin. CCK radioligand binding to membrane-enriched preparations of porcine and bovine gallbladder muscularis was specific and of high affinity. The equilibrium data revealed that binding of CCK and gastrin peptides best fit a single site. CCK-8 and cionin displayed similar affinities [Kd 0.5 nM (porcine), 0.5 nM (bovine, CCK) vs. Kd 0.8 and 0.9 nM (cionin), respectively]. These differed again significantly from Kd 0.6 and 1.5 microM (sulfated gastrin) and 0.7 and 0.2 microM (nonsulfated gastrin). The results show that cionin behaves like CCK rather than gastrin in mammals.

  7. Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins

    PubMed Central

    Kralt, Annemarie; Jagalur, Noorjahan B.; van den Boom, Vincent; Lokareddy, Ravi K.; Steen, Anton; Cingolani, Gino; Fornerod, Maarten; Veenhoff, Liesbeth M.

    2015-01-01

    Endoplasmic reticulum–synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2β are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2ΔNLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins. PMID:26179916

  8. Retrograde fibroblast growth factor 22 (FGF22) signaling regulates insulin-like growth factor 2 (IGF2) expression for activity-dependent synapse stabilization in the mammalian brain

    PubMed Central

    Terauchi, Akiko; Johnson-Venkatesh, Erin M; Bullock, Brenna; Lehtinen, Maria K; Umemori, Hisashi

    2016-01-01

    Communication between pre- and postsynaptic cells promotes the initial organization of synaptic specializations, but subsequent synaptic stabilization requires transcriptional regulation. Here we show that fibroblast growth factor 22 (FGF22), a target-derived presynaptic organizer in the mouse hippocampus, induces the expression of insulin-like growth factor 2 (IGF2) for the stabilization of presynaptic terminals. FGF22 is released from CA3 pyramidal neurons and organizes the differentiation of excitatory nerve terminals formed onto them. Local application of FGF22 on the axons of dentate granule cells (DGCs), which are presynaptic to CA3 pyramidal neurons, induces IGF2 in the DGCs. IGF2, in turn, localizes to DGC presynaptic terminals and stabilizes them in an activity-dependent manner. IGF2 application rescues presynaptic defects of Fgf22-/- cultures. IGF2 is dispensable for the initial presynaptic differentiation, but is required for the following presynaptic stabilization both in vitro and in vivo. These results reveal a novel feedback signal that is critical for the activity-dependent stabilization of presynaptic terminals in the mammalian hippocampus. DOI: http://dx.doi.org/10.7554/eLife.12151.001 PMID:27083047

  9. Wound healing complications and the use of mammalian target of rapamycin inhibitors in kidney transplantation: a critical review of the literature.

    PubMed

    Nashan, Björn; Citterio, Franco

    2012-09-27

    Surgical complications, including events such as lymphocele and urological complications that affect wound healing, are reported with an incidence of 15% to 32% after kidney transplantation. The experience of the surgeon and comorbidities play an important role in determining the risk of such complications occurring. Since the introduction of the inosine 5'-monophosphate dehydrogenase inhibitors (mycophenolate mofetil) to the immunosuppressive armamentarium, replacing the antimetabolite prodrug azathioprine, reports have associated certain forms of wound healing complications (wound dehiscence, impaired healing, lymphocele, and incisional hernia) with the use of these agents. When mammalian target of rapamycin (mTOR) inhibitors (sirolimus, everolimus) became available, these findings were observed increasingly, particularly in direct comparisons with inosine 5'-monophosphate dehydrogenase inhibitors. The purpose of this article was to review the reported incidence of wound healing complications from randomized clinical trials that investigated the use of sirolimus- and everolimus-based treatment regimens in de novo kidney transplantation and the information available from the U.S. Food and Drug Administration database. The clinical trials included were primarily identified using biomedical literature database searches, with additional studies added at the authors' discretion. This review summarizes these studies to consider whether modern mTOR inhibitor-based immunosuppressive regimens exert and affect wound healing after kidney transplantation.

  10. Inhibition of Mammalian Target of Rapamycin Complex 1 (mTORC1) Downregulates ELOVL1 Gene Expression and Fatty Acid Synthesis in Goat Fetal Fibroblasts

    PubMed Central

    Wang, Weipeng; He, Qiburi; Guo, Zhixin; Yang, Limin; Bao, Lili; Bao, Wenlei; Zheng, Xu; Wang, Yanfeng; Wang, Zhigang

    2015-01-01

    Elongation of very-long-chain fatty acids 1 (ELOVL1) is a ubiquitously expressed gene that belongs to the ELOVL family and regulates the synthesis of very-long-chain fatty acids (VLCFAs) and sphingolipids, from yeast to mammals. Mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of cell metabolism and is associated with fatty acids synthesis. In this study, we cloned the cDNA that encodes Cashmere goat (Capra hircus) ELOVL1 (GenBank Accession number KF549985) and investigated its expression in 10 tissues. ELOVL1 cDNA was 840 bp, encoding a deduced protein of 279 amino acids, and ELOVL1 mRNA was expressed in a wide range of tissues. Inhibition of mTORC1 by rapamycin decreased ELOVL1 expression and fatty acids synthesis in Cashmere goat fetal fibroblasts. These data show that ELOVL1 expression is regulated by mTORC1 and that mTORC1 has significant function in fatty acids synthesis in Cashmere goat. PMID:26204830

  11. Targeted Inhibition of Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Sensitizes Pancreatic Cancer Cells to Doxorubicin without Exacerbating Cardiac Toxicity

    PubMed Central

    Durrant, David E.; Das, Anindita; Dyer, Samya; Tavallai, Seyedmehrad; Dent, Paul

    2015-01-01

    Pancreatic cancer has the lowest 5-year survival rate of all major cancers despite decades of effort to design and implement novel, more effective treatment options. In this study, we tested whether the dual phosphoinositide 3-kinase/mechanistic target of rapamycin inhibitor BEZ235 (BEZ) potentiates the antitumor effects of doxorubicin (DOX) against pancreatic cancer. Cotreatment of BEZ235 with DOX resulted in dose-dependent inhibition of the phosphoinositide 3-kinase/mechanistic target of rapamycin survival pathway, which corresponded with an increase in poly ADP ribose polymerase cleavage. Moreover, BEZ cotreatment significantly improved the effects of DOX toward both cell viability and cell death in part through reduced Bcl-2 expression and increased expression of the shorter, more cytotoxic forms of BIM. BEZ also facilitated intracellular accumulation of DOX, which led to enhanced DNA damage and reactive oxygen species generation. Furthermore, BEZ in combination with gemcitabine reduced MiaPaca2 cell proliferation but failed to increase reactive oxygen species generation or BIM expression, resulting in reduced necrosis and apoptosis. Treatment with BEZ and DOX in mice bearing tumor xenographs significantly repressed tumor growth as compared with BEZ, DOX, or gemcitabine. Additionally, in contrast to the enhanced expression seen in MiaPaca2 cells, BEZ and DOX cotreatment reduced BIM expression in H9C2 cardiomyocytes. Also, the Bcl-2/Bax ratio was increased, which was associated with a reduction in cell death. In vivo echocardiography showed decreased cardiac function with DOX treatment, which was not improved by combination treatment with BEZ. Thus, we propose that combining BEZ with DOX would be a better option for patients than current standard of care by providing a more effective tumor response without the associated increase in toxicity. PMID:26101222

  12. The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain

    SciTech Connect

    Lechner, Mark S. . E-mail: msl27@drexel.edu; Schultz, David C.; Negorev, Dmitri; Maul, Gerd G.; Rauscher, Frank J.

    2005-06-17

    The HP1 proteins regulate epigenetic gene silencing by promoting and maintaining chromatin condensation. The HP1 chromodomain binds to methylated histone H3. More enigmatic is the chromoshadow domain (CSD), which mediates dimerization, transcription repression, and interaction with multiple nuclear proteins. Here we show that KAP-1, CAF-1 p150, and NIPBL carry a canonical amino acid motif, PxVxL, which binds directly to the CSD with high affinity. We also define a new class of variant PxVxL CSD-binding motifs in Sp100A, LBR, and ATRX. Both canonical and variant motifs recognize a similar surface of the CSD dimer as demonstrated by a panel of CSD mutants. These in vitro binding results were confirmed by the analysis of polypeptides found associated with nuclear HP1 complexes and we provide the first evidence of the NIPBL/delangin protein in human cells, a protein recently implicated in the developmental disorder, Cornelia de Lange syndrome. NIPBL is related to Nipped-B, a factor participating in gene activation by remote enhancers in Drosophila melanogaster. Thus, this spectrum of direct binding partners suggests an expanded role for HP1 as factor participating in promoter-enhancer communication, chromatin remodeling/assembly, and sub-nuclear compartmentalization.

  13. Cortical energy demands of signaling and nonsignaling components in brain are conserved across mammalian species and activity levels

    PubMed Central

    Hyder, Fahmeed; Rothman, Douglas L.; Bennett, Maxwell R.

    2013-01-01

    The continuous need for ion gradient restoration across the cell membrane, a prerequisite for synaptic transmission and conduction, is believed to be a major factor for brain’s high oxidative demand. However, do energy requirements of signaling and nonsignaling components of cortical neurons and astrocytes vary with activity levels and across species? We derived oxidative ATP demand associated with signaling (Ps) and nonsignaling (Pns) components in the cerebral cortex using species-specific physiologic and anatomic data. In rat, we calculated glucose oxidation rates from layer-specific neuronal activity measured across different states, spanning from isoelectricity to awake and sensory stimulation. We then compared these calculated glucose oxidation rates with measured glucose metabolic data for the same states as reported by 2-deoxy-glucose autoradiography. Fixed values for Ps and Pns were able to predict the entire range of states in the rat. We then calculated glucose oxidation rates from human EEG data acquired under various conditions using fixed Ps and Pns values derived for the rat. These calculated metabolic data in human cerebral cortex compared well with glucose metabolism measured by PET. Independent of species, linear relationship was established between neuronal activity and neuronal oxidative demand beyond isoelectricity. Cortical signaling requirements dominated energy demand in the awake state, whereas nonsignaling requirements were ∼20% of awake value. These predictions are supported by 13C magnetic resonance spectroscopy results. We conclude that mitochondrial energy support for signaling and nonsignaling components in cerebral cortex are conserved across activity levels in mammalian species. PMID:23319606

  14. The leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) does not activate transcription in mammalian mitochondria.

    PubMed

    Harmel, Julia; Ruzzenente, Benedetta; Terzioglu, Mügen; Spåhr, Henrik; Falkenberg, Maria; Larsson, Nils-Göran

    2013-05-31

    Regulation of mtDNA expression is critical for controlling oxidative phosphorylation capacity and has been reported to occur at several different levels in mammalian mitochondria. LRPPRC (leucine-rich pentatricopeptide repeat-containing protein) has a key role in this regulation and acts at the post-transcriptional level to stabilize mitochondrial mRNAs, to promote mitochondrial mRNA polyadenylation, and to coordinate mitochondrial translation. However, recent studies have suggested that LRPPRC may have an additional intramitochondrial role by directly interacting with the mitochondrial RNA polymerase POLRMT to stimulate mtDNA transcription. In this study, we have further examined the intramitochondrial roles for LRPPRC by creating bacterial artificial chromosome transgenic mice with moderately increased LRPPRC expression and heterozygous Lrpprc knock-out mice with moderately decreased LRPPRC expression. Variation of LRPPRC levels in mice in vivo, occurring within a predicted normal physiological range, strongly affected the levels of an unprocessed mitochondrial precursor transcript (ND5-cytochrome b) but had no effect on steady-state levels of mitochondrial transcripts or de novo transcription of mtDNA. We further assessed the role of LRPPRC in mitochondrial transcription by performing size exclusion chromatography and immunoprecipitation experiments in human cell lines and mice, but we found no interaction between LRPPRC and POLRMT. Furthermore, addition of purified LRPPRC to a recombinant human in vitro transcription system did not activate mtDNA transcription. On the basis of these data, we conclude that LRPPRC does not directly regulate mtDNA transcription but rather acts as a post-transcriptional regulator of mammalian mtDNA expression. PMID:23599432

  15. Solution structure and alanine scan of a spider toxin that affects the activation of mammalian voltage-gated sodium channels.

    PubMed

    Corzo, Gerardo; Sabo, Jennifer K; Bosmans, Frank; Billen, Bert; Villegas, Elba; Tytgat, Jan; Norton, Raymond S

    2007-02-16

    Magi 5, from the hexathelid spider Macrothele gigas, is a 29-residue polypeptide containing three disulfide bridges. It binds specifically to receptor site 4 on mammalian voltage-gated sodium channels and competes with scorpion beta-toxins, such as Css IV from Centruroides suffusus suffusus. As a consequence, Magi 5 shifts the activation voltage of the mammalian rNav1.2a channel to more hyperpolarized voltages, whereas the insect channel, DmNav1, is not affected. To gain insight into toxin-channel interactions, Magi 5 and 23 analogues were synthesized. The three-dimensional structure of Magi 5 in aqueous solution was determined, and its voltage-gated sodium channel-binding surfaces were mapped onto this structure using data from electrophysiological measurements on a series of Ala-substituted analogues. The structure clearly resembles the inhibitor cystine knot structural motif, although the triple-stranded beta-sheet typically found in that motif is partially distorted in Magi 5. The interactive surface of Magi 5 toward voltage-gated sodium channels resembles in some respects the Janus-faced atracotoxins, with functionally important charged residues on one face of the toxin and hydrophobic residues on the other. Magi 5 also resembles the scorpion beta-toxin Css IV, which has distinct nonpolar and charged surfaces that are critical for channel binding and has a key Glu involved in voltage sensor trapping. These two distinct classes of toxin, with different amino acid sequences and different structures, may utilize similar groups of residues on their surface to achieve the common end of modifying voltage-gated sodium channel function.

  16. Neuronal activity promotes oligodendrogenesis and adaptive myelination in the mammalian brain.

    PubMed

    Gibson, Erin M; Purger, David; Mount, Christopher W; Goldstein, Andrea K; Lin, Grant L; Wood, Lauren S; Inema, Ingrid; Miller, Sarah E; Bieri, Gregor; Zuchero, J Bradley; Barres, Ben A; Woo, Pamelyn J; Vogel, Hannes; Monje, Michelle

    2014-05-01

    Myelination of the central nervous system requires the generation of functionally mature oligodendrocytes from oligodendrocyte precursor cells (OPCs). Electrically active neurons may influence OPC function and selectively instruct myelination of an active neural circuit. In this work, we use optogenetic stimulation of the premotor cortex in awake, behaving mice to demonstrate that neuronal activity elicits a mitogenic response of neural progenitor cells and OPCs, promotes oligodendrogenesis, and increases myelination within the deep layers of the premotor cortex and subcortical white matter. We further show that this neuronal activity-regulated oligodendrogenesis and myelination is associated with improved motor function of the corresponding limb. Oligodendrogenesis and myelination appear necessary for the observed functional improvement, as epigenetic blockade of oligodendrocyte differentiation and myelin changes prevents the activity-regulated behavioral improvement.

  17. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing

    PubMed Central

    Lee, Ciaran M; Cradick, Thomas J; Fine, Eli J; Bao, Gang

    2016-01-01

    The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. PMID:26750397

  18. The C. elegans apoptotic nuclease NUC-1 is related in sequence and activity to mammalian DNase II.

    PubMed

    Lyon, C J; Evans, C J; Bill, B R; Otsuka, A J; Aguilera, R J

    2000-07-11

    The Caenorhabditis elegans nuc-1 gene has previously been implicated in programmed cell death due to the presence of persistent undegraded apoptotic DNA in nuc-1 mutant animals. In this report, we describe the cloning and characterization of nuc-1, which encodes an acidic nuclease with significant sequence similarity to mammalian DNase II. Database searches performed with human DNase II protein sequence revealed a significant similarity with the predicted C. elegans C07B5.5 ORF. Subsequent analysis of crude C. elegans protein extracts revealed that wild-type animals contained a potent endonuclease activity with a cleavage preference similar to DNase II, while nuc-1 mutant worms demonstrated a marked reduction in this nuclease activity. Sequence analysis of C07B5.5 DNA and mRNA also revealed that nuc-1(e1392), but not wild-type animals contained a nonsense mutation within the CO7B5.5 coding region. Furthermore, nuc-1 transgenic lines carrying the wild-type C07B5.5 locus demonstrated a complete complementation of the nuc-1 mutant phenotype. Our results therefore provide compelling evidence that the C07B5.5 gene encodes the NUC-1 apoptotic nuclease and that this nuclease is related in sequence and activity to DNase II.

  19. Targeted, noninvasive blockade of cortical neuronal activity

    NASA Astrophysics Data System (ADS)

    McDannold, Nathan; Zhang, Yongzhi; Power, Chanikarn; Arvanitis, Costas D.; Vykhodtseva, Natalia; Livingstone, Margaret

    2015-11-01

    Here we describe a novel method to noninvasively modulate targeted brain areas through the temporary disruption of the blood-brain barrier (BBB) via focused ultrasound, enabling focal delivery of a neuroactive substance. Ultrasound was used to locally disrupt the BBB in rat somatosensory cortex, and intravenous administration of GABA then produced a dose-dependent suppression of somatosensory-evoked potentials in response to electrical stimulation of the sciatic nerve. No suppression was observed 1-5 days afterwards or in control animals where the BBB was not disrupted. This method has several advantages over existing techniques: it is noninvasive; it is repeatable via additional GABA injections; multiple brain regions can be affected simultaneously; suppression magnitude can be titrated by GABA dose; and the method can be used with freely behaving subjects. We anticipate that the application of neuroactive substances in this way will be a useful tool for noninvasively mapping brain function, and potentially for surgical planning or novel therapies.

  20. Simple and sensitive fluorescence detection of the RNA endonuclease activity of mammalian argonaute2 protein based on an RNA molecular beacon.

    PubMed

    Li, Feng; Li, Peng; Yang, Limin; Tang, Bo

    2012-12-28

    A new strategy for determining the RNA endonuclease activity of mammalian argonaute2 (Ago2) protein has been developed, which combines the unique cleavage function of Ago2 protein with an RNA molecular beacon (RMB). Through the fluorescence restoration of the RMB, simple and sensitive detection of Ago2 is achieved.

  1. Survival Benefit With Proapoptotic Molecular and Pathologic Responses From Dual Targeting of Mammalian Target of Rapamycin and Epidermal Growth Factor Receptor in a Preclinical Model of Pancreatic Neuroendocrine Carcinogenesis

    PubMed Central

    Chiu, Christopher W.; Nozawa, Hiroaki; Hanahan, Douglas

    2010-01-01

    Purpose Pancreatic neuroendocrine tumors (PNETs), although rare, often metastasize, such that surgery, the only potentially curative therapy, is not possible. There is no effective systemic therapy for patients with advanced PNETs. Therefore, new strategies are needed. Toward that end, we investigated the potential benefit of dual therapeutic targeting of the epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) kinases, using a preclinical mouse model of PNET. Materials and Methods Rapamycin and erlotinib, inhibitors of mTOR and EGFR, respectively, were used to treat RIP-Tag2 transgenic mice bearing advanced multifocal PNET. Tumor growth and survival were monitored, and tumors were surveyed for potential biomarkers of response to the therapeutics. Results Rapamycin monotherapy was notably efficacious, prolonging survival concomitant with tumor stasis (stable disease). However, the tumors developed resistance, as evidenced by eventual relapse to progressive tumor growth. Erlotinib monotherapy slowed tumor growth and elicited a marginal survival benefit. In combination, there was an unprecedented survival benefit in the face of this aggressive multifocal cancer and, in contrast to either monotherapy, the development of adaptive resistance was not apparent. Additionally, the antiapoptotic protein survivin was implicated as a biomarker of sensitivity and beneficial responses to the dual targeted therapy. Conclusion Preclinical trials in a mouse model of endogenous PNET suggest that combined targeting of the mTOR and EGFR signaling pathways could have potential clinical benefit in treating PNET. These results have encouraged development of an ongoing phase II clinical trial aimed to evaluate the efficacy of this treatment regimen in human neuroendocrine tumors. PMID:20823411

  2. Activation of p53 Facilitates the Target Search in DNA by Enhancing the Target Recognition Probability.

    PubMed

    Itoh, Yuji; Murata, Agato; Sakamoto, Seiji; Nanatani, Kei; Wada, Takehiko; Takahashi, Satoshi; Kamagata, Kiyoto

    2016-07-17

    Tumor suppressor p53 binds to the target in a genome and regulates the expression of downstream genes. p53 searches for the target by combining three-dimensional diffusion and one-dimensional sliding along the DNA. To examine the regulation mechanism of the target binding, we constructed the pseudo-wild type (pseudo-WT), activated (S392E), and inactive (R248Q) mutants of p53 and observed their target binding in long DNA using single-molecule fluorescence imaging. The pseudo-WT sliding along the DNA showed many pass events over the target and possessed target recognition probability (TRP) of 7±2%. The TRP increased to 18±2% for the activated mutant but decreased to 0% for the inactive mutant. Furthermore, the fraction of the target binding by the one-dimensional sliding among the total binding events increased from 63±9% for the pseudo-WT to 87±2% for the activated mutant. Control of TRP upon activation, as demonstrated here for p53, might be a general activation mechanism of transcription factors.

  3. Insights into Substrate Specificity and Metal Activation of Mammalian Tetrahedral Aspartyl Aminopeptidase

    SciTech Connect

    Chen, Yuanyuan; Farquhar, Erik R.; Chance, Mark R.; Palczewski, Krzysztof; Kiser, Philip D.

    2012-07-11

    Aminopeptidases are key enzymes involved in the regulation of signaling peptide activity. Here, we present a detailed biochemical and structural analysis of an evolutionary highly conserved aspartyl aminopeptidase called DNPEP. We show that this peptidase can cleave multiple physiologically relevant substrates, including angiotensins, and thus may play a key role in regulating neuron function. Using a combination of x-ray crystallography, x-ray absorption spectroscopy, and single particle electron microscopy analysis, we provide the first detailed structural analysis of DNPEP. We show that this enzyme possesses a binuclear zinc-active site in which one of the zinc ions is readily exchangeable with other divalent cations such as manganese, which strongly stimulates the enzymatic activity of the protein. The plasticity of this metal-binding site suggests a mechanism for regulation of DNPEP activity. We also demonstrate that DNPEP assembles into a functionally relevant tetrahedral complex that restricts access of peptide substrates to the active site. These structural data allow rationalization of the enzyme's preference for short peptide substrates with N-terminal acidic residues. This study provides a structural basis for understanding the physiology and bioinorganic chemistry of DNPEP and other M18 family aminopeptidases.

  4. Transposable elements become active and mobile in the genomes of aging mammalian somatic tissues.

    PubMed

    De Cecco, Marco; Criscione, Steven W; Peterson, Abigail L; Neretti, Nicola; Sedivy, John M; Kreiling, Jill A

    2013-12-01

    Transposable elements (TEs) were discovered by Barbara McClintock in maize and have since been found to be ubiquitous in all living organisms. Transposition is mutagenic and organisms have evolved mechanisms to repress the activity of their endogenous TEs. Transposition in somatic cells is very low, but recent evidence suggests that it may be derepressed in some cases, such as cancer development. We have found that during normal aging several families of retrotransposable elements (RTEs) start being transcribed in mouse tissues. In advanced age the expression culminates in active transposition. These processes are counteracted by calorie restriction (CR), an intervention that slows down aging. Retrotransposition is also activated in age-associated, naturally occurring cancers in the mouse. We suggest that somatic retrotransposition is a hitherto unappreciated aging process. Mobilization of RTEs is likely to be an important contributor to the progressive dysfunction of aging cells. PMID:24323947

  5. Transposable elements become active and mobile in the genomes of aging mammalian somatic tissues.

    PubMed

    De Cecco, Marco; Criscione, Steven W; Peterson, Abigail L; Neretti, Nicola; Sedivy, John M; Kreiling, Jill A

    2013-12-01

    Transposable elements (TEs) were discovered by Barbara McClintock in maize and have since been found to be ubiquitous in all living organisms. Transposition is mutagenic and organisms have evolved mechanisms to repress the activity of their endogenous TEs. Transposition in somatic cells is very low, but recent evidence suggests that it may be derepressed in some cases, such as cancer development. We have found that during normal aging several families of retrotransposable elements (RTEs) start being transcribed in mouse tissues. In advanced age the expression culminates in active transposition. These processes are counteracted by calorie restriction (CR), an intervention that slows down aging. Retrotransposition is also activated in age-associated, naturally occurring cancers in the mouse. We suggest that somatic retrotransposition is a hitherto unappreciated aging process. Mobilization of RTEs is likely to be an important contributor to the progressive dysfunction of aging cells.

  6. Functional differences between junctional and extrajunctional adrenergic receptor activation in mammalian ventricle

    PubMed Central

    Ajijola, Olujimi A.; Vaseghi, Marmar; Zhou, Wei; Yamakawa, Kentaro; Benharash, Peyman; Hadaya, Joseph; Lux, Robert L.; Mahajan, Aman

    2013-01-01

    Increased cardiac sympathetic activation worsens dispersion of repolarization and is proarrhythmic. The functional differences between intrinsic nerve stimulation and adrenergic receptor activation remain incompletely understood. This study was undertaken to determine the functional differences between efferent cardiac sympathetic nerve stimulation and direct adrenergic receptor activation in porcine ventricles. Female Yorkshire pigs (n = 13) underwent surgical exposure of the heart and stellate ganglia. A 56-electrode sock was placed over the ventricles to record epicardial electrograms. Animals underwent bilateral sympathetic stimulation (BSS) (n = 8) or norepinephrine (NE) administration (n = 5). Activation recovery intervals (ARIs) were measured at each electrode before and during BSS or NE. The degree of ARI shortening during BSS or NE administration was used as a measure of functional nerve or adrenergic receptor density. During BSS, ARI shortening was nonuniform across the epicardium (F value 9.62, P = 0.003), with ARI shortening greatest in the mid-basal lateral right ventricle and least in the midposterior left ventricle (LV) (mean normalized values: 0.9 ± 0.08 vs. 0.56 ± 0.08; P = 0.03). NE administration resulted in greater ARI shortening in the LV apex than basal segments [0.91 ± 0.04 vs. 0.63 ± 0.05 (averaged basal segments); P = 0.003]. Dispersion of ARIs increased in 50% and 60% of the subjects undergoing BSS and NE, respectively, but decreased in the others. There is nonuniform response to cardiac sympathetic activation of both porcine ventricles, which is not fully explained by adrenergic receptor density. Different pools of adrenergic receptors may mediate the cardiac electrophysiological effects of efferent sympathetic nerve activity and circulating catecholamines. PMID:23241324

  7. Active calibration target for bistatic radar cross-section measurements

    NASA Astrophysics Data System (ADS)

    Pienaar, M.; Odendaal, J. W.; Joubert, J.; Cilliers, J. E.; Smit, J. C.

    2016-05-01

    Either passive calibration targets are expensive and complex to manufacture or their bistatic radar cross section (RCS) levels are significantly lower than the monostatic RCS levels of targets such as spheres, dihedral, and trihedral corner reflectors. In this paper the performance of an active calibration target with relative high bistatic RCS values is illustrated as a reference target for bistatic RCS measurements. The reference target is simple to manufacture, operates over a wide frequency range, and can be configured to calibrate all four polarizations (VV, HH, HV, and VH). Bistatic RCS measurements of canonical targets, performed in a controlled environment, are calibrated with the reference target and the results are compared to simulated results using FEKO.

  8. Target Fishing for Chemical Compounds using Target-Ligand Activity data and Ranking based Methods

    PubMed Central

    Wale, Nikil; Karypis, George

    2009-01-01

    In recent years the development of computational techniques that identify all the likely targets for a given chemical compound, also termed as the problem of Target Fishing, has been an active area of research. Identification of likely targets of a chemical compound helps to understand problems such as toxicity, lack of efficacy in humans, and poor physical properties associated with that compound in the early stages of drug discovery. In this paper we present a set of techniques whose goal is to rank or prioritize targets in the context of a given chemical compound such that most targets that this compound may show activity against appear higher in the ranked list. These methods are based on our extensions to the SVM and Ranking Perceptron algorithms for this problem. Our extensive experimental study shows that the methods developed in this work outperform previous approaches by 2% to 60% under different evaluation criterions. PMID:19764745

  9. Simultaneous blockade of the epidermal growth factor receptor/mammalian target of rapamycin pathway by epidermal growth factor receptor inhibitors and rapamycin results in reduced cell growth and survival in biliary tract cancer cells.

    PubMed

    Herberger, Beata; Berger, Walter; Puhalla, Harald; Schmid, Katharina; Novak, Sabine; Brandstetter, Anita; Pirker, Christine; Gruenberger, Thomas; Filipits, Martin

    2009-06-01

    The prognosis of patients with biliary tract adenocarcinomas (BTA) is still poor due to lack of effective systemic treatment options. Knowledge of the molecular mechanisms involved in the pathogenesis of this disease is of importance for the development of new treatment strategies. We determined the expression of epidermal growth factor receptor (EGFR) and activated mammalian target of rapamycin (p-mTOR) in paraffin-embedded surgical specimens of BTA (n = 89) by immunohistochemistry. Overall survival was analyzed with Cox models adjusted for clinical and pathologic factors. Combined EGFR/p-mTOR expression was significantly associated with relapse-free survival [adjusted hazard ratio for relapse, 2.20; 95% confidence interval (95% CI), 1.45-3.33; P < 0.001] and overall survival (adjusted hazard ratio for death, 2.32; 95% CI, 1.50-3.58; P < 0.001) of the patients. The effect of the EGFR inhibitors erlotinib or cetuximab and the mTOR inhibitor rapamycin on growth and survival of five BTA cell lines was tested in short-term 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and long-term colony formation assays. Simultaneous blockade of EGFR and mTOR in biliary tract cancer cell lines results in a synergistic inhibition of both phosphatidylinositol-3-kinase and mitogen-activated protein kinase pathways, leading to reduced cell growth and survival. These results suggest that combined targeted therapy with EGFR and mTOR inhibitors may potentially benefit patients with BTAs and should be further evaluated in clinical trials.

  10. The Herbicide Atrazine Activates Endocrine Gene Networks via Non-Steroidal NR5A Nuclear Receptors in Fish and Mammalian Cells

    PubMed Central

    Suzawa, Miyuki; Ingraham, Holly A.

    2008-01-01

    Atrazine (ATR) remains a widely used broadleaf herbicide in the United States despite the fact that this s-chlorotriazine has been linked to reproductive abnormalities in fish and amphibians. Here, using zebrafish we report that environmentally relevant ATR concentrations elevated zcyp19a1 expression encoding aromatase (2.2 µg/L), and increased the ratio of female to male fish (22 µg/L). ATR selectively increased zcyp19a1, a known gene target of the nuclear receptor SF-1 (NR5A1), whereas zcyp19a2, which is estrogen responsive, remained unchanged. Remarkably, in mammalian cells ATR functions in a cell-specific manner to upregulate SF-1 targets and other genes critical for steroid synthesis and reproduction, including Cyp19A1, StAR, Cyp11A1, hCG, FSTL3, LHß, INHα, αGSU, and 11ß-HSD2. Our data appear to eliminate the possibility that ATR directly affects SF-1 DNA- or ligand-binding. Instead, we suggest that the stimulatory effects of ATR on the NR5A receptor subfamily (SF-1, LRH-1, and zff1d) are likely mediated by receptor phosphorylation, amplification of cAMP and PI3K signaling, and possibly an increase in the cAMP-responsive cellular kinase SGK-1, which is known to be upregulated in infertile women. Taken together, we propose that this pervasive and persistent environmental chemical alters hormone networks via convergence of NR5A activity and cAMP signaling, to potentially disrupt normal endocrine development and function in lower and higher vertebrates. PMID:18461179

  11. Isolation of Mammalian Oogonial Stem Cells by Antibody-Based Fluorescence-Activated Cell Sorting.

    PubMed

    Navaroli, Deanna M; Tilly, Jonathan L; Woods, Dori C

    2016-01-01

    The ability to isolate and subsequently culture mitotically active female germ cells from adult ovaries, referred to as either oogonial stem cells (OSCs) or adult female germline stem cells (aFGSCs), has provided a robust system to study female germ cell development under multiple experimental conditions, and in many species. Flow cytometry or fluorescence-activated cell sorting (FACS) is an integral part of many isolation and characterization protocols. Here, we provide methodological details for antibody-based flow cytometric isolation of OSCs using antibodies specific for external epitopes of the proteins Ddx4 or Ifitm3, alone or in combination with the use of fluorescent reporter mice. Beginning with sample preparation, we provide point-by-point instructions to guide researchers on how to isolate OSCs using flow cytometry. PMID:27557587

  12. Quantitation of Na+, K+-atpase Enzymatic Activity in Tissues of the Mammalian Vestibular System

    NASA Technical Reports Server (NTRS)

    Kerr, T. P.

    1985-01-01

    In order to quantify vestibular Na(+), K(+)-ATPase, a microassay technique was developed which is sufficiently sensitive to measure the enzymatic activity in tissue from a single animal. The assay was used to characterize ATPase in he vestibular apparatus of the Mongolian gerbil. The quantitative procedure employs NPP (5 mM) as synthetic enzyme substrate. The assay relies upon spectrophotometric measurement (410 nm) of nitrophenol (NP) released by enzymatic hydrolysis of the substrate. Product formation in the absence of ouabain reflects both specific (Na(+), K(+)-ATPase) and non-specific (Mg(++)-ATPase) enzymatic activity. By measuring the accumulation of reaction product (NP) at three-minute intervals during the course of incubation, it is found that the overall enzymatic reaction proceeds linearly for at least 45 minutes. It is therefore possible to determine two separate reaction rates from a single set of tissues. Initial results indicate that total activity amounts to 53.3 + or - 11.2 (S.E.M.) nmol/hr/mg dry tissue, of which approximately 20% is ouabain-sensitive.

  13. Mammalian airborne allergens.

    PubMed

    Aalberse, Rob C

    2014-01-01

    Historically, horse dandruff was a favorite allergen source material. Today, however, allergic symptoms due to airborne mammalian allergens are mostly a result of indoor exposure, be it at home, at work or even at school. The relevance of mammalian allergens in relation to the allergenic activity of house dust extract is briefly discussed in the historical context of two other proposed sources of house dust allergenic activity: mites and Maillard-type lysine-sugar conjugates. Mammalian proteins involved in allergic reactions to airborne dust are largely found in only 2 protein families: lipocalins and secretoglobins (Fel d 1-like proteins), with a relatively minor contribution of serum albumins, cystatins and latherins. Both the lipocalin and the secretoglobin family are very complex. In some instances this results in a blurred separation between important and less important allergenic family members. The past 50 years have provided us with much detailed information on the genomic organization and protein structure of many of these allergens. However, the complex family relations, combined with the wide range of post-translational enzymatic and non-enzymatic modifications, make a proper qualitative and quantitative description of the important mammalian indoor airborne allergens still a significant proteomic challenge. PMID:24925404

  14. Structural characterization and anti-aging activity of a novel extracellular polysaccharide from fungus Phellinus sp. in a mammalian system.

    PubMed

    Ma, Xiao-Kui; Guo, Dan Dan; Peterson, Eric Charles; Dun, Ying; Li, Dan Yang

    2016-08-10

    Little is known about the chemical structure of purified extracellular polysaccharides from Phellinus sp., a fungal species with known medicinal properties. A combination of IR spectroscopy, methylation analysis and NMR were performed for the structural analysis of a purified extracellular polysaccharide derived from Phellinus sp. culture, denoted as SHP-1, along with an evaluation of the anti-aging effect in vivo of the polysaccharide supplementation. The structure of SHP-1 was established, with a backbone composed of →2,4)-α-d-glucopyranose-(1→ and →2)-β-d-mannopyranose-(1→ and two terminal glucopyranose branches. Biochemical analysis from mammalian animal experiments demonstrated that SHP-1 possesses the ability to enhance antioxidant enzyme activities, such as catalase (CAT) and superoxide dismutase (SOD) activities, Trolox equivalent antioxidant capacity (TEAC) in serum of d-galactose-aged mice, while reducing lipofuscin levels, another indicator of cell aging, indicating a potential association with anti-aging activities in a dose dependent manner. This compound had a favourable influence on immune organ indices, and a marked amelioration ability of histopathological hepatic lesions such as necrosis, karyolysis and reduced inflammation and apoptosis in mouse hepatocytes. These results suggest that SHP-1 has strong antioxidant activities and a significant protective effect against oxidative stress or hepatotoxicity induced by d-galactose in mice and it could be developed as a food ingredient or a pharmaceutical to prevent many age-associated diseases such as major depressive disorder and hepatotoxicity. To our knowledge, this is the first report on the antioxidant effects of a novel purified exopolysaccharide derived from Phellinus sp. PMID:27405813

  15. Cancer active targeting by nanoparticles: a comprehensive review of literature

    PubMed Central

    Bazak, Remon; Houri, Mohamad; Achy, Samar El; Kamel, Serag

    2016-01-01

    Purpose Cancer is one of the leading causes of death, and thus, the scientific community has but great efforts to improve cancer management. Among the major challenges in cancer management is development of agents that can be used for early diagnosis and effective therapy. Conventional cancer management frequently lacks accurate tools for detection of early tumors and has an associated risk of serious side effects of chemotherapeutics. The need to optimize therapeutic ratio as the difference with which a treatment affects cancer cells versus healthy tissues lead to idea that it is needful to have a treatment that could act a the “magic bullet”—recognize cancer cells only. Nanoparticle platforms offer a variety of potentially efficient solutions for development of targeted agents that can be exploited for cancer diagnosis and treatment. There are two ways by which targeting of nanoparticles can be achieved, namely passive and active targeting. Passive targeting allows for the efficient localization of nanoparticles within the tumor microenvironment. Active targeting facilitates the active uptake of nanoparticles by the tumor cells themselves. Methods Relevant English electronic databases and scientifically published original articles and reviews were systematically searched for the purpose of this review. Results In this report, we present a comprehensive review of literatures focusing on the active targeting of nanoparticles to cancer cells, including antibody and antibody fragment-based targeting, antigen-based targeting, aptamer-based targeting, as well as ligand-based targeting. Conclusion To date, the optimum targeting strategy has not yet been announced, each has its own advantages and disadvantages even though a number of them have found their way for clinical application. Perhaps, a combination of strategies can be employed to improve the precision of drug delivery, paving the way for a more effective personalized therapy. PMID:25005786

  16. Reversible oxidation controls the activity and oligomeric state of the mammalian phosphoglycolate phosphatase AUM.

    PubMed

    Seifried, Annegrit; Bergeron, Alexandre; Boivin, Benoit; Gohla, Antje

    2016-08-01

    Redox-dependent switches of enzyme activity are emerging as important fine-tuning mechanisms in cell signaling. For example, protein tyrosine phosphatases employ a conserved cysteine residue for catalysis, which also renders them highly susceptible to reversible inactivation by oxidation. In contrast, haloacid dehalogenase (HAD)-type phosphatases perform catalysis via a phosphoaspartyltransferase reaction. The potential regulation of HAD phosphatases by reversible oxidation has not yet been explored. Here, we investigate the redox-sensitivity of the HAD-type phosphoglycolate phosphatase PGP, also known as AUM or glycerol-3-phosphate phosphatase. We show that recombinant, purified murine PGP is inhibited by oxidation and re-activated by reduction. We identify three reactive cysteine residues in the catalytic core domain of PGP (Cys35, Cys104 and Cys243) that mediate the reversible inhibition of PGP activity and the associated, redox-dependent conformational changes. Structural analysis suggests that Cys35 oxidation weakens van-der-Waals interactions with Thr67, a conserved catalytic residue required for substrate coordination. Cys104 and Cys243 form a redox-dependent disulfide bridge between the PGP catalytic core and cap domains, which may impair the open/close-dynamics of the catalytic cycle. In addition, we demonstrate that Cys297 in the PGP cap domain is essential for redox-dependent PGP oligomerization, and that PGP oxidation/oligomerization occurs in response to stimulation of cells with EGF. Finally, employing a modified cysteinyl-labeling assay, we show that cysteines of cellular PGP are transiently oxidized to sulfenic acids. Taken together, our findings establish that PGP, an aspartate-dependent HAD phosphatase, is transiently inactivated by reversible oxidation in cells.

  17. Body size and activity times mediate mammalian responses to climate change.

    PubMed

    McCain, Christy M; King, Sarah R B

    2014-06-01

    Model predictions of extinction risks from anthropogenic climate change are dire, but still overly simplistic. To reliably predict at-risk species we need to know which species are currently responding, which are not, and what traits are mediating the responses. For mammals, we have yet to identify overarching physiological, behavioral, or biogeographic traits determining species' responses to climate change, but they must exist. To date, 73 mammal species in North America and eight additional species worldwide have been assessed for responses to climate change, including local extirpations, range contractions and shifts, decreased abundance, phenological shifts, morphological or genetic changes. Only 52% of those species have responded as expected, 7% responded opposite to expectations, and the remaining 41% have not responded. Which mammals are and are not responding to climate change is mediated predominantly by body size and activity times (phylogenetic multivariate logistic regressions, P < 0.0001). Large mammals respond more, for example, an elk is 27 times more likely to respond to climate change than a shrew. Obligate diurnal and nocturnal mammals are more than twice as likely to respond as mammals with flexible activity times (P < 0.0001). Among the other traits examined, species with higher latitudinal and elevational ranges were more likely to respond to climate change in some analyses, whereas hibernation, heterothermy, burrowing, nesting, and study location did not influence responses. These results indicate that some mammal species can behaviorally escape climate change whereas others cannot, analogous to paleontology's climate sheltering hypothesis. Including body size and activity flexibility traits into future extinction risk forecasts should substantially improve their predictive utility for conservation and management. PMID:24449019

  18. Body size and activity times mediate mammalian responses to climate change.

    PubMed

    McCain, Christy M; King, Sarah R B

    2014-06-01

    Model predictions of extinction risks from anthropogenic climate change are dire, but still overly simplistic. To reliably predict at-risk species we need to know which species are currently responding, which are not, and what traits are mediating the responses. For mammals, we have yet to identify overarching physiological, behavioral, or biogeographic traits determining species' responses to climate change, but they must exist. To date, 73 mammal species in North America and eight additional species worldwide have been assessed for responses to climate change, including local extirpations, range contractions and shifts, decreased abundance, phenological shifts, morphological or genetic changes. Only 52% of those species have responded as expected, 7% responded opposite to expectations, and the remaining 41% have not responded. Which mammals are and are not responding to climate change is mediated predominantly by body size and activity times (phylogenetic multivariate logistic regressions, P < 0.0001). Large mammals respond more, for example, an elk is 27 times more likely to respond to climate change than a shrew. Obligate diurnal and nocturnal mammals are more than twice as likely to respond as mammals with flexible activity times (P < 0.0001). Among the other traits examined, species with higher latitudinal and elevational ranges were more likely to respond to climate change in some analyses, whereas hibernation, heterothermy, burrowing, nesting, and study location did not influence responses. These results indicate that some mammal species can behaviorally escape climate change whereas others cannot, analogous to paleontology's climate sheltering hypothesis. Including body size and activity flexibility traits into future extinction risk forecasts should substantially improve their predictive utility for conservation and management.

  19. Reversible oxidation controls the activity and oligomeric state of the mammalian phosphoglycolate phosphatase AUM.

    PubMed

    Seifried, Annegrit; Bergeron, Alexandre; Boivin, Benoit; Gohla, Antje

    2016-08-01

    Redox-dependent switches of enzyme activity are emerging as important fine-tuning mechanisms in cell signaling. For example, protein tyrosine phosphatases employ a conserved cysteine residue for catalysis, which also renders them highly susceptible to reversible inactivation by oxidation. In contrast, haloacid dehalogenase (HAD)-type phosphatases perform catalysis via a phosphoaspartyltransferase reaction. The potential regulation of HAD phosphatases by reversible oxidation has not yet been explored. Here, we investigate the redox-sensitivity of the HAD-type phosphoglycolate phosphatase PGP, also known as AUM or glycerol-3-phosphate phosphatase. We show that recombinant, purified murine PGP is inhibited by oxidation and re-activated by reduction. We identify three reactive cysteine residues in the catalytic core domain of PGP (Cys35, Cys104 and Cys243) that mediate the reversible inhibition of PGP activity and the associated, redox-dependent conformational changes. Structural analysis suggests that Cys35 oxidation weakens van-der-Waals interactions with Thr67, a conserved catalytic residue required for substrate coordination. Cys104 and Cys243 form a redox-dependent disulfide bridge between the PGP catalytic core and cap domains, which may impair the open/close-dynamics of the catalytic cycle. In addition, we demonstrate that Cys297 in the PGP cap domain is essential for redox-dependent PGP oligomerization, and that PGP oxidation/oligomerization occurs in response to stimulation of cells with EGF. Finally, employing a modified cysteinyl-labeling assay, we show that cysteines of cellular PGP are transiently oxidized to sulfenic acids. Taken together, our findings establish that PGP, an aspartate-dependent HAD phosphatase, is transiently inactivated by reversible oxidation in cells. PMID:27179418

  20. piggyBac is a flexible and highly active transposon as compared to sleeping beauty, Tol2, and Mos1 in mammalian cells.

    PubMed

    Wu, Sareina Chiung-Yuan; Meir, Yaa-Jyuhn James; Coates, Craig J; Handler, Alfred M; Pelczar, Pawel; Moisyadi, Stefan; Kaminski, Joseph M

    2006-10-10

    A nonviral vector for highly efficient site-specific integration would be desirable for many applications in transgenesis, including gene therapy. In this study we directly compared the genomic integration efficiencies of piggyBac, hyperactive Sleeping Beauty (SB11), Tol2, and Mos1 in four mammalian cell lines. piggyBac demonstrated significantly higher transposition activity in all cell lines whereas Mos1 had no activity. Furthermore, piggyBac transposase coupled to the GAL4 DNA-binding domain retains transposition activity whereas similarly manipulated gene products of Tol2 and SB11 were inactive. The high transposition activity of piggyBac and the flexibility for molecular modification of its transposase suggest the possibility of using it routinely for mammalian transgenesis. PMID:17005721

  1. Probable nature of higher-dimensional symmetries underlying mammalian grid-cell activity patterns

    PubMed Central

    Mathis, Alexander; Stemmler, Martin B; Herz, Andreas VM

    2015-01-01

    Lattices abound in nature—from the crystal structure of minerals to the honey-comb organization of ommatidia in the compound eye of insects. These arrangements provide solutions for optimal packings, efficient resource distribution, and cryptographic protocols. Do lattices also play a role in how the brain represents information? We focus on higher-dimensional stimulus domains, with particular emphasis on neural representations of physical space, and derive which neuronal lattice codes maximize spatial resolution. For mammals navigating on a surface, we show that the hexagonal activity patterns of grid cells are optimal. For species that move freely in three dimensions, a face-centered cubic lattice is best. This prediction could be tested experimentally in flying bats, arboreal monkeys, or marine mammals. More generally, our theory suggests that the brain encodes higher-dimensional sensory or cognitive variables with populations of grid-cell-like neurons whose activity patterns exhibit lattice structures at multiple, nested scales. DOI: http://dx.doi.org/10.7554/eLife.05979.001 PMID:25910055

  2. Effects of trimebutine maleate on electrical activities of isolated mammalian cardiac preparations.

    PubMed

    Igawa, O; Kotake, H; Hirai, S; Hisatome, I; Hasegawa, J; Mashiba, H

    1989-05-01

    The effects of trimebutine maleate on electrical activity in guinea-pig isolated papillary muscles and rabbit sino-atrial nodes have been studied by means of a standard microelectrode method. In papillary muscles, trimebutine (above 10 microM) decreased the maximum rate of rise (Vmax) and the action potential duration at 90% repolarization (APD90), whereas the resting potential was not significantly altered. As to a decrease in Vmax, trimebutine produced a negative shift of the curve relating Vmax to the resting potential along the voltage axis. Trimebutine also depressed the slow action potentials of papillary muscles produced by 27 mM K and 0.2 mM Ba. In spontaneously beating sino-atrial node preparations, trimebutine (above 10 microM) decreased the heart rate, Vmax and the rate of diastolic depolarization. These results indicate that trimebutine maleate possesses a depressant action on the electrical activities of the fast- and slow-response fibres of the heart mainly due to inhibitions of both fast Na+ and slow Ca2+ channels. PMID:2569517

  3. Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells

    PubMed Central

    Descostes, Nicolas; Heidemann, Martin; Spinelli, Lionel; Schüller, Roland; Maqbool, Muhammad Ahmad; Fenouil, Romain; Koch, Frederic; Innocenti, Charlène; Gut, Marta; Gut, Ivo; Eick, Dirk; Andrau, Jean-Christophe

    2014-01-01

    In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5′ associated) Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability. DOI: http://dx.doi.org/10.7554/eLife.02105.001 PMID:24842994

  4. Activity assays of mammalian thioredoxin and thioredoxin reductase: fluorescent disulfide substrates, mechanisms, and use with tissue samples.

    PubMed

    Montano, Sergio J; Lu, Jun; Gustafsson, Tomas N; Holmgren, Arne

    2014-03-15

    Thioredoxin (Trx) is a protein disulfide reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin-glutathione disulfide (Di-E-GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC-insulin), which displayed higher fluorescence on disulfide reduction. Di-E-GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC-insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.

  5. Designing quantitative structure activity relationships to predict specific toxic endpoints for polybrominated diphenyl ethers in mammalian cells.

    PubMed

    Rawat, S; Bruce, E D

    2014-01-01

    Polybrominated diphenyl ethers (PBDEs) are known as effective flame retardants and have vast industrial application in products like plastics, building materials and textiles. They are found to be structurally similar to thyroid hormones that are responsible for regulating metabolism in the body. Structural similarity with the hormones poses a threat to human health because, once in the system, PBDEs have the potential to affect thyroid hormone transport and metabolism. This study was aimed at designing quantitative structure-activity relationship (QSAR) models for predicting toxic endpoints, namely cell viability and apoptosis, elicited by PBDEs in mammalian cells. Cell viability was evaluated quantitatively using a general cytotoxicity bioassay using Janus Green dye and apoptosis was evaluated using a caspase assay. This study has thus modelled the overall cytotoxic influence of PBDEs at an early and a late endpoint by the Genetic Function Approximation method. This research was a twofold process including running in vitro bioassays to collect data on the toxic endpoints and modeling the evaluated endpoints using QSARs. Cell viability and apoptosis responses for Hep G2 cells exposed to PBDEs were successfully modelled with an r(2) of 0.97 and 0.94, respectively. PMID:24738916

  6. Expression of active human clotting factor IX from recombinant DNA clones in mammalian cells.

    PubMed

    Anson, D S; Austen, D E; Brownlee, G G

    Haemophilia B, or Christmas disease, is an inherited X-chromosome-linked bleeding disorder caused by a defect in clotting factor IX and occurs in about 1 in 30,000 males in the United Kingdom. Injection of factor IX concentrate obtained from blood donors allows most patients to be successfully managed. However, because of impurities in the factor IX concentrate presently in use, this treatment involves some risk of infection by blood-borne viruses such as non-A, non-B hepatitis and the virus causing acquired immune deficiency syndrome (AIDS). Because of the recent concern about the increasing incidence of AIDS amongst haemophiliacs, a factor IX preparation derived from a source other than blood is desirable. Here, we report that after introduction of human factor IX DNA clones into a rat hepatoma cell line using recombinant DNA methods, we were able to isolate small amounts of biologically active human factor IX.

  7. The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities.

    PubMed

    Hageman, Jurre; van Waarde, Maria A W H; Zylicz, Alicja; Walerych, Dawid; Kampinga, Harm H

    2011-04-01

    Humans contain many HSP (heat-shock protein) 70/HSPA- and HSP40/DNAJ-encoding genes and most of the corresponding proteins are localized in the cytosol. To test for possible functional differences and/or substrate specificity, we assessed the effect of overexpression of each of these HSPs on refolding of heat-denatured luciferase and on the suppression of aggregation of a non-foldable polyQ (polyglutamine)-expanded Huntingtin fragment. Overexpressed chaperones that suppressed polyQ aggregation were found not to be able to stimulate luciferase refolding. Inversely, chaperones that supported luciferase refolding were poor suppressors of polyQ aggregation. This was not related to client specificity itself, as the polyQ aggregation inhibitors often also suppressed heat-induced aggregation of luciferase. Surprisingly, the exclusively heat-inducible HSPA6 lacks both luciferase refolding and polyQ aggregation-suppressing activities. Furthermore, whereas overexpression of HSPA1A protected cells from heat-induced cell death, overexpression of HSPA6 did not. Inversely, siRNA (small interfering RNA)-mediated blocking of HSPA6 did not impair the development of heat-induced thermotolerance. Yet, HSPA6 has a functional substrate-binding domain and possesses intrinsic ATPase activity that is as high as that of the canonical HSPA1A when stimulated by J-proteins. In vitro data suggest that this may be relevant to substrate specificity, as purified HSPA6 could not chaperone heat-unfolded luciferase but was able to assist in reactivation of heat-unfolded p53. So, even within the highly sequence-conserved HSPA family, functional differentiation is larger than expected, with HSPA6 being an extreme example that may have evolved to maintain specific critical functions under conditions of severe stress.

  8. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    SciTech Connect

    Roper, Katherine; Coverley, Dawn

    2012-03-10

    In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naieve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naieve nuclei. At the same time, H2AX is phosphorylated in naieve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naieve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: Black-Right-Pointing-Pointer A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. Black-Right-Pointing-Pointer Damage-activated extracts impose the cellular response to DNA damage on naieve nuclei. Black-Right-Pointing-Pointer PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. Black-Right-Pointing-Pointer Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. Black-Right-Pointing-Pointer LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening

  9. The mammalian guanine nucleotide exchange factor mSec12 is essential for activation of the Sar1 GTPase directing endoplasmic reticulum export.

    PubMed

    Weissman, J T; Plutner, H; Balch, W E

    2001-07-01

    The Sar1 GTPase is an essential component of COPII vesicle coats involved in export of cargo from the endoplasmic reticulum of mammalian cells. To begin to elucidate its mechanism of action, we now report the identity of the mammalian homolog to the yeast Sec12 guanine nucleotide exchange factor (18% identity) that promotes Sar1 activation. Mammalian Sec12 (mSec12) is a type II transmembrane protein with a large cytosolic domain, a fragment of which has previously been reported as the transcription factor prolactin regulatory element binding protein (PREB). mSec12 promotes efficient guanine nucleotide exchange on Sar1, but not Arf1 or Rab GTPases. mSec12 is localized to the endoplasmic reticulum and an antibody to the cytosolic domain of mSec12 potently inhibits Sar1 recruitment and the formation of COPII vesicles in vitro. The dominant negative GDP-restricted mutant Sar1[T39N] is shown to be a potent inhibitor of mSec12 activity, consistent with its role in preventing COPII vesicle formation in vitro and during transient expression in vivo. We propose that mSec12 is an evolutionarily distant guanine nucleotide exchange factor directing Sar1 GTPase activation in mammalian cells. Its divergence from yeast Sec12p may reflect the specialized needs of the mammalian endoplasmic reticulum involving the formation of Sar1-dependent transitional elements (Aridor M, et al. J Cell Biol 2001;152:213-229) and selection of cargo into prebudding complexes.

  10. Thermal stress and Ca-independent contractile activation in mammalian skeletal muscle fibers at high temperatures.

    PubMed Central

    Ranatunga, K W

    1994-01-01

    Temperature dependence of the isometric tension was examined in chemically skinned, glycerinated, rabbit Psoas, muscle fibers immersed in relaxing solution (pH approximately 7.1 at 20 degrees C, pCa approximately 8, ionic strength 200 mM); the average rate of heating/cooling was 0.5-1 degree C/s. The resting tension increased reversibly with temperature (5-42 degrees C); the tension increase was slight in warming to approximately 25 degrees C (a linear thermal contraction, -alpha, of approximately 0.1%/degree C) but became more pronounced above approximately 30 degrees C (similar behavior was seen in intact rat muscle fibers). The extra tension rise at the high temperatures was depressed in acidic pH and in the presence of 10 mM inorganic phosphate; it was absent in rigor fibers in which the tension decreased with heating (a linear thermal expansion, alpha, of approximately 4 x 10(-5)/degree C). Below approximately 20 degrees C, the tension response after a approximately 1% length increase (complete < 0.5 ms) consisted of a fast decay (approximately 150.s-1 at 20 degrees C) and a slow decay (approximately 10.s-1) of tension. The rate of fast decay increased with temperature (Q10 approximately 2.4); at 35-40 degrees C, it was approximately 800.s-1, and it was followed by a delayed tension rise (stretch-activation) at 30-40.s-1. The linear rise of passive tension in warming to approximately 25 degrees C may be due to increase of thermal stress in titin (connectin)-myosin composite filament, whereas the extra tension above approximately 30 degrees C may arise from cycling cross-bridges; based on previous findings from regulated actomyosin in solution (Fuchs, 1975), it is suggested that heating reversibly inactivates the troponin-tropomyosin control mechanism and leads to Ca-independent thin filament activation at high temperatures. Additionally, we propose that the heating-induced increase of endo-sarcomeric stress within titin-myosin composite filament makes the

  11. Discovery and optimization of 2-(4-substituted-pyrrolo[2,3-b]pyridin-3-yl)methylene-4-hydroxybenzofuran-3(2H)-ones as potent and selective ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR)

    SciTech Connect

    Tsoua, Hwei-Ru; MacEwana, Gloria; Birnberga, Gary; Grosua, George; Bursavicha, Matthew G.; Barda, Joel; Brooijmansa, Natasja; Toral-Barzab, Lourdes; Hollanderb, Irwin; Mansoura, Tarek S.; Ayral-Kaloustiana, Semiramis; Yub, Ker

    2010-07-19

    We discovered 2-(4-substituted-pyrrolo[2,3-b]pyridin-3-yl)methylene-4-hydroxybenzofuran-3(2H)-ones as potent and selective ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR). Since phenolic OH groups pose metabolic liability, one of the two hydroxyl groups was selectively removed. The SAR data showed the structural features necessary for subnanomolar inhibitory activity against mTOR kinase as well as selectivity over PI3K?. An X-ray co-crystal structure of one inhibitor with the mTOR-related PI3K? revealed the key hydrogen bonding interactions.

  12. Targeting Energy Metabolic and Oncogenic Signaling Pathways in Triple-negative Breast Cancer by a Novel Adenosine Monophosphate-activated Protein Kinase (AMPK) Activator*

    PubMed Central

    Lee, Kuen-Haur; Hsu, En-Chi; Guh, Jih-Hwa; Yang, Hsiao-Ching; Wang, Dasheng; Kulp, Samuel K.; Shapiro, Charles L.; Chen, Ching-Shih

    2011-01-01

    The antitumor activities of the novel adenosine monophosphate-activated protein kinase (AMPK) activator, OSU-53, were assessed in in vitro and in vivo models of triple-negative breast cancer. OSU-53 directly stimulated recombinant AMPK kinase activity (EC50, 0.3 μm) and inhibited the viability and clonogenic growth of MDA-MB-231 and MDA-MB-468 cells with equal potency (IC50, 5 and 2 μm, respectively) despite lack of LKB1 expression in MDA-MB-231 cells. Nonmalignant MCF-10A cells, however, were unaffected. Beyond AMPK-mediated effects on mammalian target of rapamycin signaling and lipogenesis, OSU-53 also targeted multiple AMPK downstream pathways. Among these, the protein phosphatase 2A-dependent dephosphorylation of Akt is noteworthy because it circumvents the feedback activation of Akt that results from mammalian target of rapamycin inhibition. OSU-53 also modulated energy homeostasis by suppressing fatty acid biosynthesis and shifting the metabolism to oxidation by up-regulating the expression of key regulators of mitochondrial biogenesis, such as a peroxisome proliferator-activated receptor γ coactivator 1α and the transcription factor nuclear respiratory factor 1. Moreover, OSU-53 suppressed LPS-induced IL-6 production, thereby blocking subsequent Stat3 activation, and inhibited hypoxia-induced epithelial-mesenchymal transition in association with the silencing of hypoxia-inducible factor 1a and the E-cadherin repressor Snail. In MDA-MB-231 tumor-bearing mice, daily oral administration of OSU-53 (50 and 100 mg/kg) suppressed tumor growth by 47–49% and modulated relevant intratumoral biomarkers of drug activity. However, OSU-53 also induced protective autophagy that attenuated its antiproliferative potency. Accordingly, cotreatment with the autophagy inhibitor chloroquine increased the in vivo tumor-suppressive activity of OSU-53. OSU-53 is a potent, orally bioavailable AMPK activator that acts through a broad spectrum of antitumor activities. PMID

  13. Mammalian cAMP-responsive element can activate transcription in yeast and binds a yeast factor(s) that resembles the mammalian transcription factor ANF.

    PubMed Central

    Jones, R H; Jones, N C

    1989-01-01

    The human ATF and AP1 transcription factors bind to highly related DNA sequences. Their consensus binding sites differ by a single nucleotide, but this single change is crucial in determining factor binding specificity. We have previously identified an AP1 (yAP1) binding activity in yeast. In this report we identify a yeast ATF (yATF) binding activity whose specificity can be distinguished from that of yAP1 by the same crucial nucleotide that distinguishes binding of human ATF and AP1. The ATF binding site can act as an efficient upstream activating sequence in vivo, suggesting that yATF is a transcriptional activator. The yATF DNA-binding complex is phosphorylated and the binding activity of partially purified yATF can be enhanced in vitro by the addition of protein kinase A, indicating that the phosphorylation state of yATF may be important in determining its ability to bind DNA. Images PMID:2538834

  14. Histone H4 Lys 20 monomethylation by histone methylase SET8 mediates Wnt target gene activation.

    PubMed

    Li, Zhenfei; Nie, Fen; Wang, Sheng; Li, Lin

    2011-02-22

    Histone methylation has an important role in transcriptional regulation. However, unlike H3K4 and H3K9 methylation, the role of H4K20 monomethylation (H4K20me-1) in transcriptional regulation remains unclear. Here, we show that Wnt3a specifically stimulates H4K20 monomethylation at the T cell factor (TCF)-binding element through the histone methylase SET8. Additionally, SET8 is crucial for activation of the Wnt reporter gene and target genes in both mammalian cells and zebrafish. Furthermore, SET8 interacts with lymphoid enhancing factor-1 (LEF1)/TCF4 directly, and this interaction is regulated by Wnt3a. Therefore, we conclude that SET8 is a Wnt signaling mediator and is recruited by LEF1/TCF4 to regulate the transcription of Wnt-activated genes, possibly through H4K20 monomethylation at the target gene promoters. Our findings also indicate that H4K20me-1 is a marker for gene transcription activation, at least in canonical Wnt signaling. PMID:21282610

  15. Targeted Deficiency of the Transcriptional Activator Hnf1α Alters Subnuclear Positioning of Its Genomic Targets

    PubMed Central

    Sadoni, Nicolas; Zink, Daniele; Ferrer, Jorge

    2008-01-01

    DNA binding transcriptional activators play a central role in gene-selective regulation. In part, this is mediated by targeting local covalent modifications of histone tails. Transcriptional regulation has also been associated with the positioning of genes within the nucleus. We have now examined the role of a transcriptional activator in regulating the positioning of target genes. This was carried out with primary β-cells and hepatocytes freshly isolated from mice lacking Hnf1α, an activator encoded by the most frequently mutated gene in human monogenic diabetes (MODY3). We show that in Hnf1a−/− cells inactive endogenous Hnf1α-target genes exhibit increased trimethylated histone H3-Lys27 and reduced methylated H3-Lys4. Inactive Hnf1α-targets in Hnf1a−/− cells are also preferentially located in peripheral subnuclear domains enriched in trimethylated H3-Lys27, whereas active targets in wild-type cells are positioned in more central domains enriched in methylated H3-Lys4 and RNA polymerase II. We demonstrate that this differential positioning involves the decondensation of target chromatin, and show that it is spatially restricted rather than a reflection of non-specific changes in the nuclear organization of Hnf1a-deficient cells. This study, therefore, provides genetic evidence that a single transcriptional activator can influence the subnuclear location of its endogenous genomic targets in primary cells, and links activator-dependent changes in local chromatin structure to the spatial organization of the genome. We have also revealed a defect in subnuclear gene positioning in a model of a human transcription factor disease. PMID:18497863

  16. Selenite-induced variation in glutathione peroxidase activity of three mammalian cell lines: no effect on radiation-induced cell killing or DNA strand breakage

    SciTech Connect

    Sandstroem, B.E.C.; Carlsson, J.; Marklund, S.L.

    1989-02-01

    The selenium-dependent glutathione peroxidase activities of three mammalian cell lines, HT29, P31, and N-18, cultured in medium with low serum content, increased about 2-, 5-, and 40-fold, respectively, after supplementation with 100 nM selenite. Catalase, CuZn superoxide dismutase, and Mn superoxide dismutase activities were not generally influenced by selenite supplementation, and there was only a minor nonselenium-dependent glutathione peroxidase activity in the investigated cell lines. Gamma-irradiated control and selenite-supplemented cells showed no changes in the surviving fractions, as estimated by clonogenic survival or (/sup 3/H)-thymidine uptake, nor were there any significant differences between the two groups in the induction of DNA strand breaks after gamma irradiation under repairing (37 degrees C) or nonrepairing (0 degrees C) conditions. The results suggest that selenium-dependent glutathione peroxidase does not contribute significantly to the radiation resistance of cultured mammalian cells.

  17. Enhanced Active Targeting via Cooperative Binding of Ligands on Liposomes to Target Receptors

    PubMed Central

    Sugiyama, Tomoki; Asai, Tomohiro; Nedachi, Yuki Murase; Katanasaka, Yasufumi; Shimizu, Kosuke; Maeda, Noriyuki; Oku, Naoto

    2013-01-01

    To achieve effective active targeting in a drug delivery system, we previously developed dual-targeting (DT) liposomes decorated with both vascular endothelial growth factor receptor-1 (VEGFR-1)-targeted APRPG and CD13-targeted GNGRG peptide ligands for tumor neovessels, and observed the enhanced suppression of tumor growth in Colon26 NL-17 tumor-bearing mice by the treatment with the DT liposomes encapsulating doxorubicin. In this present study, we examined the binding characteristics of DT liposomes having a different couple of ligands, namely, APRPG and integrin αvβ3-targeted GRGDS peptides. These DT liposomes synergistically associated to stimulated human umbilical vein endothelial cells compared with single-targeting (ST) liposomes decorated with APRPG or GRGDS. The results of a surface plasmon resonance assay showed that ST liposomes modified with APRPG or GRGDS peptide selectively bound to immobilized VEGFR-1 or integrin αvβ3, respectively. DT liposomes showed a higher affinity for a mixture of VEGFR-1 and integrin αvβ3 compared with ST liposomes, suggesting the cooperative binding of these 2 kinds of ligand on the liposomal surface. In a biodistribution assay, the DT liposomes accumulated to a significantly greater extent in the tumors of Colon26 NL-17 tumor-bearing mice compared with other liposomes. Moreover, the intratumoral distribution of the liposomes examined by confocal microscopy suggested that the DT liposomes targeted not only angiogenic endothelial cells but also tumor cells due to GRGDS-decoration. These findings suggest that "dual-targeting" augmented the affinity of the liposomes for the target cells and would thus be useful for active-targeting drug delivery for cancer treatment. PMID:23840738

  18. Mammalian Target of Rapamycin (mTOR) Tagging Promotes Dendritic Branch Variability through the Capture of Ca2+/Calmodulin-dependent Protein Kinase II α (CaMKIIα) mRNAs by the RNA-binding Protein HuD*

    PubMed Central

    Sosanya, Natasha M.; Cacheaux, Luisa P.; Workman, Emily R.; Niere, Farr; Perrone-Bizzozero, Nora I.; Raab-Graham, Kimberly F.

    2015-01-01

    The fate of a memory, whether stored or forgotten, is determined by the ability of an active or tagged synapse to undergo changes in synaptic efficacy requiring protein synthesis of plasticity-related proteins. A synapse can be tagged, but without the “capture” of plasticity-related proteins, it will not undergo long lasting forms of plasticity (synaptic tagging and capture hypothesis). What the “tag” is and how plasticity-related proteins are captured at tagged synapses are unknown. Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) is critical in learning and memory and is synthesized locally in neuronal dendrites. The mechanistic (mammalian) target of rapamycin (mTOR) is a protein kinase that increases CaMKIIα protein expression; however, the mechanism and site of dendritic expression are unknown. Herein, we show that mTOR activity mediates the branch-specific expression of CaMKIIα, favoring one secondary, daughter branch over the other in a single neuron. mTOR inhibition decreased the dendritic levels of CaMKIIα protein and mRNA by shortening its poly(A) tail. Overexpression of the RNA-stabilizing protein HuD increased CaMKIIα protein levels and preserved its selective expression in one daughter branch over the other when mTOR was inhibited. Unexpectedly, deleting the third RNA recognition motif of HuD, the domain that binds the poly(A) tail, eliminated the branch-specific expression of CaMKIIα when mTOR was active. These results provide a model for one molecular mechanism that may underlie the synaptic tagging and capture hypothesis where mTOR is the tag, preventing deadenylation of CaMKIIα mRNA, whereas HuD captures and promotes its expression in a branch-specific manner. PMID:25944900

  19. High efficiency cell-specific targeting of cytokine activity

    NASA Astrophysics Data System (ADS)

    Garcin, Geneviève; Paul, Franciane; Staufenbiel, Markus; Bordat, Yann; van der Heyden, José; Wilmes, Stephan; Cartron, Guillaume; Apparailly, Florence; de Koker, Stefaan; Piehler, Jacob; Tavernier, Jan; Uzé, Gilles

    2014-01-01

    Systemic toxicity currently prevents exploiting the huge potential of many cytokines for medical applications. Here we present a novel strategy to engineer immunocytokines with very high targeting efficacies. The method lies in the use of mutants of toxic cytokines that markedly reduce their receptor-binding affinities, and that are thus rendered essentially inactive. Upon fusion to nanobodies specifically binding to marker proteins, activity of these cytokines is selectively restored for cell populations expressing this marker. This ‘activity-by-targeting’ concept was validated for type I interferons and leptin. In the case of interferon, activity can be directed to target cells in vitro and to selected cell populations in mice, with up to 1,000-fold increased specific activity. This targeting strategy holds promise to revitalize the clinical potential of many cytokines.

  20. Protein Folding Activity of Ribosomal RNA Is a Selective Target of Two Unrelated Antiprion Drugs

    PubMed Central

    Tribouillard-Tanvier, Déborah; Dos Reis, Suzana; Gug, Fabienne; Voisset, Cécile; Béringue, Vincent; Sabate, Raimon; Kikovska, Ema; Talarek, Nicolas; Bach, Stéphane; Huang, Chenhui; Desban, Nathalie; Saupe, Sven J.; Supattapone, Surachai; Thuret, Jean-Yves; Chédin, Stéphane; Vilette, Didier; Galons, Hervé; Sanyal, Suparna; Blondel, Marc

    2008-01-01

    Background 6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases. Methodology/Principal Findings Here we report the identification of cellular targets of these drugs. Using affinity chromatography matrices for both drugs, we demonstrate an RNA-dependent interaction of 6AP and GA with the ribosome. These specific interactions have no effect on the peptidyl transferase activity of the ribosome or on global translation. In contrast, 6AP and GA specifically inhibit the ribosomal RNA-mediated protein folding activity of the ribosome. Conclusion/Significance 6AP and GA are therefore the first compounds to selectively inhibit the protein folding activity of the ribosome. They thus constitute precious tools to study the yet largely unexplored biological role of this protein folding activity. PMID:18478094

  1. Mammalian sleep

    NASA Astrophysics Data System (ADS)

    Staunton, Hugh

    2005-05-01

    This review examines the biological background to the development of ideas on rapid eye movement sleep (REM sleep), so-called paradoxical sleep (PS), and its relation to dreaming. Aspects of the phenomenon which are discussed include physiological changes and their anatomical location, the effects of total and selective sleep deprivation in the human and animal, and REM sleep behavior disorder, the latter with its clinical manifestations in the human. Although dreaming also occurs in other sleep phases (non-REM or NREM sleep), in the human, there is a contingent relation between REM sleep and dreaming. Thus, REM is taken as a marker for dreaming and as REM is distributed ubiquitously throughout the mammalian class, it is suggested that other mammals also dream. It is suggested that the overall function of REM sleep/dreaming is more important than the content of the individual dream; its function is to place the dreamer protagonist/observer on the topographical world. This has importance for the developing infant who needs to develop a sense of self and separateness from the world which it requires to navigate and from which it is separated for long periods in sleep. Dreaming may also serve to maintain a sense of ‘I’ness or “self” in the adult, in whom a fragility of this faculty is revealed in neurological disorders.

  2. Details in the catalytic mechanism of mammalian thioredoxin reductase 1 revealed using point mutations and juglone-coupled enzyme activities.

    PubMed

    Xu, Jianqiang; Cheng, Qing; Arnér, Elias S J

    2016-05-01

    The mammalian selenoprotein thioredoxin reductase 1 (TrxR1) is a key enzyme in redox regulation, antioxidant defense, and cellular growth. TrxR1 can catalyze efficient reduction of juglone (5-hydroxy-1,4-naphthoquinone; walnut toxin) in a reaction which, in contrast to reduction of most other substrates of TrxR1, is not dependent upon an intact selenocysteine (Sec, U) residue of the enzyme. Using a number of TrxR1 mutant variants, we here found that a sole Cys residue at the C-terminal tail of TrxR1 is required for high-efficiency juglone-coupled NADPH oxidase activity of Sec-deficient enzyme, occurring with mixed one- and two-electron reactions producing superoxide. The activity also utilizes the FAD and the N-terminal redox active disulfide/dithiol motif of TrxR1. If a sole Cys residue at the C-terminal tail of TrxR1, in the absence of Sec, was moved further towards the C-terminal end of the protein compared to its natural position at residue 497, juglone reduction was, surprisingly, further increased. Ala substitutions of Trp407, Asn418 and Asn419 in a previously described "guiding bar", thought to mediate interactions of the C-terminal tail of TrxR1 with the FAD/dithiol site at the N-terminal domain of the other subunit in the dimeric enzyme, lowered turnover with juglone about 4.5-fold. Four residues of Sec-deficient TrxR1 were found to be easily arylated by juglone, including the Cys residue at position 497. Based upon our observations we suggest a model for involvement of the juglone-arylated C-terminal motif of TrxR1 to explain its high activity with juglone. This study thus provides novel insights into the catalytic mechanisms of TrxR1. One-electron juglone reduction by TrxR1 producing superoxide should furthermore contribute to the well-known prooxidant cytotoxicity of juglone. PMID:26898501

  3. Proper regulation of Cdc42 activity is required for tight actin concentration at the equator during cytokinesis in adherent mammalian cells.

    PubMed

    Zhu, Xiaodong; Wang, Junxia; Moriguchi, Kazuki; Liow, Lu Ting; Ahmed, Sohail; Kaverina, Irina; Murata-Hori, Maki

    2011-10-01

    Cytokinesis in mammalian cells requires actin assembly at the equatorial region. Although functions of RhoA in this process have been well established, additional mechanisms are likely involved. We have examined if Cdc42 is involved in actin assembly during cytokinesis. Depletion of Cdc42 had no apparent effects on the duration of cytokinesis, while overexpression of constitutively active Cdc42 (CACdc42) caused cytokinesis failure in normal rat kidney epithelial cells. Cells depleted of Cdc42 displayed abnormal cell morphology and caused a failure of tight accumulation of actin and RhoA at the equator. In contrast, in cells overexpressing CACdc42, actin formed abnormal bundles and RhoA was largely eliminated from the equator. Our results suggest that accurate regulation of Cdc42 activity is crucial for proper equatorial actin assembly and RhoA localization during cytokinesis. Notably, our observations also suggest that tight actin concentration is not essential for cytokinesis in adherent mammalian cells.

  4. A Comparison of Ci/Gli Activity as Regulated by Sufu in Drosophila and Mammalian Hedgehog Response

    PubMed Central

    Zhang, Chi; Guo, Yurong; Beachy, Philip A.

    2015-01-01

    Suppressor of fused (Su(fu)/Sufu), one of the most conserved components of the Hedgehog (Hh) signaling pathway, binds Ci/Gli transcription factors and impedes activation of target gene expression. In Drosophila, the Su(fu) mutation has a minimal phenotype, and we show here that Ci transcriptional activity in large part is regulated independently of Su(fu) by other pathway components. Mutant mice lacking Sufu in contrast show excessive pathway activity and die as embryos with patterning defects. Here we show that in cultured cells Hh stimulation can augment transcriptional activity of a Gli2 variant lacking Sufu interaction and, surprisingly, that regulation of Hh pathway targets is nearly normal in the neural tube of Sufu-/- mutant embryos that also lack Gli1 function. Some degree of Hh-induced transcriptional activation of Ci/Gli thus can occur independently of Sufu in both flies and mammals. We further note that Sufu loss can also reduce Hh induction of high-threshold neural tube fates, such as floor plate, suggesting a possible positive pathway role for Sufu. PMID:26271100

  5. Crystal structures of glutaminyl cyclases (QCs) from Drosophila melanogaster reveal active site conservation between insect and mammalian QCs.

    PubMed

    Koch, Birgit; Kolenko, Petr; Buchholz, Mirko; Carrillo, David Ruiz; Parthier, Christoph; Wermann, Michael; Rahfeld, Jens-Ulrich; Reuter, Gunter; Schilling, Stephan; Stubbs, Milton T; Demuth, Hans-Ulrich

    2012-09-18

    Glutaminyl cyclases (QCs), which catalyze the formation of pyroglutamic acid (pGlu) at the N-terminus of a variety of peptides and proteins, have attracted particular attention for their potential role in Alzheimer's disease. In a transgenic Drosophila melanogaster (Dm) fruit fly model, oral application of the potent competitive QC inhibitor PBD150 was shown to reduce the burden of pGlu-modified Aβ. In contrast to mammals such as humans and rodents, there are at least three DmQC species, one of which (isoDromeQC) is localized to mitochondria, whereas DromeQC and an isoDromeQC splice variant possess signal peptides for secretion. Here we present the recombinant expression, characterization, and crystal structure determination of mature DromeQC and isoDromeQC, revealing an overall fold similar to that of mammalian QCs. In the case of isoDromeQC, the putative extended substrate binding site might be affected by the proximity of the N-terminal residues. PBD150 inhibition of DromeQC is roughly 1 order of magnitude weaker than that of the human and murine QCs. The inhibitor binds to isoDromeQC in a fashion similar to that observed for human QCs, whereas it adopts alternative binding modes in a DromeQC variant lacking the conserved cysteines near the active center and shows a disordered dimethoxyphenyl moiety in wild-type DromeQC, providing an explanation for the lower affinity. Our biophysical and structural data suggest that isoDromeQC and human QC are similar with regard to functional aspects. The two Dm enzymes represent a suitable model for further in-depth analysis of the catalytic mechanism of animal QCs, and isoDromeQC might serve as a model system for the structure-based design of potential AD therapeutics. PMID:22897232

  6. Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC).

    PubMed

    Niere, Farr; Namjoshi, Sanjeev; Song, Ehwang; Dilly, Geoffrey A; Schoenhard, Grant; Zemelman, Boris V; Mechref, Yehia; Raab-Graham, Kimberly F

    2016-02-01

    Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer's disease, and autism spectrum disorder-neurological disorders that exhibit elevated mTORC1 activity. Through a protein-protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson's disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes

  7. Use of mammalian target of rapamycin inhibitors after failure of tyrosine kinase inhibitors in patients with metastatic renal cell carcinoma undergoing hemodialysis: A single-center experience with four cases.

    PubMed

    Omae, Kenji; Kondo, Tsunenori; Takagi, Toshio; Iizuka, Junpei; Kobayashi, Hirohito; Hashimoto, Yasunobu; Tanabe, Kazunari

    2016-07-01

    We retrospectively identified patients with end-stage renal disease undergoing hemodialysis treated with the mammalian target of rapamycin inhibitors as a second- and/or third-line targeted therapy after treatment failure with the tyrosine kinase inhibitors for metastatic renal cell carcinoma. Patient medical records were reviewed to evaluate the response to therapies and treatment-related toxicities. Four patients were identified. All patients had undergone nephrectomy, and one had received immunotherapy before targeted therapy. Two patients had clear cell histology, and the other two had papillary histology. All patients were classified into the intermediate risk group according to the Memorial Sloan-Kettering Cancer Center risk model. All patients were treated with everolimus as a second- or third-line therapy, and two patients were treated with temsirolimus as a second- or third-line therapy after treatment failure with sorafenib or sunitinib. The median duration of everolimus therapy was 6.7 months, whereas that of temsirolimus was 9.5 months. All patients had stable disease as the best response during each period of therapy. There were no severe adverse events. The use of mammalian target of rapamycin inhibitors in patients who previously failed to respond to tyrosine kinase inhibitors appears to be feasible in patients with end-stage renal disease requiring hemodialysis.

  8. Mitogen-activated protein kinase kinase activity is required for the G2/M transition of the cell cycle in mammalian fibroblasts

    PubMed Central

    Wright, Jocelyn H.; Munar, Erlynda; Jameson, Damon R.; Andreassen, Paul R.; Margolis, Robert L.; Seger, Rony; Krebs, Edwin G.

    1999-01-01

    The mitogen-activated protein kinase (MAPK) cascade is required for mitogenesis in somatic mammalian cells and is activated by a wide variety of oncogenic stimuli. Specific roles for this signaling module in growth were dissected by inhibiting MAPK kinase 1 (MAPKK1) activity in highly synchronized NIH 3T3 cells. In addition to the known role of this kinase in cell-cycle entry from G0, the level of MAPKK activity was observed to affect the kinetics of progression through both the G1 and G2 phases of the cell cycle in NIH 3T3 cells. Ectopic expression of dominant-negative forms of MAPKK1, which was previously shown to inhibit G0/G1 progression, was found to also delay progression of cells through G2. In addition, treatment of cells with the specific MAPKK inhibitor PD 98059 during a synchronous S phase arrested the cells in the following G2 phase. These data demonstrate a novel role for the MAPK cascade in progression from G2 into mitosis in NIH 3T3 cells. PMID:10500177

  9. Amino acid activation in mammalian brain. Purification and characterization of tryptophan-activating enzyme from buffalo brain.

    PubMed

    Liu, C C; Chung, C H; Lee, M L

    1973-10-01

    l-Tryptophan-activating enzyme [l-tryptophan-tRNA ligase (AMP), EC 6.1.1.2] of water-buffalo brain was purified to near homogeneity by heat and pH treatments, ammonium sulphate fractionation, column chromatography on DEAE-cellulose, hydroxyapatite and Amberlite CG-50, and gel filtration on Sephadex G-200. The purified enzyme catalyses tryptophanyl-tRNA formation with yeast tRNA, but not with Escherichia coli tRNA. The enzyme exhibits multiple peaks of activity in Sephadex gel filtration with molecular weights corresponding to 155000, 105000 and 50000. However, only one peak of activity with molecular weight of 155000 can be detected when the enzyme is subjected to gel filtration at high concentration. Disc gel electrophoresis in the presence of sodium dodecyl sulphate reveals a single band with molecular weight of 55000. The activity of the enzyme is concentration dependent. Different K(m) and V(max.) values are obtained at different enzyme concentrations. These data suggest that this enzyme may exist in different quaternary structures, each with its own kinetic constants. The enzyme activity is inhibited by p-chloromercuribenzoate, and is not protected by the presence of the substrates, l-tryptophan, Mg(2+), ATP, in any combination.

  10. Representation of multi-target activity landscapes through target pair-based compound encoding in self-organizing maps.

    PubMed

    Iyer, Preeti; Bajorath, Jürgen

    2011-11-01

    Activity landscape representations provide access to structure-activity relationships information in compound data sets. In general, activity landscape models integrate molecular similarity relationships with biological activity data. Typically, activity against a single target is monitored. However, for steadily increasing numbers of compounds, activity against multiple targets is reported, resulting in an opportunity, and often a need, to explore multi-target structure-activity relationships. It would be attractive to utilize activity landscape representations to aid in this process, but the design of activity landscapes for multiple targets is a complicated task. Only recently has a first multi-target landscape model been introduced, consisting of an annotated compound network focused on the systematic detection of activity cliffs. Herein, we report a conceptually different multi-target activity landscape design that is based on a 2D projection of chemical reference space using self-organizing maps and encodes compounds as arrays of pair-wise target activity relationships. In this context, we introduce the concept of discontinuity in multi-target activity space. The well-ordered activity landscape model highlights centers of discontinuity in activity space and is straightforward to interpret. It has been applied to analyze compound data sets with three, four, and five target annotations and identify multi-target structure-activity relationships determinants in analog series.

  11. Integration of Transcriptomics, Proteomics, and MicroRNA Analyses Reveals Novel MicroRNA Regulation of Targets in the Mammalian Inner Ear

    PubMed Central

    Elkan-Miller, Tal; Ulitsky, Igor; Hertzano, Ronna; Rudnicki, Anya; Dror, Amiel A.; Lenz, Danielle R.; Elkon, Ran; Irmler, Martin; Beckers, Johannes; Shamir, Ron; Avraham, Karen B.

    2011-01-01

    We have employed a novel approach for the identification of functionally important microRNA (miRNA)-target interactions, integrating miRNA, transcriptome and proteome profiles and advanced in silico analysis using the FAME algorithm. Since miRNAs play a crucial role in the inner ear, demonstrated by the discovery of mutations in a miRNA leading to human and mouse deafness, we applied this approach to microdissected auditory and vestibular sensory epithelia. We detected the expression of 157 miRNAs in the inner ear sensory epithelia, with 53 miRNAs differentially expressed between the cochlea and vestibule. Functionally important miRNAs were determined by searching for enriched or depleted targets in the transcript and protein datasets with an expression consistent with the dogma of miRNA regulation. Importantly, quite a few of the targets were detected only in the protein datasets, attributable to regulation by translational suppression. We identified and experimentally validated the regulation of PSIP1-P75, a transcriptional co-activator previously unknown in the inner ear, by miR-135b, in vestibular hair cells. Our findings suggest that miR-135b serves as a cellular effector, involved in regulating some of the differences between the cochlear and vestibular hair cells. PMID:21483685

  12. Synthesis of 3,3'-carbonyl-bis(chromones) and their activity as mammalian alkaline phosphatase inhibitors.

    PubMed

    Miliutina, Mariia; Ejaz, Syeda Abida; Iaroshenko, Viktor O; Villinger, Alexander; Iqbal, Jamshed; Langer, Peter

    2016-01-14

    Hitherto unknown 3,3'-carbonyl-bis(chromones) 8, dimeric chromones bridged by a carbonyl group, were prepared by reaction of chromone-3-carboxylic acid chloride with 3-(dimethylamino)-1- (2-hydroxyphenyl)-2-propen-1-ones 9. The method is generally applicable for the synthesis of novel symmetrical or non-symmetrical products which were found to inhibit mammalian alkaline phosphatases.

  13. Mammalian touch catches up

    PubMed Central

    Walsh, Carolyn M.; Bautista, Diana M.; Lumpkin, Ellen A.

    2015-01-01

    An assortment of touch receptors innervate the skin and encode different tactile features of the environment. Compared with invertebrate touch and other sensory systems, our understanding of the molecular and cellular underpinnings of mammalian touch lags behind. Two recent breakthroughs have accelerated progress. First, an arsenal of cell-type-specific molecular markers allowed the functional and anatomical properties of sensory neurons to be matched, thereby unraveling a cellular code for touch. Such markers have also revealed key roles of non-neuronal cell types, such as Merkel cells and keratinocytes, in touch reception. Second, the discovery of Piezo genes as a new family of mechanically activated channels has fueled the discovery of molecular mechanisms that mediate and mechanotransduction in mammalian touch receptors. PMID:26100741

  14. Anti-Tumor Effects of Novel 5-O-Acyl Plumbagins Based on the Inhibition of Mammalian DNA Replicative Polymerase Activity

    PubMed Central

    Kawamura, Moe; Kuriyama, Isoko; Maruo, Sayako; Kuramochi, Kouji; Tsubaki, Kazunori; Yoshida, Hiromi; Mizushina, Yoshiyuki

    2014-01-01

    We previously found that vitamin K3 (menadione, 2-methyl-1,4-naphthoquinone) inhibits the activity of human mitochondrial DNA polymerase γ (pol γ). In this study, we focused on plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), and chemically synthesized novel plumbagins conjugated with C2:0 to C22:6 fatty acids (5-O-acyl plumbagins). These chemically modified plumbagins enhanced mammalian pol inhibition and their cytotoxic activity. Plumbagin conjugated with chains consisting of more than C18-unsaturated fatty acids strongly inhibited the activities of calf pol α and human pol γ. Plumbagin conjugated with oleic acid (C18:1-acyl plumbagin) showed the strongest suppression of human colon carcinoma (HCT116) cell proliferation among the ten synthesized 5-O-acyl plumbagins. The inhibitory activity on pol α, a DNA replicative pol, by these compounds showed high correlation with their cancer cell proliferation suppressive activity. C18:1-Acyl plumbagin selectively inhibited the activities of mammalian pol species, but did not influence the activities of other pols and DNA metabolic enzymes tested. This compound inhibited the proliferation of various human cancer cell lines, and was the cytotoxic inhibitor showing strongest inhibition towards HT-29 colon cancer cells (LD50 = 2.9 µM) among the nine cell lines tested. In an in vivo anti-tumor assay conducted on nude mice bearing solid tumors of HT-29 cells, C18:1-acyl plumbagin was shown to be a promising tumor suppressor. These data indicate that novel 5-O-acyl plumbagins act as anti-cancer agents based on mammalian DNA replicative pol α inhibition. Moreover, the results suggest that acylation of plumbagin is an effective chemical modification to improve the anti-cancer activity of vitamin K3 derivatives, such as plumbagin. PMID:24520419

  15. Truncation of the A,A(∗),A' helices segment impairs the actin bundling activity of mammalian eEF1A1.

    PubMed

    Vlasenko, Dmytro O; Novosylna, Oleksandra V; Negrutskii, Boris S; El'skaya, Anna V

    2015-05-01

    Translation elongation factor eEF1A is a G-protein which has a crucial role in the ribosomal polypeptide elongation and possesses a number of non-translational functions. Here, we show that the A,A(∗),A' helices segment of mammalian eEF1A is dispensable for the eEF1A*eEF1Bα complex formation. The A,A(∗),A' helices region did not interact with actin; however, its removal eliminates the actin bundling activity of eEF1A, probably due to the destruction of a dimeric structure of eEF1A. The translation function of monomers and the actin-bundling function of dimers of mammalian eEF1A is suggested.

  16. Active Targeted Drug Delivery for Microbes Using Nano-Carriers

    PubMed Central

    Lin, Yung-Sheng; Lee, Ming-Yuan; Yang, Chih-Hui; Huang, Keng-Shiang

    2015-01-01

    Although vaccines and antibiotics could kill or inhibit microbes, many infectious diseases remain difficult to treat because of acquired resistance and adverse side effects. Nano-carriers-based technology has made significant progress for a long time and is introducing a new paradigm in drug delivery. However, it still has some challenges like lack of specificity toward targeting the infectious site. Nano-carriers utilized targeting ligands on their surface called ‘active target’ provide the promising way to solve the problems like accelerating drug delivery to infectious areas and preventing toxicity or side-effects. In this mini review, we demonstrate the recent studies using the active targeted strategy to kill or inhibit microbes. The four common nano-carriers (e.g. liposomes, nanoparticles, dendrimers and carbon nanotubes) delivering encapsulated drugs are introduced. PMID:25877093

  17. Effect of target probability on pre-stimulus brain activity.

    PubMed

    Lucci, G; Berchicci, M; Perri, R L; Spinelli, D; Di Russo, F

    2016-05-13

    Studies on perceptual decision-making showed that manipulating the proportion of target and non-target stimuli affects the behavioral performance. Tasks with high frequency of targets are associated to faster response times (RTs) conjunctively to higher number of errors (reflecting a response bias characterized by speed/accuracy trade-off) when compared to conditions with low frequency of targets. Electroencephalographic studies well described modulations of post-stimulus event-related potentials as effect of the stimulus probability; in contrast, in the present study we focused on the pre-stimulus preparatory activities subtending the response bias. Two versions of a Go/No-go task characterized by different proportion of Go stimuli (88% vs. 12%) were adopted. In the task with frequent go trials, we observed a strong enhancement in the motor preparation as indexed by the Bereitschaftspotential (BP, previously associated with activity within the supplementary motor area), faster RTs, and larger commission error rate than in the task with rare go trials. Contemporarily with the BP, a right lateralized prefrontal negativity (lateral pN, previously associated with activity within the dorsolateral prefrontal cortex) was larger in the task with rare go trial. In the post-stimulus processing stage, we confirmed that the N2 and the P3 components were larger for rare trials, irrespective of the Go/No-go stimulus category. The increase of activities recorded in the preparatory phase related to frequency of targets is consistent with the view proposed in accumulation models of perceptual decision for which target frequency affects the subjective baseline, reducing the distance between the starting-point and the response boundary, which determines the response speed. PMID:26912279

  18. Eliciting Production of L2 Target Structures through Priming Activities

    ERIC Educational Resources Information Center

    McDonough, Kim; Trofimovich, Pavel; Neumann, Heike

    2015-01-01

    This study focuses on the pedagogical applications of structural priming research in an English for academic purposes (EAP) context, investigating whether priming activities are an effective tool for eliciting production of target grammatical structures. University students across four EAP classes carried out a total of 6 information-exchange…

  19. Rapid, specific, no-wash, far-red fluorogen activation in subcellular compartments by targeted fluorogen activating proteins.

    PubMed

    Telmer, Cheryl A; Verma, Richa; Teng, Haibing; Andreko, Susan; Law, Leann; Bruchez, Marcel P

    2015-05-15

    Live cell imaging requires bright photostable dyes that can target intracellular organelles and proteins with high specificity in a no-wash protocol. Organic dyes possess the desired photochemical properties and can be covalently linked to various protein tags. The currently available fluorogenic dyes are in the green/yellow range where there is high cellular autofluorescence and the near-infrared (NIR) dyes need to be washed out. Protein-mediated activation of far-red fluorogenic dyes has the potential to address these challenges because the cell-permeant dye is small and nonfluorescent until bound to its activating protein, and this binding is rapid. In this study, three single chain variable fragment (scFv)-derived fluorogen activating proteins (FAPs), which activate far-red emitting fluorogens, were evaluated for targeting, brightness, and photostability in the cytosol, nucleus, mitochondria, peroxisomes, and endoplasmic reticulum with a cell-permeant malachite green analog in cultured mammalian cells. Efficient labeling was achieved within 20-30 min for each protein upon the addition of nM concentrations of dye, producing a signal that colocalized significantly with a linked mCerulean3 (mCer3) fluorescent protein and organelle specific dyes but showed divergent photostability and brightness properties dependent on the FAP. These FAPs and the ester of malachite green dye (MGe) can be used as specific, rapid, and wash-free labels for intracellular sites in live cells with far-red excitation and emission properties, useful in a variety of multicolor experiments. PMID:25650487

  20. A functional dissection of PTEN N-terminus: implications in PTEN subcellular targeting and tumor suppressor activity.

    PubMed

    Gil, Anabel; Rodríguez-Escudero, Isabel; Stumpf, Miriam; Molina, María; Cid, Víctor J; Pulido, Rafael

    2015-01-01

    Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization signal (NLS) and an overlapping PIP2-binding motif (PBM) involved in plasma membrane targeting. We report a comprehensive mutational and functional analysis of the PTEN N-terminus, including a panel of tumor-related mutations at this region. Nuclear/cytoplasmic partitioning in mammalian cells and PIP3 phosphatase assays in reconstituted S. cerevisiae defined categories of PTEN N-terminal mutations with distinct PIP3 phosphatase and nuclear accumulation properties. Noticeably, most tumor-related mutations that lost PIP3 phosphatase activity also displayed impaired nuclear localization. Cell proliferation and soft-agar colony formation analysis in mammalian cells of mutations with distinctive nuclear accumulation and catalytic activity patterns suggested a contribution of both properties to PTEN tumor suppressor activity. Our functional dissection of the PTEN N-terminus provides the basis for a systematic analysis of tumor-related and experimentally engineered PTEN mutations.

  1. A Functional Dissection of PTEN N-Terminus: Implications in PTEN Subcellular Targeting and Tumor Suppressor Activity

    PubMed Central

    Gil, Anabel; Rodríguez-Escudero, Isabel; Stumpf, Miriam; Molina, María; Cid, Víctor J.; Pulido, Rafael

    2015-01-01

    Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization signal (NLS) and an overlapping PIP2-binding motif (PBM) involved in plasma membrane targeting. We report a comprehensive mutational and functional analysis of the PTEN N-terminus, including a panel of tumor-related mutations at this region. Nuclear/cytoplasmic partitioning in mammalian cells and PIP3 phosphatase assays in reconstituted S. cerevisiae defined categories of PTEN N-terminal mutations with distinct PIP3 phosphatase and nuclear accumulation properties. Noticeably, most tumor-related mutations that lost PIP3 phosphatase activity also displayed impaired nuclear localization. Cell proliferation and soft-agar colony formation analysis in mammalian cells of mutations with distinctive nuclear accumulation and catalytic activity patterns suggested a contribution of both properties to PTEN tumor suppressor activity. Our functional dissection of the PTEN N-terminus provides the basis for a systematic analysis of tumor-related and experimentally engineered PTEN mutations. PMID:25875300

  2. Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation

    PubMed Central

    Bordeleau, Marie-Eve; Matthews, James; Wojnar, Joanna M.; Lindqvist, Lisa; Novac, Olivia; Jankowsky, Eckhard; Sonenberg, Nahum; Northcote, Peter; Teesdale-Spittle, Paul; Pelletier, Jerry

    2005-01-01

    RNA helicases are the largest group of enzymes in eukaryotic RNA metabolism. The DEXD/H-box putative RNA helicases form the helicase superfamily II, whose members are defined by seven highly conserved amino acid motifs, making specific targeting of selected members a challenging pharmacological problem. The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase that works in conjunction with eIF4B and eIF4H and as a subunit of eIF4F to prepare the mRNA template for ribosome binding, possibly by unwinding the secondary structure proximal to the 5′ m7GpppN cap structure. We report the identification and characterization of a small molecule inhibitor of eukaryotic translation initiation that acts in an unusual manner by stimulating eIF4A-associated activities. Our results suggest that proper control of eIF4A helicase activity is necessary for efficient ribosome binding and demonstrate the feasibility of selectively targeting DEAD-box RNA helicases with small molecules. PMID:16030146

  3. Active helium target: Neutron scalar polarizability extraction via Compton scattering

    SciTech Connect

    Morris, Meg Hornidge, David; Annand, John; Strandberg, Bruno

    2015-12-31

    Precise measurement of the neutron scalar polarizabilities has been a lasting challenge because of the lack of a free-neutron target. Led by the University of Glasgow and the Mount Allison University groups of the A2 collaboration in Mainz, Germany, preparations have begun to test a recent theoretical model with an active helium target with the hope of determining these elusive quantities with small statistical, systematic, and model-dependent errors. Apparatus testing and background-event simulations have been carried out, with the full experiment projected to run in 2015. Once determined, these values can be applied to help understand quantum chromodynamics in the nonperturbative region.

  4. Proteinase-activated receptors (PARs) as targets for antiplatelet therapy.

    PubMed

    Cunningham, Margaret; McIntosh, Kathryn; Bushell, Trevor; Sloan, Graeme; Plevin, Robin

    2016-04-15

    Since the identification of the proteinase-activated receptor (PAR) family as mediators of serine protease activity in the 1990s, there has been tremendous progress in the elucidation of their pathophysiological roles. The development of drugs that target PARs has been the focus of many laboratories for the potential treatment of thrombosis, cancer and other inflammatory diseases. Understanding the mechanisms of PAR activation and G protein signalling pathways evoked in response to the growing list of endogenous proteases has yielded great insight into receptor regulation at the molecular level. This has led to the development of new selective modulators of PAR activity, particularly PAR1. The mixed success of targeting PARs has been best exemplified in the context of inhibiting PAR1 as a new antiplatelet therapy. The development of the competitive PAR1 antagonist, vorapaxar (Zontivity), has clearly shown the value in targeting PAR1 in acute coronary syndrome (ACS); however the severity of associated bleeding with this drug has limited its use in the clinic. Due to the efficacy of thrombin acting via PAR1, strategies to selectively inhibit specific PAR1-mediated G protein signalling pathways or to target the second thrombin platelet receptor, PAR4, are being devised. The rationale behind these alternative approaches is to bias downstream thrombin activity via PARs to allow for inhibition of pro-thrombotic pathways but maintain other pathways that may preserve haemostatic balance and improve bleeding profiles for widespread clinical use. This review summarizes the structural determinants that regulate PARs and the modulators of PAR activity developed to date.

  5. Buoyancy-Activated Cell Sorting Using Targeted Biotinylated Albumin Microbubbles

    PubMed Central

    Liou, Yu-Ren; Wang, Yu-Hsin; Lee, Chia-Ying; Li, Pai-Chi

    2015-01-01

    Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including florescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs) conjugated with antibodies (i.e., targeted biotin-MBs). Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10g for 1 min, and then allowed 1 hour at 4°C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs), which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44+) and MDA-MB-453 cells (CD44–), which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44+ is a commonly used cancer-stem-cell biomarker, our targeted

  6. Mitogen-activated protein kinases: a new therapeutic target in cardiac pathology.

    PubMed

    Ravingerová, Tána; Barancík, Miroslav; Strnisková, Monika

    2003-05-01

    Eukaryotic cells respond to different external stimuli by activation of mechanisms of cell signaling. One of the major systems participating in the transduction of signal from the cell membrane to nuclear and other intracellular targets is the highly conserved mitogen-activated protein kinase (MAPK) superfamily. The members of MAPK family are involved in the regulation of a large variety of cellular processes such as cell growth, differentiation, development, cell cycle, death and survival. Several MAPK subfamilies, each with apparently unique signaling pathway, have been identified in the mammalian myocardium. These cascades differ in their upstream activation sequence and in downstream substrate specifity. Each pathway follows the same conserved three-kinase module consisting of MAPK, MAPK kinase (MAPKK, MKK or MEK), and MAPK kinase kinase (MAPKKK, MEKK). The major groups of MAPKs found in cardiac tissue include the extracellular signal-regulated kinases (ERKs), the stress-activated/c-Jun NH2-terminal kinases (SAPK/JNKs), p38-MAPK, and ERK5/big MAPK 1 (BMK1). The ERKs are strongly activated by mitogenic and growth factors and by physical stress, whereas SAPK/JNKs and p38-MAPK can be activated by various cell stresses, such as hyperosmotic shock, metabolic stress or protein synthesis inhibitors, UV radiation, heat shock, cytokines, and ischemia. Activation of MAPKs family plays a key role in the pathogenesis of various processes in the heart, e.g. myocardial hypertrophy and its transition to heart failure, in ischemic and reperfusion injury, as well in the cardioprotection conferred by ischemia- or pharmacologically-induced preconditioning. The following approaches are currently utilized to elucidate the role of MAPKs in the myocardium: (i) studies of the effects of myocardial processes on the activity of these kinases; (ii) pharmacological modulations of MAPKs activity and evaluation of their impact on the (patho)physiological processes in the heart; (iii) gene

  7. Heat-activated liposome targeting to streptavidin-coated surfaces.

    PubMed

    Jing, Yujia; Trefná, Hana Dobšíček; Persson, Mikael; Svedhem, Sofia

    2015-06-01

    There is a great need of improved anticancer drugs and corresponding drug carriers. In particular, liposomal drug carriers with heat-activated release and targeting functions are being developed for combined hyperthermia and chemotherapy treatments of tumors. The aim of this study is to demonstrate the heat-activation of liposome targeting to biotinylated surfaces, in model experiments where streptavidin is used as a pretargeting protein. The design of the heat-activated liposomes is based on liposomes assembled in an asymmetric structure and with a defined phase transition temperature. Asymmetry between the inside and the outside of the liposome membrane was generated through the enzymatic action of phospholipase D, where lipid head groups in the outer membrane leaflet, i.e. exposed to the enzyme, were hydrolyzed. The enzymatically treated and purified liposomes did not bind to streptavidin-modified surfaces. When activation heat was applied, starting from 22°C, binding of the liposomes occurred once the temperature approached 33±0.5°C. Moreover, it was observed that the asymmetric structure remained stable for at least 2 weeks. These results show the potential of asymmetric liposomes for the targeted binding to cell membranes in response to (external) temperature stimulus. By using pretargeting proteins, this approach can be further developed for personalized medicine, where tumor-specific antibodies can be selected for the conjugation of pretargeting agents.

  8. Inhibition of mammalian DNA polymerases and the suppression of inflammatory and allergic responses by tyrosol from used activated charcoal waste generated during sake production.

    PubMed

    Mizushina, Yoshiyuki; Ogawa, Yoshiaki; Onodera, Takefumi; Kuriyama, Isoko; Sakamoto, Yuka; Nishikori, Shu; Kamisuki, Shinji; Sugawara, Fumio

    2014-08-01

    The components adsorbed onto activated charcoal following the fermentation process of the Japanese rice wine "sake" have been studied with the aim of identifying suitable applications for this industrial food waste product. The absorbed materials were effectively extracted from the charcoal, and inhibited the activity of several mammalian DNA polymerases (pols). Subsequent purification of the extract afforded tyrosol [4-(2-hydroxyethyl)phenol] as the active component, which selectively inhibited the activity of 11 mammalian pols with IC50 values in the range of 34.3-46.1 μM. In contrast, this compound did not influence the activities of plant or prokaryotic pols or any of the other DNA metabolic enzymes tested. Tyrosol suppressed both anti-inflammatory and antiallergic effects in vivo, including 12-O-tetradecanoylphorbol-13-acetate-induced inflammatory mouse ear edema, and immunoglobulin E-induced passive cutaneous anaphylactic reaction in mice. These results suggested that this byproduct formed during the sake-brewing process could be used as an anti-inflammatory and/or antiallergic agent.

  9. Electroporation into Cultured Mammalian Embryos

    NASA Astrophysics Data System (ADS)

    Nomura, Tadashi; Takahashi, Masanori; Osumi, Noriko

    Over the last century, mammalian embryos have been used extensively as a common animal model to investigate fundamental questions in the field of developmental biology. More recently, the establishment of transgenic and gene-targeting systems in laboratory mice has enabled researchers to unveil the genetic mechanisms under lying complex developmental processes (Mak, 2007). However, our understanding of cell—cell interactions and their molecular basis in the early stages of mammalian embryogenesis is still very fragmentary. One of the major problems is the difficulty of precise manipulation and limited accessibility to mammalian embryos via uterus wall. Unfortunately, existing tissue and organotypic culture systems per se do not fully recapitulate three-dimensional, dynamic processes of organogenesis observed in vivo. Although transgenic animal technology and virus-mediated gene delivery are useful to manipulate gene expression, these techniques take much time and financial costs, which limit their use.

  10. Feasibility study of an active target for the MEG experiment

    NASA Astrophysics Data System (ADS)

    Papa, A.; Cavoto, G.; Ripiccini, E.

    2014-03-01

    We consider the possibility to have an active target for the upgrade of the MEG experiment (MEG II). The active target should work as (1) a beam monitoring, to continuously measure the muon stopping rate and therefore provide a direct evaluation of the detector acceptance (or an absolute normalization of the stopped muon); and as (2) an auxiliary device for the spectrometer, to improve the determination of the muon decay vertex and consequently to achieve a better positron momentum and angular resolutions, detecting the positron from the muon decay. In this work we studied the feasibility of detecting minimum ionizing particle with a single layer of 250 μm fiber and the capability to discriminate between the signal induced by either a muon or a positron.

  11. The Platin-X series: activation, targeting, and delivery.

    PubMed

    Basu, Uttara; Banik, Bhabatosh; Wen, Ru; Pathak, Rakesh K; Dhar, Shanta

    2016-08-16

    Anticancer platinum (Pt) complexes have long been considered to be one of the biggest success stories in the history of medicinal inorganic chemistry. Yet there remains the hunt for the "magic bullet" which can satisfy the requirements of an effective chemotherapeutic drug formulation. Pt(iv) complexes are kinetically more inert than the Pt(ii) congeners and offer the opportunity to append additional functional groups/ligands for prodrug activation, tumor targeting, or drug delivery. The ultimate aim of functionalization is to enhance the tumor selective action and attenuate systemic toxicity of the drugs. Moreover, an increase in cellular accumulation to surmount the resistance of the tumor against the drugs is also of paramount importance in drug development and discovery. In this review, we will address the attempts made in our lab to develop Pt(iv) prodrugs that can be activated and delivered using targeted nanotechnology-based delivery platforms. PMID:27493131

  12. Brain Activation Underlying Threat Detection to Targets of Different Races

    PubMed Central

    Senholzi, Keith B.; Depue, Brendan E.; Correll, Joshua; Banich, Marie T.; Ito, Tiffany A.

    2016-01-01

    The current study examined blood oxygen level dependent (BOLD) signal underlying racial differences in threat detection. During fMRI, participants determined whether pictures of Black or White individuals held weapons. They were instructed to make shoot responses when the picture showed armed individuals but don’t shoot responses to unarmed individuals, with the cost of not shooting armed individuals being greater than that of shooting unarmed individuals. Participants were faster to shoot armed Blacks than Whites, but faster in making don’t shoot responses to unarmed Whites than Blacks. Brain activity differed to armed versus unarmed targets depending on target race, suggesting different mechanisms underlying threat versus safety decisions. Anterior cingulate cortex was preferentially engaged for unarmed Whites than Blacks. Parietal and visual cortical regions exhibited greater activity for armed Blacks than Whites. Seed-based functional connectivity of the amygdala revealed greater coherence with parietal and visual cortices for armed Blacks than Whites. Furthermore, greater implicit Black-danger associations were associated with increased amygdala activation to armed Blacks, compared to armed Whites. Our results suggest that different neural mechanisms may underlie racial differences in responses to armed versus unarmed targets. PMID:26357911

  13. Brain activation underlying threat detection to targets of different races.

    PubMed

    Senholzi, Keith B; Depue, Brendan E; Correll, Joshua; Banich, Marie T; Ito, Tiffany A

    2015-01-01

    The current study examined blood oxygen level-dependent signal underlying racial differences in threat detection. During functional magnetic resonance imaging, participants determined whether pictures of Black or White individuals held weapons. They were instructed to make shoot responses when the picture showed armed individuals but don't shoot responses to unarmed individuals, with the cost of not shooting armed individuals being greater than that of shooting unarmed individuals. Participants were faster to shoot armed Blacks than Whites, but faster in making don't shoot responses to unarmed Whites than Blacks. Brain activity differed to armed versus unarmed targets depending on target race, suggesting different mechanisms underlying threat versus safety decisions. Anterior cingulate cortex was preferentially engaged for unarmed Whites than Blacks. Parietal and visual cortical regions exhibited greater activity for armed Blacks than Whites. Seed-based functional connectivity of the amygdala revealed greater coherence with parietal and visual cortices for armed Blacks than Whites. Furthermore, greater implicit Black-danger associations were associated with increased amygdala activation to armed Blacks, compared to armed Whites. Our results suggest that different neural mechanisms may underlie racial differences in responses to armed versus unarmed targets. PMID:26357911

  14. Active multispectral near-IR detection of small surface targets

    NASA Astrophysics Data System (ADS)

    de Jong, Arie N.; Winkel, Hans; Roos, Marco J. J.

    2001-10-01

    The detection and identification of small surface targets with Electro-Optical sensors is seriously hampered by ground clutter, leading to false alarms and reduced detection probabilities. Active ground illumination can improve the detection performance of EO sensors compared to passive skylight illumination because of the knowledge of the illumination level and of its temporal stability. Sun and sky cannot provide this due to the weather variability. In addition multispectral sensors with carefully chosen spectral bands ranging from the visual into the near IR from 400-2500 nm wavelength can take benefit of a variety of cheap active light sources, ranging from lasers to Xenon or halogen lamps. Results are presented, obtained with a two- color laser scanner with one wavelength in the chlorophyll absorption dip. Another active scanner is described operating at 4 wavebands between 1400 and 2300 nm, using tungsten halogen lamps. Finally a simple TV camera was used with either a ste of narrow band spectral filters or polarization filters in front of the lamps. The targets consisted of an array of mixed objects, most of them real mines. The results how great promise in enhancing the detection and identification probabilities of EO sensors against small surface targets.

  15. Kinome-wide selectivity profiling of ATP-competitive mammalian target of rapamycin (mTOR) inhibitors and characterization of their binding kinetics.

    PubMed

    Liu, Qingsong; Kirubakaran, Sivapriya; Hur, Wooyoung; Niepel, Mario; Westover, Kenneth; Thoreen, Carson C; Wang, Jinhua; Ni, Jing; Patricelli, Matthew P; Vogel, Kurt; Riddle, Steve; Waller, David L; Traynor, Ryan; Sanda, Takaomi; Zhao, Zheng; Kang, Seong A; Zhao, Jean; Look, A Thomas; Sorger, Peter K; Sabatini, David M; Gray, Nathanael S

    2012-03-23

    An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 μM revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.

  16. Asp-ase Activity of the Opossum Granzyme B Supports the Role of Granzyme B as Part of Anti-Viral Immunity Already during Early Mammalian Evolution.

    PubMed

    Fu, Zhirong; Thorpe, Michael; Akula, Srinivas; Hellman, Lars

    2016-01-01

    Granzyme B is one of the key effector molecules in our defense against viruses and intracellular bacteria. This serine protease together with the pore forming protein perforin, induces caspase or Bid-dependent apoptosis in target cells. Here we present the first characterization of a granzyme B homolog, the grathepsodenase, in a non-placental mammal, the American opossum (Monodelphis domestica). The recombinant enzyme was produced in a human cell line and used to study its primary and extended cleavage specificity using a panel of chromogenic substrates and recombinant protein substrates. The opossum granzyme B was found to have a specificity similar to human granzyme B, although slightly less restrictive in its extended specificity. The identification of a granzyme B homolog with asp-ase (cleaving after aspartic acid) specificity in a non-placental mammal provides strong indications that caspase or Bid-dependent apoptosis by a serine protease with a conserved primary specificity has been part of anti-viral immunity since early mammalian evolution. This finding also indicates that an asp-ase together with a chymase were the first two serine protease genes to appear in the mammalian chymase locus. PMID:27152961

  17. Asp-ase Activity of the Opossum Granzyme B Supports the Role of Granzyme B as Part of Anti-Viral Immunity Already during Early Mammalian Evolution

    PubMed Central

    Fu, Zhirong; Thorpe, Michael; Akula, Srinivas; Hellman, Lars

    2016-01-01

    Granzyme B is one of the key effector molecules in our defense against viruses and intracellular bacteria. This serine protease together with the pore forming protein perforin, induces caspase or Bid-dependent apoptosis in target cells. Here we present the first characterization of a granzyme B homolog, the grathepsodenase, in a non-placental mammal, the American opossum (Monodelphis domestica). The recombinant enzyme was produced in a human cell line and used to study its primary and extended cleavage specificity using a panel of chromogenic substrates and recombinant protein substrates. The opossum granzyme B was found to have a specificity similar to human granzyme B, although slightly less restrictive in its extended specificity. The identification of a granzyme B homolog with asp-ase (cleaving after aspartic acid) specificity in a non-placental mammal provides strong indications that caspase or Bid-dependent apoptosis by a serine protease with a conserved primary specificity has been part of anti-viral immunity since early mammalian evolution. This finding also indicates that an asp-ase together with a chymase were the first two serine protease genes to appear in the mammalian chymase locus. PMID:27152961

  18. Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD(+) biosynthetic machinery in mammalian cells.

    PubMed

    Zamporlini, Federica; Ruggieri, Silverio; Mazzola, Francesca; Amici, Adolfo; Orsomando, Giuseppe; Raffaelli, Nadia

    2014-11-01

    The redox coenzyme NAD(+) is also a rate-limiting co-substrate for several enzymes that consume the molecule, thus rendering its continuous re-synthesis indispensable. NAD(+) biosynthesis has emerged as a therapeutic target due to the relevance of NAD(+) -consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD(+) biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD(+) levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD(+) availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole-cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD(+) precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma. PMID:25223558

  19. Coordinated regulation of Myc trans-activation targets by Polycomb and the Trithorax group protein Ash1

    PubMed Central

    Goodliffe, Julie M; Cole, Michael D; Wieschaus, Eric

    2007-01-01

    Background The Myc oncoprotein is a transcriptional regulator whose function is essential for normal development. Myc is capable of binding to 10% of the mammalian genome, and it is unclear how a developing embryo controls the DNA binding of its abundant Myc proteins in order to avoid Myc's potential for inducing tumorigenesis. Results To identify chromatin binding proteins with a potential role in controlling Myc activity, we established a genetic assay for dMyc activity in Drosophila. We conducted a genome-wide screen using this assay, and identified the Trithorax Group protein Ash1 as a modifier of dMyc activity. Ash1 is a histone methyltransferase known for its role in opposing repression by Polycomb. Using RNAi in the embryo and Affymetrix microarrays, we show that ash1 RNAi causes the increased expression of many genes, suggesting that it is directly or indirectly required for repression in the embryo, in contrast to its known role in maintenance of activation. Many of these genes also respond similarly upon depletion of Pc and pho transcripts, as determined by concurrent microarray analysis of Pc and pho RNAi embryos, suggesting that the three are required for low levels of expression of a common set of targets. Further, many of these overlapping targets are also activated by Myc overexpression. We identify a second group of genes whose expression in the embryo requires Ash1, consistent with its previously established role in maintenance of activation. We find that this second group of Ash1 targets overlaps those activated by Myc and that ectopic Myc overcomes their requirement for Ash1. Conclusion Genetic, genomic and chromatin immunoprecipitation data suggest a model in which Pc, Ash1 and Pho are required to maintain a low level of expression of embryonic targets of activation by Myc, and that this occurs, directly or indirectly, by a combination of disparate chromatin modifications. PMID:17519021

  20. Factor XI and Contact Activation as Targets for Antithrombotic Therapy

    PubMed Central

    Gailani, David; Bane, Charles E.; Gruber, Andras

    2015-01-01

    Summary The most commonly used anticoagulants produce therapeutic antithrombotic effects either by inhibiting thrombin or factor Xa, or by lowering the plasma levels of the precursors of these key enzymes, prothrombin and factor X. These drugs do not distinguish between thrombin generation contributing to thrombosis from thrombin generation required for hemostasis. Thus, anticoagulants increase bleeding risk, and many patients who would benefit from therapy go untreated because of comorbidities that place them at unacceptable risk for hemorrhage. Studies in animals demonstrate that components of the plasma contact activation system contribute to experimentally-induced thrombosis, despite playing little or no role in hemostasis. Attention has focused on factor XII, the zymogen of a protease (factor XIIa) that initiates contact activation when blood is exposed to foreign surfaces; and factor XI, the zymogen of the protease factor XIa, which links contact activation to the thrombin generation mechanism. In the case of factor XI, epidemiologic data indicate this protein contributes to stroke and venous thromboembolism, and perhaps myocardial infarction, in humans. A phase 2 trial showing that reduction of factor XI may be more effective than low-molecular-weight heparin at preventing venous thrombosis during knee replacement surgery provides proof of concept for the premise that an antithrombotic effect can be uncoupled from an anticoagulant effect in humans by targeting components of contact activation. Here we review data on the role of factor XI and factor XII in thrombosis, and results of pre-clinical and human trials for therapies targeting these proteins. PMID:25976012

  1. HER2 activating mutations are targets for colorectal cancer treatment

    PubMed Central

    Kavuri, Shyam M.; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M.; Migliardi, Giorgia; Searleman, Adam C.; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A.; Bertotti, Andrea; Bose, Ron

    2015-01-01

    The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of colorectal cancer patients. Introduction of the HER2 mutations, S310F, L755S, V777L, V842I, and L866M, into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutations are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors, neratinib and afatinib. HER2 gene sequencing of 48 cetuximab resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) WT colorectal cancer patient-derived xenografts (PDX’s) identified 4 PDX’s with HER2 mutations. HER2 targeted therapies were tested on two PDX’s. Treatment with a single HER2 targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2 targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2 mutated PDX’s. PMID:26243863

  2. Mitochondria and mammalian reproduction.

    PubMed

    Ramalho-Santos, João; Amaral, Sandra

    2013-10-15

    Mitochondria are cellular organelles with crucial roles in ATP synthesis, metabolic integration, reactive oxygen species (ROS) synthesis and management, the regulation of apoptosis (namely via the intrinsic pathway), among many others. Additionally, mitochondria in different organs or cell types may have distinct properties that can decisively influence functional analysis. In terms of the importance of mitochondria in mammalian reproduction, and although there are species-specific differences, these aspects involve both energetic considerations for gametogenesis and fertilization, control of apoptosis to ensure the proper production of viable gametes, and ROS signaling, as well as other emerging aspects. Crucially, mitochondria are the starting point for steroid hormone biosynthesis, given that the conversion of cholesterol to pregnenolone (a common precursor for all steroid hormones) takes place via the activity of the cytochrome P450 side-chain cleavage enzyme (P450scc) on the inner mitochondrial membrane. Furthermore, mitochondrial activity in reproduction has to be considered in accordance with the very distinct strategies for gamete production in the male and female. These include distinct gonad morpho-physiologies, different types of steroids that are more prevalent (testosterone, estrogens, progesterone), and, importantly, the very particular timings of gametogenesis. While spermatogenesis is complete and continuous since puberty, producing a seemingly inexhaustible pool of gametes in a fixed environment; oogenesis involves the episodic production of very few gametes in an environment that changes cyclically. These aspects have always to be taken into account when considering the roles of any common element in mammalian reproduction.

  3. Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum

    PubMed Central

    Wang, Jigang; Zhang, Chong-Jing; Chia, Wan Ni; Loh, Cheryl C. Y.; Li, Zhengjun; Lee, Yew Mun; He, Yingke; Yuan, Li-Xia; Lim, Teck Kwang; Liu, Min; Liew, Chin Xia; Lee, Yan Quan; Zhang, Jianbin; Lu, Nianci; Lim, Chwee Teck; Hua, Zi-Chun; Liu, Bin; Shen, Han-Ming; Tan, Kevin S. W.; Lin, Qingsong

    2015-01-01

    The mechanism of action of artemisinin and its derivatives, the most potent of the anti-malarial drugs, is not completely understood. Here we present an unbiased chemical proteomics analysis to directly explore this mechanism in Plasmodium falciparum. We use an alkyne-tagged artemisinin analogue coupled with biotin to identify 124 artemisinin covalent binding protein targets, many of which are involved in the essential biological processes of the parasite. Such a broad targeting spectrum disrupts the biochemical landscape of the parasite and causes its death. Furthermore, using alkyne-tagged artemisinin coupled with a fluorescent dye to monitor protein binding, we show that haem, rather than free ferrous iron, is predominantly responsible for artemisinin activation. The haem derives primarily from the parasite's haem biosynthesis pathway at the early ring stage and from haemoglobin digestion at the latter stages. Our results support a unifying model to explain the action and specificity of artemisinin in parasite killing. PMID:26694030

  4. Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum.

    PubMed

    Wang, Jigang; Zhang, Chong-Jing; Chia, Wan Ni; Loh, Cheryl C Y; Li, Zhengjun; Lee, Yew Mun; He, Yingke; Yuan, Li-Xia; Lim, Teck Kwang; Liu, Min; Liew, Chin Xia; Lee, Yan Quan; Zhang, Jianbin; Lu, Nianci; Lim, Chwee Teck; Hua, Zi-Chun; Liu, Bin; Shen, Han-Ming; Tan, Kevin S W; Lin, Qingsong

    2015-01-01

    The mechanism of action of artemisinin and its derivatives, the most potent of the anti-malarial drugs, is not completely understood. Here we present an unbiased chemical proteomics analysis to directly explore this mechanism in Plasmodium falciparum. We use an alkyne-tagged artemisinin analogue coupled with biotin to identify 124 artemisinin covalent binding protein targets, many of which are involved in the essential biological processes of the parasite. Such a broad targeting spectrum disrupts the biochemical landscape of the parasite and causes its death. Furthermore, using alkyne-tagged artemisinin coupled with a fluorescent dye to monitor protein binding, we show that haem, rather than free ferrous iron, is predominantly responsible for artemisinin activation. The haem derives primarily from the parasite's haem biosynthesis pathway at the early ring stage and from haemoglobin digestion at the latter stages. Our results support a unifying model to explain the action and specificity of artemisinin in parasite killing. PMID:26694030

  5. Calcium-Activated Potassium Channels: Potential Target for Cardiovascular Diseases.

    PubMed

    Dong, De-Li; Bai, Yun-Long; Cai, Ben-Zhi

    2016-01-01

    Ca(2+)-activated K(+) channels (KCa) are classified into three subtypes: big conductance (BKCa), intermediate conductance (IKCa), and small conductance (SKCa) KCa channels. The three types of KCa channels have distinct physiological or pathological functions in cardiovascular system. BKCa channels are mainly expressed in vascular smooth muscle cells (VSMCs) and inner mitochondrial membrane of cardiomyocytes, activation of BKCa channels in these locations results in vasodilation and cardioprotection against cardiac ischemia. IKCa channels are expressed in VSMCs, endothelial cells, and cardiac fibroblasts and involved in vascular smooth muscle proliferation, migration, vessel dilation, and cardiac fibrosis. SKCa channels are widely expressed in nervous and cardiovascular system, and activation of SKCa channels mainly contributes membrane hyperpolarization. In this chapter, we summarize the physiological and pathological roles of the three types of KCa channels in cardiovascular system and put forward the possibility of KCa channels as potential target for cardiovascular diseases.

  6. A double-stranded RNA unwinding activity introduces structural alterations by means of adenosine to inosine conversions in mammalian cells and Xenopus eggs.

    PubMed Central

    Wagner, R W; Smith, J E; Cooperman, B S; Nishikura, K

    1989-01-01

    Amphibian eggs and embryos as well as mammalian cells have been reported to contain an activity that unwinds double-stranded RNA. We have now found that adenosine residues have been modified in the RNA products of this unwinding activity. Although the modified RNA remains double-stranded, the modification causes the RNA to be susceptible to single-strand-specific RNase and to migrate as a retarded smear on a native polyacrylamide electrophoresis gel. The modification is specific for double-stranded RNA. At least 40% of the adenosine residues can be modified in vitro in a given random sequence RNA molecule. By using standard two-dimensional TLC and HPLC analyses, the modified base has been identified as inosine. Mismatched base-pairing between inosine and uridine appears to be responsible for the observed characteristics of the unwound RNA. The biological significance of this modifying activity and also of the modified double-stranded RNA is discussed. Images PMID:2704740

  7. Tombusvirus P19 RNA silencing suppressor (RSS) activity in mammalian cells correlates with charged amino acids that contribute to direct RNA-binding

    PubMed Central

    2012-01-01

    Background Tombusvirus P19 is a protein encoded by tomato bushy stunt virus and related tombusviruses. Earlier studies have demonstrated that P19 is an RNA silencing suppressor (RSS) in plant cells. However, it has not been systematically investigated how P19 suppresses RNA interference in various mammalian cell settings. Results We have studied the RSS effect of P19 in mammalian cells, HEK293T, HeLa, and mouse embryonic fibroblasts. We have individually mutated 18 positively charged residues in P19 and found that 6 of these charged residues in P19 reduce its ability to suppress RNA interference. In each case, the reduction of silencing of RNA interference correlated with the reduced ability by these P19 mutants to bind siRNAs (small interfering RNAs). Conclusions Our findings characterize a class of RNA-binding proteins that function as RSS moieties. We find a tight correlation between positively charged residues in P19 accounting for siRNA-binding and their RSS activity. Because P19’s activity is conserved in plant and animal cells, we conclude that its RSS function unlikely requires cell type-specific co-factors and likely arises from direct RNA-binding. PMID:23216864

  8. Effects of slow-release urea and rumen-protected methionine and histidine on mammalian target of rapamycin (mTOR) signaling and ubiquitin proteasome-related gene expression in skeletal muscle of dairy cows.

    PubMed

    Sadri, H; Giallongo, F; Hristov, A N; Werner, J; Lang, C H; Parys, C; Saremi, B; Sauerwein, H

    2016-08-01

    The mammalian target of rapamycin (mTOR) is a major regulator of protein synthesis, whereas the ubiquitin-proteasome system (UPS) is regarded as the main proteolytic pathway in skeletal muscle. The objective of the current study was to investigate the effects of slow-release urea and rumen-protected (RP) Met and His supplementation of a metabolizable protein (MP)-deficient diet on the abundance of key components of the mTOR pathway and of the UPS in skeletal muscle of dairy cows. Sixty Holstein cows were blocked based on days in milk and milk yield and were randomly assigned within block to 1 of 5 diets in a 10-wk experiment (including the first 2 wk as covariate period) as follows: (1) MP-adequate diet (AMP; 107% of MP requirements, based on the National Research Council requirements); (2) MP-deficient diet (DMP; 95% of MP requirements); (3) DMP supplemented with slow-release urea (DMPU); (4) DMPU supplemented with RPMet (DMPUM); and (5) DMPUM supplemented with RPHis (DMPUMH). Muscle biopsies were collected from longissimus dorsi during the last week of the experiment. The mRNA abundance of key mTOR signaling genes was not affected by the treatments. The phosphorylated (P)-mTOR protein was or tended to be greater for DMP compared with DMPU and AMP, respectively. The P-mTOR protein in DMPUMH was decreased when compared against DMPUM. The P-ribosomal protein S6 tended to be increased by DMPUM compared with DMPU. The abundance of total-S6 was or tended to be greater for DMP compared with AMP and DMPU, respectively. The mRNA abundance of ubiquitin activating and conjugating enzymes was not affected by the treatments, whereas that of muscle ring-finger protein 1 (MuRF-1) was greater in DMP than DMPU. The increased abundance of mTOR-associated signaling proteins and MuRF-1 mRNA abundance indicates a higher rate of protein turnover in muscle of DMP-fed cows. The reduced abundance of P-mTOR by supplementation of RPHis may suggest that His is likely partitioned to the

  9. Anti-autophagic and anti-apoptotic effects of memantine in a SH-SY5Y cell model of Alzheimer's disease via mammalian target of rapamycin-dependent and -independent pathways

    PubMed Central

    SONG, GUIJUN; LI, YU; LIN, LULU; CAO, YUNPENG

    2015-01-01

    Memantine non-competitively blocks the N-methyl-d-aspartate receptor in order to inhibit beta-amyloid (Aβ) secretion, and has been used to treat moderate-to-severe Alzheimer's disease (AD). However, the mechanisms underlying the role of memantine in the autophagy and apoptosis of neuronal cells in AD, as well as the association between neuronal autophagy and apoptosis have yet to be elucidated. The present study aimed to establish an AD cell model overexpressing the 695-amino-acid Swedish mutant of Aβ precursor protein (APP695swe) in order to observe the effects of memantine on the cell viability, autophagy and apoptosis of SH-SY5Y cells in the AD model, and to investigate the associated underlying mechanisms. A pcDNA3.1-APP695 plasmid was transfected into the SH-SY5Y cells. Reverse transcription-quantitative polymerase chain reaction and western blot analyses demonstrated that the AD cell model was successfully established. MTT assays demonstrated that memantine was able to upregulate neuronal cell survival, and acridine orange staining and flow cytometry demonstrated that memantine (5 µM) was able to inhibit neuronal autophagy and apoptosis. Following neuronal autophagy induction by rapamycin, cell apoptosis rates increased significantly. Further experiments revealed that memantine was able to upregulate the expression of signaling molecules phosphorylated (p)-phosphoinositide 3-kinase, p-Akt and p-mammalian target of rapamycin (mTOR), and also inhibited the phosphorylation of the B-cell lymphoma 2/Beclin-1 complex via mitogen-activated protein kinase 8. In conclusion, the results of the present study demonstrated that in the AD cell model, autophagy was able to promote apoptosis. Memantine exerted anti-autophagic and anti-apoptotic functions, and mTOR-dependent as well as-independent autophagic signaling pathways were involved in this process. Therefore, these results of the present study strongly supported the use of memantine as a potential therapeutic

  10. Anti-autophagic and anti-apoptotic effects of memantine in a SH-SY5Y cell model of Alzheimer's disease via mammalian target of rapamycin-dependent and -independent pathways.

    PubMed

    Song, Guijun; Li, Yu; Lin, Lulu; Cao, Yunpeng

    2015-11-01

    Memantine non-competitively blocks the N-methyl-D-aspartate receptor in order to inhibit beta-amyloid (Aβ) secretion, and has been used to treat moderate-to-severe Alzheimer's disease (AD). However, the mechanisms underlying the role of memantine in the autophagy and apoptosis of neuronal cells in AD, as well as the association between neuronal autophagy and apoptosis have yet to be elucidated. The present study aimed to establish an AD cell model overexpressing the 695‑amino‑acid Swedish mutant of Aβ precursor protein (APP695swe) in order to observe the effects of memantine on the cell viability, autophagy and apoptosis of SH‑SY5Y cells in the AD model, and to investigate the associated underlying mechanisms. A pcDNA3.1‑APP695 plasmid was transfected into the SH‑SY5Y cells. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses demonstrated that the AD cell model was successfully established. MTT assays demonstrated that memantine was able to upregulate neuronal cell survival, and acridine orange staining and flow cytometry demonstrated that memantine (5 µM) was able to inhibit neuronal autophagy and apoptosis. Following neuronal autophagy induction by rapamycin, cell apoptosis rates increased significantly. Further experiments revealed that memantine was able to upregulate the expression of signaling molecules phosphorylated (p)-phosphoinositide 3‑kinase, p‑Akt and p‑mammalian target of rapamycin (mTOR), and also inhibited the phosphorylation of the B‑cell lymphoma 2/Beclin‑1 complex via mitogen‑activated protein kinase 8. In conclusion, the results of the present study demonstrated that in the AD cell model, autophagy was able to promote apoptosis. Memantine exerted anti‑autophagic and anti‑apoptotic functions, and mTOR‑dependent as well as ‑independent autophagic signaling pathways were involved in this process. Therefore, these results of the present study strongly supported the use of

  11. Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

    PubMed Central

    Costa, Liliana; Faustino, Maria Amparo F.; Neves, Maria Graça P. M. S.; Cunha, Ângela; Almeida, Adelaide

    2012-01-01

    Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process. PMID:22852040

  12. Photodynamic inactivation of mammalian viruses and bacteriophages.

    PubMed

    Costa, Liliana; Faustino, Maria Amparo F; Neves, Maria Graça P M S; Cunha, Angela; Almeida, Adelaide

    2012-07-01

    Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process.

  13. Recent advances in mammalian protein production

    PubMed Central

    Bandaranayake, Ashok D.; Almo, Steven C.

    2014-01-01

    Mammalian protein production platforms have had a profound impact in many areas of basic and applied research, and an increasing number of blockbuster drugs are recombinant mammalian proteins. With global sales of these drugs exceeding US$120 billion per year, both industry and academic research groups continue to develop cost effective methods for producing mammalian proteins to support preclinical and clinical evaluations of potential therapeutics. While a wide range of platforms have been successfully exploited for laboratory use, the bulk of recent biologics have been produced in mammalian cell lines due to the requirement for post translational modification and the biosynthetic complexity of the target proteins. In this review we highlight the range of mammalian expression platforms available for recombinant protein production, as well as advances in technologies for the rapid and efficient selection of highly productive clones. PMID:24316512

  14. Utilization of a BGO detector as an active oxygen target

    NASA Astrophysics Data System (ADS)

    Loveman, R.; Gozani, T.; Bendahan, J.; Krivicich, J.; Elias, E.; Altschuler, E.

    1994-12-01

    The (n, n'γx) cross section for the 6.13 MeV state in oxygen has recently become of general interest because of the possibility of using this process to assay oxygen as a part of non-intrusive inspections. Localized densities of carbon, oxygen, and nitrogen are particularly useful in determining the presence of explosives and/or drugs in containers of all sizes, from suitcases to cargo containers. The presence of oxygen in BGO (Bi 4Ge 3O 12) scintillator makes this detector suitable for use as an active target for the measurement of the energy dependence of the excitation, of the first (6.049 MeV O +) and second (6.130 MeV 3 -) excited states in 16O by fast neutron interactions. An active target functions as both a target and an active device such as a detector. The de-excitations of the 6.049 and 6.130 states take place by nuclear pair production and γ-ray emission respectively. There is a large probability of absorbing all of the de-excitation energy in the scintillator in either of these cases. Since the energies deposited in the scintillator by these transitions are very close, the de-excitations are indistinguishable. However, since the cross section for the excitation of the 6.13 MeV state is believed to be larger than that of the 6.049 MeV, the major measured features of the energy variations are those related to the second state. The validity of the technique was initially tested using (MCNP) calculations. The calculations established that the detected neutron count rate in the crystal was proportional to the cross-sections used as input for the calculations, and that the constant of proportionality did not vary with neutron energy. Subsequently, measurements were made with a BGO detector as an active oxygen target. The results clearly show a strong energy dependence including several resonances.

  15. Bat auditory cortex – model for general mammalian auditory computation or special design solution for active time perception?

    PubMed

    Kössl, Manfred; Hechavarria, Julio; Voss, Cornelia; Schaefer, Markus; Vater, Marianne

    2015-03-01

    Audition in bats serves passive orientation, alerting functions and communication as it does in other vertebrates. In addition, bats have evolved echolocation for orientation and prey detection and capture. This put a selective pressure on the auditory system in regard to echolocation-relevant temporal computation and frequency analysis. The present review attempts to evaluate in which respect the processing modules of bat auditory cortex (AC) are a model for typical mammalian AC function or are designed for echolocation-unique purposes. We conclude that, while cortical area arrangement and cortical frequency processing does not deviate greatly from that of other mammals, the echo delay time-sensitive dorsal cortex regions contain special designs for very powerful time perception. Different bat species have either a unique chronotopic cortex topography or a distributed salt-and-pepper representation of echo delay. The two designs seem to enable similar behavioural performance. PMID:25728173

  16. Regulation of Blood–Testis Barrier (BTB) Dynamics during Spermatogenesis via the “Yin” and “Yang” Effects of Mammalian Target of Rapamycin Complex 1 (mTORC1) and mTORC2

    PubMed Central

    Mok, Ka Wai; Mruk, Dolores D.; Cheng, C. Yan

    2014-01-01

    In mammalian testes, haploid spermatozoa are formed from diploid spermatogonia during spermatogenesis, which is a complicated cellular process. While these cellular events were reported in the 1960s and 1970s, the underlying molecular mechanism(s) that regulates these events remained unexplored until the past ~10 years. For instance, adhesion proteins were shown to be integrated components at the Sertoli cell–cell interface and/or the Sertoli–spermatid interface in the late 1980s. But only until recently, studies have demonstrated that some of the adhesion proteins serve as the platform for signal transduction that regulates cell adhesion. In this chapter, a brief summary and critical discussion are provided on the latest findings regarding these cell-adhesion proteins in the testis and their relationship to spermatogenesis. Moreover, antagonistic effects of two mammalian target of rapamycin (mTOR) complexes, known as mTORC1 and mTORC2, on cell-adhesion function in the testis are discussed. Finally, a hypothetic model is presented to depict how these two mTOR-signaling complexes having the “yin” and “yang” antagonistic effects on the Sertoli cell tight junction (TJ)-permeability barrier can maintain the blood–testis barrier (BTB) integrity during the epithelial cycle while preleptotene spermatocytes are crossing the BTB. PMID:23317821

  17. Activity based chemical proteomics: profiling proteases as drug targets.

    PubMed

    Heal, William Percy; Wickramasinghe, Sasala Roshinie; Tate, Edward William

    2008-09-01

    The pivotal role of proteases in many diseases has generated considerable interest in their basic biology, and in the potential to target them for chemotherapy. Although fundamental to the initiation and progression of diseases such as cancer, diabetes, arthritis and malaria, in many cases their precise role remains unknown. Activity-based chemical proteomics-an emerging field involving a combination of organic synthesis, biochemistry, cell biology, biophysics and bioinformatics-allows the detection, visualisation and activity quantification of whole families or selected sub-sets of proteases based upon their substrate specificity. This approach can be applied for drug target/lead identification and validation, the fundamentals of drug discovery. The activity-based probes discussed in this review contain three key features; a 'warhead' (binds irreversibly but selectively to the active site), a 'tag' (allowing enzyme 'handling', with a combination of fluorescent, affinity and/or radio labels), and a linker region between warhead and tag. From the design and synthesis of the linker arise some of the latest developments discussed here; not only can the physical properties (e.g., solubility, localisation) of the probe be tuned, but the inclusion of a cleavable moiety allows selective removal of tagged enzyme from affinity beads etc. The design and synthesis of recently reported probes is discussed, including modular assembly of highly versatile probes via solid phase synthesis. Recent applications of activity-based protein profiling to specific proteases (serine, threonine, cysteine and metalloproteases) are reviewed as are demonstrations of their use in the study of disease function in cancer and malaria.

  18. A Deterministic Approach to Active Debris Removal Target Selection

    NASA Astrophysics Data System (ADS)

    Lidtke, A.; Lewis, H.; Armellin, R.

    2014-09-01

    Many decisions, with widespread economic, political and legal consequences, are being considered based on space debris simulations that show that Active Debris Removal (ADR) may be necessary as the concerns about the sustainability of spaceflight are increasing. The debris environment predictions are based on low-accuracy ephemerides and propagators. This raises doubts about the accuracy of those prognoses themselves but also the potential ADR target-lists that are produced. Target selection is considered highly important as removal of many objects will increase the overall mission cost. Selecting the most-likely candidates as soon as possible would be desirable as it would enable accurate mission design and allow thorough evaluation of in-orbit validations, which are likely to occur in the near-future, before any large investments are made and implementations realized. One of the primary factors that should be used in ADR target selection is the accumulated collision probability of every object. A conjunction detection algorithm, based on the smart sieve method, has been developed. Another algorithm is then applied to the found conjunctions to compute the maximum and true probabilities of collisions taking place. The entire framework has been verified against the Conjunction Analysis Tools in AGIs Systems Toolkit and relative probability error smaller than 1.5% has been achieved in the final maximum collision probability. Two target-lists are produced based on the ranking of the objects according to the probability they will take part in any collision over the simulated time window. These probabilities are computed using the maximum probability approach, that is time-invariant, and estimates of the true collision probability that were computed with covariance information. The top-priority targets are compared, and the impacts of the data accuracy and its decay are highlighted. General conclusions regarding the importance of Space Surveillance and Tracking for the

  19. Small Molecule Inhibitors Targeting Activator Protein 1 (AP-1)

    PubMed Central

    2015-01-01

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases. PMID:24831826

  20. Semiconducting Polymer Nanobioconjugates for Targeted Photothermal Activation of Neurons.

    PubMed

    Lyu, Yan; Xie, Chen; Chechetka, Svetlana A; Miyako, Eijiro; Pu, Kanyi

    2016-07-27

    Optogenetics provides powerful means for precise control of neuronal activity; however, the requirement of transgenesis and the incapability to extend the neuron excitation window into the deep-tissue-penetrating near-infrared (NIR) region partially limit its application. We herein report a potential alternative approach to optogenetics using semiconducting polymer nanobioconjugates (SPNsbc) as the photothermal nanomodulator to control the thermosensitive ion channels in neurons. SPNsbc are designed to efficiently absorb the NIR light at 808 nm and have a photothermal conversion efficiency higher than that of gold nanorods. By virtue of the fast heating capability in conjunction with the precise targeting to the thermosensitive ion channel, SPNsbc can specifically and rapidly activate the intracellular Ca(2+) influx of neuronal cells in a reversible and safe manner. Our study provides an organic nanoparticle based strategy that eliminates the need for genetic transfection to remotely regulate cellular machinery. PMID:27404507

  1. Novel strategies for ultrahigh specific activity targeted nanoparticles

    SciTech Connect

    Zhou, Dong

    2012-12-13

    We have developed novel strategies optimized for preparing high specific activity radiolabeled nanoparticles, targeting nuclear imaging of low abundance biomarkers. Several compounds have been labeled with F-18 and Cu-64 for radiolabeling of SCK-nanoparticles via Copper(I) catalyzed or copper-free alkyne-azide cyclolization. Novel strategies have been developed to achieve ultrahigh specific activity with administrable amount of dose for human study using copper-free chemistry. Ligands for carbonic anhydrase 12 (CA12), a low abundance extracellular biomarker for the responsiveness of breast cancer to endocrine therapie, have been labeled with F-18 and Cu-64, and one of them has been evaluated in animal models. The results of this project will lead to major improvements in the use of nanoparticles in nuclear imaging and will significantly advance their potential for detecting low abundance biomarkers of medical importance.

  2. mRNA Targeting to Endoplasmic Reticulum Precedes Ago Protein Interaction and MicroRNA (miRNA)-mediated Translation Repression in Mammalian Cells.

    PubMed

    Barman, Bahnisikha; Bhattacharyya, Suvendra N

    2015-10-01

    MicroRNA (miRNA) binds to the 3'-UTR of its target mRNAs to repress protein synthesis. Extensive research was done to understand the mechanism of miRNA-mediated repression in animal cells. Considering the progress in understanding the mechanism, information about the subcellular sites of miRNA-mediated repression is surprisingly limited. In this study, using an inducible expression system for an miRNA target message, we have delineated how a target mRNA passes through polysome association and Ago2 interaction steps on rough endoplasmic reticulum (ER) before the miRNA-mediated repression sets in. From this study, de novo formed target mRNA localization to the ER-bound polysomes manifested as the earliest event, which is followed by Ago2 micro-ribonucleoprotein binding, and translation repression of target message. Compartmentalization of this process to rough ER membrane ensures enrichment of miRNA-targeted messages and micro-ribonucleoprotein components on ER upon reaching a steady state. PMID:26304123

  3. mRNA Targeting to Endoplasmic Reticulum Precedes Ago Protein Interaction and MicroRNA (miRNA)-mediated Translation Repression in Mammalian Cells.

    PubMed

    Barman, Bahnisikha; Bhattacharyya, Suvendra N

    2015-10-01

    MicroRNA (miRNA) binds to the 3'-UTR of its target mRNAs to repress protein synthesis. Extensive research was done to understand the mechanism of miRNA-mediated repression in animal cells. Considering the progress in understanding the mechanism, information about the subcellular sites of miRNA-mediated repression is surprisingly limited. In this study, using an inducible expression system for an miRNA target message, we have delineated how a target mRNA passes through polysome association and Ago2 interaction steps on rough endoplasmic reticulum (ER) before the miRNA-mediated repression sets in. From this study, de novo formed target mRNA localization to the ER-bound polysomes manifested as the earliest event, which is followed by Ago2 micro-ribonucleoprotein binding, and translation repression of target message. Compartmentalization of this process to rough ER membrane ensures enrichment of miRNA-targeted messages and micro-ribonucleoprotein components on ER upon reaching a steady state.

  4. A combination of targeted toxin technology and the piggyBac-mediated gene transfer system enables efficient isolation of stable transfectants in nonhuman mammalian cells.

    PubMed

    Sato, Masahiro; Inada, Emi; Saitoh, Issei; Matsumoto, Yuko; Ohtsuka, Masato; Miura, Hiromi; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

    2015-01-01

    Isolation of cells harboring exogenous DNA is typically achieved by the introduction of plasmids, but its efficiency remains still low. In this study, we developed a novel strategy to obtain stable transfectants efficiently. Porcine embryonic fibroblasts were transfected with two plasmids: (i) pTransIEnd, which comprises the ubiquitous promoter, the piggyBac (PB) transposase gene, an internal ribosomal entry site, the Clostridium perfringens-derived endo-β-galactosidase C (EndoGalC) gene, and a poly(A) tail and (ii) a PB-based plasmid, termed pT-EGFP, which contains enhanced green fluorescent protein (EGFP) expression unit flanked by PB acceptor sites. The PB transposase can accelerate the chromosomal integration of transposon vectors. EndoGalC expression results in removal of a cell surface α-Gal epitope, which is specifically recognized by Bandeiraea simplicifolia isolectin-B4 (IB4). Four days after transfection, cells were treated with IB4SAP (IB4 conjugated to saporin, which eliminates any α-Gal epitope-expressing cells) for a short period, followed by standard culture for approximately 10 days. Several colonies emerged, most of which were positive for EGFP expression and lacked TransIEnd. These results indicated that the proposed approach is useful and efficient for obtaining stable transfectants without the use of drug-resistance genes, and offers a novel route for gene manipulation in cultured nonhuman mammalian cells. PMID:25345906

  5. A combination of targeted toxin technology and the piggyBac-mediated gene transfer system enables efficient isolation of stable transfectants in nonhuman mammalian cells.

    PubMed

    Sato, Masahiro; Inada, Emi; Saitoh, Issei; Matsumoto, Yuko; Ohtsuka, Masato; Miura, Hiromi; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

    2015-01-01

    Isolation of cells harboring exogenous DNA is typically achieved by the introduction of plasmids, but its efficiency remains still low. In this study, we developed a novel strategy to obtain stable transfectants efficiently. Porcine embryonic fibroblasts were transfected with two plasmids: (i) pTransIEnd, which comprises the ubiquitous promoter, the piggyBac (PB) transposase gene, an internal ribosomal entry site, the Clostridium perfringens-derived endo-β-galactosidase C (EndoGalC) gene, and a poly(A) tail and (ii) a PB-based plasmid, termed pT-EGFP, which contains enhanced green fluorescent protein (EGFP) expression unit flanked by PB acceptor sites. The PB transposase can accelerate the chromosomal integration of transposon vectors. EndoGalC expression results in removal of a cell surface α-Gal epitope, which is specifically recognized by Bandeiraea simplicifolia isolectin-B4 (IB4). Four days after transfection, cells were treated with IB4SAP (IB4 conjugated to saporin, which eliminates any α-Gal epitope-expressing cells) for a short period, followed by standard culture for approximately 10 days. Several colonies emerged, most of which were positive for EGFP expression and lacked TransIEnd. These results indicated that the proposed approach is useful and efficient for obtaining stable transfectants without the use of drug-resistance genes, and offers a novel route for gene manipulation in cultured nonhuman mammalian cells.

  6. Active mechanistic target of rapamycin plays an ancillary rather than essential role in zebrafish CNS axon regeneration

    PubMed Central

    Diekmann, Heike; Kalbhen, Pascal; Fischer, Dietmar

    2015-01-01

    The developmental decrease of the intrinsic regenerative ability of the mammalian central nervous system (CNS) is associated with reduced activity of mechanistic target of rapamycin (mTOR) in mature neurons such as retinal ganglion cells (RGCs). While mTOR activity is further decreased upon axonal injury, maintenance of its pre-injury level, for instance by genetic deletion of the phosphatase and tensin homolog (PTEN), markedly promotes axon regeneration in mammals. The current study now addressed the question whether active mTOR might generally play a central role in axon regeneration by analyzing its requirement in regeneration-competent zebrafish. Remarkably, regulation of mTOR activity after optic nerve injury in zebrafish is fundamentally different compared to mammals. Hardly any activity was detected in naïve RGCs, whereas it was markedly increased upon axotomy in vivo as well as in dissociated cell cultures. After a short burst, mTOR activity was quickly attenuated, which is contrary to the requirements for axon regeneration in mammals. Surprisingly, mTOR activity was not essential for axonal growth per se, but correlated with cytokine- and PTEN inhibitor-induced neurite extension in vitro. Moreover, inhibition of mTOR using rapamycin significantly reduced axon regeneration in vivo and compromised functional recovery after optic nerve injury. Therefore, axotomy-induced mTOR activity is involved in CNS axon regeneration in zebrafish similar to mammals, although it plays an ancillary rather than essential role in this regeneration-competent species. PMID:26217179

  7. Evaluation of the genetic effects induced by vinyl chloride monomer (VCM) under mammalian metabolic activation: studies in vitro and in vivo.

    PubMed

    Loprieno, N; Barale, R; Baroncelli, S; Bauer, C; Bronzetti, G; Cammellini, A; Cercignani, G; Corsi, C; Gervasi, G; Leporini, C; Nieri, R; Rossi, A M; Stretti, G; Turchi, G

    1976-04-01

    As part of a programme of investigations on the biological effects of the industrial compound vinyl chloride monomer (VCM), the raw material for the production of polyvinyl chloride (PVC), analyses on the genetic effects by this compound have been done by experiments (in vitro) which have taken mammalian metabolism into account. Vinyl chloride in the presence of purified microsomes (sedimented at 105,000 g) obtained from mouse liver was converted into an active metabolite(s) which produced gene mutations in the yeast Schizosaccharomyces pombe (forward mutation) and gene conversions in two loci of a diploid Saccharomyces cerevisiae. Moreover, the compound was active in the host-mediated assay, when mice were treated with an oral dose of 700 mg/kg. The role is discussed of mutagenicity tests for the prediction of both genetic and carcinogenic risks of chemical compounds in industrial use.

  8. Crystal structure of mammalian acid sphingomyelinase

    PubMed Central

    Gorelik, Alexei; Illes, Katalin; Heinz, Leonhard X.; Superti-Furga, Giulio; Nagar, Bhushan

    2016-01-01

    Acid sphingomyelinase (ASMase, ASM, SMPD1) converts sphingomyelin into ceramide, modulating membrane properties and signal transduction. Inactivating mutations in ASMase cause Niemann–Pick disease, and its inhibition is also beneficial in models of depression and cancer. To gain a better understanding of this critical therapeutic target, we determined crystal structures of mammalian ASMase in various conformations. The catalytic domain adopts a calcineurin-like fold with two zinc ions and a hydrophobic track leading to the active site. Strikingly, the membrane interacting saposin domain assumes either a closed globular conformation independent from the catalytic domain, or an open conformation, which establishes an interface with the catalytic domain essential for activity. Structural mapping of Niemann–Pick mutations reveals that most of them likely destabilize the protein's fold. This study sheds light on the molecular mechanism of ASMase function, and provides a platform for the rational development of ASMase inhibitors and therapeutic use of recombinant ASMase. PMID:27435900

  9. Crystal structure of mammalian acid sphingomyelinase.

    PubMed

    Gorelik, Alexei; Illes, Katalin; Heinz, Leonhard X; Superti-Furga, Giulio; Nagar, Bhushan

    2016-01-01

    Acid sphingomyelinase (ASMase, ASM, SMPD1) converts sphingomyelin into ceramide, modulating membrane properties and signal transduction. Inactivating mutations in ASMase cause Niemann-Pick disease, and its inhibition is also beneficial in models of depression and cancer. To gain a better understanding of this critical therapeutic target, we determined crystal structures of mammalian ASMase in various conformations. The catalytic domain adopts a calcineurin-like fold with two zinc ions and a hydrophobic track leading to the active site. Strikingly, the membrane interacting saposin domain assumes either a closed globular conformation independent from the catalytic domain, or an open conformation, which establishes an interface with the catalytic domain essential for activity. Structural mapping of Niemann-Pick mutations reveals that most of them likely destabilize the protein's fold. This study sheds light on the molecular mechanism of ASMase function, and provides a platform for the rational development of ASMase inhibitors and therapeutic use of recombinant ASMase. PMID:27435900

  10. Cytotoxic activity of Bacillus thuringiensis Cry proteins on mammalian cells transfected with cadherin-like Cry receptor gene of Bombyx mori (silkworm).

    PubMed Central

    Tsuda, Yoko; Nakatani, Fumiki; Hashimoto, Keiko; Ikawa, Satoshi; Matsuura, Chikako; Fukada, Takashi; Sugimoto, Kenji; Himeno, Michio

    2003-01-01

    Cry1Aa, an insecticidal protein produced by Bacillus thuringiensis, has been shown to bind to cadherin-like protein, BtR175, in Bombyx mori (silkworm) midgut. We previously reported three variant alleles of BtR175 (BtR175a, b and c). When transiently expressed in COS7 cells, all the three BtR175 variants bound to Cry1Aa. We stably expressed BtR175b in HEK293 cells. These BtR175b-expressing cells swelled and died in the presence of activated Cry1Aa in a dose- and time-dependent manner, showing that BtR175b itself can impart Cry1Aa-susceptibility to mammalian cells. These cells were more susceptible to Cry1Aa than to Cry1Ab and Cry1Ac. Since dispersed B. mori midgut cells were reported to be highly susceptible to Cry1Ac, this result suggested that other Cry1Ac-specific receptor(s) were simultaneously working with BtR175 in the midgut cells. Advantages are also discussed of applying these transfected mammalian cells to toxicity assays of mutant Cry proteins. PMID:12403648

  11. Crowding and Follicular Fate: Spatial Determinants of Follicular Reserve and Activation of Follicular Growth in the Mammalian Ovary

    PubMed Central

    Gaytan, Francisco; Morales, Concepcion; Leon, Silvia; Garcia-Galiano, David; Roa, Juan; Tena-Sempere, Manuel

    2015-01-01

    Initiation of growth of resting ovarian follicles is a key phenomenon for providing an adequate number of mature oocytes in each ovulation, while preventing premature exhaustion of primordial follicle reserve during the reproductive lifespan. Resting follicle dynamics strongly suggest that primordial follicles are under constant inhibitory influences, by mechanisms and factors whose nature remains ill defined. In this work, we aimed to assess the influence of spatial determinants, with special attention to clustering patterns and crowding, on the fate of early follicles in the adult mouse and human ovary. To this end, detailed histological and morphometric analyses, targeting resting and early growing follicles, were conducted in ovaries from mice, either wild type (WT) or genetically modified to lack kisspeptin receptor expression (Kiss1r KO), and healthy adult women. Kiss1r KO mice were studied as model of persistent hypogonadotropism and anovulation. Different qualitative and quantitative indices of the patterns of spatial distribution of resting and early growing follicles in the mouse and human ovary, including the Morisita’s index of clustering, were obtained. Our results show that resting primordial follicles display a clear-cut clustered pattern of spatial distribution in adult mouse and human ovaries, and that resting follicle aggrupation is inversely correlated with the proportion of follicles initiating growth and entering into the growing pool. As a whole, our data suggest that resting follicle crowding, defined by changes in density and clustered pattern of distribution, is a major determinant of follicular activation and the fate of ovarian reserve. Uneven follicle crowding would constitute the structural counterpart of the major humoral regulators of early follicular growth, with potential implications in ovarian ageing and pathophysiology. PMID:26642206

  12. Composition of three essential oils, and their mammalian cell toxicity and antimycobacterial activity against drug resistant-tuberculosis and nontuberculous mycobacteria strains.

    PubMed

    Bueno, Juan; Escobar, Patricia; Martínez, Jairo René; Leal, Sandra Milena; Stashenko, Elena E

    2011-11-01

    Tuberculosis (TB) is the most ancient epidemic disease in the world and a serious opportunistic disease in HIV/AIDS patients. The increase in multidrug resistant Mycobacterium tuberculosis (MDR-TB, XDR-TB) demands the search for novel antimycobacterial drugs. Essential oils (EOs) have been widely used in medicine and some EOs and their major components have been shown to be active against M. tuberculosis. The aim of this work was to evaluate the antimycobacterial and cell toxicity activities of three EOs derived from Salvia aratocensis, Turnera diffusa and Lippia americana, aromatics plants collected in Colombia. The EOs were isolated by hydrodistillation and analyzed by GC/MS techniques. The EOs were tested against 15 Mycobacterium spp using a colorimetric macrodilution method and against mammalian Vero and THP-1 cells by MTT. The activity was expressed as minimal concentration in microg/mL that inhibits growth, and the concentration that is cytotoxic for 50 or 90% of the cells (CC50 and CC90). The major components were epi-alpha-cadinol (20.1%) and 1,10-di-epi-cubenol (14.2%) for Salvia aratocensis; drima-7,9(11)-diene (22.9%) and viridiflorene (6.6%) for Turnera diffusa; and germacrene D (15.4%) and trans-beta- caryophyllene (11.3%) for Lippia americana. The most active EO was obtained from S. aratocensis, with MIC values below 125 microg mL(-1) for M. tuberculosis Beijing genotype strains, and 200 to 500 microg mL(-1) for nontuberculous mycobacterial strains. The EOs were either partially or non toxic to Vero and THP-1 mammalian cells with CC50 values from 30 to > 100 microg mL(-1), and a CC90 > 100 microg mL(-1). The EOs obtained from the three aromatic Colombian plants are an important source of potential compounds against TB. Future studies using the major EO components are recommended. PMID:22224302

  13. Editing DNA Methylation in the Mammalian Genome.

    PubMed

    Liu, X Shawn; Wu, Hao; Ji, Xiong; Stelzer, Yonatan; Wu, Xuebing; Czauderna, Szymon; Shu, Jian; Dadon, Daniel; Young, Richard A; Jaenisch, Rudolf

    2016-09-22

    Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation. PMID:27662091

  14. Lethal Giant Larvae 1 Tumour Suppressor Activity Is Not Conserved in Models of Mammalian T and B Cell Leukaemia

    PubMed Central

    Hawkins, Edwin D.; Oliaro, Jane; Ramsbottom, Kelly M.; Ting, Stephen B.; Sacirbegovic, Faruk; Harvey, Michael; Kinwell, Tanja; Ghysdael, Jacques; Johnstone, Ricky W.; Humbert, Patrick O.; Russell, Sarah M.

    2014-01-01

    In epithelial and stem cells, lethal giant larvae (Lgl) is a potent tumour suppressor, a regulator of Notch signalling, and a mediator of cell fate via asymmetric cell division. Recent evidence suggests that the function of Lgl is conserved in mammalian haematopoietic stem cells and implies a contribution to haematological malignancies. To date, direct measurement of the effect of Lgl expression on malignancies of the haematopoietic lineage has not been tested. In Lgl1−/− mice, we analysed the development of haematopoietic malignancies either alone, or in the presence of common oncogenic lesions. We show that in the absence of Lgl1, production of mature white blood cell lineages and long-term survival of mice are not affected. Additionally, loss of Lgl1 does not alter leukaemia driven by constitutive Notch, c-Myc or Jak2 signalling. These results suggest that the role of Lgl1 in the haematopoietic lineage might be restricted to specific co-operating mutations and a limited number of cellular contexts. PMID:24475281

  15. Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells

    PubMed Central

    Hockberger, Philip E.; Skimina, Timothy A.; Centonze, Victoria E.; Lavin, Colleen; Chu, Su; Dadras, Soheil; Reddy, Janardan K.; White, John G.

    1999-01-01

    Violet-blue light is toxic to mammalian cells, and this toxicity has been linked with cellular production of H2O2. In this report, we show that violet-blue light, as well as UVA, stimulated H2O2 production in cultured mouse, monkey, and human cells. We found that H2O2 originated in peroxisomes and mitochondria, and it was enhanced in cells overexpressing flavin-containing oxidases. These results support the hypothesis that photoreduction of flavoproteins underlies light-induced production of H2O2 in cells. Because H2O2 and its metabolite, hydroxyl radicals, can cause cellular damage, these reactive oxygen species may contribute to pathologies associated with exposure to UVA, violet, and blue light. They may also contribute to phototoxicity often encountered during light microscopy. Because multiphoton excitation imaging with 1,047-nm wavelength prevented light-induced H2O2 production in cells, possibly by minimizing photoreduction of flavoproteins, this technique may be useful for decreasing phototoxicity during fluorescence microscopy. PMID:10339574

  16. Effect of GO-Fe3O4 and rotating magnetic field on cellular metabolic activity of mammalian cells.

    PubMed

    Urbas, Karolina; Jedrzejczak-Silicka, Magdalena; Rakoczy, Rafal; Zaborski, Daniel; Mijowska, Ewa

    2016-04-01

    The effect of hybrid material-graphene flakes with Fe3O4 nanospheres (GO-Fe3O4), graphene oxide (GO) and magnetite nanospheres (Fe3O4) in rotating magnetic field on mammalian cells metabolism has been studied. Several reports shown that exposure to magnetic field may have influence on cellular membrane permeability. Thus, the aim of presented study was to determine the cellular response of L929 fibroblast cells to nanomaterials and rotating magnetic field for 8-h exposure experiment. The GO had tendency to adsorb proteins, thus cell metabolism was decreased and the effect of that mechanism was enhanced by impact of nanospheres and rotating magnetic field. The highest reduction of cellular metabolism was recorded for WST-1 and NR assays at concentration 100 µg/mL of all tested nanomaterials and magnetic induction value 10.06 mT. The lactate dehydrogenase leakage assay has shown significant changes in membrane permeability. Further studies need to be carried out to precisely determine the mechanism of that process. PMID:26809700

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

    PubMed

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

    2015-09-01

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

  18. Hyaluronic acid-coated liposomes for active targeting of gemcitabine.

    PubMed

    Arpicco, Silvia; Lerda, Carlotta; Dalla Pozza, Elisa; Costanzo, Chiara; Tsapis, Nicolas; Stella, Barbara; Donadelli, Massimo; Dando, Ilaria; Fattal, Elias; Cattel, Luigi; Palmieri, Marta

    2013-11-01

    The aim of this work was the preparation, characterization, and preliminary evaluation of the targeting ability toward pancreatic adenocarcinoma cells of liposomes containing the gemcitabine lipophilic prodrug [4-(N)-lauroyl-gemcitabine, C12GEM]. Hyaluronic acid (HA) was selected as targeting agent since it is biodegradable, biocompatible, and can be chemically modified and its cell surface receptor CD44 is overexpressed on various tumors. For this purpose, conjugates between a phospholipid, the 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and HA of two different low molecular weights 4800 Da (12 disaccharidic units) and 12,000 Da (32 disaccharidic units), were prepared, characterized, and introduced in the liposomes during the preparation. Different liposomal formulations were prepared and their characteristics were analyzed: size, Z potential, and TEM analyses underline a difference in the HA-liposomes from the non-HA ones. In order to better understand the HA-liposome cellular localization and to evaluate their interaction with CD44 receptor, confocal microscopy studies were performed. The results demonstrate that HA facilitates the recognition of liposomes by MiaPaCa2 cells (CD44(+)) and that the uptake increases with increase in the polymer molecular weight. Finally, the cytotoxicity of the different preparations was evaluated and data show that incorporation of C12GEM increases their cytotoxic activity and that HA-liposomes inhibit cell growth more than plain liposomes. Altogether, the results demonstrate the specificity of C12GEM targeting toward CD44-overexpressing pancreatic adenocarcinoma cell line using HA as a ligand.

  19. Hyaluronic acid-coated liposomes for active targeting of gemcitabine.

    PubMed

    Arpicco, Silvia; Lerda, Carlotta; Dalla Pozza, Elisa; Costanzo, Chiara; Tsapis, Nicolas; Stella, Barbara; Donadelli, Massimo; Dando, Ilaria; Fattal, Elias; Cattel, Luigi; Palmieri, Marta

    2013-11-01

    The aim of this work was the preparation, characterization, and preliminary evaluation of the targeting ability toward pancreatic adenocarcinoma cells of liposomes containing the gemcitabine lipophilic prodrug [4-(N)-lauroyl-gemcitabine, C12GEM]. Hyaluronic acid (HA) was selected as targeting agent since it is biodegradable, biocompatible, and can be chemically modified and its cell surface receptor CD44 is overexpressed on various tumors. For this purpose, conjugates between a phospholipid, the 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and HA of two different low molecular weights 4800 Da (12 disaccharidic units) and 12,000 Da (32 disaccharidic units), were prepared, characterized, and introduced in the liposomes during the preparation. Different liposomal formulations were prepared and their characteristics were analyzed: size, Z potential, and TEM analyses underline a difference in the HA-liposomes from the non-HA ones. In order to better understand the HA-liposome cellular localization and to evaluate their interaction with CD44 receptor, confocal microscopy studies were performed. The results demonstrate that HA facilitates the recognition of liposomes by MiaPaCa2 cells (CD44(+)) and that the uptake increases with increase in the polymer molecular weight. Finally, the cytotoxicity of the different preparations was evaluated and data show that incorporation of C12GEM increases their cytotoxic activity and that HA-liposomes inhibit cell growth more than plain liposomes. Altogether, the results demonstrate the specificity of C12GEM targeting toward CD44-overexpressing pancreatic adenocarcinoma cell line using HA as a ligand. PMID:23791684

  20. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5'-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa).

    PubMed

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E; Gallo-Reynoso, Juan P

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5'-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5'-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), ATP, guanosine 5'-diphosphate (GDP), guanosine 5'-triphosphate (GTP), and xanthosine 5'-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

  1. FVT-1 is a mammalian 3-ketodihydrosphingosine reductase with an active site that faces the cytosolic side of the endoplasmic reticulum membrane.

    PubMed

    Kihara, Akio; Igarashi, Yasuyuki

    2004-11-19

    Sphingolipids are essential membrane components of eukaryotic cells. Their synthesis is initiated with the condensation of l-serine with palmitoyl-CoA, producing 3-ketodihydrosphingosine (KDS), followed by a reduction to dihydrosphingosine by KDS reductase. Until now, only yeast TSC10 has been identified as a KDS reductase gene. Here, we provide evidence that the human FVT-1 (hFVT-1) and mouse FVT-1 (mFVT-1) are functional mammalian KDS reductases. The forced expression of hFVT-1 or mFVT-1 in TSC10-null yeast cells suppressed growth defects, and hFVT-1 overproduced in cultured cells exhibited KDS reductase activity in vitro. Moreover, purified recombinant hFVT-1 protein exhibited NADPH-dependent KDS reductase activity. The identification of the FVT-1 genes enabled us to characterize the mammalian KDS reductase at the molecular level. Northern blot analyses demonstrated that both hFVT-1 and mFVT-1 mRNAs are ubiquitously expressed, suggesting that FVT-1 is a major KDS reductase. We also found the presence of hFVT-1 variants, which were differentially expressed among tissues. Immunofluorescence microscopic analysis revealed that hFVT-1 is localized at the endoplasmic reticulum. Moreover, a proteinase K digestion assay revealed that the large hydrophilic domain of hFVT-1, which contains putative active site residues, faces the cytosol. These results suggest that KDS is converted to dihydrosphingosine in the cytosolic side of the endoplasmic reticulum membrane. Moreover, the topology studies provide insight into the spatial organization of the sphingolipid biosynthetic pathway.

  2. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5'-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa).

    PubMed

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E; Gallo-Reynoso, Juan P

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5'-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5'-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), ATP, guanosine 5'-diphosphate (GDP), guanosine 5'-triphosphate (GTP), and xanthosine 5'-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts. PMID:26283971

  3. Active targeting schemes for nanoparticle systems in cancer therapeutics.

    PubMed

    Byrne, James D; Betancourt, Tania; Brannon-Peppas, Lisa

    2008-12-14

    The objective of this review is to outline current major cancer targets for nanoparticle systems and give insight into the direction of the field. The major targeting strategies that have been used for the delivery of therapeutic or imaging agents to cancer have been broken into three sections. These sections are angiogenesis-associated targeting, targeting to uncontrolled cell proliferation markers, and tumor cell targeting. The targeting schemes explored for many of the reported nanoparticle systems suggest the great potential of targeted delivery to revolutionize cancer treatment.

  4. Epigenetic drugs that do not target enzyme activity.

    PubMed

    Owen, Dafydd R; Trzupek, John D

    2014-06-01

    While the installation and removal of epigenetic post-translational modifications or ‘marks’ on both DNA and histone proteins are the tangible outcome of enzymatically catalyzed processes, the role of the epigenetic reader proteins looks, at first, less obvious. As they do not catalyze a chemical transformation or process as such, their role is not enzymatic. However, this does not preclude them from being potential targets for drug discovery as their function is clearly correlated to transcriptional activity and as a class of proteins, they appear to have binding sites of sufficient definition and size to be inhibited by small molecules. This suggests that this third class of epigenetic proteins that are involved in the interpretation of post-translational marks (as opposed to the creation or deletion of marks) may represent attractive targets for drug discovery efforts. This review mainly summarizes selected publications, patent literature and company disclosures on these non-enzymatic epigenetic reader proteins from 2009 to the present.

  5. Rubisco activity and regulation as targets for crop improvement.

    PubMed

    Parry, Martin A J; Andralojc, P John; Scales, Joanna C; Salvucci, Michael E; Carmo-Silva, A Elizabete; Alonso, Hernan; Whitney, Spencer M

    2013-01-01

    Rubisco (ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase) enables net carbon fixation through the carboxylation of RuBP. However, some characteristics of Rubisco make it surprisingly inefficient and compromise photosynthetic productivity. For example, Rubisco catalyses a wasteful reaction with oxygen that leads to the release of previously fixed CO(2) and NH(3) and the consumption of energy during photorespiration. Furthermore, Rubisco is slow and large amounts are needed to support adequate photosynthetic rates. Consequently, Rubisco has been studied intensively as a prime target for manipulations to 'supercharge' photosynthesis and improve both productivity and resource use efficiency. The catalytic properties of Rubiscos from diverse sources vary considerably, suggesting that changes in turnover rate, affinity, or specificity for CO(2) can be introduced to improve Rubisco performance in specific crops and environments. While attempts to manipulate plant Rubisco by nuclear transformation have had limited success, modifying its catalysis by targeted changes to its catalytic large subunit via chloroplast transformation have been much more successful. However, this technique is still in need of development for most major food crops including maize, wheat, and rice. Other bioengineering approaches for improving Rubisco performance include improving the activity of its ancillary protein, Rubisco activase, in addition to modulating the synthesis and degradation of Rubisco's inhibitory sugar phosphate ligands. As the rate-limiting step in carbon assimilation, even modest improvements in the overall performance of Rubisco pose a viable pathway for obtaining significant gains in plant yield, particularly under stressful environmental conditions.

  6. Targeting silymarin for improved hepatoprotective activity through chitosan nanoparticles

    PubMed Central

    Gupta, Swati; Singh, Shailendra Kumar; Girotra, Priti

    2014-01-01

    Introduction: Silymarin is one of the best known hepatoprotective drugs, which is obtained from the seeds of Silybum marianum L., Family: Asteraceae or Compositae. The plant has traditionally been used for centuries as a natural remedy for liver and biliary tract diseases. The aim of the present investigation was to enhance the hepatoprotective activity of silymarin by incorporating it in chitosan (Ch) nanoparticles (NPs) for passive targeted delivery, thereby prolonging its retention time. Materials and Methods: Silymarin loaded NPs were prepared by ionic gelation technique, which were then optimized using a central composite design in order to minimize the particle size and maximize the drug entrapment efficiency. The optimized formulation was evaluated for in vitro drug release study and in vitro study on Swiss Albino mice using carbon tetrachloride (CCL4) induced hepatotoxicity model. Results: In vitro dissolution studies illustrated sustained, zero order drug release from optimized formulation; also its therapeutic potential was amplified during in vitro studies on Swiss Albino mice using CCL4 induced hepatotoxicity model. Conclusion: The results suggested that NPs of silymarin could successfully enhance its hepatoprotective effect by passive targeting and sustained release. PMID:25426436

  7. Thrombin-Mediated Direct Activation of Proteinase-Activated Receptor-2: Another Target for Thrombin Signaling.

    PubMed

    Mihara, Koichiro; Ramachandran, Rithwik; Saifeddine, Mahmoud; Hansen, Kristina K; Renaux, Bernard; Polley, Danny; Gibson, Stacy; Vanderboor, Christina; Hollenberg, Morley D

    2016-05-01

    Thrombin is known to signal to cells by cleaving/activating a G-protein-coupled family of proteinase-activated receptors (PARs). The signaling mechanism involves the proteolytic unmasking of an N-terminal receptor sequence that acts as a tethered receptor-activating ligand. To date, the recognized targets of thrombin cleavage and activation for signaling are PAR1 and PAR4, in which thrombin cleaves at a conserved target arginine to reveal a tethered ligand. PAR2, which like PAR1 is also cleaved at an N-terminal arginine to unmask its tethered ligand, is generally regarded as a target for trypsin but not for thrombin signaling. We now show that thrombin, at concentrations that can be achieved at sites of acute injury or in a tumor microenvironment, can directly activate PAR2 vasorelaxation and signaling, stimulating calcium and mitogen-activated protein kinase responses along with triggeringβ-arrestin recruitment. Thus, PAR2 can be added alongside PAR1 and PAR4 to the targets, whereby thrombin can affect tissue function.

  8. Activating frataxin expression by repeat-targeted nucleic acids.

    PubMed

    Li, Liande; Matsui, Masayuki; Corey, David R

    2016-02-04

    Friedreich's ataxia is an incurable genetic disorder caused by a mutant expansion of the trinucleotide GAA within an intronic FXN RNA. This expansion leads to reduced expression of frataxin (FXN) protein and evidence suggests that transcriptional repression is caused by an R-loop that forms between the expanded repeat RNA and complementary genomic DNA. Synthetic agents that increase levels of FXN protein might alleviate the disease. We demonstrate that introducing anti-GAA duplex RNAs or single-stranded locked nucleic acids into patient-derived cells increases FXN protein expression to levels similar to analogous wild-type cells. Our data are significant because synthetic nucleic acids that target GAA repeats can be lead compounds for restoring curative FXN levels. More broadly, our results demonstrate that interfering with R-loop formation can trigger gene activation and reveal a new strategy for upregulating gene expression.

  9. Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3′ end can be exported efficiently and specifically to the cytoplasm in mammalian cells

    PubMed Central

    Kuwabara, Tomoko; Warashina, Masaki; Koseki, Shiori; Sano, Masayuki; Ohkawa, Jun; Nakayama, Kazuhisa; Taira, Kazunari

    2001-01-01

    Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNAVal-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3′ end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5′ and 3′ ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells. PMID:11433023

  10. 13 CFR 124.509 - What are non-8(a) business activity targets?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... activity targets? 124.509 Section 124.509 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION 8(a... Contractual Assistance § 124.509 What are non-8(a) business activity targets? (a) General. (1) To ensure that...) business activity targets during transitional stage—(1) General. During the transitional stage of the...

  11. 13 CFR 124.509 - What are non-8(a) business activity targets?

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... activity targets? 124.509 Section 124.509 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION 8(a... Contractual Assistance § 124.509 What are non-8(a) business activity targets? (a) General. (1) To ensure that...) business activity targets during transitional stage—(1) General. During the transitional stage of the...

  12. 13 CFR 124.509 - What are non-8(a) business activity targets?

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... activity targets? 124.509 Section 124.509 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION 8(a... Contractual Assistance § 124.509 What are non-8(a) business activity targets? (a) General. (1) To ensure that...) business activity targets during transitional stage—(1) General. During the transitional stage of the...

  13. Can in vitro mammalian cell genotoxicity test results be used to complement positive results in the Ames test and help predict carcinogenic or in vivo genotoxic activity? I. Reports of individual databases presented at an EURL ECVAM Workshop.

    PubMed

    Kirkland, David; Zeiger, Errol; Madia, Federica; Gooderham, Nigel; Kasper, Peter; Lynch, Anthony; Morita, Takeshi; Ouedraogo, Gladys; Parra Morte, Juan Manuel; Pfuhler, Stefan; Rogiers, Vera; Schulz, Markus; Thybaud, Veronique; van Benthem, Jan; Vanparys, Philippe; Worth, Andrew; Corvi, Raffaella

    2014-12-01

    Positive results in the Ames test correlate well with carcinogenic potential in rodents. This correlation is not perfect because mutations are only one of many stages in tumour development. Also, situations can be envisaged where the mutagenic response may be specific to the bacteria or the test protocol, e.g., bacterial-specific metabolism, exceeding a detoxification threshold, or the induction of oxidative damage to which bacteria may be more sensitive than mammalian cells in vitro or tissues in vivo. Since most chemicals are also tested for genotoxicity in mammalian cells, the pattern of mammalian cell results may help identify whether Ames-positive results predict carcinogenic or in vivo mutagenic activity. A workshop was therefore organised and sponsored by the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) to investigate this further. Participants presented results from other genotoxicity tests with Ames-positive compounds. Data came from published, regulatory agency, and industry sources. The question was posed whether negative results in mammalian cell tests were associated with absence of carcinogenic or in vivo genotoxic activity despite a positive Ames test. In the limited time available, the presented data were combined and an initial analysis suggested that the association of negative in vitro mammalian cell test results with lack of in vivo genotoxic or carcinogenic activity could have some significance. Possible reasons why a positive Ames test may not be associated with in vivo activity and what additional investigations/tests might contribute to a more robust evaluation were discussed. Because a considerable overlap was identified among the different databases presented, it was recommended that a consolidated database be built, with overlapping chemicals removed, so that a more robust analysis of the predictive capacity for potential carcinogenic and in vivo genotoxic activity could be derived from the patterns of mammalian

  14. Active Targeted Nanoparticles for Oral Administration of Gastric Cancer Therapy.

    PubMed

    Lin, Yu-Hsin; Chen, Zih-Rou; Lai, Chih-Ho; Hsieh, Chia-Hung; Feng, Chun-Lung

    2015-09-14

    Gastric carcinogenesis is a commonly diagnosed type of cancer and has a dismal prognosis because of the rate at which it aggressively spreads and because of the lack of effective therapies to stop its progression. This study evaluated a type of oral drug delivery system of a potential target-activated nanosizer comprising a fucose-conjugated chitosan and polyethylene glycol-conjugated chitosan complex with gelatin containing encapsulated green tea polyphenol extract epigallocatechin-3-gallate, allowing oral administration of the drug through a site-specific release in gastric cancer cells. The results demonstrated that the nanoparticles effectively reduced drug release within gastric acids and that a controlled epigallocatechin-3-gallate release inhibited gastric cancer cell growth, induced cell apoptosis, and reduced vascular endothelial growth factor protein expression. Furthermore, in vivo assay results indicated that the prepared epigallocatechin-3-gallate-loaded fucose-chitosan/polyethylene glycol-chitosan/gelatin nanoparticles significantly affected gastric tumor activity and reduced gastric and liver tissue inflammatory reaction in an orthotopic gastric tumor mouse model.

  15. Genetic influences on composite neural activations supporting visual target identification.

    PubMed

    Ethridge, Lauren E; Malone, Stephen M; Iacono, William G; Clementz, Brett A

    2013-02-01

    Behavior genetic studies of brain activity associated with complex cognitive operations may further elucidate the genetic and physiological underpinnings of basic and complex neural processing. In the present project, monozygotic (N=51 pairs) and dizygotic (N=48 pairs) twins performed a visual oddball task with dense-array EEG. Using spatial PCA, two principal components each were retained for targets and standards; wavelets were used to obtain time-frequency maps of eigenvalue-weighted event-related oscillations for each individual. Distribution of inter-trial phase coherence (ITC) and single trial power (STP) over time indicated that the early principal component was primarily associated with ITC while the later component was associated with a mixture of ITC and STP. Spatial PCA on point-by-point broad sense heritability matrices revealed data-derived frequency bands similar to those well established in EEG literature. Biometric models of eigenvalue-weighted time-frequency data suggest a link between physiology of oscillatory brain activity and patterns of genetic influence. PMID:23201034

  16. Mammalian septin function in hemostasis and beyond.

    PubMed

    Martinez, Constantino; Ware, Jerry

    2004-12-01

    Interest in the biology of mammalian septin proteins has undergone a birth in recent years. Originally identified as critical for yeast budding throughout the 1970s, the septin family is now recognized to extend from yeast to humans and is associated with a variety of events ranging from cytokinesis to vesicle trafficking. An emerging theme for septins is their presence at sites where active membrane or cytoplasmic partitioning is occurring. Here, we briefly review the mammalian septin protein family and focus on a prototypic human and mouse septin, termed SEPT5, that is expressed in the brain, heart, and megakaryocytes. Work from neurobiology laboratories has linked SEPT5 to the exocytic complex of neurons, with implications that SEPT5 regulates neurotransmitter release. Striking similarities exist between neurotransmitter release and the platelet-release reaction, which is a critical step in platelet response to vascular injury. Work from our laboratory has characterized the platelet phenotype from mice containing a targeted deletion of SEPT5. Most strikingly, platelets from SEPT5(null) animals aggregate and release granular contents in response to subthreshold levels of agonists. Thus, the characterization of a SEPT5-deficient mouse has linked SEPT5 to the platelet exocytic process and, as such, illustrates it as an important protein for regulating platelet function. Recent data suggest that platelets contain a wide repertoire of different septin proteins and assemble to form macromolecular septin complexes. The mouse platelet provides an experimental framework to define septin function in hemostasis, with implications for neurobiology and beyond.

  17. Baculovirus Stimulates Antiviral Effects in Mammalian Cells

    PubMed Central

    Gronowski, Ann M.; Hilbert, David M.; Sheehan, Kathleen C. F.; Garotta, Gianni; Schreiber, Robert D.

    1999-01-01

    Herein, we report that Autographa californica nucleopolyhedrovirus, a member of the Baculoviridae family, is capable of stimulating antiviral activity in mammalian cells. Baculoviruses are not pathogenic to mammalian cells. Nevertheless, live baculovirus is shown here to induce interferons (IFN) from murine and human cell lines and induces in vivo protection of mice from encephalomyocarditis virus infection. Monoclonal antibodies specific for the baculovirus envelope gp67 neutralize baculovirus-dependent IFN production. Moreover, UV treatment of baculovirus eliminates both infectivity and IFN-inducing activity. In contrast, the IFN-inducing activity of the baculovirus was unaffected by DNase or RNase treatment. These data demonstrate that IFN production can be induced in mammalian cells by baculovirus even though the cells fail to serve as a natural host for an active viral infection. Baculoviruses, therefore, provide a novel model in which to study at least one alternative mechanism for IFN induction in mammalian cells. PMID:10559307

  18. Transforming growth factor-beta requires its target plasminogen activator inhibitor-1 for cytostatic activity.

    PubMed

    Kortlever, Roderik M; Nijwening, Jeroen H; Bernards, René

    2008-09-01

    The cytokine transforming growth factor beta (TGFbeta) has strong antiproliferative activity in most normal cells but contributes to tumor progression in the later stages of oncogenesis. It is not fully understood which TGFbeta target genes are causally involved in mediating its cytostatic activity. We report here that suppression of the TGFbeta target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference leads to escape from the cytostatic activity of TGFbeta both in human keratinocytes (HaCaTs) and primary mouse embryo fibroblasts. Consistent with this, PAI-1 knock-out mouse embryo fibroblasts are also resistant to TGFbeta growth arrest. Conversely, we show that ectopic expression of PAI-1 in proliferating HaCaT cells induces a growth arrest. PAI-1 knockdown does not interfere with canonical TGFbeta signaling as judged by SMAD phosphorylation and induction of bona fide TGFbeta target genes. Instead, knockdown of PAI-1 results in sustained activation of protein kinase B. Significantly, we find that constitutive protein kinase B activity leads to evasion of the growth-inhibitory action of TGFbeta. Our data are consistent with a model in which induction of PAI-1 by TGFbeta is critical for the induction of proliferation arrest.

  19. Musashi Protein-directed Translational Activation of Target mRNAs Is Mediated by the Poly(A) Polymerase, Germ Line Development Defective-2*

    PubMed Central

    Cragle, Chad; MacNicol, Angus M.

    2014-01-01

    The mRNA-binding protein, Musashi, has been shown to regulate translation of select mRNAs and to control cellular identity in both stem cells and cancer cells. Within the mammalian cells, Musashi has traditionally been characterized as a repressor of translation. However, we have demonstrated that Musashi is an activator of translation in progesterone-stimulated oocytes of the frog Xenopus laevis, and recent evidence has revealed Musashi's capability to function as an activator of translation in mammalian systems. The molecular mechanism by which Musashi directs activation of target mRNAs has not been elucidated. Here, we report a specific association of Musashi with the noncanonical poly(A) polymerase germ line development defective-2 (GLD2) and map the association domain to 31 amino acids within the C-terminal domain of Musashi. We show that loss of GLD2 interaction through deletion of the binding domain or treatment with antisense oligonucleotides compromises Musashi function. Additionally, we demonstrate that overexpression of both Musashi and GLD2 significantly enhances Musashi function. Finally, we report a similar co-association also occurs between murine Musashi and GLD2 orthologs, suggesting that coupling of Musashi to the polyadenylation apparatus is a conserved mechanism to promote target mRNA translation. PMID:24644291

  20. Musashi protein-directed translational activation of target mRNAs is mediated by the poly(A) polymerase, germ line development defective-2.

    PubMed

    Cragle, Chad; MacNicol, Angus M

    2014-05-16

    The mRNA-binding protein, Musashi, has been shown to regulate translation of select mRNAs and to control cellular identity in both stem cells and cancer cells. Within the mammalian cells, Musashi has traditionally been characterized as a repressor of translation. However, we have demonstrated that Musashi is an activator of translation in progesterone-stimulated oocytes of the frog Xenopus laevis, and recent evidence has revealed Musashi's capability to function as an activator of translation in mammalian systems. The molecular mechanism by which Musashi directs activation of target mRNAs has not been elucidated. Here, we report a specific association of Musashi with the noncanonical poly(A) polymerase germ line development defective-2 (GLD2) and map the association domain to 31 amino acids within the C-terminal domain of Musashi. We show that loss of GLD2 interaction through deletion of the binding domain or treatment with antisense oligonucleotides compromises Musashi function. Additionally, we demonstrate that overexpression of both Musashi and GLD2 significantly enhances Musashi function. Finally, we report a similar co-association also occurs between murine Musashi and GLD2 orthologs, suggesting that coupling of Musashi to the polyadenylation apparatus is a conserved mechanism to promote target mRNA translation.

  1. Searching for Common Mammalian Retroviruses in Pediatric Idiopathic Diseases.

    PubMed

    Jeziorski, Eric; Foulongne, Vincent; Ludwig, Catherine; Louhaem, Djamel; Rodiere, Michel; Sitbon, Marc; Courgnaud, Valérie

    2016-03-01

    Mammalian retroviruses cause a variety of diseases in their hosts, including hematological and immunodeficiency disorders. Both human T-cell leukemia (HTLV) and human immunodeficiency (HIV) viruses originated from several independent zoonotic transmissions, indicating that cross-species transmissions from animal to humans may still occur. Thus, as the risk for retroviral transmissions from animals to humans increase, we investigated whether mammalian retroviruses are involved in selected pediatric idiopathic diseases whose symptoms evoke retroviral infections. Blood samples, sera, and synovial fluids, or bone marrow cells were collected from pediatric patients under 18 years of age with different autoimmune idiopathic diseases. Overall, we screened clinical samples from 110 children using sensitive nested and semi-nested PCR strategies targeting env genes, and a C-type retrovirus reverse transcriptase (RT) activity kit. All clinical samples were free of retroviral signatures, indicating the unlikelihood of an etiological role of the retroviruses we assessed in the pediatric diseases we tested.

  2. Searching for Common Mammalian Retroviruses in Pediatric Idiopathic Diseases.

    PubMed

    Jeziorski, Eric; Foulongne, Vincent; Ludwig, Catherine; Louhaem, Djamel; Rodiere, Michel; Sitbon, Marc; Courgnaud, Valérie

    2016-03-01

    Mammalian retroviruses cause a variety of diseases in their hosts, including hematological and immunodeficiency disorders. Both human T-cell leukemia (HTLV) and human immunodeficiency (HIV) viruses originated from several independent zoonotic transmissions, indicating that cross-species transmissions from animal to humans may still occur. Thus, as the risk for retroviral transmissions from animals to humans increase, we investigated whether mammalian retroviruses are involved in selected pediatric idiopathic diseases whose symptoms evoke retroviral infections. Blood samples, sera, and synovial fluids, or bone marrow cells were collected from pediatric patients under 18 years of age with different autoimmune idiopathic diseases. Overall, we screened clinical samples from 110 children using sensitive nested and semi-nested PCR strategies targeting env genes, and a C-type retrovirus reverse transcriptase (RT) activity kit. All clinical samples were free of retroviral signatures, indicating the unlikelihood of an etiological role of the retroviruses we assessed in the pediatric diseases we tested. PMID:27102168

  3. Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

    SciTech Connect

    Vazquez-Iglesias, Lorena; Lostale-Seijo, Irene; Martinez-Costas, Jose; Benavente, Javier

    2012-10-25

    A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells.

  4. Bats and Rodents Shape Mammalian Retroviral Phylogeny

    PubMed Central

    Cui, Jie; Tachedjian, Gilda; Wang, Lin-Fa

    2015-01-01

    Endogenous retroviruses (ERVs) represent past retroviral infections and accordingly can provide an ideal framework to infer virus-host interaction over their evolutionary history. In this study, we target high quality Pol sequences from 7,994 Class I and 8,119 Class II ERVs from 69 mammalian genomes and surprisingly find that retroviruses harbored by bats and rodents combined occupy the major phylogenetic diversity of both classes. By analyzing transmission patterns of 30 well-defined ERV clades, we corroborate the previously published observation that rodents are more competent as originators of mammalian retroviruses and reveal that bats are more capable of receiving retroviruses from non-bat mammalian origins. The powerful retroviral hosting ability of bats is further supported by a detailed analysis revealing that the novel bat gammaretrovirus, Rhinolophus ferrumequinum retrovirus, likely originated from tree shrews. Taken together, this study advances our understanding of host-shaped mammalian retroviral evolution in general. PMID:26548564

  5. The role of repair in the survival of mammalian cells from heavy ion irradiation - Approximation to the ideal case of target theory

    NASA Technical Reports Server (NTRS)

    Lett, J. T.; Cox, A. B.; Story, M. D.

    1989-01-01

    Experiments are discussed in which the cell-cycle dependency of the repair deficiency of the S/S variant of the L5178Y murine leukemic lymphoblast was examined by treatment with the heavy ions, Ne-20, Si-28, Ar-40, Fe-56, and Nb-93. Evidence from those studies provide support for the notion that as the linear energy transfer of the incident radiation increases the ability of the S/S cell to repair radiation damage decreases until it is eliminated around 500 keV/micron. In the region of the latter linear energy transfer value, the behavior of the S/S cell approximates the ideal case of target theory where post-irradiation metabolism does not influence cell survival.

  6. Increase of microRNA-210, Decrease of Raptor Gene Expression and Alteration of Mammalian Target of Rapamycin Regulated Proteins following Mithramycin Treatment of Human Erythroid Cells

    PubMed Central

    Bianchi, Nicoletta; Finotti, Alessia; Ferracin, Manuela; Lampronti, Ilaria; Zuccato, Cristina; Breveglieri, Giulia; Brognara, Eleonora; Fabbri, Enrica; Borgatti, Monica; Negrini, Massimo; Gambari, Roberto

    2015-01-01

    Expression and regulation of microRNAs is an emerging issue in erythroid differentiation and globin gene expression in hemoglobin disorders. In the first part of this study microarray analysis was performed both in mithramycin-induced K562 cells and erythroid precursors from healthy subjects or β-thalassemia patients producing low or high levels of fetal hemoglobin. We demonstrated that: (a) microRNA-210 expression is higher in erythroid precursors from β-thalassemia patients with high production of fetal hemoglobin; (b) microRNA-210 increases as a consequence of mithramycin treatment of K562 cells and human erythroid progenitors both from healthy and β-thalassemia subjects; (c) this increase is associated with erythroid induction and elevated expression of γ-globin genes; (d) an anti-microRNA against microRNA-210 interferes with the mithramycin-induced changes of gene expression. In the second part of the study we have obtained convergent evidences suggesting raptor mRNA as a putative target of microRNA-210. Indeed, microRNA-210 binding sites of its 3’-UTR region were involved in expression and are targets of microRNA-210-mediated modulation in a luciferase reporter assays. Furthermore, (i) raptor mRNA and protein are down-regulated upon mithramycin-induction both in K562 cells and erythroid progenitors from healthy and β-thalassemia subjects. In addition, (ii) administration of anti-microRNA-210 to K562 cells decreased endogenous microRNA-210 and increased raptor mRNA and protein expression. Finally, (iii) treatment of K562 cells with premicroRNA-210 led to a decrease of raptor mRNA and protein. In conclusion, microRNA-210 and raptor are involved in mithramycin-mediated erythroid differentiation of K562 cells and participate to the fine-tuning and control of γ-globin gene expression in erythroid precursor cells. PMID:25849663

  7. Target of rapamycin activation predicts lifespan in fruit flies

    PubMed Central

    Scialò, Filippo; Sriram, Ashwin; Naudí, Alba; Ayala, Victoria; Jové, Mariona; Pamplona, Reinald; Sanz, Alberto

    2015-01-01

    Aging and age-related diseases are one of the most important health issues that the world will confront during the 21st century. Only by understanding the proximal causes will we be able to find treatments to reduce or delay the onset of degenerative diseases associated with aging. Currently, the prevalent paradigm in the field is the accumulation of damage. However, a new theory that proposes an alternative explanation is gaining momentum. The hyperfunction theory proposes that aging is not a consequence of a wear and tear process, but a result of the continuation of developmental programs during adulthood. Here we use Drosophila melanogaster, where evidence supporting both paradigms has been reported, to identify which parameters that have been previously related with lifespan best predict the rate of aging in wild type flies cultured at different temperatures. We find that mitochondrial function and mitochondrial reactive oxygen species (mtROS) generation correlates with metabolic rate, but not with the rate of aging. Importantly, we find that activation of nutrient sensing pathways (i.e. insulin-PI3K/Target of rapamycin (Tor) pathway) correlates with lifespan, but not with metabolic rate. Our results, dissociate metabolic rate and lifespan in wild type flies and instead link nutrient sensing signaling with longevity as predicted by the hyperfunction theory. PMID:26259964

  8. Long hydrophilic-and-cationic polymers: a different pathway toward preferential activity against bacterial over mammalian membranes.

    PubMed

    Yang, Xin; Hu, Kan; Hu, Guantai; Shi, Danyao; Jiang, Yunjiang; Hui, Liwei; Zhu, Rui; Xie, Yuntao; Yang, Lihua

    2014-09-01

    We show that simply converting the hydrophobic moiety of an antimicrobial peptide (AMP) or synthetic mimic of AMPs (SMAMP) into a hydrophilic one could be a different pathway toward membrane-active antimicrobials preferentially acting against bacteria over host cells. Our biostatistical analysis on natural AMPs indicated that shorter AMPs tend to be more hydrophobic, and the hydrophilic-and-cationic mutants of a long AMP experimentally demonstrated certain membrane activity against bacteria. To isolate the effects of antimicrobials' hydrophobicity and systematically examine whether hydrophilic-and-cationic mutants could inherit the membrane activity of their parent AMPs/SMAMPs, we constructed a minimal prototypical system based on methacrylate-based polymer SMAMPs and compared the antibacterial membrane activity and hemolytic toxicity of analogues with and without the hydrophobic moiety. Antibacterial assays showed that the hydrophobic moiety of polymer SMAMPs consistently promoted the antibacterial activity but diminished in effectiveness for long polymers, and the resultant long hydrophilic-and-cationic polymers were also membrane active against bacteria. What distinguished these long mutants from their parent SMAMPs were their drastically reduced hemolytic toxicities and, as a result, strikingly enhanced selectivity. Similar toxicity reduction was observed with the hydrophilic-and-cationic mutants of long AMPs. Taken together, our results suggest that long hydrophilic-and-cationic polymers could offer preferential membrane activity against bacteria over host cells, which may have implications in future antimicrobial development.

  9. Olfactory sensitivity in mammalian species.

    PubMed

    Wackermannová, M; Pinc, L; Jebavý, L

    2016-07-18

    Olfaction enables most mammalian species to detect and discriminate vast numbers of chemical structures called odorants and pheromones. The perception of such chemical compounds is mediated via two major olfactory systems, the main olfactory system and the vomeronasal system, as well as minor systems, such as the septal organ and the Grueneberg ganglion. Distinct differences exist not only among species but also among individuals in terms of their olfactory sensitivity; however, little is known about the mechanisms that determine these differences. In research on the olfactory sensitivity of mammals, scientists thus depend in most cases on behavioral testing. In this article, we reviewed scientific studies performed on various mammalian species using different methodologies and target chemical substances. Human and non-human primates as well as rodents and dogs are the most frequently studied species. Olfactory threshold studies on other species do not exist with the exception of domestic pigs. Olfactory testing performed on seals, elephants, and bats focused more on discriminative abilities than on sensitivity. An overview of olfactory sensitivity studies as well as olfactory detection ability in most studied mammalian species is presented here, focusing on comparable olfactory detection thresholds. The basics of olfactory perception and olfactory sensitivity factors are also described. PMID:27070753

  10. Facilitation by intracellular carbonic anhydrase of Na+–HCO3− co-transport but not Na+/H+ exchange activity in the mammalian ventricular myocyte

    PubMed Central

    Villafuerte, Francisco C; Swietach, Pawel; Youm, Jae-Boum; Ford, Kerrie; Cardenas, Rosa; Supuran, Claudiu T; Cobden, Philip M; Rohling, Mala; Vaughan-Jones, Richard D

    2014-01-01

    Carbonic anhydrase enzymes (CAs) catalyse the reversible hydration of CO2 to H+ and HCO3− ions. This catalysis is proposed to be harnessed by acid/base transporters, to facilitate their transmembrane flux activity, either through direct protein–protein binding (a ‘transport metabolon’) or local functional interaction. Flux facilitation has previously been investigated by heterologous co-expression of relevant proteins in host cell lines/oocytes. Here, we examine the influence of intrinsic CA activity on membrane HCO3− or H+ transport via the native acid-extruding proteins, Na+–HCO3− cotransport (NBC) and Na+/H+ exchange (NHE), expressed in enzymically isolated mammalian ventricular myocytes. Effects of intracellular and extracellular (exofacial) CA (CAi and CAe) are distinguished using membrane-permeant and –impermeant pharmacological CA inhibitors, while measuring transporter activity in the intact cell using pH and Na+ fluorophores. We find that NBC, but not NHE flux is enhanced by catalytic CA activity, with facilitation being confined to CAi activity alone. Results are quantitatively consistent with a model where CAi catalyses local H+ ion delivery to the NBC protein, assisting the subsequent (uncatalysed) protonation and removal of imported HCO3− ions. In well-superfused myocytes, exofacial CA activity is superfluous, most likely because extracellular CO2/HCO3− buffer is clamped at equilibrium. The CAi insensitivity of NHE flux suggests that, in the native cell, intrinsic mobile buffer-shuttles supply sufficient intracellular H+ ions to this transporter, while intrinsic buffer access to NBC proteins is restricted. Our results demonstrate a selective CA facilitation of acid/base transporters in the ventricular myocyte, implying a specific role for the intracellular enzyme in HCO3− transport, and hence pHi regulation in the heart. PMID:24297849

  11. Facilitation by intracellular carbonic anhydrase of Na+ -HCO3- co-transport but not Na+ / H+ exchange activity in the mammalian ventricular myocyte.

    PubMed

    Villafuerte, Francisco C; Swietach, Pawel; Youm, Jae-Boum; Ford, Kerrie; Cardenas, Rosa; Supuran, Claudiu T; Cobden, Philip M; Rohling, Mala; Vaughan-Jones, Richard D

    2014-03-01

    Carbonic anhydrase enzymes (CAs) catalyse the reversible hydration of CO2 to H+ and HCO3- ions. This catalysis is proposed to be harnessed by acid/base transporters, to facilitate their transmembrane flux activity, either through direct protein-protein binding (a 'transport metabolon') or local functional interaction. Flux facilitation has previously been investigated by heterologous co-expression of relevant proteins in host cell lines/oocytes. Here, we examine the influence of intrinsic CA activity on membrane HCO3- or H+ transport via the native acid-extruding proteins, Na+ -HCO3- cotransport (NBC) and Na+ / H+ exchange (NHE), expressed in enzymically isolated mammalian ventricular myocytes. Effects of intracellular and extracellular (exofacial) CA (CAi and CAe) are distinguished using membrane-permeant and -impermeant pharmacological CA inhibitors, while measuring transporter activity in the intact cell using pH and Na+ fluorophores. We find that NBC, but not NHE flux is enhanced by catalytic CA activity, with facilitation being confined to CAi activity alone. Results are quantitatively consistent with a model where CAi catalyses local H+ ion delivery to the NBC protein, assisting the subsequent (uncatalysed) protonation and removal of imported HCO3- ions. In well-superfused myocytes, exofacial CA activity is superfluous, most likely because extracellular CO2/HCO3- buffer is clamped at equilibrium. The CAi insensitivity of NHE flux suggests that, in the native cell, intrinsic mobile buffer-shuttles supply sufficient intracellular H+ ions to this transporter, while intrinsic buffer access to NBC proteins is restricted. Our results demonstrate a selective CA facilitation of acid/base transporters in the ventricular myocyte, implying a specific role for the intracellular enzyme in HCO3- transport, and hence pHi regulation in the heart.

  12. Homologous recombination as a potential target for caffeine radiosensitization in mammalian cells: reduced caffeine radiosensitization in XRCC2 and XRCC3 mutants

    NASA Technical Reports Server (NTRS)

    Asaad, N. A.; Zeng, Z. C.; Guan, J.; Thacker, J.; Iliakis, G.

    2000-01-01

    The radiosensitizing effect of caffeine has been associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints, but several lines of evidence also implicate inhibition of DNA repair. The role of DNA repair inhibition in caffeine radiosensitization remains uncharacterized, and it is unknown which repair process, or lesion, is affected. We show that a radiosensitive cell line, mutant for the RAD51 homolog XRCC2 and defective in homologous recombination repair (HRR), displays significantly diminished caffeine radiosensitization that can be restored by expression of XRCC2. Despite the reduced radiosensitization, caffeine effectively abrogates checkpoints in S and G2 phases in XRCC2 mutant cells indicating that checkpoint abrogation is not sufficient for radiosensitization. Another radiosensitive line, mutant for XRCC3 and defective in HRR, similarly shows reduced caffeine radiosensitization. On the other hand, a radiosensitive mutant (irs-20) of DNA-PKcs with a defect in non-homologous end-joining (NHEJ) is radiosensitized by caffeine to an extent comparable to wild-type cells. In addition, rejoining of radiation-induced DNA DSBs, that mainly reflects NHEJ, remains unaffected by caffeine in XRCC2 and XRCC3 mutants, or their wild-type counterparts. These observations suggest that caffeine targets steps in HRR but not in NHEJ and that abrogation of checkpoint response is not sufficient to explain radiosensitization. Indeed, immortalized fibroblasts from AT patients show caffeine radiosensitization despite the checkpoint defects associated with ATM mutation. We propose that caffeine radiosensitization is mediated by inhibition of stages in DNA DSB repair requiring HRR and that checkpoint disruption contributes by allowing these DSBs to transit into irreparable states. Thus, checkpoints may contribute to genomic stability by promoting error-free HRR.

  13. Expression of pokeweed antiviral protein in mammalian cells activates c-Jun NH2-terminal kinase without causing apoptosis.

    PubMed

    Chan Tung, Kelvin W; Mansouri, Sheila; Hudak, Katalin A

    2008-01-01

    Pokeweed antiviral protein (PAP) is a ribosome inactivating protein isolated from the pokeweed plant (Phytolacca americana L.) that exhibits broad range antiviral activity against several human viruses including HIV and influenza. This characteristic suggests that PAP may have therapeutic applications; however, it is not known whether the protein elicits a ribotoxic stress response that would result in cell death. Therefore, we expressed PAP in 293T cells and showed that the enzyme did not inhibit protein translation even though approximately 15% of the ribosomal RNA (rRNA) was depurinated. PAP expression induced the activation of c-Jun NH2-terminal kinase (JNK), which was specific to rRNA depurination, as the enzymatically inactive mutant PAPx did not affect kinase activity. Moreover, incubation of PAP-expressing cells with translation inhibitors diminished JNK activation, indicating that the signal for induction of the kinase pathway originated from ribosomes. JNK activation did not result in apoptosis as demonstrated by the absence of caspase-3 and poly(ADP-ribose) polymerase cleavage and by the lack of cell staining for morphological changes in membrane permeability. Unlike all ribosome inactivating proteins tested thus far, the stress response triggered by PAP expression did not result in cell death, which supports further investigation of the enzyme in the design of novel antiviral agents.

  14. Ethanol modulation of mammalian BK channels in excitable tissues: molecular targets and their possible contribution to alcohol-induced altered behavior

    PubMed Central

    Dopico, Alex M.; Bukiya, Anna N.; Martin, Gilles E.

    2014-01-01

    In most tissues, the function of Ca2+- and voltage-gated K+ (BK) channels is modified in response to ethanol concentrations reached in human blood during alcohol intoxication. In general, modification of BK current from ethanol-naïve preparations in response to brief ethanol exposure results from changes in channel open probability without modification of unitary conductance or change in BK protein levels in the membrane. Protracted and/or repeated ethanol exposure, however, may evoke changes in BK expression. The final ethanol effect on BK open probability leading to either BK current potentiation or BK current reduction is determined by an orchestration of molecular factors, including levels of activating ligand (Ca2+i), BK subunit composition and post-translational modifications, and the channel's lipid microenvironment. These factors seem to allosterically regulate a direct interaction between ethanol and a recognition pocket of discrete dimensions recently mapped to the channel-forming (slo1) subunit. Type of ethanol exposure also plays a role in the final BK response to the drug: in several central nervous system regions (e.g., striatum, primary sensory neurons, and supraoptic nucleus), acute exposure to ethanol reduces neuronal excitability by enhancing BK activity. In contrast, protracted or repetitive ethanol administration may alter BK subunit composition and membrane expression, rendering the BK complex insensitive to further ethanol exposure. In neurohypophyseal axon terminals, ethanol potentiation of BK channel activity leads to a reduction in neuropeptide release. In vascular smooth muscle, however, ethanol inhibition of BK current leads to cell contraction and vascular constriction. PMID:25538625

  15. 78 FR 35612 - Agency Information Collection Activities; Comment Request; Targeted Teacher Shortage Areas...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-13

    ... collection of information from Chief State School Officers to support and document the request for teacher... Agency Information Collection Activities; Comment Request; Targeted Teacher Shortage Areas Nationwide... to this notice will be considered public records. Title of Collection: Targeted Teacher...

  16. Development of a new screening assay to identify proteratogenic substances using zebrafish danio rerio embryo combined with an exogenous mammalian metabolic activation system (mDarT).

    PubMed

    Busquet, François; Nagel, Roland; von Landenberg, Friedrich; Mueller, Stefan O; Huebler, Nicole; Broschard, Thomas H

    2008-07-01

    The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays, for example, in rats or rabbits. We have developed an in vitro teratogenicity assay using the zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide (CPA) and ethanol were used as proteratogens to test the efficiency of this assay. Briefly, the zebrafish embryos were cocultured at 2 hpf (hours postfertilization) with the test material at varying concentrations, induced male rat liver microsomes and nicotinamide adenine dinucleotide phosphate (reduced) for 60 min at 32 degrees C under moderate agitation in Tris-buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterward fish embryos were transferred individually into 24-well plates filled with fish medium for 48 h at 26 degrees C with a 12-h light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. No teratogenic effects were observed in fish embryos exposed to the proteratogens alone, that is, without metabolic activation. In contrast, CPA and ethanol induced abnormalities in fish embryos when coincubated with microsomes. The severity of malformations increased with increasing concentrations of the proteratogens. We conclude that the application of microsomes will improve and refine the D. rerio teratogenicity assay as a predictive and valuable alternative method to screen teratogenic substances.

  17. Bypassing Protein Corona Issue on Active Targeting: Zwitterionic Coatings Dictate Specific Interactions of Targeting Moieties and Cell Receptors.

    PubMed

    Safavi-Sohi, Reihaneh; Maghari, Shokoofeh; Raoufi, Mohammad; Jalali, Seyed Amir; Hajipour, Mohammad J; Ghassempour, Alireza; Mahmoudi, Morteza

    2016-09-01

    Surface functionalization strategies for targeting nanoparticles (NP) to specific organs, cells, or organelles, is the foundation for new applications of nanomedicine to drug delivery and biomedical imaging. Interaction of NPs with biological media leads to the formation of a biomolecular layer at the surface of NPs so-called as "protein corona". This corona layer can shield active molecules at the surface of NPs and cause mistargeting or unintended scavenging by the liver, kidney, or spleen. To overcome this corona issue, we have designed biotin-cysteine conjugated silica NPs (biotin was employed as a targeting molecule and cysteine was used as a zwitterionic ligand) to inhibit corona-induced mistargeting and thus significantly enhance the active targeting capability of NPs in complex biological media. To probe the targeting yield of our engineered NPs, we employed both modified silicon wafer substrates with streptavidin (i.e., biotin receptor) to simulate a target and a cell-based model platform using tumor cell lines that overexpress biotin receptors. In both cases, after incubation with human plasma (thus forming a protein corona), cellular uptake/substrate attachment of the targeted NPs with zwitterionic coatings were significantly higher than the same NPs without zwitterionic coating. Our results demonstrated that NPs with a zwitterionic surface can considerably facilitate targeting yield of NPs and provide a promising new type of nanocarriers in biological applications.

  18. Bypassing Protein Corona Issue on Active Targeting: Zwitterionic Coatings Dictate Specific Interactions of Targeting Moieties and Cell Receptors.

    PubMed

    Safavi-Sohi, Reihaneh; Maghari, Shokoofeh; Raoufi, Mohammad; Jalali, Seyed Amir; Hajipour, Mohammad J; Ghassempour, Alireza; Mahmoudi, Morteza

    2016-09-01

    Surface functionalization strategies for targeting nanoparticles (NP) to specific organs, cells, or organelles, is the foundation for new applications of nanomedicine to drug delivery and biomedical imaging. Interaction of NPs with biological media leads to the formation of a biomolecular layer at the surface of NPs so-called as "protein corona". This corona layer can shield active molecules at the surface of NPs and cause mistargeting or unintended scavenging by the liver, kidney, or spleen. To overcome this corona issue, we have designed biotin-cysteine conjugated silica NPs (biotin was employed as a targeting molecule and cysteine was used as a zwitterionic ligand) to inhibit corona-induced mistargeting and thus significantly enhance the active targeting capability of NPs in complex biological media. To probe the targeting yield of our engineered NPs, we employed both modified silicon wafer substrates with streptavidin (i.e., biotin receptor) to simulate a target and a cell-based model platform using tumor cell lines that overexpress biotin receptors. In both cases, after incubation with human plasma (thus forming a protein corona), cellular uptake/substrate attachment of the targeted NPs with zwitterionic coatings were significantly higher than the same NPs without zwitterionic coating. Our results demonstrated that NPs with a zwitterionic surface can considerably facilitate targeting yield of NPs and provide a promising new type of nanocarriers in biological applications. PMID:27526263

  19. An Active Learning Mammalian Skeletal Muscle Lab Demonstrating Contractile and Kinetic Properties of Fast- and Slow-Twitch Muscle

    ERIC Educational Resources Information Center

    Head, S. I.; Arber, M. B.

    2013-01-01

    The fact that humans possess fast and slow-twitch muscle in the ratio of approximately 50% has profound implications for designing exercise training strategies for power and endurance activities. With the growth of exercise and sport science courses, we have seen the need to develop an undergraduate student laboratory that demonstrates the basic…

  20. Dicer1 activity in the stromal compartment regulates nephron differentiation and vascular patterning during mammalian kidney organogenesis.

    PubMed

    Nakagawa, Naoki; Xin, Cuiyan; Roach, Allie M; Naiman, Natalie; Shankland, Stuart J; Ligresti, Giovanni; Ren, Shuyu; Szak, Suzanne; Gomez, Ivan G; Duffield, Jeremy S

    2015-06-01

    MicroRNAs, activated by the enzyme Dicer1, control post-transcriptional gene expression. Dicer1 has important roles in the epithelium during nephrogenesis, but its function in stromal cells during kidney development is unknown. To study this, we inactivated Dicer1 in renal stromal cells. This resulted in hypoplastic kidneys, abnormal differentiation of the nephron tubule and vasculature, and perinatal mortality. In mutant kidneys, genes involved in stromal cell migration and activation were suppressed as were those involved in epithelial and endothelial differentiation and maturation. Consistently, polarity of the proximal tubule was incorrect, distal tubule differentiation was diminished, and elongation of Henle's loop attenuated resulting in lack of inner medulla and papilla in stroma-specific Dicer1 mutants. Glomerular maturation and capillary loop formation were abnormal, whereas peritubular capillaries, with enhanced branching and increased diameter, formed later. In Dicer1-null renal stromal cells, expression of factors associated with migration, proliferation, and morphogenic functions including α-smooth muscle actin, integrin-α8, -β1, and the WNT pathway transcriptional regulator LEF1 were reduced. Dicer1 mutation in stroma led to loss of expression of distinct microRNAs. Of these, miR-214, -199a-5p, and -199a-3p regulate stromal cell functions ex vivo, including WNT pathway activation, migration, and proliferation. Thus, Dicer1 activity in the renal stromal compartment regulates critical stromal cell functions that, in turn, regulate differentiation of the nephron and vasculature during nephrogenesis. PMID:25651362

  1. Utilizing the folate receptor for active targeting of cancer nanotherapeutics

    PubMed Central

    Zwicke, Grant L.; Mansoori, G. Ali; Jeffery, Constance J.

    2012-01-01

    The development of specialized nanoparticles for use in the detection and treatment of cancer is increasing. Methods are being proposed and tested that could target treatments more directly to cancer cells, which could lead to higher efficacy and reduced toxicity, possibly even eliminating the adverse effects of damage to the immune system and the loss of quick replicating cells. In this mini-review we focus on recent studies that employ folate nanoconjugates to target the folate receptor. Folate receptors are highly overexpressed on the surface of many tumor types. This expression can be exploited to target imaging molecules and therapeutic compounds directly to cancerous tissues. PMID:23240070

  2. In vitro Neurons in Mammalian Cortical Layer 4 Exhibit Intrinsic Oscillatory Activity in the 10- to 50-Hz Frequency Range

    NASA Astrophysics Data System (ADS)

    Llinas, Rodolfo R.; Grace, Anthony A.; Yarom, Yosef

    1991-02-01

    We report here the presence of fast subthreshold oscillatory potentials recorded in vitro from neurons within layer 4 of the guinea pig frontal cortex. Two types of oscillatory neurons were recorded: (i) One type exhibited subthreshold oscillations whose frequency increased with membrane depolarization and encompassed a range of 10-45 Hz. Action potentials in this type of neuron demonstrated clear after-hyperpolarizations. (ii) The second type of neuron was characterized by narrow-frequency oscillations near 35-50 Hz. These oscillations often outlasted the initiating depolarizing stimulus. No calcium component could be identified in their action potential. In both types of cell the subthreshold oscillations were tetrodotoxin-sensitive, indicating that the depolarizing phase of the oscillation was generated by a voltage-dependent sodium conductance. The initial depolarizing phase was followed by a potassium conductance responsible for the falling phase of the oscillatory wave. In both types of cell, the subthreshold oscillation could trigger spikes at the oscillatory frequency, if the membrane was sufficiently depolarized. Combining intracellular recordings with Lucifer yellow staining showed that the narrow-frequency oscillatory activity was produced by a sparsely spinous interneuron located in layer 4 of the cortex. This neuron has extensive local axonal collaterals that ramify in layers 3 and 4 such that they may contribute to the columnar synchronization of activity in the 40- to 50-Hz range. Cortical activity in this frequency range has been proposed as the basis for the "conjunctive properties" of central nervous system networks.

  3. Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+

    PubMed Central

    Hakimjavadi, Hesamedin; Lingrel, Jerry B.

    2015-01-01

    The Na,K-ATPase α2 isoform is the predominant Na,K-ATPase in adult skeletal muscle and the sole Na,K-ATPase in the transverse tubules (T-tubules). In quiescent muscles, the α2 isozyme operates substantially below its maximal transport capacity. Unlike the α1 isoform, the α2 isoform is not required for maintaining resting ion gradients or the resting membrane potential, canonical roles of the Na,K-ATPase in most other cells. However, α2 activity is stimulated immediately upon the start of contraction and, in working muscles, its contribution is crucial to maintaining excitation and resisting fatigue. Here, we show that α2 activity is determined in part by the K+ concentration in the T-tubules, through its K+ substrate affinity. Apparent K+ affinity was determined from measurements of the K1/2 for K+ activation of pump current in intact, voltage-clamped mouse flexor digitorum brevis muscle fibers. Pump current generated by the α2 Na,K-ATPase, Ip, was identified as the outward current activated by K+ and inhibited by micromolar ouabain. Ip was outward at all potentials studied (−90 to −30 mV) and increased with depolarization in the subthreshold range, −90 to −50 mV. The Q10 was 2.1 over the range of 22–37°C. The K1/2,K of Ip was 4.3 ± 0.3 mM at −90 mV and was relatively voltage independent. This K+ affinity is lower than that reported for other cell types but closely matches the dynamic range of extracellular K+ concentrations in the T-tubules. During muscle contraction, T-tubule luminal K+ increases in proportion to the frequency and duration of action potential firing. This K1/2,K predicts a low fractional occupancy of K+ substrate sites at the resting extracellular K+ concentration, with occupancy increasing in proportion to the frequency of membrane excitation. The stimulation of preexisting pumps by greater K+ site occupancy thus provides a rapid mechanism for increasing α2 activity in working muscles. PMID:26371210

  4. Differential Antitumoral Properties and Renal-Associated Tissue Damage Induced by Tacrolimus and Mammalian Target of Rapamycin Inhibitors in Hepatocarcinoma: In Vitro and In Vivo Studies.

    PubMed

    Navarro-Villarán, Elena; Tinoco, José; Jiménez, Granada; Pereira, Sheila; Wang, Jize; Aliseda, Sara; Rodríguez-Hernández, María A; González, Raúl; Marín-Gómez, Luís M; Gómez-Bravo, Miguel A; Padillo, Francisco J; Álamo-Martínez, José M; Muntané, Jordi

    2016-01-01

    Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with potential portal hypertension and/or bilirubinemia, but without vascular-associated diseases. The patients are receiving immunosuppressive therapy to reduce graft rejection, but differential side effects have been related to calcineurin and mTOR inhibitor administration regarding tumor recurrence and nephrotoxicity. The in vitro studies showed that Tacrolimus exerted a more potent pro-apoptotic effect than Everolimus (Huh 7>Hep 3B>HepG2), being sirolimus only active in Hep3B cell line. Tacrolimus and Everolimus exerted potent antiproliferative properties in Huh 7 and Hep3B in which cells Sirolimus was inactive. Interestingly, Tacrolimus- and Everolimus-dependent G0/G1 cell accumulation occurred as a consequence of drastic reduction in S, as well as in S and G2+M phases, respectively. The in vivo studies support data on the more effective antitumoral properties of Everolimus, eventual risk of pro-angiogenic tumoral properties and nephrotoxicity of Tacrolimus, and pro-proliferative properties of Sirolimus in tumors developed in nude mice. PMID:27518575

  5. Differential Antitumoral Properties and Renal-Associated Tissue Damage Induced by Tacrolimus and Mammalian Target of Rapamycin Inhibitors in Hepatocarcinoma: In Vitro and In Vivo Studies

    PubMed Central

    Pereira, Sheila; Wang, Jize; Aliseda, Sara; Rodríguez-Hernández, María A.; González, Raúl; Marín-Gómez, Luís M.; Gómez-Bravo, Miguel A.; Padillo, Francisco J.; Álamo-Martínez, José M.; Muntané, Jordi

    2016-01-01

    Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with potential portal hypertension and/or bilirubinemia, but without vascular-associated diseases. The patients are receiving immunosuppressive therapy to reduce graft rejection, but differential side effects have been related to calcineurin and mTOR inhibitor administration regarding tumor recurrence and nephrotoxicity. The in vitro studies showed that Tacrolimus exerted a more potent pro-apoptotic effect than Everolimus (Huh 7>Hep 3B>HepG2), being sirolimus only active in Hep3B cell line. Tacrolimus and Everolimus exerted potent antiproliferative properties in Huh 7 and Hep3B in which cells Sirolimus was inactive. Interestingly, Tacrolimus- and Everolimus-dependent G0/G1 cell accumulation occurred as a consequence of drastic reduction in S, as well as in S and G2+M phases, respectively. The in vivo studies support data on the more effective antitumoral properties of Everolimus, eventual risk of pro-angiogenic tumoral properties and nephrotoxicity of Tacrolimus, and pro-proliferative properties of Sirolimus in tumors developed in nude mice. PMID:27518575

  6. Target Assembly to Check Boresight Alignment of Active Sensors

    NASA Technical Reports Server (NTRS)

    Ramos-Izquierdo, Luis; Scott, V. Stanley; Riris, Haris; Cavanaugh, John; Liiva, Peter; Rodriguez, Michael

    2011-01-01

    A compact and portable target assembly (Fig. 1) has been developed to measure the boresite alignment of LRO's Lunar Orbiter Laser Altimeter (LOLA) instrument at the spacecraft level. The concept for this target assembly has evolved over many years with earlier versions used to test the Mars Observer Laser Altimeter (MOLA), the Geoscience Laser Altimeter System (GLAS), and the Mercury Laser Altimeter (MLA) space-based instruments.

  7. Myeloma cell growth inhibition is augmented by synchronous inhibition of the insulin-like growth factor-1 receptor by NVP-AEW541 and inhibition of mammalian target of rapamycin by Rad001.

    PubMed

    Baumann, Philipp; Hagemeier, Hilke; Mandl-Weber, Sonja; Franke, Daniel; Schmidmaier, Ralf

    2009-04-01

    Multiple myeloma is still incurable. Myeloma cells become resistant to common drugs and patients eventually die of tumour progression. Therefore, new targets and drugs are needed immediately. NVP-AEW541 is a new, orally bioavailable small molecule inhibitor of the insulin-like growth factor-1 receptor (IGF-1R). Here, we show that NVP-AEW541 inhibits cell growth in myeloma cells at low concentrations in a time-dependent and a dose-dependent manner. Further experiments using the annexin-V-fluorescein isothiocyanate/propidium iodide assay revealed induction of apoptosis in common myeloma cell lines, but not in peripheral blood mononuclear cell from healthy donors. Stimulation of myeloma cells with IGF-1 led to a vast increase of cell growth and this was blocked by low doses of NVP-AEW541. Stimulation of myeloma cells with conditioned medium obtained from a 48-h-old HS-5 stromal cell culture was only partly blocked by NVP-AEW541. Western blotting experiments revealed that NVP-AEW541 decreased the phosphorylation status of P70S6 kinase and 4E-BP-1 but not of mammalian target of rapamycin (mTOR). Combined inhibition of IGF-1R and mTOR using the novel mTOR inhibitor Rad001 led to additive/synergistic increase of cell growth inhibition in multiple myeloma cells, which was accompanied by a stronger dephosphorylation of P70S6 kinase and 4E-BP-1. Taken together, we show that the combined inhibition of IGF-1R and mTOR by combining NVP-AEW541 and Rad001 is highly effective in multiple myeloma and might represent a potential new treatment strategy.

  8. Mammalian α arrestins link activated seven transmembrane receptors to Nedd4 family e3 ubiquitin ligases and interact with β arrestins.

    PubMed

    Shea, Fortune F; Rowell, Jennie L; Li, Yechaowei; Chang, Tien-Hsien; Alvarez, Carlos E

    2012-01-01

    The complement of fungal cell surface proteins is widely regulated by ubiquitination of membrane proteins, which results in their endocytosis and vacuolar degradation. For diverse fungal transporters, the specificity of ubiquitination is conferred by alpha arrestin adaptors, which recruit the Nedd4 family E3 ubiquitin ligase Rsp5. A recent study showed that one mammalian alpha arrestin also mediates ubiquitination and lysosomal trafficking of an activated plasma membrane receptor. Here we first screen all five widely-expressed human alpha arrestins for subcellular localization in ligand-stimulated and -unstimulated cells overexpressing the seven transmembrane receptor vasopressin 2. We then characterize the effects of alpha arrestins ARRDC3 and ARRDC4 upon activation of the seven transmembrane receptors vasopressin 2 and beta adrenergic 2. Using biochemical and imaging approaches, we show that ligand-activated receptors interact with alpha arrestins, and this results in recruitment of Nedd4 family E3 ubiquitin ligases and receptor ubiquitination - which are known to result in lysosomal trafficking. Our time course studies show these effects occur in the first 1-5 minutes after ligand activation, the same time that beta arrestins are known to have roles in receptor endocytic trafficking and kinase signaling. We tested the possibility that alpha and beta arrestins function coordinately and found co-immunoprecipitation and colocalization evidence to support this. Others recently reported that Arrdc3 knockout mice are lean and resistant to obesity. In the course of breeding our own Arrdc3-deficient mice, we observed two novel phenotypes in homozygotes: skin abnormalities, and embryonic lethality on normal chow diet, but not on high fat diet. Our findings suggest that alpha and beta arrestins function coordinately to maintain the optimal complement and function of cell surface proteins according to cellular physiological context and external signals. We discuss the

  9. Mammalian Wax Biosynthesis

    PubMed Central

    Cheng, Jeffrey B.; Russell, David W.

    2009-01-01

    Wax monoesters are synthesized by the esterification of fatty alcohols and fatty acids. A mammalian enzyme that catalyzes this reaction has not been isolated. We used expression cloning to identify cDNAs encoding a wax synthase in the mouse preputial gland. The wax synthase gene is located on the X chromosome and encodes a member of the acyltransferase family of enzymes that synthesize neutral lipids. Expression of wax synthase in cultured cells led to the formation of wax monoesters from straight chain saturated, unsaturated, and polyunsaturated fatty alcohols and acids. Polyisoprenols also were incorporated into wax monoesters by the enzyme. The wax synthase had little or no ability to synthesize cholesteryl esters, diacylglycerols, or triacylglycerols, whereas other acyltransferases, including the acyl-CoA:monoacylglycerol acyltransferase 1 and 2 enzymes and the acyl-CoA:diacylglycerol acyltransferase 1 and 2 enzymes, exhibited modest wax monoester synthesis activities. Confocal light microscopy indicated that the wax synthase was localized in membranes of the endoplasmic reticulum. Wax synthase mRNA was abundant in tissues rich in sebaceous glands such as the preputial gland and eyelid and was present at lower levels in other tissues. Coexpression of cDNAs specifying fatty acyl-CoA reductase 1 and wax synthase led to the synthesis of wax monoesters. The data suggest that wax monoester synthesis in mammals involves a two step biosynthetic pathway catalyzed by fatty acyl-CoA reductase and wax synthase enzymes. PMID:15220349

  10. Pilocarpine modulates the cellular electrical properties of mammalian hearts by activating a cardiac M3 receptor and a K+ current

    PubMed Central

    Wang, Huizhen; Shi, Hong; Lu, Yanjie; Yang, Baofeng; Wang, Zhiguo

    1999-01-01

    Pilocarpine, a muscarinic acetylcholine receptor (mAChR) agonist, is widely used for treatment of xerostomia and glaucoma. It can also cause many other cellular responses by activating different subtypes of mAChRs in different tissues. However, the potential role of pilocarpine in modulating cardiac function remained unstudied.We found that pilocarpine produced concentration-dependent (0.1–10 μM) decrease in sinus rhythm and action potential duration, and hyperpolarization of membrane potential in guinea-pig hearts. The effects were nearly completely reversed by 1 μM atropine or 2 nM 4DAMP methiodide (an M3-selective antagonist).Patch-clamp recordings in dispersed myocytes from guinea-pig and canine atria revealed that pilocarpine induces a novel K+ current with delayed rectifying properties. The current was suppressed by low concentrations of M3-selective antagonists 4DAMP methiodide (2–10 nM), 4DAMP mustard (4–20 nM, an ackylating agent) and p-F-HHSiD (20–200 nM). Antagonists towards other subtypes (M1, M2 or M4) all failed to alter the current.The affinity of pilocarpine (KD) at mAChRs derived from displacement binding of [3H]-NMS in the homogenates from dog atria was 2.2 μM (65% of the total binding) and that of 4DAMP methiodide was 2.8 nM (70% of total binding), consistent with the concentration of pilocarpine needed for the current induction and for the modulation of the cardiac electrical activity and the concentration of 4DAMP to block pilocarpine effects.Our data indicate, for the first time, that pilocarpine modulates the cellular electrical properties of the hearts, likely by activating a K+ current mediated by M3 receptors. PMID:10372814

  11. Ionizing Radiation Activates AMP-Activated Kinase (AMPK): A Target for Radiosensitization of Human Cancer Cells

    SciTech Connect

    Sanli, Toran; Rashid, Ayesha; Liu Caiqiong

    2010-09-01

    Purpose: Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells. Methods and Materials: Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis. Results: IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK {alpha} subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21{sup waf/cip} as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further. Conclusions: We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21{sup waf/cip} and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21{sup waf/cip} pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.

  12. Addition of Selenium Nanoparticles to Electrospun Silk Scaffold Improves the Mammalian Cell Activity While Reducing Bacterial Growth.

    PubMed

    Chung, Stanley; Ercan, Batur; Roy, Amit K; Webster, Thomas J

    2016-01-01

    Silk possesses many beneficial wound healing properties, and electrospun scaffolds are especially applicable for skin applications, due to their smaller interstices and higher surface areas. However, purified silk promotes microbial growth. Selenium nanoparticles have shown excellent antibacterial properties and are a novel antimicrobial chemistry. Here, electrospun silk scaffolds were doped with selenium nanoparticles to impart antibacterial properties to the silk scaffolds. Results showed significantly improved bacterial inhibition and mild improvement in human dermal fibroblast metabolic activity. These results suggest that the addition of selenium nanoparticles to electrospun silk is a promising approach to improve wound healing with reduced infection, without relying on antibiotics. PMID:27471473

  13. Addition of Selenium Nanoparticles to Electrospun Silk Scaffold Improves the Mammalian Cell Activity While Reducing Bacterial Growth

    PubMed Central

    Chung, Stanley; Ercan, Batur; Roy, Amit K.; Webster, Thomas J.

    2016-01-01

    Silk possesses many beneficial wound healing properties, and electrospun scaffolds are especially applicable for skin applications, due to their smaller interstices and higher surface areas. However, purified silk promotes microbial growth. Selenium nanoparticles have shown excellent antibacterial properties and are a novel antimicrobial chemistry. Here, electrospun silk scaffolds were doped with selenium nanoparticles to impart antibacterial properties to the silk scaffolds. Results showed significantly improved bacterial inhibition and mild improvement in human dermal fibroblast metabolic activity. These results suggest that the addition of selenium nanoparticles to electrospun silk is a promising approach to improve wound healing with reduced infection, without relying on antibiotics. PMID:27471473

  14. The role of Rad51 in safeguarding mitochondrial activity during the meiotic cell cycle in mammalian oocytes

    PubMed Central

    Kim, Kyeoung-Hwa; Park, Ji-Hoon; Kim, Eun-Young; Ko, Jung-Jae; Park, Kyung-Soon; Lee, Kyung-Ah

    2016-01-01

    Rad51 is a conserved eukaryotic protein that mediates the homologous recombination repair of DNA double-strand breaks that occur during mitosis and meiosis. In addition, Rad51 promotes mitochondrial DNA synthesis when replication stress is increased. Rad51 also regulates cell cycle progression by preserving the G2/M transition in embryonic stem cells. In this study, we report a novel function of Rad51 in regulating mitochondrial activity during in vitro maturation of mouse oocytes. Suppression of Rad51 by injection of Rad51 dsRNA into germinal vesicle-stage oocytes resulted in arrest of meiosis in metaphase I. Rad51-depleted oocytes showed chromosome misalignment and failures in spindle aggregation, affecting the completion of cytokinesis. We found that Rad51 depletion was accompanied by decreased ATP production and mitochondrial membrane potential and increased DNA degradation. We further demonstrated that the mitochondrial defect activated autophagy in Rad51-depleted oocytes. Taken together, we concluded that Rad51 functions to safeguard mitochondrial integrity during the meiotic maturation of oocytes. PMID:27677401

  15. Urinary neopterin, a non-invasive marker of mammalian cellular immune activation, is highly stable under field conditions.

    PubMed

    Heistermann, Michael; Higham, James P

    2015-01-01

    Studying immunity and immune function in ecology and evolution requires field studies, but there has been a dearth of non-invasive markers of immune activation available for studying large wild mammals. Recently, we analytically and biologically validated the measurement of urinary neopterin (NEO), a biomarker of cellular immune activation, in captive macaques. However, applying this to free-ranging settings is complicated by issues involving sample collection, processing, storage, and transport. Here, we collected urine samples from captive macaques and undertook experiments simulating common field issues. We tested the effects on urinary NEO sample measurements following: dirt and faecal contamination; storage at room temperature; differences in processing and long-term storage methods (freezing, lyophilising, blotting onto filter paper); and freeze-thaw cycles. Our results show that concentrations of urinary NEO are highly stable--they are not affected by soil or faecal contamination, can be collected on filter paper and stored for many months frozen or lyophilised with minimal effect, and are resistant to multiple 24 hr freeze-thaws. With the addition of a biocidal preservative, concentrations are even stable at room temperature for long periods. Urinary NEO is remarkably resilient, and is highly suitable for non-invasive field studies of cellular immune responses in wild large mammals.

  16. Urinary neopterin, a non-invasive marker of mammalian cellular immune activation, is highly stable under field conditions

    PubMed Central

    Heistermann, Michael; Higham, James P.

    2015-01-01

    Studying immunity and immune function in ecology and evolution requires field studies, but there has been a dearth of non-invasive markers of immune activation available for studying large wild mammals. Recently, we analytically and biologically validated the measurement of urinary neopterin (NEO), a biomarker of cellular immune activation, in captive macaques. However, applying this to free-ranging settings is complicated by issues involving sample collection, processing, storage, and transport. Here, we collected urine samples from captive macaques and undertook experiments simulating common field issues. We tested the effects on urinary NEO sample measurements following: dirt and faecal contamination; storage at room temperature; differences in processing and long-term storage methods (freezing, lyophilising, blotting onto filter paper); and freeze-thaw cycles. Our results show that concentrations of urinary NEO are highly stable – they are not affected by soil or faecal contamination, can be collected on filter paper and stored for many months frozen or lyophilised with minimal effect, and are resistant to multiple 24 hr freeze-thaws. With the addition of a biocidal preservative, concentrations are even stable at room temperature for long periods. Urinary NEO is remarkably resilient, and is highly suitable for non-invasive field studies of cellular immune responses in wild large mammals. PMID:26549509

  17. Activation and propagation of Ca2+ release from inside the sarcoplasmic reticulum network of mammalian skeletal muscle.

    PubMed

    Cully, Tanya R; Edwards, Joshua N; Launikonis, Bradley S

    2014-09-01

    Skeletal muscle fibres are large and highly elongated cells specialized for producing the force required for posture and movement. The process of controlling the production of force within the muscle, known as excitation-contraction coupling, requires virtually simultaneous release of large amounts of Ca(2+) from the sarcoplasmic reticulum (SR) at the level of every sarcomere within the muscle fibre. Here we imaged Ca(2+) movements within the SR, tubular (t-) system and in the cytoplasm to observe that the SR of skeletal muscle is a connected network capable of allowing diffusion of Ca(2+) within its lumen to promote the propagation of Ca(2+) release throughout the fibre under conditions where inhibition of SR ryanodine receptors (RyRs) was reduced. Reduction of cytoplasmic [Mg(2+)] ([Mg(2+)]cyto) induced a leak of Ca(2+) through RyRs, causing a reduction in SR Ca(2+) buffering power argued to be due to a breakdown of SR calsequestrin polymers, leading to a local elevation of [Ca(2+)]SR. The local rise in [Ca(2+)]SR, an intra-SR Ca(2+) transient, induced a local diffusely rising [Ca(2+)]cyto. A prolonged Ca(2+) wave lasting tens of seconds or more was generated from these events. Ca(2+) waves were dependent on the diffusion of Ca(2+) within the lumen of the SR and ended as [Ca(2+)]SR dropped to low levels to inactivate RyRs. Inactivation of RyRs allowed re-accumulation of [Ca(2+)]SR and the activation of secondary Ca(2+) waves in the persistent presence of low [Mg(2+)]cyto if the threshold [Ca(2+)]SR for RyR opening could be reached. Secondary Ca(2+) waves occurred without an abrupt reduction in SR Ca(2+) buffering power. Ca(2+) release and wave propagation occurred in the absence of Ca(2+)-induced Ca(2+) release. These observations are consistent with the activation of Ca(2+) release through RyRs of lowered cytoplasmic inhibition by [Ca(2+)]SR or store overload-induced Ca(2+) release. Restitution of SR Ca(2+) buffering power to its initially high value required

  18. Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

    PubMed

    Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

    2015-10-20

    Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

  19. Toxins vapC and pasB from prokaryotic TA modules remain active in mammalian cancer cells.

    PubMed

    Wieteska, Łukasz; Skulimowski, Aleksander; Cybula, Magdalena; Szemraj, Janusz

    2014-10-01

    Among the great number of addictive modules which have been discovered, only a few have been characterized. However, research concerning the adoption of toxins from these systems shows their great potential as a tool for molecular biology and medicine. In our study, we tested two different toxins derived from class II addictive modules, pasAB from plasmid pTF-FC2 (Thiobacillus ferrooxidans) and vapBC 2829Rv (Mycobacterium tuberculosis), in terms of their usefulness as growth inhibitors of human cancer cell lines, namely KYSE 30, MCF-7 and HCT 116. Transfection of the pasB and vapC genes into the cells was conducted with the use of two different expression systems. Cellular effects, such as apoptosis, necrosis and changes in the cell cycle, were tested by applying flow cytometry with immunofluorescence staining. Our findings demonstrated that toxins VapC and PasB demonstrate proapoptotic activity in the human cancer cells, regardless of the expression system used. As for the toxin PasB, observed changes were more subtle than for the VapC. The level of expression for both the genes was monitored by QPCR and did not reveal statistically significant differences within the same cell line.

  20. Mammalian synthetic biology: emerging medical applications

    PubMed Central

    Kis, Zoltán; Pereira, Hugo Sant'Ana; Homma, Takayuki; Pedrigi, Ryan M.; Krams, Rob

    2015-01-01

    In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON–OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes. PMID:25808341

  1. Mammalian synthetic biology: emerging medical applications.

    PubMed

    Kis, Zoltán; Pereira, Hugo Sant'Ana; Homma, Takayuki; Pedrigi, Ryan M; Krams, Rob

    2015-05-01

    In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON-OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes.

  2. 27 CFR 478.35 - Skeet, trap, target, and similar shooting activities.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 3 2013-04-01 2013-04-01 false Skeet, trap, target, and... FIREARMS AND AMMUNITION Administrative and Miscellaneous Provisions § 478.35 Skeet, trap, target, and... records, for skeet, trap, target, and similar organized activities shall be determined by the Director...

  3. 27 CFR 478.35 - Skeet, trap, target, and similar shooting activities.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 3 2010-04-01 2010-04-01 false Skeet, trap, target, and... FIREARMS AND AMMUNITION Administrative and Miscellaneous Provisions § 478.35 Skeet, trap, target, and... records, for skeet, trap, target, and similar organized activities shall be determined by the Director...

  4. 27 CFR 478.35 - Skeet, trap, target, and similar shooting activities.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 3 2012-04-01 2010-04-01 true Skeet, trap, target, and... FIREARMS AND AMMUNITION Administrative and Miscellaneous Provisions § 478.35 Skeet, trap, target, and... records, for skeet, trap, target, and similar organized activities shall be determined by the Director...

  5. 27 CFR 478.35 - Skeet, trap, target, and similar shooting activities.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 3 2014-04-01 2014-04-01 false Skeet, trap, target, and... FIREARMS AND AMMUNITION Administrative and Miscellaneous Provisions § 478.35 Skeet, trap, target, and... records, for skeet, trap, target, and similar organized activities shall be determined by the Director...

  6. 27 CFR 478.35 - Skeet, trap, target, and similar shooting activities.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 3 2011-04-01 2010-04-01 true Skeet, trap, target, and... FIREARMS AND AMMUNITION Administrative and Miscellaneous Provisions § 478.35 Skeet, trap, target, and... records, for skeet, trap, target, and similar organized activities shall be determined by the Director...

  7. 13 CFR 124.509 - What are non-8(a) business activity targets?

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... competitive marketplace after graduating from the 8(a) BD program, Par