Sample records for a27 protein synergizes

  1. Postjunctional synergism of norepinephrine with ATP and diadenosine tetraphosphate in Guinea pig vas deferens. Role of protein kinase C and Myosin light chain phosphatase.

    PubMed

    Khattab, Mahmoud M; Al-Rawi, Mahmood B; Aleisa, Abdulaziz M

    2007-01-01

    In isolated guinea pig vas deferens, prior addition of norepinephrine (NE) significantly potentiated the contractile responses to adenosine-5'-triphosphate (ATP) and diadenosine tetraphosphate (AP4A) in a dose-dependent manner up to 240% of the control purine dose. The myosin light chain phosphatase (MLCP) inhibitor cantharidin at a dose of 10 micromol/l caused significant enhancement of ATP at concentrations of 1 and 3 mmol/l by 91 and 95% respectively. Similarly, cantharidin enhanced the contraction to AP4A, 30 and 100 micromol/l by 92 and 100% respectively. Inhibition of protein kinase C (PKC) by the use of chelerythrine (10 micromol/l), incubated at the vas deferens for 60 min, inhibited the NE-induced enhancement of purine-induced contraction. Chelerythrine reversed the NE-ATP and NE-AP4A synergism back close to control ATP and AP4A contraction values respectively. It can be concluded that postjunctional synergism becomes evident not only for adenine mononucleotides and NE but also for diadenosine polyphosphates presented here by AP4A in the guinea pig vas deferens. This synergism involves receptor-mediated activation of PKC and possibly PKC-induced inhibition of MLCP. Copyright (c) 2007 S. Karger AG, Basel.

  2. Heat shock protein 27 as a prognostic and predictive biomarker in pancreatic ductal adenocarcinoma

    PubMed Central

    Schäfer, Claus; Seeliger, Hendrik; Bader, Dominik C; Assmann, Gerald; Buchner, Denise; Guo, Yang; Ziesch, Andreas; Palagyi, Andreas; Ochs, Stephanie; Laubender, Rüdiger P; Jung, Andreas; De Toni, Enrico N; Kirchner, Thomas; Göke, Burkhard; Bruns, Christiane; Gallmeier, Eike

    2012-01-01

    Abstract A role of heat shock protein 27 (HSP27) as a potential biomarker has been reported in various tumour entities, but comprehensive studies in pancreatic cancer are lacking. Applying tissue microarray (TMA) analysis, we correlated HSP27 protein expression status with clinicopathologic parameters in pancreatic ductal adenocarcinoma specimens from 86 patients. Complementary, we established HSP27 overexpression and RNA-interference models to assess the impact of HSP27 on chemo- and radiosensitivity directly in pancreatic cancer cells. In the TMA study, HSP27 expression was found in 49% of tumour samples. Applying univariate analyses, a significant correlation was found between HSP27 expression and survival. In the multivariate Cox-regression model, HSP27 expression emerged as an independent prognostic factor. HSP27 expression also correlated inversely with nuclear p53 accumulation, indicating either protein interactions between HSP27 and p53 or TP53 mutation-dependent HSP27-regulation in pancreatic cancer. In the sensitivity studies, HSP27 overexpression rendered HSP27 low-expressing PL5 pancreatic cancer cells more susceptible towards treatment with gemcitabine. Vice versa, HSP27 protein depletion in HSP27 high-expressing AsPC-1 cells caused increased gemcitabine resistance. Importantly, HSP27 expression was inducible in pancreatic cancer cell lines as well as primary cells. Taken together, our study suggests a role for HSP27 as a prognostic and predictive marker in pancreatic cancer. Assessment of HSP27 expression could thus facilitate the identification of specific patient subpopulations that might benefit from individualized treatment options. Additional studies need to clarify whether modulation of HSP27 expression could represent an attractive concept to support the incorporation of hyperthermia in clinical treatment protocols for pancreatic cancer. PMID:22004109

  3. Synergism and Antagonism between Bacillus thuringiensis Vip3A and Cry1 Proteins in Heliothis virescens, Diatraea saccharalis and Spodoptera frugiperda

    PubMed Central

    Lemes, Ana Rita Nunes; Davolos, Camila Chiaradia; Legori, Paula Cristina Brunini Crialesi; Fernandes, Odair Aparecido; Ferré, Juan; Lemos, Manoel Victor Franco; Desiderio, Janete Apparecida

    2014-01-01

    Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC50, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC90, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC90 level than at the LC50 level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested. PMID:25275646

  4. Plant Endocytosis Requires the ER Membrane-Anchored Proteins VAP27-1 and VAP27-3.

    PubMed

    Stefano, Giovanni; Renna, Luciana; Wormsbaecher, Clarissa; Gamble, Jessie; Zienkiewicz, Krzysztof; Brandizzi, Federica

    2018-05-22

    Through yet-undefined mechanisms, the plant endoplasmic reticulum (ER) has a critical role in endocytosis. The plant ER establishes a close association with endosomes and contacts the plasma membrane (PM) at ER-PM contact sites (EPCSs) demarcated by the ER membrane-associated VAMP-associated-proteins (VAP). Here, we investigated two plant VAPs, VAP27-1 and VAP27-3, and found an interaction with clathrin and a requirement for the homeostasis of clathrin dynamics at endocytic membranes and endocytosis. We also demonstrated direct interaction of VAP27-proteins with phosphatidylinositol-phosphate lipids (PIPs) that populate endocytic membranes. These results support that, through interaction with PIPs, VAP27-proteins bridge the ER with endocytic membranes and maintain endocytic traffic, likely through their interaction with clathrin. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  5. Protective effect of surfactant protein d in pulmonary vaccinia virus infection: implication of A27 viral protein.

    PubMed

    Perino, Julien; Thielens, Nicole M; Crouch, Erika; Spehner, Danièle; Crance, Jean-Marc; Favier, Anne-Laure

    2013-03-21

    Vaccinia virus (VACV) was used as a surrogate of variola virus (VARV) (genus Orthopoxvirus), the causative agent of smallpox, to study Orthopoxvirus infection. VARV is principally transmitted between humans by aerosol droplets. Once inhaled, VARV first infects the respiratory tract where it could encounter surfactant components, such as soluble pattern recognition receptors. Surfactant protein D (SP-D), constitutively present in the lining fluids of the respiratory tract, plays important roles in innate host defense against virus infection. We investigated the role of SP-D in VACV infection and studied the A27 viral protein involvement in the interaction with SP-D. Interaction between SP-D and VACV caused viral inhibition in a lung cell model. Interaction of SP-D with VACV was mediated by the A27 viral protein. Binding required Ca2+ and interactions were blocked in the presence of excess of SP-D saccharide ligands. A27, which lacks glycosylation, directly interacted with SP-D. The interaction between SP-D and the viral particle was also observed using electron microscopy. Infection of mice lacking SP-D (SP-D-/-) resulted in increased mortality compared to SP-D+/+ mice. Altogether, our data show that SP-D participates in host defense against the vaccinia virus infection and that the interaction occurs with the viral surface protein A27.

  6. Effects of protein transduction domain (PTD) selection and position for improved intracellular delivery of PTD-Hsp27 fusion protein formulations.

    PubMed

    Ul Ain, Qurrat; Lee, Jong Hwan; Woo, Young Sun; Kim, Yong-Hee

    2016-09-01

    Protein drugs have attracted considerable attention as therapeutic agents due to their diversity and biocompatibility. However, hydrophilic proteins possess difficulty in penetrating lipophilic cell membrane. Although protein transduction domains (PTDs) have shown effectiveness in protein delivery, the importance of selection and position of PTDs in recombinant protein vector constructs has not been investigated. This study intends to investigate the significance of PTD selection and position for therapeutic protein delivery. Heat shock protein 27 (Hsp27) would be a therapeutic protein for the treatment of ischemic heart diseases, but itself is insufficient to prevent systemic degradation and overcoming biochemical barriers during cellular transport. Among all PTD-Hsp27 fusion proteins we cloned, Tat-Hsp27 fusion protein showed the highest efficacy. Nona-arginine (9R) conjugation to the N-terminal of Hsp27 (Hsp27-T) showed higher efficacy than C-terminal. To test the synergistic effect of two PTDs, Tat was inserted to the N-terminal of Hsp27-9R. Tat-Hsp27-9R exhibited enhanced transduction efficiency and significant improvement against oxidative stress and apoptosis. PTD-Hsp27 fusion proteins have strong potential to be developed as therapeutic proteins for the treatment of ischemic heart diseases and selection and position of PTDs for improved efficacy of PTD-fusion proteins need to be optimized considering protein's nature, transduction efficiency and stability.

  7. Reconceptualizing synergism and antagonism among multiple stressors.

    PubMed

    Piggott, Jeremy J; Townsend, Colin R; Matthaei, Christoph D

    2015-04-01

    The potential for complex synergistic or antagonistic interactions between multiple stressors presents one of the largest uncertainties when predicting ecological change but, despite common use of the terms in the scientific literature, a consensus on their operational definition is still lacking. The identification of synergism or antagonism is generally straightforward when stressors operate in the same direction, but if individual stressor effects oppose each other, the definition of synergism is paradoxical because what is synergistic to one stressor's effect direction is antagonistic to the others. In their highly cited meta-analysis, Crain et al. (Ecology Letters, 11, 2008: 1304) assumed in situations with opposing individual effects that synergy only occurs when the cumulative effect is more negative than the additive sum of the opposing individual effects. We argue against this and propose a new systematic classification based on an additive effects model that combines the magnitude and response direction of the cumulative effect and the interaction effect. A new class of "mitigating synergism" is identified, where cumulative effects are reversed and enhanced. We applied our directional classification to the dataset compiled by Crain et al. (Ecology Letters, 11, 2008: 1304) to determine the prevalence of synergistic, antagonistic, and additive interactions. Compared to their original analysis, we report differences in the representation of interaction classes by interaction type and we document examples of mitigating synergism, highlighting the importance of incorporating individual stressor effect directions in the determination of synergisms and antagonisms. This is particularly pertinent given a general bias in ecology toward investigating and reporting adverse multiple stressor effects (double negative). We emphasize the need for reconsideration by the ecological community of the interpretation of synergism and antagonism in situations where

  8. Synergism of aspirin and heparin with a low-frequency non-invasive ultrasound system for augmentation of in-vitro clot lysis.

    PubMed

    Atar, Shaul; Neuman, Yoram; Miyamoto, Takashi; Chen, Ming; Birnbaum, Yochai; Luo, Huai; Kobal, Sergio; Siegel, Robert J

    2003-06-01

    Aspirin, glycoprotein IIb/IIIa inhibitors and heparin are routinely used in acute coronary syndromes. Previously we showed that there is synergism between ultrasound and heparin and tirofiban in augmenting blood clot disruption. However, there is a little data on a possible synergism of low-frequency ultrasound with aspirin for in-vitro clot dissolution, and especially on the combination of aspirin with heparin and/or glycoprotein IIb/IIIa inhibitors. Human blood clots (n = 320) were incubated for 10 or 20 minutes in saline containing aspirin alone or combined with heparin and/or tirofiban and/or eptifibatide. Clots were randomly treated with low-frequency ultrasound (27.3 kHz) or incubation only. The percent clot weight loss and the incremental effect of ultrasound were calculated. The most significant incremental effect of ultrasound on clot weight reduction was detected with aspirin alone (5.2 +/- 2.3% and 5.2 +/- 2.6% after 10' and 20', p = 0.04 and p = 0.06, respectively) and in combination with heparin (8.8 +/- 2.5% and 11.5 +/- 2.7% after 10' and 20', p = 0.01 and p = 0.0001, respectively). The greatest absolute magnitude of clot weight reduction was observed with ultrasound combined with aspirin and heparin (48.5 +/- 9.5% after 20'). The addition of tirofiban or eptifibatide to aspirin, heparin and ultrasound did not increase clot lysis. However, eptifibatide had significantly better synergism than tirofiban (p = 0.025 and p = 0.015, after 10 and 20 minutes, respectively). Aspirin alone or in combination with heparin results in significant augmentation of clot lysis and is synergistic with application of low-frequency ultrasound for 10 and 20 minutes only. These results may have important implications for a possible use of low-frequency ultrasound in treatment algorithms of acute coronary syndromes.

  9. Discovery of Small Molecules that Inhibit the Disordered Protein, p27Kip1

    PubMed Central

    Iconaru, Luigi I.; Ban, David; Bharatham, Kavitha; Ramanathan, Arvind; Zhang, Weixing; Shelat, Anang A.; Zuo, Jian; Kriwacki, Richard W.

    2015-01-01

    Disordered proteins are highly prevalent in biological systems, they control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27Kip1 (p27). Two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groups of small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule:disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of-principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A). PMID:26507530

  10. Heat shock protein-27 (HSP27) regulates STAT3 and eIF4G levels in first trimester human placenta.

    PubMed

    Shochet, Gali Epstein; Komemi, Oded; Sadeh-Mestechkin, Dana; Pomeranz, Meir; Fishman, Ami; Drucker, Liat; Lishner, Michael; Matalon, Shelly Tartakover

    2016-12-01

    During placental implantation, cytotrophoblast cells differentiate to extravillous trophoblast (EVT) cells that invade from the placenta into the maternal uterine blood vessels. The heat shock protein-27 (HSP27), the signal transducer and activator of transcription-3 (STAT3) and the eukaryotic translation initiation factor 4E (EIF4E) are involved in regulating EVT cell differentiation/migration. EIF4E and EIF4G compose the translation initiation complex, which is a major control point in protein translation. The molecular chaperone distinctiveness of HSP27 implies that it directly interferes with many target proteins. STAT3, EIF4E, and EIF4G were found to be HSP27 client proteins in tumor cells. We aimed to analyze if HSP27 regulate STAT3 and EIF4G levels in first trimester human placenta. We found that like STAT3, EIF4G is highly expressed in the EVT cells (immunohistochemistry). Silencing HSP27 in HTR-8/SVneo cells (siRNA, EVT cell line) and in placental explants reduced STAT3 level (47 and 33 %, respectively, p < 0.05). HSP27 silencing reduced the levels of STAT3 phosphorylation (33 % reduction, p < 0.05) and targets (IRF1, MUC1, MMP2/9 and EIF4E, 30-49 % reduction, p < 0.05) in the HTR-8/SVneo cells. Moreover, HSP27 silencing significantly reduced EIF4G level and elevated the level of its fragments in HTR-8/SVneo cells and in the placental explants (p < 0.05). In conclusion, Placental implantation and development are accompanied by trophoblast cell proliferation and differentiation, which necessitates intense protein translation and STAT3 activation. HSP27 was found to be regulator of translation initiation and STAT3 level. Therefore, it suggests that HSP27 is a key protein during placental development and trophoblast cell differentiation.

  11. Knock Down of Heat Shock Protein 27 (HspB1) Induces Degradation of Several Putative Client Proteins

    PubMed Central

    Gibert, Benjamin; Eckel, Bénédicte; Fasquelle, Lydie; Moulin, Maryline; Bouhallier, Frantz; Gonin, Vincent; Mellier, Gregory; Simon, Stéphanie; Kretz-Remy, Carole; Arrigo, André-Patrick; Diaz-Latoud, Chantal

    2012-01-01

    Hsp27 belongs to the heat shock protein family and displays chaperone properties in stress conditions by holding unfolded polypeptides, hence avoiding their inclination to aggregate. Hsp27 is often referenced as an anti-cancer therapeutic target, but apart from its well-described ability to interfere with different stresses and apoptotic processes, its role in non-stressed conditions is still not well defined. In the present study we report that three polypeptides (histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3) were degraded in human cancerous cells displaying genetically decreased levels of Hsp27. In addition, these proteins interacted with Hsp27 complexes of different native size. Altogether, these findings suggest that HDAC6, STAT2 and procaspase-3 are client proteins of Hsp27. Hence, in non stressed cancerous cells, the structural organization of Hsp27 appears to be a key parameter in the regulation by this chaperone of the level of specific polypeptides through client-chaperone type of interactions. PMID:22238643

  12. Discovery of Small Molecules that Inhibit the Disordered Protein, p27 Kip1

    DOE PAGES

    Iconaru, Luigi I.; Ban, David; Bharatham, Kavitha; ...

    2015-10-28

    In disordered proteins we see that they are highly prevalent in biological systems. They control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27 Kip1 (p27). Moreover, two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groupsmore » of small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule: disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of- principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A).« less

  13. Rab27a negatively regulates CFTR chloride channel function in colonic epithelia: Involvement of the effector proteins in the regulatory mechanism

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

    Saxena, Sunil K.; Kaur, Simarna

    Cystic fibrosis, an autosomal recessive disorder, is caused by the disruption of biosynthesis or function of CFTR. CFTR regulatory mechanisms include channel transport to plasma membrane and protein-protein interactions. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. The colorectal epithelial HT-29 cells natively express CFTR and respond to cAMP with an increase in CFTR-mediated currents. DPC-inhibited currents could be completely eliminated with CFTR-specific SiRNA. Over-expression of Rab27a inhibited, while isoform specific SiRNA and Rab27a antibody stimulated CFTR-mediated currents in HT-29 cells. CFTR activity is inhibited both by Rab27a (Q78L) (constitutive active GTP-bound form of Rab27a) andmore » Rab27a (T23N) (constitutive negative form that mimics the GDP-bound form). Rab27a mediated effects could be reversed by Rab27a-binding proteins, the synaptotagmin-like protein (SLP-5) and Munc13-4 accessory protein (a putative priming factor for exocytosis). The SLP reversal of Rab27a effect was restricted to C2A/C2B domains while the SHD motif imparted little more inhibition. The CFTR-mediated currents remain unaffected by Rab3 though SLP-5 appears to weakly bind it. The immunoprecipitation experiments suggest protein-protein interactions between Rab27a and CFTR. Rab27a appears to impair CFTR appearance at the cell surface by trapping CFTR in the intracellular compartments. Munc13-4 and SLP-5, on the other hand, limit Rab27a availability to CFTR, thus minimizing its effect on channel function. These observations decisively prove that Rab27a is involved in CFTR channel regulation through protein-protein interactions involving Munc13-4 and SLP-5 effector proteins, and thus could be a potential target for cystic fibrosis therapy.« less

  14. Membrane Curvature and Lipid Composition Synergize To Regulate N-Ras Anchor Recruitment.

    PubMed

    Larsen, Jannik B; Kennard, Celeste; Pedersen, Søren L; Jensen, Knud J; Uline, Mark J; Hatzakis, Nikos S; Stamou, Dimitrios

    2017-09-19

    Proteins anchored to membranes through covalently linked fatty acids and/or isoprenoid groups play crucial roles in all forms of life. Sorting and trafficking of lipidated proteins has traditionally been discussed in the context of partitioning to membrane domains of different lipid composition. We recently showed that membrane shape/curvature can in itself mediate the recruitment of lipidated proteins. However, exactly how membrane curvature and composition synergize remains largely unexplored. Here we investigated how three critical structural parameters of lipids, namely acyl chain saturation, headgroup size, and acyl chain length, modulate the capacity of membrane curvature to recruit lipidated proteins. As a model system we used the lipidated minimal membrane anchor of the GTPase, N-Ras (tN-Ras). Our data revealed complex synergistic effects, whereby tN-Ras binding was higher on planar DOPC than POPC membranes, but inversely higher on curved POPC than DOPC membranes. This variation in the binding to both planar and curved membranes leads to a net increase in the recruitment by membrane curvature of tN-Ras when reducing the acyl chain saturation state. Additionally, we found increased recruitment by membrane curvature of tN-Ras when substituting PC for PE, and when decreasing acyl chain length from 14 to 12 carbons (DMPC versus DLPC). However, these variations in recruitment ability had different origins, with the headgroup size primarily influencing tN-Ras binding to planar membranes whereas the change in acyl chain length primarily affected binding to curved membranes. Molecular field theory calculations recapitulated these findings and revealed lateral pressure as an underlying biophysical mechanism dictating how curvature and composition synergize to modulate recruitment of lipidated proteins. Our findings suggest that the different compositions of cellular compartments could modulate the potency of membrane curvature to recruit lipidated proteins and

  15. The 29-kDa proteins phosphorylated ion thrombin-activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein

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

    Mendelsohn, M.E.; Yan Zhu; O'Neill, S.

    Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. The authors have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, they isolatedmore » and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the estrogen receptor-related protein is HSP27, and the three major 20-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data suggest a role for HSP27 in the signal transduction events of platelet activation.« less

  16. Proteomic Characterization of Annexin l (ANX1) and Heat Shock Protein 27 (HSP27) as Biomarkers for Invasive Hepatocellular Carcinoma Cells.

    PubMed

    Wang, Ruo-Chiau; Huang, Chien-Yu; Pan, Tai-Long; Chen, Wei-Yu; Ho, Chun-Te; Liu, Tsan-Zon; Chang, Yu-Jia

    2015-01-01

    To search for reliable biomarkers and drug targets for management of hepatocellular carcinoma (HCC), we performed a global proteomic analysis of a pair of HCC cell lines with distinct differentiation statuses using 2-DE coupled with MALDI-TOF MS. In total, 106 and 55 proteins were successfully identified from the total cell lysate and the cytosolic, nuclear and membrane fractions in well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) HCC clonal variants, respectively. Among these proteins, nine spots corresponding to proteins differentially expressed between HCC cell types were selected and confirmed by immunofluorescence staining and western blotting. Notably, Annexin 1 (ANX1), ANX-2, vimentin and stress-associated proteins, such as GRP78, HSP75, HSC-70, protein disulfide isomerase (PDI), and heat shock protein-27 (HSP27), were exclusively up-regulated in SK-Hep-1 cells. Elevated levels of ANX-4 and antioxidant/metabolic enzymes, such as MnSOD, peroxiredoxin, NADP-dependent isocitrate dehydrogenase, α-enolase and UDP-glucose dehydrogenase, were observed in HepG2 cells. We functionally demonstrated that ANX1 and HSP27 were abundantly overexpressed only in highly invasive types of HCC cells, such as Mahlavu and SK-Hep-1. Knockdown of ANX1 or HSP27 in HCC cells resulted in a severe reduction in cell migration. The in-vitro observations of ANX1 and HSP27 expressions in HCC sample was demonstrated by immunohistochemical stains performed on HCC tissue microarrays. Poorly differentiated HCC tended to have stronger ANX1 and HSP27 expressions than well-differentiated or moderately differentiated HCC. Collectively, our findings suggest that ANX1 and HSP27 are two novel biomarkers for predicting invasive HCC phenotypes and could serve as potential treatment targets.

  17. A comparison of the toxicity of synergized and technical formulations of permethrin, sumithrin, and resmethrin to trout.

    PubMed

    Paul, E A; Simonin, H A; Tomajer, T M

    2005-02-01

    Synthetic pyrethroids often have synergists added to improve effectiveness, yet decisions regarding the use of these pesticides are often based upon toxicity tests using technical material without the synergist, piperonyl butoxide. We conducted toxicity tests with brook trout (Salvelinus fontinalis) and brown trout (Salmo trutta) to compare the toxicity of synergized and technical formulations of permethrin, sumithrin, and resmethrin. We found a significant increase in toxicity in the synergized permethrin formulation using traditional 24, 48, and 96-h tests, relative to tests with the technical formulation. However, there was little difference in toxicity between synergized and technical sumithrin until 48 h had elapsed. Many test fish were strongly intoxicated by either formulation of permethrin or sumithrin, but the synergized formulations of both chemicals affected fish at lower concentrations. Intoxication was potentially severe enough to reduce the survival of these fish in the wild. Following short (6-h) exposures, we also found a larger difference in the number of fish that died or became intoxicated between the synergized and technical formulations of permethrin and sumithrin. Finally, we tested the ability of exposed fish to swim against a current. Fish exposed for 6 h to synergized permethrin and resmethrin had far less swimming stamina than those exposed to technical formulations. We found no difference in the effect on swimming between the synergized and technical formulation of sumithrin. In general, the synergized formulations of these chemicals appeared to cause a faster response than the technical formulations. This response increases the lethal and sublethal impacts of the insecticides. We also found that sumithrin was the least toxic of the three pyrethroids. Since the maximum application rate of sumithrin is half that of the other two pyrethroids, the potential risk to wild trout in streams may be reduced.

  18. Isolation and initial structural characterization of a 27 kDa protein from Zingiber officinale

    NASA Astrophysics Data System (ADS)

    Rasheed, Saima; Malik, Shoaib Ahmad; Falke, Sven; Arslan, Ali; Fazel, Ramin; Schlüter, Hartmut; Betzel, Christian; Choudhary, M. Iqbal

    2018-03-01

    Zingiber officinale Roscoe (Ginger) is a widely used traditional medicinal plant (for different ailments such as arthritis, constipation, and hypertension). This article describes the isolation and characterization of a so far unknown protein from ginger rhizomes applying ion exchange, affinity, size-exclusion chromatography, small angle X-ray scattering (SAXS), and mass spectrometry techniques. One-dimensional Coomassie-stained SDS-PAGE was performed under non-reducing conditions, showing one band corresponding to approx. 27 kDa. Dynamic light scattering (DLS) analysis of the protein solution revealed monodispersity and a monomeric state of the purified protein. Circular dichroism (CD) spectroscopy strongly indicated a β-sheet-rich protein, and disordered regions. MALDI-TOF-MS, and LC-MS/MS analysis resulted in the identification of 27.29 kDa protein, having 32.13% and 25.34% sequence coverage with Zingipain-1 and 2, respectively. The monomeric state and molecular weight were verified by small angle X-ray scattering (SAXS) studies. An elongated ab-initio model was calculated based on the scattering intensity distribution.

  19. Intraneuronal accumulation of misfolded tau protein induces overexpression of Hsp27 in activated astrocytes.

    PubMed

    Filipcik, Peter; Cente, Martin; Zilka, Norbert; Smolek, Tomas; Hanes, Jozef; Kucerak, Juraj; Opattova, Alena; Kovacech, Branislav; Novak, Michal

    2015-07-01

    Accumulation of misfolded forms of microtubule associated, neuronal protein tau causes neurofibrillary degeneration typical of Alzheimer's disease and other tauopathies. This process is accompanied by elevated cellular stress and concomitant deregulation of heat-shock proteins. We used a transgenic rat model of tauopathy to study involvement of heat shock protein 27 (Hsp27) in the process of neurofibrillary degeneration, its cell type specific expression and correlation with the amount of insoluble tau protein aggregates. The expression of Hsp27-mRNA is more than doubled and levels of Hsp27 protein tripled in aged transgenic animals with tau pathology. The data revealed a strong positive and highly significant correlation between Hsp27-mRNA and amount of sarkosyl insoluble tau. Interestingly, intracellular accumulation of insoluble misfolded tau protein in neurons was associated with overexpression of Hsp27 almost exclusively in reactive astrocytes, not in neurons. The topological dissociation of neuronally expressed pathological tau and the induction of astrocytic Hsp27, GFAP, and Vimentin along with up-regulation of microglia specific markers such as CD18, CD68 and C3 point to cooperation of astrocytes, microglia and neurons in response to intra-neuronal accumulation of insoluble tau. Our data suggest that over expression of Hsp27 represents a part of microglia-mediated astrocytic response mechanism in the process of neurofibrillary degeneration, which is not necessarily associated with neuroprotection and which in contrary may accelerate neurodegeneration in late stage of the disease. This phenomenon should be considered during development of disease modifying strategies for treatment of tauopathies and AD via regulation of activity of Hsp27. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Extracellular Release and Signaling by Heat Shock Protein 27: Role in Modifying Vascular Inflammation

    PubMed Central

    Batulan, Zarah; Pulakazhi Venu, Vivek Krishna; Li, Yumei; Koumbadinga, Geremy; Alvarez-Olmedo, Daiana Gisela; Shi, Chunhua; O’Brien, Edward R.

    2016-01-01

    Heat shock protein 27 (HSP27) is traditionally viewed as an intracellular chaperone protein with anti-apoptotic properties. However, recent data indicate that a number of heat shock proteins, including HSP27, are also found in the extracellular space where they may signal via membrane receptors to alter gene transcription and cellular function. Therefore, there is increasing interest in better understanding how HSP27 is released from cells, its levels and composition in the extracellular space, and the cognate cell membrane receptors involved in effecting cell signaling. In this paper, the knowledge to date, as well as some emerging paradigms about the extracellular function of HSP27 is presented. Of particular interest is the role of HSP27 in attenuating atherogenesis by modifying lipid uptake and inflammation in the plaque. Moreover, the abundance of HSP27 in serum is an emerging new biomarker for ischemic events. Finally, HSP27 replacement therapy may represent a novel therapeutic opportunity for chronic inflammatory disorders, such as atherosclerosis. PMID:27507972

  1. Computational Analyses of Synergism in Small Molecular Network Motifs

    PubMed Central

    Zhang, Yili; Smolen, Paul; Baxter, Douglas A.; Byrne, John H.

    2014-01-01

    Cellular functions and responses to stimuli are controlled by complex regulatory networks that comprise a large diversity of molecular components and their interactions. However, achieving an intuitive understanding of the dynamical properties and responses to stimuli of these networks is hampered by their large scale and complexity. To address this issue, analyses of regulatory networks often focus on reduced models that depict distinct, reoccurring connectivity patterns referred to as motifs. Previous modeling studies have begun to characterize the dynamics of small motifs, and to describe ways in which variations in parameters affect their responses to stimuli. The present study investigates how variations in pairs of parameters affect responses in a series of ten common network motifs, identifying concurrent variations that act synergistically (or antagonistically) to alter the responses of the motifs to stimuli. Synergism (or antagonism) was quantified using degrees of nonlinear blending and additive synergism. Simulations identified concurrent variations that maximized synergism, and examined the ways in which it was affected by stimulus protocols and the architecture of a motif. Only a subset of architectures exhibited synergism following paired changes in parameters. The approach was then applied to a model describing interlocked feedback loops governing the synthesis of the CREB1 and CREB2 transcription factors. The effects of motifs on synergism for this biologically realistic model were consistent with those for the abstract models of single motifs. These results have implications for the rational design of combination drug therapies with the potential for synergistic interactions. PMID:24651495

  2. Rab27a regulates epithelial sodium channel (ENaC) activity through synaptotagmin-like protein (SLP-5) and Munc13-4 effector mechanism

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

    Saxena, Sunil K.; Horiuchi, Hisanori; Fukuda, Mitsunori

    Liddle's syndrome (excessive absorption of sodium ions) and PHA-1 (pseudohypoaldosteronism type 1) with decreased sodium absorption are caused by the mutations in the amiloride-sensitive epithelial sodium channel ENaC. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. Earlier, we reported that Rab27a inhibits ENaC-mediated currents through protein-protein interaction in HT-29 cells. We hereby report that Rab27a-dependent inhibition is associated with the GTP/GDP status as constitutively active or GTPase-deficient mutant Q78L inhibits amiloride-sensitive currents whereas GDP-locked inactive mutant T23N showed no effect. In order to further explore the molecular mechanism of this regulation, we performed competitive assays withmore » two Rab27a-binding proteins: synaptotagmin-like protein (SLP-5) and Munc13-4 (a putative priming factor for exocytosis). Both proteins eliminate negative modulation of Rab27a on ENaC function. The SLP-5 reversal of Rab27a effect was restricted to C-terminal C2A/C2B domains assigned for putative phospholipids-binding function while the Rab27a-binding SHD motif imparted higher inhibition. The ENaC-mediated currents remain unaffected by Rab27a though SLP-5 appears to strongly bind it. The immunoprecipitation experiments suggest that in the presence of excessive Munc13-4 and SLP-5 proteins, Rab27a interaction with ENaC is diminished. Munc13-4 and SLP-5 limit the Rab27a availability to ENaC, thus minimizing its effect on channel function. These observations decisively prove that Rab27a inhibits ENaC function through a complex mechanism that involves GTP/GDP status, and protein-protein interactions involving Munc13-4 and SLP-5 effector proteins.« less

  3. Identification of a 27.8 kDa protein from flounder gill cells involved in lymphocystis disease virus binding and infection.

    PubMed

    Wang, Mu; Sheng, Xiu-Zhen; Xing, Jing; Tang, Xiao-Qian; Zhan, Wen-Bin

    2011-03-16

    In vitro, lymphocystis disease virus (LCDV) infection of flounder gill (FG) cell cultures causes obvious cytopathic effect (CPE). We describe attempts to isolate and characterize the LCDV-binding molecule(s) on the plasma membrane of FG cells that were responsible for virus entry. The results showed that the co-immunoprecipitation assay detected a 27.8 kDa molecule from FG cells that bound to LCDV. In a blocking ELISA, pre-incubation of FG cell membrane proteins with the specific antiserum developed against the 27.8 kDa protein could block LCDV binding. Similarly, antiserum against 27.8 kDa protein could also inhibit LCDV infection of FG cells in vitro. Mass spectrometric analysis established that the 27.8 kDa protein and beta-actin had a strong association. These results strongly supported the possibility that the 27.8 kDa protein was the putative receptor specific for LCDV infection of FG cells.

  4. Silencing heat shock protein 27 (HSP27) inhibits the proliferation and migration of vascular smooth muscle cells in vitro.

    PubMed

    Huang, Jie; Xie, Liang-di; Luo, Li; Zheng, Su-Li; Wang, Hua-Jun; Xu, Chang-Sheng

    2014-05-01

    The objective of this study was to examine the role of heat shock protein 27 (HSP27) in proliferation and migration of vascular smooth muscle cells (VSMCs). Three complementary DNA sequences targeting rat HSP27 gene were designed, synthesized, and subcloned into lentiviral vector. The interfering efficiency was detected by reverse transcriptase-polymerase chain reaction and Western blot. Methyl thiazolyl tetrazolium bromide assay was used for examining cell proliferation. F-actin polymerization was detected by FITC-Phalloidin staining using confocal microscopy. Modified Boyden chamber technique was used to assess VSMCs migration. The recombinant lentivirus containing RNAi targeting HSP27 gene significantly inhibited expression of HSP27 at both mRNA and protein levels. The interfering efficiencies of pNL-HSP27-EGFP-1, pNL-HSP27-EGFP-2, and pNL-HSP27-EGFP-3 were 71 %, 77 %, and 43 %, respectively. Reorganization of actin stimulated by PDGF-BB was markedly blocked by pretreatment with pNL-HSP27-EGFP-2. Proliferation and migration rates of VSMCs induced by PDGF-BB were inhibited by 30.8 % and 45.6 %, respectively, by pNL-HSP27-EGFP-2 (all P < 0.01). To conclude, these data indicate that HSP27 may regulate the proliferation, actin reorganization, and the migration of VSMCs. RNAi targeting at HSP27 may be a potential approach for inhibition of cell migration involved in pathogenesis of proliferative vascular diseases.

  5. Cloning, characterization, and heat stress-induced redistribution of a protein homologous to human hsp27 in the zebrafish Danio rerio

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

    Mao Li; Bryantsev, Anton L.; Chechenova, Maria B.

    Hsp27 is a small heat shock protein (shsp) regulating stress tolerance and increasingly thought to play roles in tissue homeostasis and differentiation. The zebrafish Danio rerio is an important model for the study of developmental processes, but little is known regarding shsps in this animal. Here, we report the sequence, expression, regulation, and function of a zebrafish protein (zfHsp27) homologous to human Hsp27. zfHsp27 contains three conserved phosphorylatable serines and a cysteine important for regulation of apoptosis, but it lacks much of a C-terminal tail domain and shows low homology in two putative actin interacting domains that are features ofmore » mammalian Hsp27. zfHsp27 mRNA is most abundant in adult skeletal muscle and heart and is upregulated during early embryogenesis. zfHsp27 expressed in mammalian fibroblasts was phosphorylated in response to heat stress and anisomycin, and this phosphorylation was prevented by treatment with SB202190, an inhibitor of p38 MAPK. Expression of zfHsp27 and human Hsp27 in mammalian fibroblasts promoted a similar degree of tolerance to heat stress. zfHsp27 fusion proteins entered the nucleus and associated with the cytoskeleton of heat stressed cells in vitro and in zebrafish embryos. These results reveal conservation in regulation and function of mammalian and teleost Hsp27 proteins and define zebrafish as a new model for the study of Hsp27 function.« less

  6. EPI64B Acts as a GTPase-activating Protein for Rab27B in Pancreatic Acinar Cells*

    PubMed Central

    Hou, Yanan; Chen, Xuequn; Tolmachova, Tatyana; Ernst, Stephen A.; Williams, John A.

    2013-01-01

    The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells. PMID:23671284

  7. A General Bioinspired, Metals-Based Synergic Cross-Linking Strategy toward Mechanically Enhanced Materials.

    PubMed

    Chen, Ke; Ding, Jin; Zhang, Shuhao; Tang, Xuke; Yue, Yonghai; Guo, Lin

    2017-03-28

    Creating lightweight engineering materials combining high strength and great toughness remains a significant challenge. Despite possessing-enhanced strength and stiffness, bioinspired/polymeric materials usually suffer from clearly reduced extensibility and toughness when compared to corresponding bulk polymer materials. Herein, inspired by tiny amounts of various inorganic impurities for mechanical improvement in natural materials, we present a versatile and effective metal ion (M n+ )-based synergic cross-linking (MSC) strategy incorporating eight types of metal ions into material bulks that can drastically enhance the tensile strength (∼24.1-70.8%), toughness (∼18.6-110.1%), modulus (∼21.6-66.7%), and hardness (∼6.4-176.5%) of multiple types of pristine materials (from hydrophilic to hydrophobic and from unary to binary). More importantly, we also explore the primarily elastic-plastic deformation mechanism and brittle fracture behavior (indentation strain of >5%) of the synergic cross-linked graphene oxide (Syn-GO) paper by means of in situ nanoindentation SEM. The MSC strategy for mechanically enhanced integration can be readily attributed to the formation of the complicated metals-based cross-linking/complex networks in the interfaces and intermolecules between functional groups of materials and various metal ions that give rise to efficient energy dissipation. This work suggests a promising MSC strategy for designing advanced materials with outstanding mechanical properties by adding low amounts (<1.0 wt %) of synergic metal ions serving as synergic ion-bonding cross-linkers.

  8. Effect of the heat shock protein HSP27 on androgen receptor expression and function in prostate cancer cells.

    PubMed

    Stope, Matthias B; Schubert, Tina; Staar, Doreen; Rönnau, Cindy; Streitbörger, Andreas; Kroeger, Nils; Kubisch, Constanze; Zimmermann, Uwe; Walther, Reinhard; Burchardt, Martin

    2012-06-01

    Heat shock proteins (HSP) are involved in processes of folding, activation, trafficking and transcriptional activity of most steroid receptors including the androgen receptor (AR). Accumulating evidence links rising heat shock protein 27 (HSP27) levels with the development of castration-resistant prostate cancer. In order to study the functional relationship between HSP27 and the AR, we modulated the expression of the small heat shock protein HSP27 in human prostate cancer (PC) cell lines. HSP27 protein concentrations in LNCaP and PC-3 cells were modulated by over-expression or silencing of HSP27. The effects of HSP27 on AR protein and mRNA levels were monitored by Western blotting and quantitative RT-PCR. Treatment for the AR-positive LNCaP with HSP27-specific siRNA resulted in a down-regulation of AR levels. This down-regulation of protein was paralleled by a decrease in AR mRNA. Most interestingly, over-expression of HSP27 in PC-3 cells led to a significant increase in AR mRNA although the cells were unable to produce functional AR protein. The observation that HSP27 is involved in the regulation of AR mRNA by a yet unknown mechanism highlights the complexity of HSP27-AR signaling network.

  9. 27-Hydroxycholesterol upregulates the production of heat shock protein 60 of monocytic cells.

    PubMed

    Kim, Bo-Young; Son, Yonghae; Choi, Jeongyoon; Eo, Seong-Kug; Park, Young Chul; Kim, Koanhoi

    2017-09-01

    Investigating differentially expressed proteins in a milieu rich in cholesterol oxidation products, we found via mass spectrometry-based proteomics that surface levels of heat shock protein 60 (HSP60) were upregulated on monocytic cells in the presence of 27-hydroxycholesterol (27OHChol). The elevated levels of cytoplasmic membrane HSP60 were verified via Western blot analysis and visualized by confocal microscopy. Treatment with 27OHChol also resulted in increased levels of cellular HSP60 without altering its transcription. Cholesterol, however, did not affect cell-surface levels and cellular amount of HSP60. GSK 2033, an LXR antagonist, inhibited expression of live X receptor α, but not of HSP60, induced by 27OHChol. Treatment with 27OHChol also resulted in increased release of HSP60 from monocytic cells, but the release was significantly reduced by inhibitors of endoplasmic reticulum-Golgi protein trafficking, brefeldin A and monensin. Results of the current study indicate that 27OHChol upregulates not only cell-surface and cellular levels of HSP60 but also its release from monocytic cells, thereby contributing to activation of the immune system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Antibodies to the A27 protein of vaccinia virus neutralize and protect against infection but represent a minor component of Dryvax vaccine--induced immunity.

    PubMed

    He, Yong; Manischewitz, Jody; Meseda, Clement A; Merchlinsky, Michael; Vassell, Russell A; Sirota, Lev; Berkower, Ira; Golding, Hana; Weiss, Carol D

    2007-10-01

    The smallpox vaccine Dryvax, which consists of replication-competent vaccinia virus, elicits antibodies that play a major role in protection. Several vaccinia proteins generate neutralizing antibodies, but their importance for protection is unknown. We investigated the potency of antibodies to the A27 protein of the mature virion in neutralization and protection experiments and the contributions of A27 antibodies to Dryvax-induced immunity. Using a recombinant A27 protein (rA27), we confirmed that A27 contains neutralizing determinants and that vaccinia immune globulin (VIG) derived from Dryvax recipients contains reactivity to A27. However, VIG neutralization was not significantly reduced when A27 antibodies were removed, and antibodies elicited by an rA27 enhanced the protection conferred by VIG in passive transfer experiments. These findings demonstrate that A27 antibodies do not represent the major fraction of neutralizing activity in VIG and suggest that immunity may be augmented by vaccines and immune globulins that include strong antibody responses to A27.

  11. Identification and biochemical analysis of Slac2-c/MyRIP as a Rab27A-, myosin Va/VIIa-, and actin-binding protein.

    PubMed

    Kuroda, Taruho S; Fukuda, Mitsunori

    2005-01-01

    Slac2-c/MyRIP is a specific Rab27A-binding protein that contains an N-terminal synaptotagmin-like protein (Slp) homology domain (SHD, a newly identified GTP-Rab27A-binding motif), but in contrast to the Slp family proteins, it lacks C-terminal tandem C2 domains. In vitro Slac2-c simultaneously directly interacts with both Rab27A and an actin-based motor protein, myosin Va, via its N-terminal SHD and middle region, respectively, consistent with the fact that the overall structure of Slac2-c is similar to that of Slac2-a/melanophilin, a linker protein between Rab27A and myosin Va in the melanosome transport in melanocytes. Unlike Slac2-a, however, the middle region of Slac2-c interacts with two types of myosins, myosin Va and myosin VIIa. In addition, the most C-terminal part of both Slac2-a and Slac2-c functions as an actin-binding domain: it directly interacts with globular and fibrous actin in vitro, and the actin-binding domain of Slac2-a and Slac2-c colocalizes with actin filaments when it is expressed in living cells (i.e., PC12 cells and mouse melanocytes). In this chapter we describe the methods that have been used to analyze the protein-protein interactions of Slac2-c, specifically with Rab27A, myosin Va/VIIa, and actin.

  12. Recombinant heat shock protein 27 (HSP27/HSPB1) protects against cadmium-induced oxidative stress and toxicity in human cervical cancer cells.

    PubMed

    Alvarez-Olmedo, Daiana G; Biaggio, Veronica S; Koumbadinga, Geremy A; Gómez, Nidia N; Shi, Chunhua; Ciocca, Daniel R; Batulan, Zarah; Fanelli, Mariel A; O'Brien, Edward R

    2017-05-01

    Cadmium (Cd) is a carcinogen with several well-described toxicological effects in humans, but its molecular mechanisms are still not fully understood. Overexpression of heat shock protein 27 (HSP27/HSPB1)-a multifunctional protein chaperone-has been shown to protect cells from oxidative damage and apoptosis triggered by Cd exposure. The aims of this work were to investigate the potential use of extracellular recombinant HSP27 to prevent/counteract Cd-induced cellular toxicity and to evaluate if peroxynitrite was involved in the development of Cd-induced toxicity. Here, we report that the harmful effects of Cd correlated with changes in oxidative stress markers: upregulation of reactive oxygen species, reduction in nitric oxide (NO) bioavailability, increment in lipid peroxidation, peroxynitrite (PN), and protein nitration; intracellular HSP27 was reduced. Treatments with Cd (100 μM) for 24 h or with the peroxynitrite donor, SIN-1, decreased HSP27 levels (~50%), suggesting that PN formation is responsible for the reduction of HSP27. Pre-treatments of the cells either with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (a pharmacological inhibitor of NO synthase) or with recombinant HSP27 (rHSP27) attenuated the disruption of the cellular metabolism induced by Cd, increasing in a 55 and 52%, respectively, the cell viability measured by CCK-8. Cd induced necrotic cell death pathways, although apoptosis was also activated; pre-treatment with L-NAME or rHSP27 mitigated cell death. Our findings show for the first time a direct relationship between Cd-induced toxicity and PN production and a role for rHSP27 as a potential therapeutic agent that may counteract Cd toxicity.

  13. Cell cycle regulation by the intrinsically disordered proteins p21 and p27.

    PubMed

    Yoon, Mi-Kyung; Mitrea, Diana M; Ou, Li; Kriwacki, Richard W

    2012-10-01

    Today, it is widely accepted that proteins that lack highly defined globular three-dimensional structures, termed IDPs (intrinsically disordered proteins), play key roles in myriad biological processes. Our understanding of how intrinsic disorder mediates biological function is, however, incomplete. In the present paper, we review disorder-mediated cell cycle regulation by two intrinsically disordered proteins, p21 and p27. A structural adaptation mechanism involving a stretchable dynamic linker helix allows p21 to promiscuously recognize the various Cdk (cyclin-dependent kinase)-cyclin complexes that regulate cell division. Disorder within p27 mediates transmission of an N-terminal tyrosine phosphorylation signal to a C-terminal threonine phosphorylation, constituting a signalling conduit. These mechanisms are mediated by folding upon binding p21/p27's regulatory targets. However, residual disorder within the bound state contributes critically to these functional mechanisms. Our studies provide insights into how intrinsic protein disorder mediates regulatory processes and opportunities for designing drugs that target cancer-associated IDPs.

  14. Heat shock protein 27 is a potential indicator for response to YangZheng XiaoJi and chemotherapy agents in cancer cells.

    PubMed

    Owen, Sioned; Zhao, Huishan; Dart, Alwyn; Wang, Yamei; Ruge, Fiona; Gao, Yong; Wei, Cong; Wu, Yiling; Jiang, Wen G

    2016-11-01

    Heat shock protein 27 (HSP27) is a member of the heat shock protein family which has been linked to tumour progression and, most interestingly, to chemotherapy resistance in cancer patients. The present study examined the potential interplay between HSP27 and YangZheng XiaoJi, a traditional Chinese medicine used in cancer treatment. A range of cell lines from different tumour types including pancreatic, lung, gastric, colorectal, breast, prostate and ovarian cancer (both wild-type and resistant) were used. Levels and activation of HSP27 and its potential associated signalling pathways were evaluated by protein array and western blotting. Knockdown of HSP27 in cancer cells was achieved using siRNA. Localisation and co-localisation of HSP27 and other proteins were carried out by immunofluorescence. Cell growth and migration were evaluated in their response to a range of chemotherapeutic agents. The present study first identified, by way of protein array, that YangZheng XiaoJi was able to inhibit the phosphorylation of HSP27 protein in cancer cells. We further demonstrated that HSP27, which is co-localised with caspase-9, can be blocked from localising in focal adhesions and co-localising with caspase-9 by YangZheng XiaoJi. The study also demonstrated that YangZheng XiaoJi was able to sensitise cancer cells including those cells that were resistant to chemotherapy, to chemotherapeutic agents. Finally, knocking down HSP27 markedly reduced the migration of cancer cells and increased the sensitivity of cancer cells to the inhibitory effect on cellular migration by YangZheng XiaoJi. YangZheng XiaoJi can act as an agent in first sensitising cancer cells to chemotherapy and secondly to overcome, to some degree, chemoresistance when used in an appropriate fashion in patients who have active HSP27.

  15. Levamisole and cocaine synergism: a prevalent adulterant enhances cocaine's action in vivo.

    PubMed

    Tallarida, Christopher S; Egan, Erin; Alejo, Gissel D; Raffa, Robert; Tallarida, Ronald J; Rawls, Scott M

    2014-04-01

    Levamisole is estimated by the Drug Enforcement Agency (DEA) to be present in about 80% of cocaine seized in the United States and linked to debilitating, and sometimes fatal, immunologic effects in cocaine abusers. One explanation for the addition of levamisole to cocaine is that it increases the amount of product and enhances profits. An alternative possibility, and one investigated here, is that levamisole alters cocaine's action in vivo. We specifically investigated effects of levamisole on cocaine's stereotypical and place-conditioning effects in an established invertebrate (planarian) assay. Acute exposure to levamisole or cocaine produced concentration-dependent increases in stereotyped movements. For combined administration of the two agents, isobolographic analysis revealed that the observed stereotypical response was enhanced relative to the predicted effect, indicating synergism for the interaction. In conditioned place preference (CPP) experiments, cocaine produced a significant preference shift; in contrast, levamisole was ineffective at all concentrations tested. For combination experiments, a submaximal concentration of cocaine produced CPP that was enhanced by inactive concentrations of levamisole, indicating synergism. The present results provide the first experimental evidence that levamisole enhances cocaine's action in vivo. Most important is the identification of synergism for the levamisole/cocaine interaction, which now requires further study in mammals. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models

    PubMed Central

    Ramalho, José S; Anders, Ross; Jaissle, Gesine B; Seeliger, Mathias W; Huxley, Clare; Seabra, Miguel C

    2002-01-01

    Background Transgenic mice have proven to be a powerful system to study normal and pathological gene functions. Here we describe an attempt to generate a transgenic mouse model for choroideremia (CHM), a slow-onset X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP1) gene. REP1 is part of the Rab geranylgeranylation machinery, a modification that is essential for Rab function in membrane traffic. The loss of REP1 in CHM patients may trigger retinal degeneration through its effects on Rab proteins. We have previously reported that Rab27a is the Rab most affected in CHM lymphoblasts and hypothesised that the selective dysfunction of Rab27a (and possibly a few other Rab GTPases) plays an essential role in the retinal degenerative process. Results To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b) transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous β-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal. Conclusions We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases. PMID:12401133

  17. Cellular Hsp27 interacts with classical swine fever virus NS5A protein and negatively regulates viral replication by the NF-κB signaling pathway.

    PubMed

    Ling, Shifeng; Luo, Mingyang; Jiang, Shengnan; Liu, Jiayu; Ding, Chunying; Zhang, Qinghuan; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

    2018-05-01

    Classical swine fever virus (CSFV) nonstructural protein NS5A is a multifunctional protein functioning in regulation of viral genome replication, protein translation and assembly by interaction with viral or host proteins. Here, heat shock protein 27 (Hsp27) has been identified as a novel binding partner of NS5A by using His tag "pull down" coupled with shotgun LC-MS/MS, with interaction of both proteins further confirmed by co-immunoprecipitation and laser confocal assays. In PK-15 cells, silencing of Hsp27 expression by siRNA enhanced CSFV replication, and upregulation of Hsp27 inhibited viral proliferation. Additionally, we have shown that overexpression of Hsp27 increased NF-κB signaling induced by TNFα. Blocking NF-κB signaling in PK-15 cells overexpressing Hsp27 by ammonium pyrrolidinedithiocarbamate (PDTC) eliminated the inhibition of CSFV replication by Hsp27. These findings clearly demonstrate that the inhibition of CSFV replication by Hsp27 is mediated via the NF-κB signaling pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Regulation of fat specific protein 27 by isoproterenol and TNF-alpha to control lipolysis in murine adipocytes

    USDA-ARS?s Scientific Manuscript database

    The lipid droplet-associated fat specific protein 27 (FSP27) suppresses lipolysis and thereby enhances triglyceride accumulation in adipocytes. We and others have recently found FSP27 to be a remarkably short-lived protein (half-life, 15 min) due to its rapid ubiquitination and proteasomal degradati...

  19. Anti-vRE and anti-MRSA activities of new quinolones and their synergism with commercial antibiotics. Part 2.

    PubMed

    Sakagami, Yoshikazu; Komemushi, Sadao; Tsukamoto, Goro; Kondo, Hirosato; Yoshikawa, Akiko; Muraoka, Osamu

    2008-09-01

    Anti-VRE and anti-MRSA activities of new quinolone derivatives [The two quinolone derivatives are 8- [3-[(ethylamino) methyl]-1-pyrrodinyl] -7-fluoro-9, 1-[(N-methylimino)methano]-5-oxo-5H-thiazolo[3,2-a]quinolone-4-carboxylic acid (compound A) and 7-fluoro-8-morpholino-9,1-[(N-methylimino) methanol-5-oxo-5H-thiazolo [3,2-a] quinolone-4-carboxylic acid (compound B)] and their synergism with commercial antibiotics were investigated. Compound A exhibited potent antibacterial activity against VRE and MRSA among the five new quinolone compounds tested, and showed superior activity to pefloxacin, ofloxacin and levofloxacin, which are clinically in use these days. With respect to the anti-VRE activity, compound A showed synergism with fosfomycin (FOM), and partial synergism with ampicillin (ABPC), gentaicin (GM), minocycline (MINO) and vancomycin hydrochloride (VCM). Partial synergism in anti-VRE activity was also observed between compound B and GM, MINO, FOM and VCM. Compound A also showed synergism with MINO and FOM in anti-MASA activity. Partial synergism was observed with ABPC, GM and VCM. Synergism with ABPC was not detected in anti-MRSA activity. On the other hand, the synergism of compound B with FOM, and the partial synergisms with ABPC, GM and MINO were also found against MRSA. No synergism with ABPC was found against MRSA. These results suggested that compound A and B could possibly reduce the daily administration dose of these antibiotics in the treatment of nosocomial infections, and also reduce the possibility of the occurrence of nosocomial infections caused by VRE and/or MRSA.

  20. BRD4 inhibitor IBET upregulates p27kip/cip protein stability in neuroendocrine tumor cells.

    PubMed

    Wang, Lei; Matkar, Smita; Xie, Gengchen; An, Chiying; He, Xin; Kong, Xiangchen; Liu, Xiuheng; Hua, Xianxin

    2017-04-03

    The prevalence of neuroendocrine tumors (NETs) has recently been increasing. Although various drugs such as Octreotide and its analogs show certain efficacy, NETs in many patients progress and metastasize. It is desirable to develop new interventions to improve the therapy. Here we show that human neuroendocrine tumor BON cells are resistant to several drugs commonly used for NET therapy, including Octreotide that activates somatostatin receptor-induced anti-proliferation, and Capecitabine and Temozolimide that damage DNA. In contrast, an inhibitor (IBET) to an epigenetic regulator, Brd4 that binds acetylated histones and upregulates transcription of multiple genes including protooncogene c-Myc, potently inhibited the NET cells. We found that IBET increased the protein levels of cyclin-dependent kinase (CDK) inhibitor p27 kip/cip (or p27), but not its mRNA levels. Moreover, the p27 induction at protein level by IBET was at least partly through increasing the protein stability of p27. The increased protein stability of p27 likely resulted from IBET-mediated suppression of Skp2, an E3 ligase that can mediate p27 degradation by increasing its ubiquitinylation. These findings unravel a new mechanism whereby the IBET-induced repression of proliferation of neuroendocrine cells.

  1. The small heat shock protein Hsp27: Present understanding and future prospects.

    PubMed

    Singh, Manish Kumar; Sharma, Bechan; Tiwari, Pramod K

    2017-10-01

    Heat shock proteins are important for maintaining protein homeostasis and cell survival. Among different classes of highly conserved Hsps, low molecular weight Hsps (sHsps) have significant place, particularly Hsp27, whose role has been demonstrated in wide range of biological processes, including development, immunity, diseases and therapy. In this review, the structure and functions of Hsp27 and related genes, their role in different cellular processes as well as in stress tolerance, is highlighted. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Hsp27 as a therapeutic target in cancers.

    PubMed

    Acunzo, Julie; Andrieu, Claudia; Baylot, Virginie; So, Alan; Rocchi, Palma

    2014-04-01

    Heat shock protein 27 (Hsp27), induced by heat shock, environmental and pathophysiological stressors, is a multidimensional protein that acts as a protein chaperone and an antioxidant. This protein plays a major role in the inhibition of apoptosis and actin cytoskeletal remodeling. This stress-activated protein is up-regulated in many cancers and is associated with poor prognosis as well as treatment resistance by protecting cells from therapeutic agent that normally induces apoptosis. This review highlights the most recent findings and role of Hsp27 in cancer and the different strategies to target and inhibit Hsp27 for clinical purposes.

  3. Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress

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

    Bryantsev, A.L.; Chechenova, M.B.; Shelden, E.A.

    During stress, the mammalian small heat shock protein Hsp27 enters cell nuclei. The present study examines the requirements for entry of Hsp27 into nuclei of normal rat kidney (NRK) renal epithelial cells, and for its interactions with specific nuclear structures. We find that phosphorylation of Hsp27 is necessary for the efficient entry into nuclei during heat shock but not sufficient for efficient nuclear entry under control conditions. We further report that Hsp27 is recruited to an RNAse sensitive fraction of SC35 positive nuclear speckles, but not other intranuclear structures, in response to heat shock. Intriguingly, Hsp27 phosphorylation, in the absencemore » of stress, is sufficient for recruitment to speckles found in post-anaphase stage mitotic cells. Additionally, pseudophosphorylated Hsp27 fused to a nuclear localization peptide (NLS) is recruited to nuclear speckles in unstressed interphase cells, but wildtype and nonphosphorylatable Hsp27 NLS fusion proteins are not. The expression of NLS-Hsp27 mutants does not enhance colony forming abilities of cells subjected to severe heat shock, but does regulate nuclear speckle morphology. These data demonstrate that phosphorylation, but not stress, mediates Hsp27 recruitment to an RNAse soluble fraction of nuclear speckles and support a site-specific role for Hsp27 within the nucleus.« less

  4. Small heat shock protein 27: An effective adjuvant for enhancement of HIV-1 Nef antigen-specific immunity.

    PubMed

    Milani, Alireza; Bolhassani, Azam; Shahbazi, Sepideh; Motevalli, Fatemeh; Sadat, Seyed Mehdi; Soleymani, Sepehr

    2017-11-01

    Novel vaccine modalities have been designed to improve the efficiency of vaccines against HIV infections. In this way, the HIV-1 Nef protein has been known as an attractive antigenic candidate in therapeutic vaccine development. Moreover, the endogenous adjuvants such as heat shock proteins (HSPs) and high mobility group box 1 protein (HMGB1) have been suggested effectively to induce antigen-specific humoral and cellular immune responses. In this study, different Nef DNA and protein constructs were produced in eukaryotic and prokaryotic expression systems, and their immunostimulatory properties were evaluated using small heat shock protein 27 (Hsp27) and the HMGB1-derived peptide (Hp91) in a mouse model. Generally, our results indicated that the Hsp27-Nef fusion DNA or protein could significantly elicit higher humoral and cellular immune responses than Nef DNA or protein, respectively. Analysis of the immune responses demonstrated that the Hsp27-Nef fusion protein, and also the mixture of Nef and Hp91 significantly enhanced the Nef-specific T cell responses. Indeed, these regimens induced high levels of IgG2a and IFN-γ directed toward Th1 responses and also Granzyme B secretion as compared to other immunization strategies. The immunostimulatory properties of Freund's adjuvant were significantly less than Hsp27 and Hp91 peptide in various immunization strategies. These findings showed that the use of Hsp27 and Hp91 in protein strategy could improve HIV-1 Nef-specific B- and T-cell immune responses, and also represent a promising HIV-1 vaccine candidate in future. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  5. Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.

    PubMed

    Johnson, Jennifer L; Pacquelet, Sandrine; Lane, William S; Eam, Boreth; Catz, Sergio D

    2005-08-01

    Here, we show that the Rab27a-binding protein JFC1/Slp1 (synaptotagmin-like protein) is regulated by Akt-mediated phosphorylation. Using the phosphatase and tensin homolog-null LNCaP cells and the phosphatidylinositol 3-kinase inhibitor LY294002, we show that the phosphorylation of endogenous JFC1 is dependent on the phosphatidylinositol 3-kinase/Akt pathway. JFC1 was phosphorylated in cells expressing a constitutively active Akt, confirming that it is an Akt substrate in vivo. Direct phosphorylation of JFC1 by Akt was confirmed in vitro. Using microcapillary high-performance liquid chromatography tandem mass spectrometry, we identified five Akt-phosphorylation sites in JFC1. By mutagenesis analysis and subsequent immunoprecipitation (IP), we established that Akt phosphorylates JFC1 at serine 241. JFC1 and Rab27a colocalize in the proximity of the plasma membrane in LNCaP cells. The interaction was confirmed by IP analysis and was abolished by the point mutation W83S in JFC1. Phosphorylation did not alter the ability of JFC1 to bind to Rab27a. Instead, phosphorylation by Akt dramatically decreased when JFC1 was bound to Rab27a. Finally, we show that as a consequence of in vivo phosphorylation, JFC1 dissociates from the membrane, promoting JFC1 redistribution to the cytosol. Our results suggest that Akt regulates JFC1/Slp1 function by phosphorylation and may have implications on Rab27a-containing vesicle secretion.

  6. 27-Hydroxycholesterol increases Myc protein stability via suppressing PP2A, SCP1 and FBW7 transcription in MCF-7 breast cancer cells

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

    Ma, Li-Ming; Liang, Zi-Rui; Zhou, Ke-Ren

    27-hydroxycholesterol (27-HC), the most abundant metabolite of cholesterol, is a risk factor for breast cancer. It can increase the proliferation of breast cancer cells and promote the metastasis of breast tumours in mouse models. Myc is a critical oncoprotein overexpressed in breast cancer. However, whether 27-HC affects Myc expression has not been reported. In the current study, we aimed to investigate the effects of 27-HC on Myc and the underlying mechanisms in MCF-7 breast cancer cells. Our data demonstrated that 27-HC activated Myc via increasing its protein stability. Three key negative modulators of Myc protein stability, PP2A, SCP1 and FBW7,more » were suppressed by 27-HC at the transcriptional level. We performed a data-mining analysis of the chromatin immunoprecipitation with next-generation DNA sequencing (ChIP-Seq) data in the ChIPBase, and discovered that a number of putative transcription factors (TFs), including Myc itself, were involved in the transcriptional regulation of PP2A, SCP1 and FBW7. Our results provide a novel mechanistic insight into the activation of Myc by 27-HC via transcriptional repression of PP2A, SCP1 and FBW7 to increase Myc protein stability in breast cancer cells. - Highlights: • 27-Hydroxycholesterol (27-HC) activates Myc via increasing its protein stability. • 27-HC inhibits PP2A and SCP1 transcription to block pS62-Myc dephosphorylation. • 27-HC suppresses FBW7 transcription to prevent pT58-Myc degradation.« less

  7. [Apoptosis-modulating effects of heat shock proteins: the influence of Hsp27 chaperone on TBA Bcl-2 family proteins in Jurkat cell line].

    PubMed

    Riazantseva, N V; Kaĭgorodova, E V; Maroshkina, A N; Belkina, M V; Novitskiĭ, V V

    2012-01-01

    The in vitro phosphorylated and non-phosphorylated Hsp27 forms concentrations and Bcl-2 proteins affected by Hsp27 inhibition were studied in Jurkat-line tumor cells and healthy donor mononuclear lymphocytes by Western blotting technique. The Hsp27 inhibition causes the increase of intracellular Bax protein concentration and the decrease of Bcl-2 level leading to an increase of apoptotic changes in Jurkat line cells.

  8. A synergic approach to terraforming Mars

    NASA Astrophysics Data System (ADS)

    Fogg, Martyn J.

    1992-08-01

    A two-stage terraforming scenario is outlined for Mars. The approach adopted differs from past methodology in two ways. It adopts a more conservative and plausible Martian volatile inventory. Possible planetary engineering solutions, including possible synergic use of terraforming techniques, are examined in detail. In the first stage, the Martian environment is modified to a state where it can support microbial and hardy plant life in approximately 200 years. While this step is conceptually similar to past scenarios, it differs greatly in detail. The second stage deals with the creation of conditions tolerable for human beings over a period of approximately 21,000 years. It is concluded that terraforming Mars is possible but not by the passive, or near-spontaneous, methods favored by some workers. A powerful industrial effort is required both on the planet's surface and in space as will be continuing technological intervention to stabilize the postterraformed regime.

  9. Association between circulating levels of heat-shock protein 27 and aggressive periodontitis.

    PubMed

    Kaiser, Frank; Donos, Nikos; Henderson, Brian; Alagarswamy, Rajesh; Pelekos, George; Boniface, David; Nibali, Luigi

    2018-05-15

    Heat-shock protein (Hsp) 27 is a major intracellular molecular chaperone and controller of intracellular responses to inflammatory signals. In the extracellular space, recombinant Hsp27 has been described to exert anti-inflammatory activities. The aim of this study was to assess the association between circulating levels of Hsp27 and different types of periodontitis. Pro- and anti-inflammatory cytokines and the stress proteins Hsp27 and Hsp60 with proposed anti- and pro-inflammatory properties, respectively, were measured by two-site ELISA in the serum of patients with aggressive periodontitis (AgP, n = 30), chronic periodontitis (CP, n = 29) and periodontally healthy controls (H, n = 28). Furthermore, Hsp27 and Hsp60 levels were also measured longitudinally in 12 AgP patients at 6 time points up to 3 months after treatment. AgP patients had lower levels of Hsp27 compared to CP patients and healthy subjects (adjusted one-way ANOVA, p < 0.001, followed by post hoc Tukey HSD comparisons), while no differences in levels of Hsp60 or cytokines between the three groups were detected. In CP patients and H subjects, the systemic Hsp27 levels correlated with Hsp60 (r = 0.43, p < 0.001; r = 0.59, p < 0.001, respectively) and with pro-inflammatory cytokines TNF-α (r = 0.48, p < 0.001; r = 0.55, p < 0.001, respectively) and IL-6 (r = 0.44, p < 0.01). However, no such correlations were detected in AgP cases. No consistent temporal patterns of changes of Hsp27 concentration were detected across AgP patients following periodontal treatment. This study provides the first evidence that Hsp27 may be differentially expressed and regulated in AgP patients as compared with CP patients and healthy individuals.

  10. Heat-shock protein 27 (HSP27, HSPB1) is up-regulated by MET kinase inhibitors and confers resistance to MET-targeted therapy

    PubMed Central

    Musiani, Daniele; Konda, John David; Pavan, Simona; Torchiaro, Erica; Sassi, Francesco; Noghero, Alessio; Erriquez, Jessica; Perera, Timothy; Olivero, Martina; Di Renzo, Maria Flavia

    2014-01-01

    The tyrosine kinase encoded by the MET oncogene is activated by gene mutation or amplification in tumors, which in most instances maintain addiction, i.e., dependency, to MET activation. This makes MET an attractive candidate for targeted therapies. Here we show that, in 3/3 MET-addicted human gastric cancer cell lines, MET kinase inhibition resulted in a 3- to 4-fold increased expression of the antiapoptotic small heat-shock protein of 27 kDa (HSP27, HSPB1). HSP27 increase depended on the inhibition of the MEK/ERK pathway and on heat-shock factor 1 (HSF1) and hypoxia-inducible factor-1α (HIF-1α) regulation. Importantly, HSP27-silenced MET-addicted cells underwent 2- and 3-fold more apoptosis following MET inhibition in vitro and in vivo, respectively. Likewise, in human cancer cells susceptible to epidermal growth factor receptor (EGFR) inhibition, EGFR inhibitors induced HSP27 expression and were strengthened by HSP27 suppression. In control cell lines that were not affected by drugs targeting MET or EGFR, these drugs did not induce HSP27 increase. Therefore, in cancer therapies targeting the MET pathway, the induction of HSP27 might limit the efficacy of anti-MET agents. As HSP27 increase also impairs the effectiveness of EGFR inhibitors and is known to protect cells from chemotherapeutics, the induction of HSP27 by targeted agents might strongly affect the success of combination treatments.—Musiani, D., Konda, J. D., Pavan, S., Torchiaro, E., Sassi, F., Noghero, A., Erriquez, J., Perera, T., Olivero, M., Di Renzo, M. F. Heat-shock protein 27 (HSP27, HSPB1) is up-regulated by MET kinase inhibitors and confers resistance to MET-targeted therapy. PMID:24903273

  11. Human Myocardium Releases Heat Shock Protein 27 (HSP27) after Global Ischemia: The Proinflammatory Effect of Extracellular HSP27 through Toll-like Receptor (TLR)-2 and TLR4

    PubMed Central

    Jin, Chunhua; Cleveland, Joseph C; Ao, Lihua; Li, Jilin; Zeng, Qingchun; Fullerton, David A; Meng, Xianzhong

    2014-01-01

    The myocardial inflammatory response contributes to cardiac functional injury associated with heart surgery obligating global ischemia/reperfusion (I/R). Toll-like receptors (TLRs) play an important role in the mechanism underlying myocardial I/R injury. The aim of this study was to examine the release of small constitutive heat shock proteins (HSPs) from human and mouse myocardium after global ischemia and examine the role of extracellular small HSP in myocardial injury. HSP27 release was assessed by enzyme-linked immunosorbent assay. Anti-HSP27 was applied to evaluate the role of extracellular HSP27 in the postischemic inflammatory response and functional injury in mouse hearts. Isolated hearts and cultured coronary vascular endothelial cells were exposed to recombinant HSP27 to determine its effect on proinflammatory signaling and production of proinflammatory mediators. HSP27 levels were markedly elevated in coronary sinus blood of patients and in coronary effluent of mouse hearts after global ischemia. Neutralizing extracellular HSP27 suppressed myocardial nuclear factor (NF)-κB activation and interleukin (IL)-6 production and improved cardiac function in mouse hearts. Perfusion of HSP27 to mouse hearts induced NF-κB activation and IL-6 production and depressed contractility. Further, recombinant HSP27 induced NF-κB phosphorylation and upregulated monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1 production in both human and mouse coronary vascular endothelial cells. TLR2 knockout (KO) or TLR4 mutation abolished NF-κB phosphorylation and reduced MCP-1 and ICAM-1 production induced by extracellular HSP27 in endothelial cells. In conclusion, these results show that the myocardium releases HSP27 after global ischemia and that extracellular HSP27 is proinflammatory and contributes to the inflammatory mechanism of myocardial functional injury. Both TLR2 and TLR4 are involved in mediating the proinflammatory effect of

  12. Human myocardium releases heat shock protein 27 (HSP27) after global ischemia: the proinflammatory effect of extracellular HSP27 through toll-like receptor (TLR)-2 and TLR4.

    PubMed

    Jin, Chunhua; Cleveland, Joseph C; Ao, Lihua; Li, Jilin; Zeng, Qingchun; Fullerton, David A; Meng, Xianzhong

    2014-06-09

    The myocardial inflammatory response contributes to cardiac functional injury associated with heart surgery obligating global ischemia/reperfusion (I/R). Toll-like receptors (TLRs) play an important role in the mechanism underlying myocardial I/R injury. The aim of this study was to examine the release of small constitutive heat shock proteins (HSPs) from human and mouse myocardium after global ischemia and examine the role of extracellular small HSP in myocardial injury. HSP27 release was assessed by enzyme-linked immunosorbent assay. Anti-HSP27 was applied to evaluate the role of extracellular HSP27 in the postischemic inflammatory response and functional injury in mouse hearts. Isolated hearts and cultured coronary vascular endothelial cells were exposed to recombinant HSP27 to determine its effect on proinflammatory signaling and production of proinflammatory mediators. HSP27 levels were markedly elevated in coronary sinus blood of patients and in coronary effluent of mouse hearts after global ischemia. Neutralizing extracellular HSP27 suppressed myocardial nuclear factor (NF)-κB activation and interleukin (IL)-6 production and improved cardiac function in mouse hearts. Perfusion of HSP27 to mouse hearts induced NF-κB activation and IL-6 production and depressed contractility. Further, recombinant HSP27 induced NF-κB phosphorylation and upregulated monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1 production in both human and mouse coronary vascular endothelial cells. TLR2 knockout (KO) or TLR4 mutation abolished NF-κB phosphorylation and reduced MCP-1 and ICAM-1 production induced by extracellular HSP27 in endothelial cells. In conclusion, these results show that the myocardium releases HSP27 after global ischemia and that extracellular HSP27 is proinflammatory and contributes to the inflammatory mechanism of myocardial functional injury. Both TLR2 and TLR4 are involved in mediating the proinflammatory effect of

  13. HSP27, 70 and 90, anti-apoptotic proteins, in clinical cancer therapy (Review).

    PubMed

    Wang, Xiaoxia; Chen, Meijuan; Zhou, Jing; Zhang, Xu

    2014-07-01

    Among the heat shock proteins (HSP), HSP27, HSP70 and HSP90 are the most studied stress-inducible HSPs, and are induced in response to a wide variety of physiological and environmental insults, thus allowing cells to survive to lethal conditions based on their powerful cytoprotective functions. Different functions of HSPs have been described to explain their cytoprotective functions, including their most basic role as molecular chaperones, that is to regulate protein folding, transport, translocation and assembly, especially helping in the refolding of misfolded proteins, as well as their anti-apoptotic properties. In cancer cells, the expression and/or activity of the three HSPs is abnormally high, and is associated with increased tumorigenicity, metastatic potential of cancer cells and resistance to chemotherapy. Associating with key apoptotic factors, they are powerful anti-apoptotic proteins, having the capacity to block the cell death process at different levels. Altogether, the properties suggest that HSP27, HSP70 and HSP90 are appropriate targets for modulating cell death pathways. In this review, we summarize the role of HSP90, HSP70 and HSP27 in apoptosis and the emerging strategies that have been developed for cancer therapy based on the inhibition of the three HSPs.

  14. A 32 kDa viral attachment protein of lymphocystis disease virus (LCDV) specifically interacts with a 27.8 kDa cellular receptor from flounder (Paralichthys olivaceus).

    PubMed

    Zhong, Ying; Fei, Chenjie; Tang, Xiaoqian; Zhan, Wenbin; Sheng, Xiuzhen

    2017-06-01

    The 27.8 kDa protein in flounder gill (FG) cells was previously proved to be a receptor specific for lymphocystis disease virus (LCDV) entry and infection. In this paper, a 32 kDa viral attachment protein (VAP) of LCDV specifically binding to the 27.8 kDa receptor (27.8R) was found by far-Western blotting coupled with monoclonal antibodies (MAbs) against 27.8R. The 32 kDa protein was confirmed to be encoded by the open reading frame (ORF) 038 gene in LCDV-C, and predicted to contain a putative transmembrane region, multiple N-myristoylation and glycosylation sites and phosphorylation motifs. The expression plasmid of pET-32a-ORF038 was constructed and the recombinant VAP (rVAP) was obtained. Rabbit polyclonal antibodies against the rVAP were prepared and could recognize the rVAP and 32 kDa protein in LCDV. Immunogold electron microscopy showed that the 32 kDa protein was located on the surface of LCDV particles. Immunofluorescence assay demonstrated that the rVAP could bind to the 27.8R on the cell membrane of the FG monolayer and the anti-27.8R MAbs could block the rVAP binding. Pre-incubation of the rVAP with FG cells before LCDV infection, or pre-incubation of LCDV with the antibodies against the rVAP, could significantly decrease the LCDV copy numbers (P<0.05) and delay the emergence of cytopathic effects in FG cells in a dose-dependent manner. These results indicated for the first time that the 32 kDa protein functioned as an attachment protein for the initial attachment and entry of LCDV, and the interaction of the 32 kDa VAP with the 27.8R-initiated LCDV infection.

  15. Differential Roles of AC2 and AC4 of Cassava Geminiviruses in Mediating Synergism and Suppression of Posttranscriptional Gene Silencing

    PubMed Central

    Vanitharani, Ramachandran; Chellappan, Padmanabhan; Pita, Justin S.; Fauquet, Claude M.

    2004-01-01

    Posttranscriptional gene silencing (PTGS) in plants is a natural defense mechanism against virus infection. In mixed infections, virus synergism is proposed to result from suppression of the host defense mechanism by the viruses. Synergistic severe mosaic disease caused by simultaneous infection with isolates of the Cameroon strain of African cassava mosaic virus (ACMV-[CM]) and East African cassava mosaic Cameroon virus (EACMCV) in cassava and tobacco is characterized by a dramatic increase in symptom severity and a severalfold increase in viral-DNA accumulation by both viruses compared to that in singly infected plants. Here, we report that synergism between ACMV-[CM] and EACMCV is a two-way process, as the presence of the DNA-A component of ACMV-[CM] or EACMCV in trans enhanced the accumulation of viral DNA of EACMCV and ACMV-[CM], respectively, in tobacco BY-2 protoplasts. Furthermore, transient expression of ACMV-[CM] AC4 driven by the Cauliflower mosaic virus 35S promoter (p35S-AC4) enhanced EACMCV DNA accumulation by ∼8-fold in protoplasts, while p35S-AC2 of EACMCV enhanced ACMV-[CM] DNA accumulation, also by ∼8-fold. An Agrobacterium-based leaf infiltration assay determined that ACMV-[CM] AC4 and EACMCV AC2, the putative synergistic genes, were able to suppress PTGS induced by green fluorescent protein (GFP) and eliminated the short interfering RNAs associated with PTGS, with a correlated increase in GFP mRNA accumulation. In addition, we have identified AC4 of Sri Lankan cassava mosaic virus and AC2 of Indian cassava mosaic virus as suppressors of PTGS, indicating that geminiviruses evolved differently in regard to interaction with the host. The specific and different roles played by these AC2 and AC4 proteins of cassava geminiviruses in regulating anti-PTGS activity and their relation to synergism are discussed. PMID:15308741

  16. Mechanism underlying selective regulation of G protein-gated inwardly rectifying potassium channels by the psychostimulant-sensitive sorting nexin 27

    PubMed Central

    Balana, Bartosz; Maslennikov, Innokentiy; Kwiatkowski, Witek; Stern, Kalyn M.; Bahima, Laia; Choe, Senyon; Slesinger, Paul A.

    2011-01-01

    G protein-gated inwardly rectifying potassium (GIRK) channels are important gatekeepers of neuronal excitability. The surface expression of neuronal GIRK channels is regulated by the psychostimulant-sensitive sorting nexin 27 (SNX27) protein through a class I (-X-Ser/Thr-X-Φ, where X is any residue and Φ is a hydrophobic amino acid) PDZ-binding interaction. The G protein-insensitive inward rectifier channel (IRK1) contains the same class I PDZ-binding motif but associates with a different synaptic PDZ protein, postsynaptic density protein 95 (PSD95). The mechanism by which SNX27 and PSD95 discriminate these channels was previously unclear. Using high-resolution structures coupled with biochemical and functional analyses, we identified key amino acids upstream of the channel's canonical PDZ-binding motif that associate electrostatically with a unique structural pocket in the SNX27-PDZ domain. Changing specific charged residues in the channel's carboxyl terminus or in the PDZ domain converts the selective association and functional regulation by SNX27. Elucidation of this unique interaction site between ion channels and PDZ-containing proteins could provide a therapeutic target for treating brain diseases. PMID:21422294

  17. A PARP1-ERK2 synergism is required for the induction of LTP.

    PubMed

    Visochek, L; Grigoryan, G; Kalal, A; Milshtein-Parush, H; Gazit, N; Slutsky, I; Yeheskel, A; Shainberg, A; Castiel, A; Seger, R; Langelier, M F; Dantzer, F; Pascal, J M; Segal, M; Cohen-Armon, M

    2016-04-28

    Unexpectedly, a post-translational modification of DNA-binding proteins, initiating the cell response to single-strand DNA damage, was also required for long-term memory acquisition in a variety of learning paradigms. Our findings disclose a molecular mechanism based on PARP1-Erk synergism, which may underlie this phenomenon. A stimulation induced PARP1 binding to phosphorylated Erk2 in the chromatin of cerebral neurons caused Erk-induced PARP1 activation, rendering transcription factors and promoters of immediate early genes (IEG) accessible to PARP1-bound phosphorylated Erk2. Thus, Erk-induced PARP1 activation mediated IEG expression implicated in long-term memory. PARP1 inhibition, silencing, or genetic deletion abrogated stimulation-induced Erk-recruitment to IEG promoters, gene expression and LTP generation in hippocampal CA3-CA1-connections. Moreover, a predominant binding of PARP1 to single-strand DNA breaks, occluding its Erk binding sites, suppressed IEG expression and prevented the generation of LTP. These findings outline a PARP1-dependent mechanism required for LTP generation, which may be implicated in long-term memory acquisition and in its deterioration in senescence.

  18. Heat-shock protein 27 (HSP27, HSPB1) is synthetic lethal to cells with oncogenic activation of MET, EGFR and BRAF.

    PubMed

    Konda, John D; Olivero, Martina; Musiani, Daniele; Lamba, Simona; Di Renzo, Maria F

    2017-06-01

    The small heat-shock protein of 27 kDa (HSP27) is highly expressed in many cancers and is associated with aggressive tumour behaviour, metastasis, poor prognosis and resistance to chemotherapy. We aimed at assessing the role of HSP27 in modulating responses to target therapies. We selected several oncogene-addicted cancer cell lines, which undergo either cell cycle blockade or cell death in response to agents that target the specific oncogene. Surprisingly, HSP27 suppression alone resulted in the apoptotic death of MET-addicted EBC-1 lung cancer cells, epidermal growth factor receptor (EGFR)-addicted colorectal carcinoma (CRC) DiFi cells and BRAF-addicted CRC COLO205 and OXCO-1 and melanoma COLO741 cells, all of which also undergo death when treated with the specific targeted agent. In other cell lines, such as MET-addicted gastric carcinoma MKN45 and EGFR-addicted CRC SW48 lines, where oncogene inhibition only blocked proliferation, HSP27 knockdown made targeted agents switch from cytostatic to cytotoxic activity. Mechanistically, the more the cells were susceptible to HSP27 suppression, the more they were primed for death, as demonstrated by increased levels of mitochondrial outer membrane permeabilization. Priming for death was accompanied by the increase in pro-apoptotic proteins of the BCL2 family and of active caspase-3 and lamin B. Together, these data suggest that oncogene-addicted cells require HSP27 for survival and that HSP27 might interfere with the effectiveness of targeted agents. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  19. Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation

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

    Yu, Jungeun; Shin, Bongjin; Park, Eui-Soon

    2010-01-01

    Protein arginine methylation is involved in viral infection and replication through the modulation of diverse cellular processes including RNA metabolism, cytokine signaling, and subcellular localization. It has been suggested previously that the protein arginine methylation of the RGG-box of ICP27 is required for herpes simplex virus type-1 (HSV-1) viral replication and gene expression in vivo. However, a cellular mediator for this process has not yet been identified. In our current study, we show that the protein arginine methyltransferase 1 (PRMT1) is a cellular mediator of the arginine methylation of ICP27 RGG-box. We generated arginine substitution mutants in this domain andmore » examined which arginine residues are required for methylation by PRMT1. R138, R148 and R150 were found to be the major sites of this methylation but additional arginine residues serving as minor methylation sites are still required to sustain the fully methylated form of ICP27 RGG. We also demonstrate that the nuclear foci-like structure formation, SRPK interactions, and RNA-binding activity of ICP27 are modulated by the arginine methylation of the ICP27 RGG-box. Furthermore, HSV-1 replication is inhibited by hypomethylation of this domain resulting from the use of general PRMT inhibitors or arginine mutations. Our data thus suggest that the PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.« less

  20. Phosphorylation of Heat Shock Protein 27 is Increased by Cast Immobilization and by Serum-free Starvation in Skeletal Muscles

    PubMed Central

    Kim, Mee-Young; Lee, Jeong-Uk; Kim, Ju-Hyun; Lee, Lim-Kyu; Park, Byoung-Sun; Yang, Seung-Min; Jeon, Hye-Joo; Lee, Won-Deok; Noh, Ji-Woong; Kwak, Taek-Yong; Jang, Sung-Ho; Lee, Tae-Hyun; Kim, Ju-Young; Kim, Bokyung; Kim, Junghwan

    2014-01-01

    [Purpose] Cast immobilization- and cell starvation-induced loss of muscle mass are closely associated with a dramatic reduction in the structural muscle proteins. Heat shock proteins are molecular chaperones that are constitutively expressed in several eukaryotic cells and have been shown to protect against various stressors. However, the changes in the phosphorylation of atrophy-related heat shock protein 27 (HSP27) are still poorly understood in skeletal muscles. In this study, we examine whether or not phosphorylation of HSP27 is changed in the skeletal muscles after cast immobilization and serum-free starvation with low glucose in a time-dependent manner. [Methods] We undertook a HSP27 expression and high-resolution differential proteomic analysis in skeletal muscles. Furthermore, we used western blotting to examine protein expression and phosphorylation of HSP27 in atrophied gastrocnemius muscle strips and L6 myoblasts. [Results] Cast immobilization and starvation significantly upregulated the phosphorylation of HSP27 in a time-dependent manner, respectively. [Conclusion] Our results suggest that cast immobilization- and serum-free starvation-induced atrophy may be in part related to changes in the phosphorylation of HSP27 in rat skeletal muscles. PMID:25540511

  1. Crystal structure of the sweet-tasting protein thaumatin II at 1.27 A

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

    Masuda, Tetsuya, E-mail: t2masuda@kais.kyoto-u.ac.jp; Department Natural Resources, Graduate School of Global Environmental Studies, Kyoto University, Uji, Kyoto 611-0011; Ohta, Keisuke

    2011-07-08

    Highlights: {yields} X-ray crystallographic structure of sweet-tasting protein, thaumatin II, was determined at a resolution of 1.27 A. {yields} The overall structure of thaumatin II is similar to that of thaumatin I, but a slight shift of the C{alpha} atom of G96 in thaumatin II was observed. {yields} The side chain of two critical residues, 67 and 82, for sweetness was modeled in two alternative conformations. {yields} The flexibility and fluctuation of side chains at 67 and 82 seems to be suitable for interaction of thaumatin molecules with sweet receptors. -- Abstract: Thaumatin, an intensely sweet-tasting protein, elicits a sweetmore » taste sensation at 50 nM. Here the X-ray crystallographic structure of one of its variants, thaumatin II, was determined at a resolution of 1.27 A. Overall structure of thaumatin II is similar to thaumatin I, but a slight shift of the C{alpha} atom of G96 in thaumatin II was observed. Furthermore, the side chain of residue 67 in thaumatin II is highly disordered. Since residue 67 is one of two residues critical to the sweetness of thaumatin, the present results suggested that the critical positive charges at positions 67 and 82 are disordered and the flexibility and fluctuation of these side chains would be suitable for interaction of thaumatin molecules with sweet receptors.« less

  2. Clostridium perfringens iota toxin: synergism between two proteins.

    PubMed

    Stiles, B G; Wilkins, T D

    1986-01-01

    The iota toxin of Clostridium perfringens type E is a guinea pig dermonecrotic, mouse lethal toxin which cross-reacts with the iota-like toxin of Clostridium spiroforme. Antiserum raised against C. spiroforme or C. perfringens type E neutralizes the toxin from both species. By using C. spiroforme antiserum and crossed immunoelectrophoresis, we have found that there are two cross-reacting proteins, designated iota a (ia) and iota b (ib) in the culture filtrate of C. perfringens type E. Both proteins of C. perfringens were separated by preparative isoelectric focusing and had very little toxic activity when tested alone. However, when they were recombined there were 8- and 25-fold increases in bioactivity as determined by mouse lethal and guinea pig dermonecrotic assays, respectively. These results demonstrate that the iota toxin of C. perfringens requires two immunologically and biochemically different proteins for maximum activity.

  3. Heat-shock protein-25/27 phosphorylation by the delta isoform of protein kinase C.

    PubMed Central

    Maizels, E T; Peters, C A; Kline, M; Cutler, R E; Shanmugam, M; Hunzicker-Dunn, M

    1998-01-01

    Small heat-shock proteins (sHSPs) are widely expressed 25-28 kDa proteins whose functions are dynamically regulated by phosphorylation. While recent efforts have clearly delineated a stress-responsive p38 mitogen-activated protein-kinase (MAPK)-dependent kinase pathway culminating in activation of the heat-shock (HSP)-kinases, mitogen-activated protein-kinase-activated protein kinase-2 and -3, not all sHSP phosphorylation events can be explained by the p38 MAPK-dependent pathway. The contribution of protein kinase C (PKC) to sHSP phosphorylation was suggested by early studies but later questioned on the basis of the reported poor ability of purified PKC to phosphorylate sHSP in vitro. The current study re-evaluates the role of PKC in sHSP phosphorylation in the light of the isoform complexity of the PKC family. We evaluated the sHSP phosphorylation status in rat corpora lutea obtained from two stages of pregnancy, mid-pregnancy and late-pregnancy, which express different levels of the novel PKC isoform, PKC-delta. Two-dimensional Western blot analysis showed that HSP-27 was more highly phosphorylated in vivo in corpora lutea of late pregnancy, corresponding to the developmental stage in which PKC-delta is abundant and active. Late-pregnant luteal extracts contained a lipid-sensitive HSP-kinase activity which exactly co-purified with PKC-delta using hydroxyapatite and S-Sepharose column chromatography. To determine whether there might be preferential phosphorylation of sHSP by a particular PKC isoform, purified recombinant PKC isoforms corresponding to those PKC isoforms detected in rat corpora lutea were evaluated for HSP-kinase activity in vitro. Recombinant PKC-delta effectively catalysed the phosphorylation of sHSP in vitro, and PKC-alpha was 30-50% as effective as an HSP-kinase; other PKCs tested (beta1, beta2, epsilon and zeta) were poor HSP-kinases. These results show that select PKC family members can function as direct HSP-kinases in vitro. Moreover, the

  4. A Novel Marker for Purkinje Cells, Ribosomal Protein MPS1/S27: Expression of MPS1 in Human Cerebellum.

    PubMed

    Fernandez-Pol, J Alberto

    2016-01-01

    The ribosomal protein metallopanstimulin-1 (MPS1/S27) serves critical survival purposes in cell division, in normal and cancerous cells; for this reason, selective pressures of evolution have conserved the DNA sequences encoding MPS1/S27 in Archaea and Eukariotic cells. The expression of MPS1/S27 protein in human adult cerebellum has not been established. The presence of MPS1/S27, was screened in paraffin-embedded human adult brain specimens processed for tissue inmunohistochemistry. Affinity-purified specific antibodies were directed against the N-terminus of MPS1. The antibodies to MPS1 detected Purkinje cells (PC) and their dendrites. In PC, MPS1 antigen-positive staining was found in: the nucleolus, which was strongly stained; ribosomes attached to the external nuclear membrane; cytoplasm of PC, with strong staining in a punctuate fashion; the soma-attached large dendrite trunks of PC, which were MPS1 antigen-positive; and the granular cell layer, where cellular staining in a few cells that appeared to resemble smaller PC was observed. Since MPS1 is involved in cell division, DNA repair, and ribosomal biogenesis, it may be a useful antigen for studying processes such as protein synthesis, oncogenesis, regeneration, aging, and perhaps diseases of the human cerebellum. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

  5. Multiple Protein Kinases Determine the Phosphorylated State of the Small Heat Shock Protein, HSP27, in SH-SY5Y Neuroblastoma Cells

    PubMed Central

    Dokas, Linda A.; Malone, Amy M.; Williams, Frederick E.; Nauli, Surya M.; Messer, William S.

    2011-01-01

    In SH-SY5Y human neuroblastoma cells, the cholinergic agonist, carbachol, stimulates phosphorylation of the small heat shock protein 27 (HSP27). Carbachol increases phosphorylation of both Ser-82 and Ser-78 while the phorbol ester, phorbol-12, 13-dibutyrate (PDB) affects only Ser-82. Muscarinic receptor activation by carbachol was confirmed by sensitivity of Ser-82 phosphorylation to hyoscyamine with no effect of nicotine or bradykinin. This response to carbachol is partially reduced by inhibition of protein kinase C (PKC) with GF 109203X and p38 mitogen-activated protein kinase (MAPK) with SB 203580. In contrast, phosphorylation produced by PDB is completely reversed by GF 109203X or CID 755673, an inhibitor of PKD. Inhibition of phosphatidylinositol 3-kinase or Akt with LY 294002 or Akti-1/2 stimulates HSP27 phosphorylation while rapamycin, which inhibits mTORC1, does not. The stimulatory effect of Akti-1/2 is reversed by SB 203580 and correlates with increased p38 MAPK phosphorylation. SH-SY5Y cells differentiated with a low concentration of PDB and basic fibroblast growth factor to a more neuronal phenotype retain carbachol-, PDB- and Akti-1/2-responsive HSP27 phosphorylation. Immunofluorescence microscopy confirms increased HSP27 phosphorylation in response to carbachol or PDB. At cell margins, PDB causes f-actin to reorganize forming lamellipodial structures from which phospho-HSP27 is segregated. The resultant phenotypic change in cell morphology is dependent upon PKC, but not PKD, activity. The major conclusion from this study is that the phosphorylated state of HSP27 in SH-SY5Y cells results from integrated signaling involving PKC, p38 MAPK and Akt. PMID:21338617

  6. Endothelium-targeted overexpression of heat shock protein 27 ameliorates blood–brain barrier disruption after ischemic brain injury

    PubMed Central

    Jiang, Xiaoyan; Zhang, Lili; Pu, Hongjian; Hu, Xiaoming; Zhang, Wenting; Cai, Wei; Gao, Yanqin; Leak, Rehana K.; Keep, Richard F.; Bennett, Michael V. L.; Chen, Jun

    2017-01-01

    The damage borne by the endothelial cells (ECs) forming the blood–brain barrier (BBB) during ischemic stroke and other neurological conditions disrupts the structure and function of the neurovascular unit and contributes to poor patient outcomes. We recently reported that structural aberrations in brain microvascular ECs—namely, uncontrolled actin polymerization and subsequent disassembly of junctional proteins, are a possible cause of the early onset BBB breach that arises within 30–60 min of reperfusion after transient focal ischemia. Here, we investigated the role of heat shock protein 27 (HSP27) as a direct inhibitor of actin polymerization and protectant against BBB disruption after ischemia/reperfusion (I/R). Using in vivo and in vitro models, we found that targeted overexpression of HSP27 specifically within ECs—but not within neurons—ameliorated BBB impairment 1–24 h after I/R. Mechanistically, HSP27 suppressed I/R-induced aberrant actin polymerization, stress fiber formation, and junctional protein translocation in brain microvascular ECs, independent of its protective actions against cell death. By preserving BBB integrity after I/R, EC-targeted HSP27 overexpression attenuated the infiltration of potentially destructive neutrophils and macrophages into brain parenchyma, thereby improving long-term stroke outcome. Notably, early poststroke administration of HSP27 attached to a cell-penetrating transduction domain (TAT-HSP27) rapidly elevated HSP27 levels in brain microvessels and ameliorated I/R-induced BBB disruption and subsequent neurological deficits. Thus, the present study demonstrates that HSP27 can function at the EC level to preserve BBB integrity after I/R brain injury. HSP27 may be a therapeutic agent for ischemic stroke and other neurological conditions involving BBB breakdown. PMID:28137866

  7. Integromics network meta-analysis on cardiac aging offers robust multi-layer modular signatures and reveals micronome synergism.

    PubMed

    Dimitrakopoulou, Konstantina; Vrahatis, Aristidis G; Bezerianos, Anastasios

    2015-03-04

    The avalanche of integromics and panomics approaches shifted the deciphering of aging mechanisms from single molecular entities to communities of them. In this orientation, we explore the cardiac aging mechanisms - risk factor for multiple cardiovascular diseases - by capturing the micronome synergism and detecting longevity signatures in the form of communities (modules). For this, we developed a meta-analysis scheme that integrates transcriptome expression data from multiple cardiac-specific independent studies in mouse and human along with proteome and micronome interaction data in the form of multiple independent weighted networks. Modularization of each weighted network produced modules, which in turn were further analyzed so as to define consensus modules across datasets that change substantially during lifespan. Also, we established a metric that determines - from the modular perspective - the synergism of microRNA-microRNA interactions as defined by significantly functionally associated targets. The meta-analysis provided 40 consensus integromics modules across mouse datasets and revealed microRNA relations with substantial collective action during aging. Three modules were reproducible, based on homology, when mapped against human-derived modules. The respective homologs mainly represent NADH dehydrogenases, ATP synthases, cytochrome oxidases, Ras GTPases and ribosomal proteins. Among various observations, we corroborate to the involvement of miR-34a (included in consensus modules) as proposed recently; yet we report that has no synergistic effect. Moving forward, we determined its age-related neighborhood in which HCN3, a known heart pacemaker channel, was included. Also, miR-125a-5p/-351, miR-200c/-429, miR-106b/-17, miR-363/-92b, miR-181b/-181d, miR-19a/-19b, let-7d/-7f, miR-18a/-18b, miR-128/-27b and miR-106a/-291a-3p pairs exhibited significant synergy and their association to aging and/or cardiovascular diseases is supported in many cases by a

  8. IL-27 Regulates IL-18 binding protein in skin resident cells.

    PubMed

    Wittmann, Miriam; Doble, Rosella; Bachmann, Malte; Pfeilschifter, Josef; Werfel, Thomas; Mühl, Heiko

    2012-01-01

    IL-18 is an important mediator involved in chronic inflammatory conditions such as cutaneous lupus erythematosus, psoriasis and chronic eczema. An imbalance between IL-18 and its endogenous antagonist IL-18 binding protein (BP) may account for increased IL-18 activity. IL-27 is a cytokine with dual function displaying pro- and anti-inflammatory properties. Here we provide evidence for a yet not described anti-inflammatory mode of action on skin resident cells. Human keratinocytes and surprisingly also fibroblasts (which do not produce any IL-18) show a robust, dose-dependent and highly inducible mRNA expression and secretion of IL-18BP upon IL-27 stimulation. Other IL-12 family members failed to induce IL-18BP. The production of IL-18BP peaked between 48-72 h after stimulation and was sustained for up to 96 h. Investigation of the signalling pathway showed that IL-27 activates STAT1 in human keratinocytes and that a proximal GAS site at the IL-18BP promoter is of importance for the functional activity of IL-27. The data are in support of a significant anti-inflammatory effect of IL-27 on skin resident cells. An important novel property of IL-27 in skin pathobiology may be to counter-regulate IL-18 activities by acting on keratinocytes and importantly also on dermal fibroblasts.

  9. Role of Krüppel-like factor 4 and heat shock protein 27 in cancer of the larynx

    PubMed Central

    Karam, Jihad; Fadous-Khalifé, Marie Claude; Tannous, Rita; Fakhreddine, Sally; Massoud, Marcel; Hadchity, Joseph; Aftimos, Georges; Hadchity, Elie

    2017-01-01

    Late detection and lack of standard treatment strategies in larynx cancer patients result in high levels of mortality and poor prognosis. Prognostic stratification of larynx cancer patients based on molecular prognostic tumor biomarkers may lead to more efficient clinical management. Krüppel-like factor 4 (KLF4) and Heat Shock Protein 27 (HSP27) have an important role in tumorigenesis and are considered promising candidate biomarkers for various types of cancer. However, their role in larynx carcinoma remains to be elucidated. The present study aimed to determine KLF4 and HSP27 expression profiles in laryngeal tumors. The protein and mRNA expression levels of KLF4 and HSP27 were evaluated by immunohistochemical and reverse transcription-polymerase chain reaction analyses in 44 larynx carcinoma samples and 21 normal tissue samples, and then correlated with clinical characteristics. A differential expression of KLF4 and HSP27 was observed between normal and tumor tissues. The protein and mRNA expression levels of KLF4 were significantly decreased in larynx squamous cell carcinoma (LSCC) compared with normal tissue, whereas HSP27 was significantly overexpressed in tumor tissues compared with normal tissues, at the protein and mRNA levels. KLF4 expression decreased gradually with tumor progression whereas HSP27 expression increased. A significant difference was observed between stages I and IV. KLF4 and HSP27 exhibit opposite functions and roles in the carcinogenic process of LSCC. Their role in laryngeal cancer initiation and progression emphasizes their use as potential future targets for prognosis and treatment. KLF4 and HSP27 expression levels may act as potential biomarkers in patients with cancer of the larynx. PMID:29181170

  10. Protective Role of Hsp27 Protein Against Gamma Radiation-Induced Apoptosis and Radiosensitization Effects of Hsp27 Gene Silencing in Different Human Tumor Cells

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

    Aloy, Marie-Therese; Hospices Civils de Lyon, Service de Radiotherapie, Centre Hospitalier Lyon-Sud, Pierre-Benite; Hadchity, Elie

    Purpose: The ability of heat shock protein 27 (Hsp27) to protect cells from stressful stimuli and its increased levels in tumors resistant to anticancer therapeutics suggest that it may represent a target for sensitization to radiotherapy. In this study, we investigate the protective role of Hsp27 against radiation-induced apoptosis and the effect of its attenuation in highly expressing radioresistant cancer cell lines. Methods and Materials: We examined clonogenic death and the kinetics of apoptotic events in different tumor cell lines overexpressing or underexpressing Hsp27 protein irradiated with photons. The radiosensitive Jurkat cell line, which does not express Hsp27 constitutively ormore » in response to {gamma}-rays, was stably transfected with Hsp27 complementary DNA. Attenuation of Hsp27 expression was accomplished by antisense or RNAi (interfering RNA) strategies in SQ20B head-and-neck squamous carcinoma, PC3 prostate cancer, and U87 glioblastoma radioresistant cells. Results: We measured concentration-dependent protection against the cytotoxic effects of radiation in Jurkat-Hsp27 cells, which led to a 50% decrease in apoptotic cells at 48 hours in the highest expressing cells. Underlying mechanisms leading to radiation resistance involved a significant increase in glutathione levels associated with detoxification of reactive oxygen species, a delay in mitochondrial collapse, and caspase activation. Conversely, attenuation of Hsp27 in SQ20B cells, characterized by their resistance to apoptosis, sensitizes cells to irradiation. This was emphasized by increased apoptosis, decreased glutathione basal level, and clonogenic cell death. Sensitization to irradiation was confirmed in PC3 and U87 radioresistant cells. Conclusion: Hsp27 gene therapy offers a potential adjuvant to radiation-based therapy of resistant tumors.« less

  11. The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and enhances apoptosis.

    PubMed

    Weber, Arnim; Heinlein, Melanie; Dengjel, Jörn; Alber, Claudia; Singh, Prafull Kumar; Häcker, Georg

    2016-05-01

    Bim is a pro-apoptotic Bcl-2 family member of the BH3-only protein subgroup. Expression levels of Bim determine apoptosis susceptibility in non-malignant and in tumour cells. Bim protein expression is downregulated by proteasomal degradation following ERK-dependent phosphorylation and ubiquitination. Here, we report the identification of a deubiquitinase, Usp27x, that binds Bim upon its ERK-dependent phosphorylation and can upregulate its expression levels. Overexpression of Usp27x reduces ERK-dependent Bim ubiquitination, stabilizes phosphorylated Bim, and induces apoptosis in PMA-stimulated cells, as well as in tumour cells with a constitutively active Raf/ERK pathway. Loss of endogenous Usp27x enhances the Bim-degrading activity of oncogenic Raf. Overexpression of Usp27x induces low levels of apoptosis in melanoma and non-small cell lung cancer (NSCLC) cells and substantially enhances apoptosis induced in these cells by the inhibition of ERK signalling. Finally, deletion of Usp27x reduces apoptosis in NSCLC cells treated with an EGFR inhibitor. Thus, Usp27x can trigger via its proteolytic activity the deubiquitination of Bim and enhance its levels, counteracting the anti-apoptotic effects of ERK activity, and therefore acts as a tumour suppressor. © 2016 The Authors.

  12. Enhancing the pH sensitivity by laterally synergic modulation in dual-gate electric-double-layer transistors

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

    Liu, Ning; Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo 315201; Hui Liu, Yang

    2015-02-16

    The sensitivity of a standard ion-sensitive field-effect transistor is limited to be 59.2 mV/pH (Nernst limit) at room temperature. Here, a concept based on laterally synergic electric-double-layer (EDL) modulation is proposed in order to overcome the Nernst limit. Indium-zinc-oxide EDL transistors with two laterally coupled gates are fabricated, and the synergic modulation behaviors of the two asymmetric gates are investigated. A high sensitivity of ∼168 mV/pH is realized in the dual-gate operation mode. Laterally synergic modulation in oxide-based EDL transistors is interesting for high-performance bio-chemical sensors.

  13. Potent In Vitro Synergism of Fluconazole and Osthole against Fluconazole-Resistant Candida albicans

    PubMed Central

    Li, De-Dong; Chai, Dong; Huang, Xiao-Wen; Guan, Shao-Xing; Du, Jiang; Zhang, Hao-Yu

    2017-01-01

    ABSTRACT Osthole is a natural coumarin that exhibits wide biological and pharmacological activities such as neuroprotective, osteogenic, immunomodulation, antitumor, and anti-inflammatory effects. In this study, we investigated the antifungal effects of osthole in vitro. A checkerboard microdilution assay showed that osthole has significant synergistic effect with fluconazole against fluconazole-resistant Candida albicans. Similar results were obtained from a growth curve assay. Meanwhile, XTT reduction assay demonstrated the synergism of fluconazole and osthole against C. albicans biofilm formation. Microarray results showed that the expression of genes involved in the oxidation-reduction process, energy metabolism, and transportation changed significantly after the combined treatment with fluconazole and osthole, and further results showed that endogenous reactive oxygen species (ROS) was significantly increased in the combination group. In conclusion, these results demonstrate the synergism of fluconazole and osthole against fluconazole-resistant C. albicans and indicate that endogenous ROS augmentation might contribute to the synergism of fluconazole and osthole. PMID:28607012

  14. Potent In Vitro Synergism of Fluconazole and Osthole against Fluconazole-Resistant Candida albicans.

    PubMed

    Li, De-Dong; Chai, Dong; Huang, Xiao-Wen; Guan, Shao-Xing; Du, Jiang; Zhang, Hao-Yu; Sun, Yan; Jiang, Yuan-Ying

    2017-08-01

    Osthole is a natural coumarin that exhibits wide biological and pharmacological activities such as neuroprotective, osteogenic, immunomodulation, antitumor, and anti-inflammatory effects. In this study, we investigated the antifungal effects of osthole in vitro A checkerboard microdilution assay showed that osthole has significant synergistic effect with fluconazole against fluconazole-resistant Candida albicans Similar results were obtained from a growth curve assay. Meanwhile, XTT reduction assay demonstrated the synergism of fluconazole and osthole against C. albicans biofilm formation. Microarray results showed that the expression of genes involved in the oxidation-reduction process, energy metabolism, and transportation changed significantly after the combined treatment with fluconazole and osthole, and further results showed that endogenous reactive oxygen species (ROS) was significantly increased in the combination group. In conclusion, these results demonstrate the synergism of fluconazole and osthole against fluconazole-resistant C. albicans and indicate that endogenous ROS augmentation might contribute to the synergism of fluconazole and osthole. Copyright © 2017 American Society for Microbiology.

  15. Characterization of a Viral Synergism in the Monocot Brachypodium distachyon Reveals Distinctly Altered Host Molecular Processes Associated with Disease1[C][W][OA

    PubMed Central

    Mandadi, Kranthi K.; Scholthof, Karen-Beth G.

    2012-01-01

    Panicum mosaic virus (PMV) and its satellite virus (SPMV) together infect several small grain crops, biofuel, and forage and turf grasses. Here, we establish the emerging monocot model Brachypodium (Brachypodium distachyon) as an alternate host to study PMV- and SPMV-host interactions and viral synergism. Infection of Brachypodium with PMV+SPMV induced chlorosis and necrosis of leaves, reduced seed set, caused stunting, and lowered biomass, more than PMV alone. Toward gaining a molecular understanding of PMV- and SPMV-affected host processes, we used a custom-designed microarray and analyzed global changes in gene expression of PMV- and PMV+SPMV-infected plants. PMV infection by itself modulated expression of putative genes functioning in carbon metabolism, photosynthesis, metabolite transport, protein modification, cell wall remodeling, and cell death. Many of these genes were additively altered in a coinfection with PMV+SPMV and correlated to the exacerbated symptoms of PMV+SPMV coinfected plants. PMV+SPMV coinfection also uniquely altered expression of certain genes, including transcription and splicing factors. Among the host defenses commonly affected in PMV and PMV+SPMV coinfections, expression of an antiviral RNA silencing component, SILENCING DEFECTIVE3, was suppressed. Several salicylic acid signaling components, such as pathogenesis-related genes and WRKY transcription factors, were up-regulated. By contrast, several genes in jasmonic acid and ethylene responses were down-regulated. Strikingly, numerous protein kinases, including several classes of receptor-like kinases, were misexpressed. Taken together, our results identified distinctly altered immune responses in monocot antiviral defenses and provide insights into monocot viral synergism. PMID:22961132

  16. A PARP1-ERK2 synergism is required for the induction of LTP

    PubMed Central

    Visochek, L.; Grigoryan, G.; Kalal, A.; Milshtein-Parush, H.; Gazit, N.; Slutsky, I.; Yeheskel, A.; Shainberg, A.; Castiel, A.; Seger, R.; Langelier, M. F.; Dantzer, F.; Pascal, J. M.; Segal, M.; Cohen-Armon, M.

    2016-01-01

    Unexpectedly, a post-translational modification of DNA-binding proteins, initiating the cell response to single-strand DNA damage, was also required for long-term memory acquisition in a variety of learning paradigms. Our findings disclose a molecular mechanism based on PARP1-Erk synergism, which may underlie this phenomenon. A stimulation induced PARP1 binding to phosphorylated Erk2 in the chromatin of cerebral neurons caused Erk-induced PARP1 activation, rendering transcription factors and promoters of immediate early genes (IEG) accessible to PARP1-bound phosphorylated Erk2. Thus, Erk-induced PARP1 activation mediated IEG expression implicated in long-term memory. PARP1 inhibition, silencing, or genetic deletion abrogated stimulation-induced Erk-recruitment to IEG promoters, gene expression and LTP generation in hippocampal CA3-CA1-connections. Moreover, a predominant binding of PARP1 to single-strand DNA breaks, occluding its Erk binding sites, suppressed IEG expression and prevented the generation of LTP. These findings outline a PARP1-dependent mechanism required for LTP generation, which may be implicated in long-term memory acquisition and in its deterioration in senescence. PMID:27121568

  17. Knocking down of heat-shock protein 27 directs differentiation of functional glutamatergic neurons from placenta-derived multipotent cells

    PubMed Central

    Cheng, Yu-Che; Huang, Chi-Jung; Lee, Yih-Jing; Tien, Lu-Tai; Ku, Wei-Chi; Chien, Raymond; Lee, Fa-Kung; Chien, Chih-Cheng

    2016-01-01

    This study presents human placenta-derived multipotent cells (PDMCs) as a source from which functional glutamatergic neurons can be derived. We found that the small heat-shock protein 27 (HSP27) was downregulated during the neuronal differentiation process. The in vivo temporal and spatial profiles of HSP27 expression were determined and showed inverted distributions with neuronal proteins during mouse embryonic development. Overexpression of HSP27 in stem cells led to the arrest of neuronal differentiation; however, the knockdown of HSP27 yielded a substantially enhanced ability of PDMCs to differentiate into neurons. These neurons formed synaptic networks and showed positive staining for multiple neuronal markers. Additionally, cellular phenomena including the absence of apoptosis and rare proliferation in HSP27-silenced PDMCs, combined with molecular events such as cleaved caspase-3 and the loss of stemness with cleaved Nanog, indicated that HSP27 is located upstream of neuronal differentiation and constrains that process. Furthermore, the induced neurons showed increasing intracellular calcium concentrations upon glutamate treatment. These differentiated cells co-expressed the N-methyl-D-aspartate receptor, vesicular glutamate transporter, and synaptosomal-associated protein 25 but did not show expression of tyrosine hydroxylase, choline acetyltransferase or glutamate decarboxylase 67. Therefore, we concluded that HSP27-silenced PDMCs differentiated into neurons possessing the characteristics of functional glutamatergic neurons. PMID:27444754

  18. Enhance-Synergism and Suppression Effects in Multiple Regression

    ERIC Educational Resources Information Center

    Lipovetsky, Stan; Conklin, W. Michael

    2004-01-01

    Relations between pairwise correlations and the coefficient of multiple determination in regression analysis are considered. The conditions for the occurrence of enhance-synergism and suppression effects when multiple determination becomes bigger than the total of squared correlations of the dependent variable with the regressors are discussed. It…

  19. Introducing differential expression of human heat shock protein 27 in hepatocellular carcinoma: moving toward identification of cancer biomarker.

    PubMed

    Khan, Rizma; Siddiqui, Nadir Naveed; Ul Haq, Ahtesham; Rahman, M Ataur

    2016-01-01

    Previously, it has to be acknowledged that overexpressed heat shock protein B27 (HSPB27) have been implicated in the etiology of wide range of human cancers. However, the molecular mechanism leading to the disease initiation to progression in liver cancer is still unknown. Present work was undertaken to investigate the differentially expressed HSPB27 in association with those damages that lead to liver cancer development. For the identification of liver cancer biomarker, samples were subjected to comparative proteomic analysis using two-dimensional gel electrophoresis (2-DE) and were further validated by Western blot and immunohistochemical analysis. After validation, in silico studies were applied to demonstrate the significantly induced phosphorylated and S-nitrosylated signals. The later included the interacting partner of HSPB27, i.e., mitogen-activated protein kinase-3 and 5 (MAPK3 and 5), ubiquitin C (UBC), v-akt murine thymoma viral oncogene homolog 1 (AKT1), mitogen-activated protein kinase 14 (MAPK14), and tumor protein p53 (TP53), which bestowed with critical capabilities, namely, apoptosis, cell cycling, stress activation, tumor suppression, cell survival, angiogenesis, proliferation, and stress resistance. Taking together, these results shed new light on the potential biomarker HSPB27 that overexpression of HSPB27 did lead to upregulation of their interacting partner that together demonstrate their possible role as a novel tumor progressive agent for the treatment of metastasis in liver cancer. HSPB27 is a promising diagnostic marker for liver cancer although further large-scale studies are required. Also, molecular profiling may help pave the road to the discovery of new therapies.

  20. Heat shock protein 27 regulates human prostate cancer cell motility and metastatic progression

    PubMed Central

    Voll, Eric A; Ogden, Irene M; Pavese, Janet M; Huang, XiaoKe; Xu, Li; Jovanovic, Borko D; Bergan, Raymond C

    2014-01-01

    Prostate cancer (PCa) is the most common form of cancer in American men. Mortality from PCa is caused by the movement of cancer cells from the primary organ to form metastatic tumors at distant sites. Heat shock protein 27 (HSP27) is known to increase human PCa cell invasion and its overexpression is associated with metastatic disease. The role of HSP27 in driving PCa cell movement from the prostate to distant metastatic sites is unknown. Increased HSP27 expression increased metastasis as well as primary tumor mass. In vitro studies further examined the mechanism of HSP27-induced metastatic behavior. HSP27 did not affect cell detachment, adhesion, or migration, but did increase cell invasion. Cell invasion was dependent upon matrix metalloproteinase 2 (MMP-2), whose expression was increased by HSP27. In vivo, HSP27 induced commensurate changes in MMP-2 expression in tumors. These findings demonstrate that HSP27 drives metastatic spread of cancer cells from the prostate to distant sites, does so across a continuum of expression levels, and identifies HSP27-driven increases in MMP-2 expression as functionally relevant. These findings add to prior studies demonstrating that HSP27 increases PCa cell motility, growth and survival. Together, they demonstrate that HSP27 plays an important role in PCa progression. PMID:24798191

  1. Phosphorylation of HSP27 by Protein Kinase D is Essential for Mediating Neuroprotection Against Ischemic Neuronal Injury

    PubMed Central

    Stetler, R. Anne; Gao, Yanqin; Zhang, Lili; Weng, Zhongfang; Zhang, Feng; Hu, Xiaoming; Wang, Suping; Vosler, Peter; Cao, Guodong; Sun, Dandan; Graham, Steven H.; Chen, Jun

    2012-01-01

    Heat shock protein 27 (HSP27, or HSPB1) exerts cytoprotection against many cellular insults, including cerebral ischemia. We previously identified apoptosis signal-regulating kinase 1 (ASK1) as a critical downstream target of HSP27 conferring the neuroprotective effects of HSP27 against neuronal ischemia. However, the function of HSP27 is highly influenced by post-translational modification, with differential cellular effects based on phosphorylation at specific serine residues. The role of phosphorylation in neuronal ischemic neuroprotection is currently unknown. We have created transgenic mice and viral vectors containing HSP27 mutated at three critical serine residues (Ser15, Ser78 and Ser82) to either alanine (HSP27-A, non-phosphorylatable) or aspartate (HSP27-D, phospho-mimetic) residues. Under both in vitro and in vivo neuronal ischemic settings, overexpression of wild-type HSP27 (HSP27) and HSP27-D, but not HSP27-A, was neuroprotective and inhibited downstream ASK1 signaling pathways. Consistently, overexpressed HSP27 was phosphorylated by endogenous mechanisms when neurons were under ischemic stress, and single point mutations identified Ser15 and Ser82 as critical for neuroprotection. Using a panel of inhibitors and gene knockdown approaches, we identified the upstream kinase protein kinase D (PKD) as the primary kinase targeting HSP27 directly for phosphorylation. PKD and HSP27 co-immunoprecipitated, and inhibition or knockdown of PKD abrogated the neuroprotective effects of HSP27 as well as the interaction with and inhibition of ASK1 signaling. Taken together, these data demonstrate that HSP27 requires PKD-mediated phosphorylation for its suppression of ASK1 cell death signaling and neuroprotection against ischemic injury. PMID:22357851

  2. In silico evaluation of human small heat shock protein HSP27: homology modeling, mutation analyses and docking studies.

    PubMed

    Fossa, Paola; Cichero, Elena

    2015-07-01

    Small heat-shock proteins, possessing chaperone-like activity, represented crucial proteins actively involved in maintain protein homeostasis, which act to prevent improper polypeptide aggregation and deposition of misfolded proteins. In this context, a number of mutations concerning the HspB1 protein proved to be associated with the development of several neuropathologies. Unfortunately, molecular mechanisms underlying the onset of these diseases and in particular the changes induced by the mutations in HspB1 structure, remain poorly characterized. On the other hand, more recent studies demonstrated that HspB1 overexpression leads to an overactive chaperone activity, which in turn contributes to the anticancer agent resistance. On these basis, Hsp27 could represent a good innovative target for development of novel cancer therapy. Therefore, in this work a computational study, based on the homology model of the complete Hsp27 protein and of several pathological mutant forms, was developed. Finally, the derived model was employed to perform, for the first time, docking simulations on a recently identified Hsp27 inhibitor, disclosing a new useful panorama to be exploited for the further development of new compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 1 targets misfolded HLA-B27 dimers for endoplasmic reticulum-associated degradation.

    PubMed

    Guiliano, David B; Fussell, Helen; Lenart, Izabela; Tsao, Edward; Nesbeth, Darren; Fletcher, Adam J; Campbell, Elaine C; Yousaf, Nasim; Williams, Sarah; Santos, Susana; Cameron, Amy; Towers, Greg J; Kellam, Paul; Hebert, Daniel N; Gould, Keith G; Powis, Simon J; Antoniou, Antony N

    2014-11-01

    HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR-induced ER-associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers. HeLa cell lines expressing only 2 copies of a carboxy-terminally Sv5-tagged HLA-B27 were generated. The ER stress-induced protein ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) was overexpressed by transfection, and dimer levels were monitored by immunoblotting. EDEM1, the UPR-associated transcription factor X-box binding protein 1 (XBP-1), the E3 ubiquitin ligase hydroxymethylglutaryl-coenzyme A reductase degradation 1 (HRD1), and the degradation-associated proteins derlin 1 and derlin 2 were inhibited using either short hairpin RNA or dominant-negative mutants. The UPR-associated ERAD of HLA-B27 was confirmed using ER stress-inducing pharamacologic agents in kinetic and pulse chase assays. We demonstrated that UPR-induced machinery can target HLA-B27 dimers and that dimer formation can be controlled by alterations to expression levels of components of the UPR-induced ERAD pathway. HLA-B27 dimers and misfolded major histocompatibility complex class I monomeric molecules bound to EDEM1 were detected, and overexpression of EDEM1 led to inhibition of HLA-B27 dimer formation. EDEM1 inhibition resulted in up-regulation of HLA-B27 dimers, while UPR-induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1, and derlins 1 and 2. The present findings indicate that the UPR ERAD pathway can dispose of HLA-B27 dimers, thus presenting a potential novel therapeutic target for modulation of HLA-B27-associated inflammatory disease. Copyright © 2014 by the American College of Rheumatology.

  4. Heat Shock Protein HSP27 Secretion by Ovarian Cancer Cells Is Linked to Intracellular Expression Levels, Occurs Independently of the Endoplasmic Reticulum Pathway and HSP27's Phosphorylation Status, and Is Mediated by Exosome Liberation

    PubMed Central

    Klinkmann, Gerd; Diesing, Karoline; Koensgen, Dominique; Burchardt, Martin

    2017-01-01

    The heat shock protein HSP27 has been correlated in ovarian cancer (OC) patients with aggressiveness and chemoresistance and, therefore, represents a promising potential biomarker for OC diagnosis, prognosis, and treatment response. Notably, secretion of soluble HSP27 has been described by a few cell types and may take place as well in OC cells. Therefore, we studied HSP27 secretion mechanisms under diverse cellular conditions in an OC cell model system. Secretion of HSP27 was characterized after overexpression of HSP27 by transfected plasmids and after heat shock. Intra- and extracellular HSP27 amounts were assessed by Western blotting and ELISA. Protein secretion was blocked by brefeldin A and the impact of the HSP27 phosphorylation status was analyzed overexpressing HSP27 phosphomutants. The present study demonstrated that HSP27 secretion by OVCAR-3 and SK-OV-3 cells depends on intracellular HSP27 concentrations. Moreover, HSP27 secretion is independent of the endoplasmic reticulum secretory pathway and HSP27 phosphorylation. Notably, analysis of OC cell-born exosomes not only confirmed the concentration-dependent correlation of HSP27 expression and secretion but also demonstrated a concentration-dependent incorporation of HSP27 protein into exosomes. Thus, secreted HSP27 may become more important as an extracellular factor which controls the tumor microenvironment and might be a noninvasive biomarker. PMID:28325957

  5. Heat Shock Protein HSP27 Secretion by Ovarian Cancer Cells Is Linked to Intracellular Expression Levels, Occurs Independently of the Endoplasmic Reticulum Pathway and HSP27's Phosphorylation Status, and Is Mediated by Exosome Liberation.

    PubMed

    Stope, Matthias B; Klinkmann, Gerd; Diesing, Karoline; Koensgen, Dominique; Burchardt, Martin; Mustea, Alexander

    2017-01-01

    The heat shock protein HSP27 has been correlated in ovarian cancer (OC) patients with aggressiveness and chemoresistance and, therefore, represents a promising potential biomarker for OC diagnosis, prognosis, and treatment response. Notably, secretion of soluble HSP27 has been described by a few cell types and may take place as well in OC cells. Therefore, we studied HSP27 secretion mechanisms under diverse cellular conditions in an OC cell model system. Secretion of HSP27 was characterized after overexpression of HSP27 by transfected plasmids and after heat shock. Intra- and extracellular HSP27 amounts were assessed by Western blotting and ELISA. Protein secretion was blocked by brefeldin A and the impact of the HSP27 phosphorylation status was analyzed overexpressing HSP27 phosphomutants. The present study demonstrated that HSP27 secretion by OVCAR-3 and SK-OV-3 cells depends on intracellular HSP27 concentrations. Moreover, HSP27 secretion is independent of the endoplasmic reticulum secretory pathway and HSP27 phosphorylation. Notably, analysis of OC cell-born exosomes not only confirmed the concentration-dependent correlation of HSP27 expression and secretion but also demonstrated a concentration-dependent incorporation of HSP27 protein into exosomes. Thus, secreted HSP27 may become more important as an extracellular factor which controls the tumor microenvironment and might be a noninvasive biomarker.

  6. Synergism of cellulase, xylanase, and pectinase on hydrolyzing sugarcane bagasse resulting from different pretreatment technologies.

    PubMed

    Li, Jingbo; Zhou, Pengfei; Liu, Hongmei; Xiong, Chunjiang; Lin, Jianghai; Xiao, Wenjuan; Gong, Yingxue; Liu, Zehuan

    2014-03-01

    Sugarcane bagasse (SCB) resulting from different pretreatments was hydrolyzed by enzyme cocktails based on replacement of cellulase (Celluclast 1.5 L:Novozym 188=1FPU:4pNPGU) by xylanase or pectinase at different proportions. Lignin content of NaOH pretreated SCB and hemicellulose content of H2SO4 pretreated SCB were the lowest. NaOH pretreatment showed the best for monosaccharide production among the four pretreatments. Synergism was apparently observed between cellulase and xylanase for monosaccharide production from steam exploded SCB (SESB), NaOH, and H2O2 pretreated SCB. No synergism was observed between cellulase and pectinase for producing glucose. Additionally, no synergism was present when H2SO4 pretreated SCB was used. Replacement of 20% of the cellulase by xylanase enhanced the glucose yield by 6.6%, 8.8%, and 9.5% from SESB, NaOH, and H2O2 pretreated SCB, respectively. Degree of synergism between cellulase and xylanase had positive relationship with xylan content and was affected by hydrolysis time. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Cloning & sequence identification of Hsp27 gene and expression analysis of the protein on thermal stress in Lucilia cuprina.

    PubMed

    Singh, Manish K; Tiwari, Pramod K

    2016-08-01

    Hsp27, a highly conserved small molecular weight heat shock protein, is widely known to be developmentally regulated and heat inducible. Its role in thermotolerance is also implicated. This study is a sequel of our earlier studies to understand the molecular organization of heat shock genes/proteins and their role in development and thermal adaptation in a sheep pest, Lucilia cuprina (blowfly), which exhibits unusually high adaptability to a variety of environmental stresses, including heat and chemicals. In this report our aim was to understand the evolutionary relationship of Lucilia hsp27 gene/protein with those of other species and its role in thermal adaptation. We sequence characterized the Lchsp27 gene (coding region) and analyzed its expression in various larval and adult tissues under normal as well as heat shock conditions. The nucleotide sequence analysis of 678 bps long-coding region of Lchsp27 exhibited closest evolutionary proximity with Drosophila (90.09%), which belongs to the same order, Diptera. Heat shock caused significant enhancement in the expression of Lchsp27 gene in all the larval and adult tissues examined, however, in a tissue specific manner. Significantly, in Malpighian tubules, while the heat-induced level of hsp27 transcript (mRNA) appeared increased as compared to control, the protein level remained unaltered and nuclear localized. We infer that Lchsp27 may have significant role in the maintenance of cellular homeostasis, particularly, during summer months, when the fly remains exposed to high heat in its natural habitat. © 2015 Institute of Zoology, Chinese Academy of Sciences.

  8. Alteration of the Tumor Stroma Using a Consensus DNA Vaccine Targeting Fibroblast Activation Protein (FAP) Synergizes with Antitumor Vaccine Therapy in Mice.

    PubMed

    Duperret, Elizabeth K; Trautz, Aspen; Ammons, Dylan; Perales-Puchalt, Alfredo; Wise, Megan C; Yan, Jian; Reed, Charles; Weiner, David B

    2018-03-01

    Purpose: Fibroblast activation protein (FAP) is overexpressed in cancer-associated fibroblasts and is an interesting target for cancer immune therapy, with prior studies indicating a potential to affect the tumor stroma. Our aim was to extend this earlier work through the development of a novel FAP immunogen with improved capacity to break tolerance for use in combination with tumor antigen vaccines. Experimental Design: We used a synthetic consensus (SynCon) sequence approach to provide MHC class II help to support breaking of tolerance. We evaluated immune responses and antitumor activity of this novel FAP vaccine in preclinical studies, and correlated these findings to patient data. Results: This SynCon FAP DNA vaccine was capable of breaking tolerance and inducing both CD8 + and CD4 + immune responses. In genetically diverse, outbred mice, the SynCon FAP DNA vaccine was superior at breaking tolerance compared with a native mouse FAP immunogen. In several tumor models, the SynCon FAP DNA vaccine synergized with other tumor antigen-specific DNA vaccines to enhance antitumor immunity. Evaluation of the tumor microenvironment showed increased CD8 + T-cell infiltration and a decreased macrophage infiltration driven by FAP immunization. We extended this to patient data from The Cancer Genome Atlas, where we find high FAP expression correlates with high macrophage and low CD8 + T-cell infiltration. Conclusions: These results suggest that immune therapy targeting tumor antigens in combination with a microconsensus FAP vaccine provides two-fisted punch-inducing responses that target both the tumor microenvironment and tumor cells directly. Clin Cancer Res; 24(5); 1190-201. ©2018 AACR . ©2018 American Association for Cancer Research.

  9. Synergic effect studies of the bi-enzymatic system laccase-peroxidase in a voltammetric biosensor for catecholamines.

    PubMed

    Leite, Oldair D; Lupetti, Karina O; Fatibello-Filho, Orlando; Vieira, Iolanda C; Barbosa, Aneli de M

    2003-04-10

    Several bi-enzymatic carbon paste biosensors modified with enzymes laccase from Pleurotus ostreatus fungi and peroxidase from zucchini (Cucurbita pepo) were constructed for evaluating the synergic effect of the two enzymes on the voltammetric biosensor response for various catecholamines. Initially was investigated the effect of pH from 5.0 to 7.5, temperature from 25 to 50 degrees C, initial stirring time from 30 to 150 s, scan rate from 10 to 60 mVs(-1) and potential pulse amplitude from 10 to 60 mV on the biosensor response for several catecholamines such as dopamine, adrenaline, isoprenaline and l-dopa. It was observed a biosensor signal increase employing both enzymes, indicating thus there is a synergic effect between laccase and peroxidase, verified also in spectrophotometric studies, in the determination of these catecholamines.

  10. Canine gastric carcinoma: immunohistochemical expression of cell cycle proteins (p53, p21, and p16) and heat shock proteins (Hsp27 and Hsp70).

    PubMed

    Carrasco, V; Canfrán, S; Rodríguez-Franco, F; Benito, A; Sáinz, A; Rodríguez-Bertos, A

    2011-01-01

    Immunohistochemical staining for cell cycle proteins and heat shock proteins was performed on 17 canine gastric carcinomas. The immunoexpression of p53, p21, p16, Hsp27, and Hsp70 was investigated. A study was conducted to determine the histological type and parameters related to tumor malignancy. Possible associations and trends were assessed between the immunoexpression of each protein and tumor type as well as specific parameters of malignancy. High intratumor frequency of cellular p53 immunostaining was observed (61.96% average), but lower frequencies of p21 and p16 expression were present (34.65% and 10.41%, respectively). The p53 overexpression was associated with tumor infiltration (P = .0258). Expression of p21 was lower in undifferentiated carcinomas, and the loss of expression was associated with histopathological parameters characteristic of a poor prognosis such as lymphatic vessel invasion (P = .0258). The lack of p16 immunoreactivity was related to histopathological characteristics of malignancy such as the presence of evident and multiple nucleoli (P = .0475). In contrast, deep tumor infiltration was observed in those carcinomas with a high p16 index (P = .0475). Hsp70 appeared to be overexpressed in all gastric neoplasms included in this study. This is in contrast to Hsp27, because a group of tumors showed complete lack of Hsp27 immunoexpression, whereas the others displayed extensive Hsp27 immunostaining. The differences in Hsp27 did not correlate with any of the histopathological parameters, but Hsp27 immunoexpression was higher in the undifferentiated carcinoma. No significant differences in the expression of the proteins were found in canine gastric carcinomas according to their histological type. These findings may be useful for establishing a prognosis for canine gastric carcinoma.

  11. The Society of Surgical Oncology and the Commission on Cancer: progress through synergism.

    PubMed

    Winchester, D P

    1998-09-01

    The 1998 Presidential Address highlights the history of The Society of Surgical Oncology and the Commission on Cancer of the American College of Surgeons, cites specific examples of progress through synergism, and discusses some of the many challenges facing surgical oncologists in the future. These include the necessity to synergize in clinical trials, to accelerate the diffusion of knowledge into the practicing community, and to redefine surgical oncology and its relation to general surgery and the American Board of Surgery.

  12. The effects of wet cupping on serum high-sensitivity C-reactive protein and heat shock protein 27 antibody titers in patients with metabolic syndrome.

    PubMed

    Farahmand, Seyed Kazem; Gang, Li Zhi; Saghebi, Seyed Ahmad; Mohammadi, Maryam; Mohammadi, Shabnam; Mohammadi, Ghazaleh; Ferns, Gordan A; Ghanbarzadeh, Majid; Razmgah, Gholamreza Ghayour; Ramazani, Zahra; Ghayour-Mobarhan, Majid; Esmaily, Habibollah; Bahrami Taghanaki, Hamidreza; Azizi, Hoda

    2014-08-01

    It has previously been reported that increased level of serum heat shock proteins (Hsps) antibody in patients with metabolic syndrome. It is possible that the expression of Hsp and inflammatory markers can be affected by cupping and traditional Chinese medicine. There is a little data investigating the effects of cupping on markers of inflammation and Hsp proteins, hence, the objective of this study was evaluation of the effects of wet cupping on serum high-sensitivity C-reactive protein (hs-CRP) and Hsp27 antibody titers in patients with metabolic syndrome. Serum Hs-CRP and Hsp27 antibody titers were assessed in samples from 126 patients with metabolic syndrome (18-65 years of age) at baseline, and after 6 and 12 weeks after treatment. One hundred and twenty-six patients were randomly divided into the experimental group treated with wet cupping combined with dietary advice, and the control group treated with dietary advice alone using a random number table. Eight patients in case group and five subjects in control groups were excluded from the study. Data were analyzed using SPSS 15.0 software and a repeated measure ANCOVA. Serum hs-CRP titers did not change significantly between groups (p>0.05) and times (p=0.27). The same result was found for Hsp27 titers (p>0.05). Wet-cupping on the interscapular region has no effect on serum hs-CRP and Hsp27 patients with metabolic syndrome. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Protective Effect of Tat PTD-Hsp27 Fusion Protein on Tau Hyperphosphorylation Induced by Okadaic Acid in the Human Neuroblastoma Cell Line SH-SY5Y.

    PubMed

    Choi, Sunghyun; Oh, Jae Hoon; Kim, Hyeseon; Nam, So Hee; Shin, Jeehae; Park, Jong-Sang

    2015-10-01

    Alzheimer's disease (AD) is an age-related disorder that causes a loss of brain function. Hyperphosphorylation of tau and the subsequent formation of intracellular neurofibrillary tangles (NFTs) are implicated in the pathogenesis of AD. Hyperphosphorylated tau accumulates into insoluble paired helical filaments that aggregate into NFTs; therefore, regulation of tau phosphorylation represents an important treatment approach for AD. Heat shock protein 27 (Hsp27) plays a specific role in human neurodegenerative diseases; however, few studies have examined its therapeutic effect. In this study, we induced tau hyperphosphorylation using okadaic acid, which is a protein phosphatase inhibitor, and generated a fusion protein of Hsp27 and the protein transduction domain of the HIV Tat protein (Tat-Hsp27) to enhance the delivery of Hsp27. We treated Tat-Hsp27 to SH-SY5Y neuroblastoma cells for 2 h; the transduction level was proportional to the Tat-hsp27 concentration. Additionally, Tat-Hsp27 reduced the level of hyperphosphorylated tau and protected cells from apoptotic cell death caused by abnormal tau aggregates. These results reveal that Hsp27 represents a valuable protein therapeutic for AD.

  14. MicroRNA 27a-3p Regulates Antimicrobial Responses of Murine Macrophages Infected by Mycobacterium avium subspecies paratuberculosis by Targeting Interleukin-10 and TGF-β-Activated Protein Kinase 1 Binding Protein 2

    PubMed Central

    Hussain, Tariq; Zhao, Deming; Shah, Syed Zahid Ali; Wang, Jie; Yue, Ruichao; Liao, Yi; Sabir, Naveed; Yang, Lifeng; Zhou, Xiangmei

    2018-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) persistently survive and replicate in mononuclear phagocytic cells by adopting various strategies to subvert host immune response. Interleukin-10 (IL-10) upregulation via inhibition of macrophage bactericidal activity is a critical step for MAP survival and pathogenesis within the host cell. Mitogen-activated protein kinase p38 signaling cascade plays a crucial role in the elevation of IL-10 and progression of MAP pathogenesis. The contribution of microRNAs (miRNAs) and their influence on the activation of macrophages during MAP pathogenesis are still unclear. In the current study, we found that miRNA-27a-3p (miR-27a) expression is downregulated during MAP infection both in vivo and in vitro. Moreover, miR-27a is also downregulated in toll-like receptor 2 (TLR2)-stimulated murine macrophages (RAW264.7 and bone marrow-derived macrophage). ELISA and real-time qRT-PCR results confirm that overexpression of miR-27a inhibited MAP-induced IL-10 production in macrophages and upregulated pro-inflammatory cytokines, while miR-27a inhibitor counteracted these effects. Luciferase reporter assay results revealed that IL-10 and TGF-β-activated protein kinase 1 binding protein 2 (TAB 2) are potential targets of miR-27a. In addition, we demonstrated that miR-27a negatively regulates TAB 2 expression and diminishes TAB 2-dependent p38/JNK phosphorylation, ultimately downregulating IL-10 expression in MAP-infected macrophages. Furthermore, overexpression of miR-27a significantly inhibited the intracellular survival of MAP in infected macrophages. Our data show that miR-27a augments antimicrobial activities of macrophages and inhibits the expression of IL-10, demonstrating that miR-27a regulates protective innate immune responses during MAP infection and can be exploited as a novel therapeutic target in the control of intracellular pathogens, including paratuberculosis. PMID:29375563

  15. 75 FR 42743 - Synergics Roth Rock North Wind Energy, LLC; Supplemental Notice That Initial Market-Based Rate...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-22

    ... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission [Docket No. ER10-1673-000] Synergics Roth Rock North Wind Energy, LLC; Supplemental Notice That Initial Market-Based Rate Filing Includes...-referenced proceeding of Synergics Roth Rock North Wind Energy, LLC's application for market- based rate...

  16. 75 FR 42744 - Synergics Roth Rock Wind Energy, LLC; Supplemental Notice That Initial Market-Based Rate Filing...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-22

    ... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission [Docket No. ER10-1637-000] Synergics Roth Rock Wind Energy, LLC; Supplemental Notice That Initial Market-Based Rate Filing Includes Request...-referenced proceeding of Synergics Roth Rock Wind Energy, LLC's application for market-based rate authority...

  17. Novel synergic antidiabetic effects of Astragalus polysaccharides combined with Crataegus flavonoids via improvement of islet function and liver metabolism.

    PubMed

    Cui, Kai; Zhang, Shaobo; Jiang, Xin; Xie, Weidong

    2016-06-01

    The present study investigated the synergic effects and potential mechanisms of action of Astragalus polysaccharides (APS) combined with Crataegus flavonoids (CF) in the treatment of type 1 diabetes. Diabetes was induced by intraperitoneal injection of 100 mg/kg streptozotocin in mice. Normal and untreated diabetic control mice were used, and CF‑treated (200 mg/kg/day), APS‑treated (200 mg/kg/day), APS + CF (AC)‑treated (200 mg/kg/day of each) and metformin‑treated (200 mg/kg/day) diabetic mice were orally administrated the appropriate therapeutic agent for 4 weeks. The results demonstrated that AC treatment significantly reduced the fasting blood glucose, food and water intake in the diabetic mice. The AC group demonstrated increased serum insulin levels and islet cell function was restored. Furthermore, the AC‑treated mice demonstrated significant increases in the protein expression levels of pancreatic and duodenal homeobox‑1 and phosphorylated adenosine 5'‑monophosphate‑activated protein kinase in the pancreatic and liver tissue samples, respectively. In addition, AC significantly increased the mRNA expression levels of neurogenin 3, v‑maf musculoaponeurotic fibrosarcoma oncogene family, protein A and insulin, and simultaneously decreased the expressions of interleukin 6, tumor necrosis factor‑α and chemokine (C‑C motif) ligand 2 in the pancreatic islet cells of diabetic mice. The anti‑inflammatory activity of APS and the islet‑restoring effect of CF may contribute to the improvement of islet function. AC exerted greater antidiabetic effects compared with APS or CF treatments alone. These results indicated that AC treatment had a synergic antidiabetic effect, which may involve improvements in islet function and liver metabolism. These effects of AC may facilitate the treatment of type 1 or 2 diabetes, as these patients frequently experience impaired islet function and disordered extrapancreatic metabolism.

  18. Heat-shock protein 27 (HSP27, HSPB1) is up-regulated by MET kinase inhibitors and confers resistance to MET-targeted therapy.

    PubMed

    Musiani, Daniele; Konda, John David; Pavan, Simona; Torchiaro, Erica; Sassi, Francesco; Noghero, Alessio; Erriquez, Jessica; Perera, Timothy; Olivero, Martina; Di Renzo, Maria Flavia

    2014-09-01

    The tyrosine kinase encoded by the MET oncogene is activated by gene mutation or amplification in tumors, which in most instances maintain addiction, i.e., dependency, to MET activation. This makes MET an attractive candidate for targeted therapies. Here we show that, in 3/3 MET-addicted human gastric cancer cell lines, MET kinase inhibition resulted in a 3- to 4-fold increased expression of the antiapoptotic small heat-shock protein of 27 kDa (HSP27, HSPB1). HSP27 increase depended on the inhibition of the MEK/ERK pathway and on heat-shock factor 1 (HSF1) and hypoxia-inducible factor-1α (HIF-1α) regulation. Importantly, HSP27-silenced MET-addicted cells underwent 2- and 3-fold more apoptosis following MET inhibition in vitro and in vivo, respectively. Likewise, in human cancer cells susceptible to epidermal growth factor receptor (EGFR) inhibition, EGFR inhibitors induced HSP27 expression and were strengthened by HSP27 suppression. In control cell lines that were not affected by drugs targeting MET or EGFR, these drugs did not induce HSP27 increase. Therefore, in cancer therapies targeting the MET pathway, the induction of HSP27 might limit the efficacy of anti-MET agents. As HSP27 increase also impairs the effectiveness of EGFR inhibitors and is known to protect cells from chemotherapeutics, the induction of HSP27 by targeted agents might strongly affect the success of combination treatments. © FASEB.

  19. Brief Report: Functional Interaction of Endoplasmic Reticulum Aminopeptidase 2 and HLA-B27 Activates the Unfolded Protein Response.

    PubMed

    Zhang, Zhenbo; Ciccia, Francesco; Zeng, Fanxing; Guggino, Giuliana; Yee, Kirby; Abdullah, Hasan; Silverberg, Mark S; Alessandro, Riccardo; Triolo, Giovanni; Haroon, Nigil

    2017-05-01

    The basic mechanisms underlying the pathogenesis of ankylosing spondylitis (AS) remain unresolved. We previously reported an association of the single-nucleotide polymorphism (SNP) rs2549782 in the endoplasmic reticulum aminopeptidase 2 gene (ERAP2) with AS. It is known that patients homozygous for the G allele (GG) of another ERAP2 SNP, rs2248374, lack expression of ERAP2 (ERAP2 null). The present study utilized this information to study the impact of ERAP2 deficiency on HLA-B27 expression in patients with AS, specifically focusing on the functional interaction of ERAP2 and HLA-B27 in peripheral blood mononuclear cells (PBMCs) from patients with AS and assessing the effects in vitro in specific cell lines. Expression of intact peptide HLA-B27 (pB27) or the major histocompatibility complex class I free heavy chains (FHCs) was assessed in PBMCs isolated from HLA-B27-positive patients with AS. ERAP2-suppressed, stable B27-expressing C1R cells (C1R-B27) were tested for the expression levels of pB27 and FHCs, as well as for markers of the unfolded protein response (UPR). Distribution of the ERAP2 SNPs rs2549782 and rs2248374 in patients with AS and in patients with Crohn's disease was assessed. PBMCs from AS patients lacking ERAP2 expressed higher levels of FHCs than did PBMCs from patients positive for ERAP2. This finding was replicated in C1R-B27 cells after suppression of ERAP2. In addition, ERAP2 suppression led to increased levels of the UPR markers BiP, CCAAT/enhancer binding protein homologous protein 10, and X-box binding protein 1 [spliced] as compared to that in short hairpin RNA-treated control cells. There was strong linkage disequilibrium in the ERAP2 locus. All patients with the rs2549782 T allele (which reportedly increases the function of the ERAP-2 protein) were homozygous for the G allele of rs2248374, leading to absence of ERAP2. ERAP2 deficiency causes increased FHC expression and up-regulation of the UPR pathway. © 2016, American College of

  20. Plant Essential Oils Synergize and Antagonize Toxicity of Different Conventional Insecticides against Myzus persicae (Hemiptera: Aphididae)

    PubMed Central

    Faraone, Nicoletta; Hillier, N. Kirk; Cutler, G. Christopher

    2015-01-01

    Plant-derived products can play an important role in pest management programs. Essential oils from Lavandula angustifolia (lavender) and Thymus vulgaris (thyme) and their main constituents, linalool and thymol, respectively, were evaluated for insecticidal activity and synergistic action in combination with insecticides against green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae). The essential oils and their main constituents exerted similar insecticidal activity when aphids were exposed by direct sprays, but were non-toxic by exposure to treated leaf discs. In synergism experiments, the toxicity of imidacloprid was synergized 16- to 20-fold by L. angustifolia and T. vulgaris essential oils, but far less synergism occurred with linalool and thymol, indicating that secondary constituents of the oils were probably responsible for the observed synergism. In contrast to results with imidacloprid, the insecticidal activity of spirotetramat was antagonized by L. angustifolia and T. vulgaris essential oils, and linalool and thymol. Our results demonstrate the potential of plant essential oils as synergists of insecticides, but show that antagonistic action against certain insecticides may occur. PMID:26010088

  1. Chronic Inflammatory Microenvironment in Epidermodysplasia Verruciformis Skin Lesions: Role of the Synergism Between HPV8 E2 and C/EBPβ to Induce Pro-Inflammatory S100A8/A9 Proteins.

    PubMed

    Podgórska, Marta; Ołdak, Monika; Marthaler, Anna; Fingerle, Alina; Walch-Rückheim, Barbara; Lohse, Stefan; Müller, Cornelia S L; Vogt, Thomas; Ustav, Mart; Wnorowski, Artur; Malejczyk, Magdalena; Majewski, Sławomir; Smola, Sigrun

    2018-01-01

    synergizes with the HPV8 E2 protein in differentiation-dependent gene expression. Potent synergistic up-regulation of S100A8/A9 was seen at transcriptional and protein levels. S100A8/A9 containing supernatants from keratinocytes co-expressing HPV8 E2 and C/EBPβ significantly induced chemotaxis of granulocytes in migration assays supporting the relevance of our finding. In conclusion, our data suggest that the HPV8 E2 protein actively contributes to the recruitment of myeloid cells into EV skin lesions, which may support chronic inflammation and progression to skin cancer.

  2. 27-hydroxycholesterol and the expression of three estrogen-sensitive proteins in MCF7 cells.

    PubMed

    Cruz, Pamela; Epuñán, María José; Ramírez, María Eugenia; Torres, Cristian G; Valladares, Luis E; Sierralta, Walter D

    2012-09-01

    The principal aim of this study was to analyze in estrogen receptor-positive MCF7 cells the response of three estrogen-dependent proteins to 27-hydroxycholesterol (27OHC), a major circulating cholesterol metabolite. Immunofluorescence, immunoblotting and immunogold labelling analyses of MCF7 cells exposed for up to 72 h to 2 nM estradiol (E2) or to 2 µM 27OHC demonstrated similar responses in the expression of MnSOD and ERβ compared to the non-stimulated cells. Thus, the results confirm 27OHC's function as a novel selective estrogen receptor modulator (SERM). The epithelial to mesenchymal transition (EMT), observed in MCF7 cells stimulated for longer than 48 h with 2 µM 27OHC, was accompanied by lower immunoreactive levels of nuclear FOXM1 in comparison to E2-treated cells. The results presented in this study are discussed taking into consideration the relationship of hypercholesterolemia, 27OHC production, ROS synthesis and macrophage infiltration, potentially occurring in obese patients with ERα-positive, infiltrated mammary tumors.

  3. Identification of Interleukin-27 (IL-27)/IL-27 Receptor Subunit Alpha as a Critical Immune Axis for In Vivo HIV Control.

    PubMed

    Ruiz-Riol, M; Berdnik, D; Llano, A; Mothe, B; Gálvez, C; Pérez-Álvarez, S; Oriol-Tordera, B; Olvera, A; Silva-Arrieta, S; Meulbroek, M; Pujol, F; Coll, J; Martinez-Picado, J; Ganoza, C; Sanchez, J; Gómez, G; Wyss-Coray, T; Brander, C

    2017-08-15

    Intact and broad immune cell effector functions and specific individual cytokines have been linked to HIV disease outcome, but their relative contribution to HIV control remains unclear. We asked whether the proteome of secreted cytokines and signaling factors in peripheral blood can be used to discover specific pathways critical for host viral control. A custom glass-based microarray, able to measure >600 plasma proteins involved in cell-to-cell communication, was used to measure plasma protein profiles in 96 HIV-infected, treatment-naive individuals with high (>50,000) or low (<10,000 HIV RNA copies/ml) viral loads. Univariate and regression model analysis demonstrate that plasma levels of soluble interleukin-27 (IL-27) are significantly elevated in individuals with high plasma viremia ( P < 0.0001) and are positively correlated with proviral HIV-DNA copy numbers in peripheral blood mononuclear cells (PBMC) (Rho = 0.4011; P = 0.0027). Moreover, soluble IL-27 plasma levels are negatively associated with the breadth and magnitude of the total virus-specific T-cell responses and directly with plasma levels of molecules involved in Wnt/β-catenin signaling. In addition to IL-27, gene expression levels of the specific IL-27 receptor ( IL27RA ) in PBMC correlated directly with both plasma viral load (Rho = 0.3531; P = 0.0218) and the proviral copy number in the peripheral blood as an indirect measure of partial viral reservoir (Rho = 0.4580; P = 0.0030). These results were validated in unrelated cohorts of early infected subjects as well as subjects before and after initiation of antiretroviral treatment, and they identify IL-27 and its specific receptor as a critical immune axis for the antiviral immune response and as robust correlates of viral load and proviral reservoir size in PBMC. IMPORTANCE The detailed knowledge of immune mechanisms that contribute to HIV control is a prerequisite for the design of effective treatment strategies to achieve HIV cure. Cells

  4. Association of polymorphisms in solute carrier family 27, isoform A6 (SLC27A6) and fatty acid-binding protein-3 and fatty acid-binding protein-4 (FABP3 and FABP4) with fatty acid composition of bovine milk.

    PubMed

    Nafikov, R A; Schoonmaker, J P; Korn, K T; Noack, K; Garrick, D J; Koehler, K J; Minick-Bormann, J; Reecy, J M; Spurlock, D E; Beitz, D C

    2013-09-01

    The main goal of this study was to develop tools for genetic selection of animals producing milk with a lower concentration of saturated fatty acids (SFA) and a higher concentration of unsaturated fatty acids (UFA). The reasons for changing milk fatty acid (FA) composition were to improve milk technological properties, such as for production of more spreadable butter, and milk nutritional value with respect to the potentially adverse effects of SFA on human health. We hypothesized that genetic polymorphisms in solute carrier family 27, isoform A6 (SLC27A6) fatty acid transport protein gene and fatty acid binding protein (FABP)-3 and FABP-4 (FABP3 and FABP4) would affect the selectivity of FA uptake into, and FA redistribution inside, mammary epithelial cells, resulting in altered FA composition of bovine milk. The objectives of our study were to discover genetic polymorphisms in SLC27A6, FABP3, and FABP4, and to test those polymorphisms for associations with milk FA composition. The results showed that after pairwise comparisons between SLC27A6 haplotypes for significantly associated traits, haplotype H3 was significantly associated with 1.37 weight percentage (wt%) lower SFA concentration, 0.091 lower SFA:UFA ratio, and 0.17 wt% lower lauric acid (12:0) concentration, but 1.37 wt% higher UFA and 1.24 wt% higher monounsaturated fatty acid (MUFA) concentrations compared with haplotype H1 during the first 3 mo of lactation. Pairwise comparisons between FABP4 haplotypes for significantly associated traits showed that haplotype H3 was significantly associated with 1.04 wt% lower SFA concentration, 0.079 lower SFA:UFA ratio, 0.15 wt% lower lauric acid (12:0), and 0.27 wt% lower myristic acid (14:0) concentrations, but 1.04 wt% higher UFA and 0.91 wt% higher MUFA concentrations compared with haplotype H1 during the first 3 mo of lactation. Percentages of genetic variance explained by H3 versus H1 haplotype substitutions for SLC27A6 and FABP4 ranged from 2.50 to 4.86% and

  5. Monoclonal antibodies against 27.8 kDa protein receptor efficiently block lymphocystis disease virus infection in flounder Paralichthys olivaceus gill cells.

    PubMed

    Sheng, Xiu-Zhen; Wang, Mu; Xing, Jing; Zhan, Wen-Bin

    2012-08-13

    In previous research using co-immunoprecipitation, a 27.8 kDa protein in flounder Paralichthys olivaceus gill (FG) cells was found to bind lymphocystis disease virus (LCDV). In this paper, 13 hybridomas secreting monoclonal antibodies (MAbs) against the 27.8 kDa protein were obtained, and 2 MAbs designated as 2G11 and 3D9 were cloned by limiting dilution. Analyzed by indirect enzyme-linked immunosorbent assay (ELISA) and western blotting, the MAbs specifically reacted with the 27.8 kDa protein of FG cells. Confocal fluorescence microscopy and immunogold electron microscopy (IEM) provided evidence that the epitopes recognized by these MAbs were located primarily on the cell membrane and occasionally in the cytoplasm near the cell membrane of FG cells. The MAbs could block LCDV binding after MAbs were pre-incubated with isolated membrane proteins of FG cells in a blocking ELISA, and MAbs also could inhibit LCDV infection of FG cells in culture. Moreover, several target tissues of LCDV in flounder, including gill, stomach, intestine and liver, displayed the presence of the LCDV receptor-27.8 kDa. These results strongly supported the possibility that the 27.8 kDa protein is the putative receptor specific for LCDV infection of FG cells in flounder.

  6. Structural and functional aspects of hetero-oligomers formed by the small heat shock proteins αB-crystallin and HSP27.

    PubMed

    Aquilina, J Andrew; Shrestha, Sudichhya; Morris, Amie M; Ecroyd, Heath

    2013-05-10

    αB-crystallin and HSP27 are mammalian intracellular small heat shock proteins. These proteins exchange subunits in a rapid and temperature-dependent manner. This facile subunit exchange suggests that differential expression could be used by the cell to regulate the response to stress. A robust technique defines parameters for the dynamic interaction between the major mammalian small heat shock proteins. Small heat shock proteins (sHSPs) exist as large polydisperse species in which there is constant dynamic subunit exchange between oligomeric and dissociated forms. Their primary role in vivo is to bind destabilized proteins and prevent their misfolding and aggregation. αB-Crystallin (αB) and HSP27 are the two most widely distributed and most studied sHSPs in the human body. They are coexpressed in different tissues, where they are known to associate with each other to form hetero-oligomeric complexes. In this study, we aimed to determine how these two sHSPs interact to form hetero-oligomers in vitro and whether, by doing so, there is an increase in their chaperone activity and stability compared with their homo-oligomeric forms. Our results demonstrate that HSP27 and αB formed polydisperse hetero-oligomers in vitro, which had an average molecular mass that was intermediate of each of the homo-oligomers and which were more thermostable than αB, but less so than HSP27. The hetero-oligomer chaperone function was found to be equivalent to that of αB, with each being significantly better in preventing the amorphous aggregation of α-lactalbumin and the amyloid fibril formation of α-synuclein in comparison with HSP27. Using mass spectrometry to monitor subunit exchange over time, we found that HSP27 and αB exchanged subunits 23% faster than the reported rate for HSP27 and αA and almost twice that for αA and αB. This represents the first quantitative evaluation of αB/HSP27 subunit exchange, and the results are discussed in the broader context of regulation of

  7. Microfluidic liquid-air dual-gradient chip for synergic effect bio-evaluation of air pollutant.

    PubMed

    Liu, Xian-Jun; Hu, Shan-Wen; Xu, Bi-Yi; Zhao, Ge; Li, Xiang; Xie, Fu-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2018-05-15

    In this paper, a novel prototype liquid-air dual gradient chip is introduced, which has paved the way for effective synergic effect bio-evaluation of air pollutant. The chip is composed of an array of the agarose liquid-air interfaces, top air gradient layer and bottom liquid gradient layer. The novel agarose liquid-air interface allows for non-biased exposure of cells to all the substances in the air and diffusive interactions with the liquid phase; while the dual liquid-air gradient provides powerful screening abilities, which well reduced errors, saved time and cost from repeated experiment. Coupling the two functions, the chip subsequently facilitates synergic effect evaluation of both liquid and air factors on cells. Here cigarette smoke was taken as the model air pollutant, and its strong synergic effects with inflammatory level of A549 lung cancer cells on their fate were successfully quantified for the first time. These results well testified that the proposed dual-gradient chip is powerful and indispensable for bio-evaluation of air pollutant. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. A novel kinase function of a nucleoside-diphosphate-kinase homologue in Porphyromonas gingivalis is critical in subversion of host cell apoptosis by targeting heat-shock protein 27.

    PubMed

    Lee, Jungnam; Roberts, JoAnn S; Atanasova, Kalina R; Chowdhury, Nityananda; Yilmaz, Özlem

    2018-05-01

    We have previously shown that a homologue of a conserved nucleoside-diphosphate-kinase (Ndk) family of multifunctional enzymes and secreted molecule in Porphyromonas gingivalis can modulate select host molecular pathways including downregulation of reactive-oxygen-species generation to promote bacterial survival in human gingival epithelial cells (GECs). In this study, we describe a novel kinase function for bacterial effector, P. gingivalis-Ndk, in abrogating epithelial cell death by phosphorylating heat-shock protein 27 (HSP27) in GECs. Infection by P. gingivalis was recently suggested to increase phosphorylation of HSP27 in cancer-epithelial cells; however, the mechanism and biological significance of antiapoptotic phospho-HSP27 during infection has never been characterised. Interestingly, using glutathione S-transferase-rNdk pull-down analysed by mass spectrometry, we identified HSP27 in GECs as a strong binder of P. gingivalis-Ndk and further verified using confocal microscopy and ELISA. Therefore, we hypothesised P. gingivalis-Ndk can phosphorylate HSP27 for inhibition of apoptosis in GECs. We further employed P. gingivalis-Ndk protein constructs and an isogenic P. gingivalis-ndk-deficient-mutant strain for functional examination. P. gingivalis-infected GECs displayed significantly increased phospho-HSP27 compared with ndk-deficient-strain during 24 hr infection. Phospho-HSP27 was significantly increased by transfection of GFP-tagged-Ndk into uninfected-GECs, and in vitro phosphorylation assays revealed direct phosphorylation of HSP27 at serines 78 and 82 by P. gingivalis-Ndk. Depletion of HSP27 via siRNA significantly reversed resistance against staurosporine-mediated-apoptosis during infection. Transfection of recombinant P. gingivalis-Ndk protein into GECs substantially decreased staurosporine-induced-apoptosis. Finally, ndk-deficient-mutant strain was unable to inhibit staurosporine-induced Cytochrome C release/Caspase-9 activation. Thus, we

  9. Synergism and foaming properties in binary mixtures of a biosurfactant derived from Camellia oleifera Abel and synthetic surfactants.

    PubMed

    Jian, Hong-lei; Liao, Xiao-xia; Zhu, Li-wei; Zhang, Wei-ming; Jiang, Jian-xin

    2011-07-15

    A biosurfactant, named tea saponin (TS), was isolated and purified from the defatted seed of Camellia oleifera Abel. The characterization of TS including molecular weight, glycosyl composition, and thermal behavior as well as the surface and foaming properties was conducted. The synergistic interactions of binary systems of CTAB-TS, SDS-TS, and Brij35-TS were investigated. The results show that TS had a weight-average molecular weight of 809.12 g mol(-1) and contained four aglycones of L-rhamnose, D-galactose, D-glucose, and D-glucuronic acid. The critical micelle concentration (cmc) of 2.242 mmol L(-1) and the minimum surface tension (γ(cmc)) of 43.5 mN m(-1) were determined for TS. Synergisms in surface tension reduction efficiency, in mixed micelle formation, and in surface tension reduction effectiveness were observed in CTAB-TS and SDS-TS systems, whereas that was not shown in Brij35-TS mixtures. The mixtures of TS with CTAB and SDS showed synergism in foaming efficiency, but this synergism did not exist in Brij35-TS system with respect to the surface properties. Nevertheless, there appears to be no significant correlation between foam stability and the surface properties. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Inhibitory effect of Campomanesia xanthocarpa in platelet aggregation: Comparison and synergism with acetylsalicylic acid.

    PubMed

    Otero, Juliana Soares; Hirsch, Gabriela Elisa; Klafke, Jonatas Zeni; Porto, Fernando Garcez; de Almeida, Amanda Spring; Nascimento, Sabrina; Schmidt, Aline; da Silva, Brenda; Pereira, Roberta Lelis Dias; Jaskulski, Mônica; Parisi, Mariana Migliorini; Dos Santos Guarda, Naiara; Moresco, Rafael Noal; Aita, Carlos Alberto Mayora; Viecili, Paulo Ricardo Nazário

    2017-06-01

    Cardiovascular diseases of thrombotic origin are related to high mortality and standard therapeutic agent used in this case is acetylsalicylic acid (ASA), but serious adverse events may occur. However, recent data has suggested the plant Campomanesia xanthocarpa has antiplatelet activity and could be a viable alternative. In this study we investigated the effects of the encapsulated powder of this plant in human platelet aggregation. 23 healthy subjects were randomly divided into three groups: (1) ASA (100mg), (2) C. xanthocarpa (1000mg) or (3) synergism (500mg of C. xanthocarpa plush 50mg of ASA); daily for five days. Antiplatelet activity was determined by turbidimetric method using ADP or arachidonic acid (AA) agonists before, 5 and 8days after treatments. Treatment with C. xanthocarpa and synergism caused a reduction of 8±13.5% and 12.5±5% in platelet aggregation induced by ADP after 5days of treatment, respectively, returning to basal levels after 8days. For AA agonist, 5days of treatment with C. xanthocarpa, ASA or synergism caused a reduction of 46±15%, 36±12% and 69.3±6% in platelet aggregation, respectively, and first two groups returned to baseline values 8days after treatment ended. Synergism group prolonged antiplatelet effect maintaining aggregation reduction after 8days the end of treatment. C. xanthocarpa showed antiplatelet action when stimulated by agonist AA, and contributed to the antiplatelet effect when associated with ASA for both agonists, allowing dose reduction to 50mg. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. WHO water quality standards Vs Synergic effect(s) of fluoride, heavy metals and hardness in drinking water on kidney tissues.

    PubMed

    Wasana, Hewa M S; Perera, Gamage D R K; Gunawardena, Panduka De S; Fernando, Palika S; Bandara, Jayasundera

    2017-02-14

    Despite WHO standards, waterborne diseases among the human being are rising alarmingly. It is known that the prolong exposure to contaminated water has major impact on public health. The effect of chemical contaminations in drinking water on human being is found to be chronic rather than acute and hence can be defined "consumption of contaminated drinking water could be a silent killer". As the WHO recommended water quality standards are only for individual element and synergic effects of trace metals and anions have not been considered, investigation of synergic effects of trace metals and anions and their effect on human being is of prime important research. By an animal trial, we investigated the synergic effect(s) of heavy metals, aluminium, arsenic, fluoride and hardness in drinking water on kidney tissues of mice. Our investigation strongly suggests existing of a synergic effect especially among Cd, F and hardness of water which could lead to severe kidney damage in mice, even at WHO maximum recommended levels. Hence, the synergic effect(s) of trace metals, fluoride and hardness present in drinking water should be investigated meticulously when stipulating the water quality at WHO maximum recommended levels.

  12. WHO water quality standards Vs Synergic effect(s) of fluoride, heavy metals and hardness in drinking water on kidney tissues

    NASA Astrophysics Data System (ADS)

    Wasana, Hewa M. S.; Perera, Gamage D. R. K.; Gunawardena, Panduka De S.; Fernando, Palika S.; Bandara, Jayasundera

    2017-02-01

    Despite WHO standards, waterborne diseases among the human being are rising alarmingly. It is known that the prolong exposure to contaminated water has major impact on public health. The effect of chemical contaminations in drinking water on human being is found to be chronic rather than acute and hence can be defined “consumption of contaminated drinking water could be a silent killer”. As the WHO recommended water quality standards are only for individual element and synergic effects of trace metals and anions have not been considered, investigation of synergic effects of trace metals and anions and their effect on human being is of prime important research. By an animal trial, we investigated the synergic effect(s) of heavy metals, aluminium, arsenic, fluoride and hardness in drinking water on kidney tissues of mice. Our investigation strongly suggests existing of a synergic effect especially among Cd, F and hardness of water which could lead to severe kidney damage in mice, even at WHO maximum recommended levels. Hence, the synergic effect(s) of trace metals, fluoride and hardness present in drinking water should be investigated meticulously when stipulating the water quality at WHO maximum recommended levels.

  13. C/EBPβ LIP and c-Jun synergize to regulate expression of the murine progesterone receptor.

    PubMed

    Wang, Weizhong; Do, Han Ngoc; Aupperlee, Mark D; Durairaj, Srinivasan; Flynn, Emily E; Miksicek, Richard J; Haslam, Sandra Z; Schwartz, Richard C

    2018-06-02

    CCAAT/enhancer binding protein β (C/EBPβ) is required for murine mammary ductal morphogenesis and alveologenesis. Progesterone is critical for proliferation and alveologenesis in adult mammary glands, and there is a similar requirement for progesterone receptor isoform B (PRB) in alveologenesis. We examined C/EBPβ regulation of PR expression. All three C/EBPβ isoforms, including typically inhibitory LIP, transactivated the PR promoter. LIP, particularly, strongly synergized with c-Jun to drive PR transcription. Endogenous C/EBPβ and c-Jun stimulated a PR promoter-reporter and these two factors showed promoter occupancy on the endogenous PR gene. Additionally, LIP overexpression elevated endogenous PR protein expression. In pregnancy, both PRB and the relative abundance of LIP among C/EBPβ isoforms increase. Consistent with a role in PRB expression, in vivo C/EBPβ and PR isoform A expression showed mutually exclusive localization in mammary epithelium, while C/EBPβ and PRB largely co-localized. We suggest a critical role for C/EBPβ, particularly LIP, in PRB expression. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  14. Localization and dynamic expression of a 27.8 kDa receptor protein for lymphocystis disease virus infection in sea bass ( Lateolabrax japonicus) tissues

    NASA Astrophysics Data System (ADS)

    Wu, Ronghua; Sheng, Xiuzhen; Tang, Xiaoqian; Xing, Jing; Zhan, Wenbin

    2017-10-01

    Lymphocystis disease virus (LCDV) infects target cells by attaching to a 27.8 kDa receptor (27.8R) protein in flounder Paralichthys olivaceus, and anti-27.8R monoclonal antibodies (MAbs) have been developed. However, the 27.8R existence in tissues of sea bass ( Lateolabrax japonicus) and its role in LCDV infection have remained unclear. In this study, the results of western blotting demonstrated that the same 27.8R was shared by flounder and sea bass. LCDV-free sea bass individuals were intramuscularly injected with LCDV, and viral copies were detected in tissues from 3 h post infection and showed a time-dependent increase during 9 days infection. Distribution and synthesis of 27.8R in sea bass tissues were investigated by using anti-27.8R MAbs as probes. It was found that 27.8R was distributed in all the tested tissues. The levels of 27.8R protein were highest in gill and skin, then a bit lowly in stomach, head kidney and heart, followed by spleen, intestine, blood cells, gonad and liver, and least in kidney and brain in healthy sea bass. Upon LCDV infection, 27.8R synthesis was up-regulated in each tissue, and higher in the tissues with higher LCDV copies. The 27.8R and LCDV were detected in some peripheral blood leukocytes but not in red blood cells. These results suggested that 27.8R was widely distributed in sea bass tissues, and it served as a receptor and correlated with tissue tropism of LCDV infection. Furthermore, leukocytes had the potential of being a LCDV carrier and were responsible for a systemic infection of LCDV in sea bass.

  15. Novel cell-penetrating peptide-loaded nanobubbles synergized with ultrasound irradiation enhance EGFR siRNA delivery for triple negative Breast cancer therapy.

    PubMed

    Jing, Hui; Cheng, Wen; Li, Shouqiang; Wu, Bolin; Leng, Xiaoping; Xu, Shouping; Tian, Jiawei

    2016-10-01

    The lack of safe and effective gene delivery strategies remains a bottleneck for cancer gene therapy. Here, we describe the synthesis, characterization, and application of cell-penetrating peptide (CPP)-loaded nanobubbles (NBs), which are characterized by their safety, strong penetrating power and high gene loading capability for gene delivery. An epidermal growth factor receptor (EGFR)-targeted small interfering RNA (siEGFR) was transfected into triple negative breast cancer (TNBC) cells via prepared CPP-NBs synergized with ultrasound-targeted microbubble destruction (UTMD) technology. Fluorescence microscopy showed that siEGFR and CPP were loaded on the shells of the NBs. The transfection efficiency and cell proliferation levels were evaluated by FACS and MTT assays, respectively. In addition, in vivo experiments showed that the expression of EGFR mRNA and protein could be efficiently downregulated and that the growth of a xenograft tumor derived from TNBC cells could be inhibited. Our results indicate that CPP-NBs carrying siEGFR could potentially be used as a promising non-viral gene vector that can be synergized with UTMD technology for efficient TNBC therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Heat Shock Protein 27 Plays a Pivotal Role in Myofibroblast Differentiation and in the Development of Bleomycin-Induced Pulmonary Fibrosis

    PubMed Central

    Park, Ah-Mee; Kanai, Kyosuke; Itoh, Tatsuki; Sato, Takao; Tsukui, Tatsuya; Inagaki, Yutaka; Selman, Moises; Matsushima, Kouji; Yoshie, Osamu

    2016-01-01

    Heat shock protein 27 (HSP27) is a member of the small molecular weight HSP family. Upon treatment with transforming growth factor β1 (TGF-β1), we observed upregulation of HSP27 along with that of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, in cultured human and mouse lung fibroblasts. Furthermore, by using siRNA knockdown, we demonstrated that HSP27 was involved in cell survival and upregulation of fibronectin, osteopontin (OPN) and type 1 collagen, all functional markers of myofibroblast differentiation, in TGF-β1-treated MRC-5 cells. In lung tissues of bleomycin-treated mice, HSP27 was strongly upregulated and substantially co-localized with α-SMA, OPN and type I collagen but not with proSP-C (a marker of type II alveolar epithelial cells), E-cadherin (a marker of epithelial cells) or F4/80 (a marker of macrophages). A similar co-localization of HSP27 and α-SMA was observed in lung tissues of patients with idiopathic pulmonary fibrosis. Furthermore, airway delivery of HSP27 siRNA effectively suppressed bleomycin-induced pulmonary fibrosis in mice. Collectively, our findings indicate that HSP27 is critically involved in myofibroblast differentiation of lung fibroblasts and may be a promising therapeutic target for lung fibrotic diseases. PMID:26859835

  17. Insect peptide CopA3-induced protein degradation of p27Kip1 stimulates proliferation and protects neuronal cells from apoptosis

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

    Nam, Seung Taek; Kim, Dae Hong; Lee, Min Bum

    2013-07-19

    Highlights: •CopA3 peptide isolated from the Korean dung beetle has antimicrobial activity. •Our study reported that CopA3 has anticancer and immunosuppressive effects. •We here demonstrated that CopA3 has neurotropic and neuroprotective effects. •CopA3 degrades p27Kip1 protein and this mediates effects of CopA3 on neuronal cells. -- Abstract: We recently demonstrated that the antibacterial peptide, CopA3 (a D-type disulfide dimer peptide, LLCIALRKK), inhibits LPS-induced macrophage activation and also has anticancer activity in leukemia cells. Here, we examined whether CopA3 could affect neuronal cell proliferation. We found that CopA3 time-dependently increased cell proliferation by up to 31 ± 2% in human neuroblastomamore » SH-SY5Y cells, and up to 29 ± 2% in neural stem cells isolated from neonatal mouse brains. In both cell types, CopA3 also significantly inhibited the apoptosis and viability losses caused by 6-hydroxy dopamine (a Parkinson disease-mimicking agent) and okadaic acid (an Alzheimer’s disease-mimicking agent). Immunoblotting revealed that the p27Kip1 protein (a negative regulator of cell cycle progression) was markedly degraded in CopA3-treated SH-SY5Y cells. Conversely, an adenovirus expressing p27Kip1 significantly inhibited the antiapoptotic effects of CopA3 against 6-hydroxy dopamine- and okadaic acid-induced apoptosis, and decreased the neurotropic effects of CopA3. These results collectively suggest that CopA3-mediated protein degradation of p27Kip1 may be the main mechanism through which CopA3 exerts neuroprotective and neurotropic effects.« less

  18. Synergic co-activation of muscles in elbow flexion via fractional Brownian motion.

    PubMed

    Chang, Shyang; Hsyu, Ming-Chun; Cheng, Hsiu-Yao; Hsieh, Sheng-Hwu

    2008-12-31

    In reflex and volitional actions, co-activations of agonist and antagonist muscles are believed to be present. Recent studies indicate that such co-activations can be either synergic or dyssynergic. The aim of this paper is to investigate if the co-activations of biceps brachii, brachialis, and triceps brachii during volitional elbow flexion are in the synergic or dyssynergic state. In this study, two groups with each containing six healthy male volunteers participated. Each person of the first group performed 30 trials of volitional elbow flexion while each of the second group performed 30 trials of passive elbow flexion as control experiments. Based on the model of fractional Brownian motion, the intensity and frequency information of the surface electromyograms (EMGs) could be extracted simultaneously. No statistically significant changes were found in the control group. As to the other group, results indicated that the surface EMGs of all five muscle groups were temporally synchronized in frequencies with persistent intensities during each elbow flexion. In addition, the mean values of fractal dimensions for rest and volitional flexion states revealed significant differences with P < 0.01. The obtained positive results suggest that these muscle groups work together synergically to facilitate elbow flexion during the co-activations.

  19. Maize 27 kDa gamma-zein is a potential allergen for early weaned pigs.

    PubMed

    Krishnan, Hari B; Kerley, Monty S; Allee, Gary L; Jang, Sungchan; Kim, Won-Seok; Fu, Chunjiang J

    2010-06-23

    Soybean and maize are extensively used in animal feed, primarily in poultry, swine, and cattle diets. Soybean meal can affect pig performance in the first few weeks following weaning and elicit specific antibodies in weaned piglets. Though maize is a major component of pig feed, it is not known if any of the maize proteins can elicit immunological response in young pigs. In this study, we have identified a prominent 27 kDa protein from maize as an immunodominant protein in young pigs. This protein, like some known allergens, exhibited resistance to pepsin digestion in vitro. Several lines of evidence identify the immunodominant 27 kDa protein as a gamma-zein, a maize seed storage protein. First, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of different solubility classes of maize seed proteins revealed the presence of an abundant 27 kDa protein in the prolamin (zein) fraction. Antibodies raised against the purified maize 27 kDa gamma-zein also reacted against the same protein recognized by the young pig serum. Additionally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the peptides generated by trypsin digestion of the immunodominant 27 kDa protein showed significant homology to the maize 27 kDa gamma-zein. Since eliminating the allergenic protein will have a great impact on the nutritive value of the maize meal and expand its use in the livestock industry, it will be highly desirable to develop maize cultivars completely lacking the 27 kDa allergenic protein.

  20. Apatinib, a novel tyrosine kinase inhibitor, suppresses tumor growth in cervical cancer and synergizes with Paclitaxel.

    PubMed

    Qiu, Haifeng; Li, Jing; Liu, Qiuli; Tang, Mei; Wang, Yuan

    2018-06-09

    Apatinib is a novel tyrosine kinase inhibitor that targets VEGFR2 signal and exhibits potent anti-tumor effects in human cancers. In this study, we aim to investigate the efficacy of Apatinib in cervical cancer. The protein expression of VEGFR2 and its relationships with clinical parameters were investigated in a panel of cervical cancer patients. In vitro, a series of experiments were performed to detect the effects of Apatinib on the proliferation, apoptosis and cell cycle in cervical cancer cells. Both the immortalized cell lines and primary cultured tissues were used to investigate the synergy between Apatinib and chemotherapeutic drugs. The in vivo effects of Apatinib were validated in a nude mouse model. Compared to that in normal cervix, VEGFR2 protein was significantly upregulated in cervical cancer tissues (P<0.001); this was positively correlated with advanced tumor stage, lymph node metastasis, and a poor prognosis. In vitro, Apatinib markedly induced apoptosis and G1-phase arrest, suppressed cell growth, and decreased colony formation ability. We also found that primary cancer tissues with higher level of VEGFR2 were much more sensitive to Apatinib. Further, we proved that Apatinib significantly increased the sensitivity to Paclitaxel in cervical cancer cells and the mouse model. Collectively, we firstly report the anti-tumor efficacy of Apatinib in cervical cancer. Moreover, Apatinib synergized with Paclitaxel to achieve more significant suppression on tumor growth, proposing that Apatinib might be a potent drug for cervical cancer.

  1. Central cavity of fructose-1,6-bisphosphatase and the evolution of AMP/fructose 2,6-bisphosphate synergism in eukaryotic organisms.

    PubMed

    Gao, Yang; Shen, Lu; Honzatko, Richard B

    2014-03-21

    The effects of AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) on porcine fructose-1,6-bisphosphatase (pFBPase) and Escherichia coli FBPase (eFBPase) differ in three respects. AMP/Fru-2,6-P2 synergism in pFBPase is absent in eFBPase. Fru-2,6-P2 induces a 13° subunit pair rotation in pFBPase but no rotation in eFBPase. Hydrophilic side chains in eFBPase occupy what otherwise would be a central aqueous cavity observed in pFBPase. Explored here is the linkage of AMP/Fru-2,6-P2 synergism to the central cavity and the evolution of synergism in FBPases. The single mutation Ser(45) → His substantially fills the central cavity of pFBPase, and the triple mutation Ser(45) → His, Thr(46) → Arg, and Leu(186) → Tyr replaces porcine with E. coli type side chains. Both single and triple mutations significantly reduce synergism while retaining other wild-type kinetic properties. Similar to the effect of Fru-2,6-P2 on eFBPase, the triple mutant of pFBPase with bound Fru-2,6-P2 exhibits only a 2° subunit pair rotation as opposed to the 13° rotation exhibited by the Fru-2,6-P2 complex of wild-type pFBPase. The side chain at position 45 is small in all available eukaryotic FBPases but large and hydrophilic in bacterial FBPases, similar to eFBPase. Sequence information indicates the likelihood of synergism in the FBPase from Leptospira interrogans (lFBPase), and indeed recombinant lFBPase exhibits AMP/Fru-2,6-P2 synergism. Unexpectedly, however, AMP also enhances Fru-6-P binding to lFBPase. Taken together, these observations suggest the evolution of AMP/Fru-2,6-P2 synergism in eukaryotic FBPases from an ancestral FBPase having a central aqueous cavity and exhibiting synergistic feedback inhibition by AMP and Fru-6-P.

  2. Central Cavity of Fructose-1,6-bisphosphatase and the Evolution of AMP/Fructose 2,6-bisphosphate Synergism in Eukaryotic Organisms*

    PubMed Central

    Gao, Yang; Shen, Lu; Honzatko, Richard B.

    2014-01-01

    The effects of AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) on porcine fructose-1,6-bisphosphatase (pFBPase) and Escherichia coli FBPase (eFBPase) differ in three respects. AMP/Fru-2,6-P2 synergism in pFBPase is absent in eFBPase. Fru-2,6-P2 induces a 13° subunit pair rotation in pFBPase but no rotation in eFBPase. Hydrophilic side chains in eFBPase occupy what otherwise would be a central aqueous cavity observed in pFBPase. Explored here is the linkage of AMP/Fru-2,6-P2 synergism to the central cavity and the evolution of synergism in FBPases. The single mutation Ser45 → His substantially fills the central cavity of pFBPase, and the triple mutation Ser45 → His, Thr46 → Arg, and Leu186 → Tyr replaces porcine with E. coli type side chains. Both single and triple mutations significantly reduce synergism while retaining other wild-type kinetic properties. Similar to the effect of Fru-2,6-P2 on eFBPase, the triple mutant of pFBPase with bound Fru-2,6-P2 exhibits only a 2° subunit pair rotation as opposed to the 13° rotation exhibited by the Fru-2,6-P2 complex of wild-type pFBPase. The side chain at position 45 is small in all available eukaryotic FBPases but large and hydrophilic in bacterial FBPases, similar to eFBPase. Sequence information indicates the likelihood of synergism in the FBPase from Leptospira interrogans (lFBPase), and indeed recombinant lFBPase exhibits AMP/Fru-2,6-P2 synergism. Unexpectedly, however, AMP also enhances Fru-6-P binding to lFBPase. Taken together, these observations suggest the evolution of AMP/Fru-2,6-P2 synergism in eukaryotic FBPases from an ancestral FBPase having a central aqueous cavity and exhibiting synergistic feedback inhibition by AMP and Fru-6-P. PMID:24436333

  3. Heat shock protein 27 is involved in PCV2 infection in PK-15 cells.

    PubMed

    Liu, Jie; Zhang, Lili; Zhu, Xuejiao; Bai, Juan; Wang, Liming; Wang, Xianwei; Jiang, Ping

    2014-08-30

    Porcine circovirus type 2 (PCV2) has been identified as the etiologic agent which causing postweaning multisystemic wasting syndrome in swine farms in the world. Some quantitative proteomic studies showed that many proteins significantly changed in PCV2-infected cells. To explore the role of cellular chaperones during PCV2 infection, cytoprotective chaperone Hsp27 was analyzed in PCV2-infected PK-15 cells in this study. The results showed that Hsp27 could up-regulate and accumulate in phosphorylated forms in the nuclear zone during PCV2 replication. Suppression of Hsp27 phosphorylation with specific chemical inhibitors or downregulation of all forms of Hsp27 via RNA interference significantly reduced the virus replication. Meanwhile, over-expression of Hsp27 enhanced PCV2 genome replication and virion production. It indicated that Hsp27 was required for PCV2 production in PK-15 cells culture. It should be helpful for understanding the mechanism of replication and pathogenesis of PCV2 and development of novel antiviral therapies in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death.

    PubMed

    Sellier, Chantal; Campanari, Maria-Letizia; Julie Corbier, Camille; Gaucherot, Angeline; Kolb-Cheynel, Isabelle; Oulad-Abdelghani, Mustapha; Ruffenach, Frank; Page, Adeline; Ciura, Sorana; Kabashi, Edor; Charlet-Berguerand, Nicolas

    2016-06-15

    An intronic expansion of GGGGCC repeats within the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). Ataxin-2 with intermediate length of polyglutamine expansions (Ataxin-2 Q30x) is a genetic modifier of the disease. Here, we found that C9ORF72 forms a complex with the WDR41 and SMCR8 proteins to act as a GDP/GTP exchange factor for RAB8a and RAB39b and to thereby control autophagic flux. Depletion of C9orf72 in neurons partly impairs autophagy and leads to accumulation of aggregates of TDP-43 and P62 proteins, which are histopathological hallmarks of ALS-FTD SMCR8 is phosphorylated by TBK1 and depletion of TBK1 can be rescued by phosphomimetic mutants of SMCR8 or by constitutively active RAB39b, suggesting that TBK1, SMCR8, C9ORF72, and RAB39b belong to a common pathway regulating autophagy. While depletion of C9ORF72 only has a partial deleterious effect on neuron survival, it synergizes with Ataxin-2 Q30x toxicity to induce motor neuron dysfunction and neuronal cell death. These results indicate that partial loss of function of C9ORF72 is not deleterious by itself but synergizes with Ataxin-2 toxicity, suggesting a double-hit pathological mechanism in ALS-FTD. © 2016 The Authors.

  5. Synergism effects of pioglitazone and Urtica dioica extract in streptozotocin-induced nephropathy via attenuation of oxidative stress.

    PubMed

    Shokrzadeh, Mohammad; Sadat-Hosseini, Sara; Fallah, Marjan; Shaki, Fatemeh

    2017-05-01

    Hyperglycemia promotes oxidative stress that plays a crucial role in the pathogenesis of Diabetic nephropathy (DN). In this study, we investigated the synergism effects of hydroalcoholic extract of Urtica dioica and pioglitazone (PIO) on the prevention of DN in streptozotocin induced-diabetic mice. Forty-two mice were divided into six groups as follows: non-diabetic control group, DMSO group (as solvent), diabetic group and four treatment groups which received U. dioica , pioglitazone, U. dioica plus pioglitazone and vitE. Diabetes was induced by a single dose of streptozotocin (STZ) (200 mg/kg body wt, IP) diluted in citrate buffer (pH= 4.6). After 4 weeks treatment, all animals were anaesthetized and blood was collected for serum urea and creatinine levels assessment in plasma and kidney tissue were excised for evaluation of oxidative stress markers. Treatment with U. dioica significantly inhibited increase in serum urea and creatinine in plasma that were observed in diabetic mice. Furthermore, the elevated level of oxidative stress markers (glutathione oxidation, lipid peroxidation (LPO), protein carbonyl) in renal supernatant of diabetic mice was inhibited by U. dioica treatment. Interestingly, U. dioica promoted beneficial effects of PIO in reducing STZ-induced hyperglycemia, renal damage and oxidative stress markers. Our findings showed that PIO plus U. dioica have synergism protective effects against STZ-induced nephropathy that can be a candidate as a therapeutic approach in order to treatment of DN.

  6. Synergism effects of pioglitazone and Urtica dioica extract in streptozotocin-induced nephropathy via attenuation of oxidative stress

    PubMed Central

    Shokrzadeh, Mohammad; Sadat-hosseini, Sara; Fallah, Marjan; Shaki, Fatemeh

    2017-01-01

    Objective(s): Hyperglycemia promotes oxidative stress that plays a crucial role in the pathogenesis of Diabetic nephropathy (DN). In this study, we investigated the synergism effects of hydroalcoholic extract of Urtica dioica and pioglitazone (PIO) on the prevention of DN in streptozotocin induced-diabetic mice. Materials and Methods: Forty-two mice were divided into six groups as follows: non-diabetic control group, DMSO group (as solvent), diabetic group and four treatment groups which received U. dioica, pioglitazone, U. dioica plus pioglitazone and vitE. Diabetes was induced by a single dose of streptozotocin (STZ) (200 mg/kg body wt, IP) diluted in citrate buffer (pH= 4.6). After 4 weeks treatment, all animals were anaesthetized and blood was collected for serum urea and creatinine levels assessment in plasma and kidney tissue were excised for evaluation of oxidative stress markers. Results: Treatment with U. dioica significantly inhibited increase in serum urea and creatinine in plasma that were observed in diabetic mice. Furthermore, the elevated level of oxidative stress markers (glutathione oxidation, lipid peroxidation (LPO), protein carbonyl) in renal supernatant of diabetic mice was inhibited by U. dioica treatment. Interestingly, U. dioica promoted beneficial effects of PIO in reducing STZ-induced hyperglycemia, renal damage and oxidative stress markers. Conclusion: Our findings showed that PIO plus U. dioica have synergism protective effects against STZ-induced nephropathy that can be a candidate as a therapeutic approach in order to treatment of DN. PMID:28656084

  7. Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine.

    PubMed

    Köhrle, J; Rasmussen, U B; Rokos, H; Leonard, J L; Hesch, R D

    1990-04-15

    125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27

  8. Chloroquine synergizes with FTS to enhance cell growth inhibition and cell death

    PubMed Central

    Schmukler, Eran; Wolfson, Eya; Haklai, Roni; Elad-Sfadia, Galit; Kloog, Yoel; Pinkas-Kramarski, Ronit

    2014-01-01

    The Ras family of small GTPases transmits extracellular signals that regulate cell growth, differentiation, motility and death. Ras signaling is constitutively active in a large number of human cancers. Ras can also regulate autophagy by affecting several signaling pathways including the mTOR pathway. Autophagy is a process that regulates the balance between protein synthesis and protein degradation. It is important for normal growth control, but may be defective in diseases. Previously, we have shown that Ras inhibition by FTS induces autophagy, which partially protects cancer cells and may limit the use of FTS as an anti-cancer drug. Since FTS is a non toxic drug we hypothesized that FTS and chloroquine (an autophagy inhibitor) will synergize in cell growth inhibition and cell death. Thus, in the present study, we explored the mechanism of each individual drug and their combined action. Our results demonstrate that in HCT-116 and in Panc-1 cells, FTS induces autophagy, which can be inhibited by chloroquine. Furthermore, the combined treatment synergistically decreased the number of viable cells. Interestingly, the combined treatment enhanced apoptotic cell death as indicated by increased sub-G1 cell population, increased Hoechst staining, activation of caspase 3, decrease in survivin expression and release of cytochrome c. Thus, chloroquine treatment may promote FTS-mediated inhibition of tumor cell growth and may stimulate apoptotic cell death. PMID:24368422

  9. Abnormal expression of p27kip1 protein in levator ani muscle of aging women with pelvic floor disorders – a relationship to the cellular differentiation and degeneration

    PubMed Central

    Bukovsky, Antonin; Copas, Pleas; Caudle, Michael R; Cekanova, Maria; Dassanayake, Tamara; Asbury, Bridgett; Van Meter, Stuart E; Elder, Robert F; Brown, Jeffrey B; Cross, Stephanie B

    2001-01-01

    Background Pelvic floor disorders affect almost 50% of aging women. An important role in the pelvic floor support belongs to the levator ani muscle. The p27/kip1 (p27) protein, multifunctional cyclin-dependent kinase inhibitor, shows changing expression in differentiating skeletal muscle cells during development, and relatively high levels of p27 RNA were detected in the normal human skeletal muscles. Methods Biopsy samples of levator ani muscle were obtained from 22 symptomatic patients with stress urinary incontinence, pelvic organ prolapse, and overlaps (age range 38–74), and nine asymptomatic women (age 31–49). Cryostat sections were investigated for p27 protein expression and type I (slow twitch) and type II (fast twitch) fibers. Results All fibers exhibited strong plasma membrane (and nuclear) p27 protein expression. cytoplasmic p27 expression was virtually absent in asymptomatic women. In perimenopausal symptomatic patients (ages 38–55), muscle fibers showed hypertrophy and moderate cytoplasmic p27 staining accompanied by diminution of type II fibers. Older symptomatic patients (ages 57–74) showed cytoplasmic p27 overexpression accompanied by shrinking, cytoplasmic vacuolization and fragmentation of muscle cells. The plasma membrane and cytoplasmic p27 expression was not unique to the muscle cells. Under certain circumstances, it was also detected in other cell types (epithelium of ectocervix and luteal cells). Conclusions This is the first report on the unusual (plasma membrane and cytoplasmic) expression of p27 protein in normal and abnormal human striated muscle cells in vivo. Our data indicate that pelvic floor disorders are in perimenopausal patients associated with an appearance of moderate cytoplasmic p27 expression, accompanying hypertrophy and transition of type II into type I fibers. The patients in advanced postmenopause show shrinking and fragmentation of muscle fibers associated with strong cytoplasmic p27 expression. PMID:11696252

  10. High Rab27A expression indicates favorable prognosis in CRC.

    PubMed

    Shi, Chuanbing; Yang, Xiaojun; Ni, Yijiang; Hou, Ning; Xu, Li; Zhan, Feng; Zhu, Huijun; Xiong, Lin; Chen, Pingsheng

    2015-06-13

    Rab27A is a peculiar member in Rab family and has been suggested to play essential roles in the development of human cancers. However, the association between Rab27A expression and clinicopathological characteristics of colorectal cancer (CRC) has not been elucidated yet. One-step quantitative real-time polymerase chain reaction (qPCR) test with 18 fresh-frozen CRC samples and immunohistochemistry (IHC) analysis in 112 CRC cases were executed to evaluate the relationship between Rab27A expression and the clinicopathological features of CRC. Cox regression and Kaplan-Meier survival analyses were performed to identify the prognostic factors for 112 CRC patients. The results specified that the expression levels of Rab27A mRNA and protein were significantly higher in CRC tissues than that in matched non-cancerous tissues, in both qPCR test (p = 0.029) and IHC analysis (p = 0.020). The IHC data indicated that the Rab27A protein expression in CRC was statistically correlated with lymph node metastasis (p = 0.022) and TNM stage (p = 0.026). Cox multi-factor analysis and Kaplan-Meier method suggested Rab27A protein expression (p = 0.012) and tumor differentiation (p = 0.004) were significantly associated with the overall survival of CRC patients. The data indicated the differentiate expression of Rab27A in CRC tissues and matched non-cancerous tissues. Rab27A may be used as a valuable prognostic biomarker for CRC patients.

  11. Synergism between permethrin and propoxur against Culex quinquefasciatus mosquito larvae.

    PubMed

    Corbel, V; Chandre, F; Darriet, F; Lardeux, F; Hougard, J-M

    2003-06-01

    To see if synergism occurs between carbamate and pyrethroid insecticides, we tested permethrin and propoxur as representatives of these two classes of compounds used for mosquito control. Larvicidal activity of both insecticides was assessed separately and together on a susceptible strain of the mosquito Culex quinquefasciatus (Diptera: Culicidae) by two methods. When mixed at a constant ratio (permethrin : propoxur 1 : 60 based on LC50) and tested at serial concentrations to plot dose/mortality regression, significant synergy occurred between them (co-toxicity coefficient = 2.2), not just an additive effect. For example, when the mixture gave 50% mortality, the same concentrations of permethrin and propoxur alone would have given merely 2 x 1% mortality. When a sublethal dose (LC0) of permethrin or propoxur was added to the other (range LC10-LC95), synergism occurred up to the LC80 level. Synergistic effects were attributed to the complementary modes of action by these two insecticide classes acting on different components of nerve impulse transmission. Apart from raising new possibilities for Culex control, it seems appropriate to consider using such mixtures or combinations for insecticide-treated mosquito nets in situations with insecticide-resistant Anopheles malaria vectors.

  12. A synergic simulation-optimization approach for analyzing biomolecular dynamics in living organisms.

    PubMed

    Sadegh Zadeh, Kouroush

    2011-01-01

    A synergic duo simulation-optimization approach was developed and implemented to study protein-substrate dynamics and binding kinetics in living organisms. The forward problem is a system of several coupled nonlinear partial differential equations which, with a given set of kinetics and diffusion parameters, can provide not only the commonly used bleached area-averaged time series in fluorescence microscopy experiments but more informative full biomolecular/drug space-time series and can be successfully used to study dynamics of both Dirac and Gaussian fluorescence-labeled biomacromolecules in vivo. The incomplete Cholesky preconditioner was coupled with the finite difference discretization scheme and an adaptive time-stepping strategy to solve the forward problem. The proposed approach was validated with analytical as well as reference solutions and used to simulate dynamics of GFP-tagged glucocorticoid receptor (GFP-GR) in mouse cancer cell during a fluorescence recovery after photobleaching experiment. Model analysis indicates that the commonly practiced bleach spot-averaged time series is not an efficient approach to extract physiological information from the fluorescence microscopy protocols. It was recommended that experimental biophysicists should use full space-time series, resulting from experimental protocols, to study dynamics of biomacromolecules and drugs in living organisms. It was also concluded that in parameterization of biological mass transfer processes, setting the norm of the gradient of the penalty function at the solution to zero is not an efficient stopping rule to end the inverse algorithm. Theoreticians should use multi-criteria stopping rules to quantify model parameters by optimization. Copyright © 2010 Elsevier Ltd. All rights reserved.

  13. Purification and characterization of ribosomal proteins L27 and L30 having antimicrobial activity produced by the Lactobacillus salivarius SGL 03.

    PubMed

    Pidutti, P; Federici, F; Brandi, J; Manna, L; Rizzi, E; Marini, U; Cecconi, D

    2018-02-01

    The aim of this study was to investigate the antimicrobial potential of proteins secreted by a new strain of Lactobacillus salivarius. The secretome of L. salivarius SGL 03 strain was analysed by gel-assisted fractionation and MS/MS to identify low-molecular-mass proteins. This strategy allowed us to identify 10 secreted proteins. Then, a combination of heterologous expression and agar well diffusion was used to characterize them as to their antimicrobial activity, mechanisms of action and stability. Our findings indicate that L27 and L30 proteins of the 50S ribosomal subunit have antimicrobial activity against Streptococcus pyogenes, Streptococcus uberis and Enterococcus faecium. In addition, both proteins are bactericidal against S. pyogenes and maintain their antimicrobial activity after different protease treatments, at acidic pH, after heat treatment, and if stored in a refrigerated ambient at least at 4°C. The overall results demonstrated that the L27 and L30 ribosomal proteins are of interest as new antimicrobial molecules to prevent the growth of S. pyogenes, S. uberis and E. faecium. Our results provide the first insight into the extra-ribosomal activity of L27 and L30 secreted proteins of L. salivarius. This study demonstrated the capacity of L. salivarius SGL 03 to produce antimicrobial molecules and suggested this strain as a promising probiotic candidate. © 2017 The Society for Applied Microbiology.

  14. Hsp27 and F-box protein β-TrCP promote degradation of mRNA decay factor AUF1.

    PubMed

    Li, Mei-Ling; Defren, Jennifer; Brewer, Gary

    2013-06-01

    Activation of the mitogen-activated protein (MAP) pathway kinases p38 and MK2 induces phosphorylation of the chaperone Hsp27 and stabilization of mRNAs containing AU-rich elements (AREs) (ARE-mRNAs). Likewise, expression of phosphomimetic mutant forms of Hsp27 also stabilizes ARE-mRNAs. It appears to perform this function by promoting degradation of the ARE-mRNA decay factor AUF1 by proteasomes. In this study, we examined the molecular mechanism linking Hsp27 phosphorylation to AUF1 degradation by proteasomes. AUF1 is a target of β-TrCP, the substrate recognition subunit of the E3 ubiquitin ligase Skp1-cullin-F-box protein complex, SCF(β-TrCP). Depletion of β-TrCP stabilized AUF1. In contrast, overexpression of β-TrCP enhanced ubiquitination and degradation of AUF1 and led to stabilization of reporter mRNAs containing cytokine AREs. Enhanced AUF1 degradation required expression of phosphomimetic mutant forms of both Hsp27 and AUF1. Our results suggest that a signaling axis composed of p38 MAP kinase-MK2-Hsp27-β-TrCP may promote AUF1 degradation by proteasomes and stabilization of cytokine ARE-mRNAs.

  15. Identification of a "glycine-loop"-like coiled structure in the 34 AA Pro,Gly,Met repeat domain of the biomineral-associated protein, PM27.

    PubMed

    Wustman, Brandon A; Santos, Rudolpho; Zhang, Bo; Evans, John Spencer

    2002-12-05

    Fracture resistance in biomineralized structures has been linked to the presence of proteins, some of which possess sequences that are associated with elastic behavior. One such protein superfamily, the Pro,Gly-rich sea urchin intracrystalline spicule matrix proteins, form protein-protein supramolecular assemblies that modify the microstructure and fracture-resistant properties of the calcium carbonate mineral phase within embryonic sea urchin spicules and adult sea urchin spines. In this report, we detail the identification of a repetitive keratin-like "glycine-loop"- or coil-like structure within the 34-AA (AA: amino acid) N-terminal domain, (PGMG)(8)PG, of the spicule matrix protein, PM27. The identification of this repetitive structural motif was accomplished using two capped model peptides: a 9-AA sequence, GPGMGPGMG, and a 34-AA peptide representing the entire motif. Using CD, NMR spectrometry, and molecular dynamics simulated annealing/minimization simulations, we have determined that the 9-AA model peptide adopts a loop-like structure at pH 7.4. The structure of the 34-AA polypeptide resembles a coil structure consisting of repeating loop motifs that do not exhibit long-range ordering. Given that loop structures have been associated with protein elastic behavior and protein motion, it is plausible that the 34-AA Pro,Gly,Met repeat sequence motif in PM27 represents a putative elastic or mobile domain. Copyright 2002 Wiley Periodicals, Inc.

  16. Novel HLA-B27-restricted epitopes from Chlamydia trachomatis generated upon endogenous processing of bacterial proteins suggest a role of molecular mimicry in reactive arthritis.

    PubMed

    Alvarez-Navarro, Carlos; Cragnolini, Juan J; Dos Santos, Helena G; Barnea, Eilon; Admon, Arie; Morreale, Antonio; López de Castro, José A

    2013-09-06

    Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na(+)-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27(+) cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330-338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211-223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211-223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA.

  17. Novel HLA-B27-restricted Epitopes from Chlamydia trachomatis Generated upon Endogenous Processing of Bacterial Proteins Suggest a Role of Molecular Mimicry in Reactive Arthritis*

    PubMed Central

    Alvarez-Navarro, Carlos; Cragnolini, Juan J.; Dos Santos, Helena G.; Barnea, Eilon; Admon, Arie; Morreale, Antonio; López de Castro, José A.

    2013-01-01

    Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na+-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27+ cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330–338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211–223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211–223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA. PMID:23867464

  18. SYNERGIC TRIAL (SYNchronizing Exercises, Remedies in Gait and Cognition) a multi-Centre randomized controlled double blind trial to improve gait and cognition in mild cognitive impairment.

    PubMed

    Montero-Odasso, Manuel; Almeida, Quincy J; Burhan, Amer M; Camicioli, Richard; Doyon, Julien; Fraser, Sarah; Li, Karen; Liu-Ambrose, Teresa; Middleton, Laura; Muir-Hunter, Susan; McIlroy, William; Morais, José A; Pieruccini-Faria, Frederico; Shoemaker, Kevin; Speechley, Mark; Vasudev, Akshya; Zou, G Y; Berryman, Nicolas; Lussier, Maxime; Vanderhaeghe, Leanne; Bherer, Louis

    2018-04-16

    Physical exercise, cognitive training, and vitamin D are low cost interventions that have the potential to enhance cognitive function and mobility in older adults, especially in pre-dementia states such as Mild Cognitive Impairment (MCI). Aerobic and progressive resistance exercises have benefits to cognitive performance, though evidence is somewhat inconsistent. We postulate that combined aerobic exercise (AE) and progressive resistance training (RT) (combined exercise) will have a better effect on cognition than a balance and toning control (BAT) intervention in older adults with MCI. We also expect that adding cognitive training and vitamin D supplementation to the combined exercise, as a multimodal intervention, will have synergistic efficacy. The SYNERGIC trial (SYNchronizing Exercises, Remedies in GaIt and Cognition) is a multi-site, double-blinded, five-arm, controlled trial that assesses the potential synergic effect of combined AE and RT on cognition and mobility, with and without cognitive training and vitamin D supplementation in older adults with MCI. Two-hundred participants with MCI aged 60 to 85 years old will be randomized to one of five arms, four of which include combined exercise plus combinations of dual-task cognitive training (real vs. sham) and vitamin D supplementation (3 × 10,000 IU/wk. vs. placebo) in a quasi-factorial design, and one arm which receives all control interventions. The primary outcome measure is the ADAS-Cog (13 and plus modalities) measured at baseline and at 6 months of follow-up. Secondary outcomes include neuroimaging, neuro-cognitive performance, gait and mobility performance, and serum biomarkers of inflammation (C reactive protein and interleukin 6), neuroplasticity (brain-derived neurotropic factor), endothelial markers (vascular endothelial growth factor 1), and vitamin D serum levels. The SYNERGIC Trial will establish the efficacy and feasibility of a multimodal intervention to improve cognitive performance

  19. Unfolded-protein response–associated stabilization of p27(Cdkn1b) interferes with lens fiber cell denucleation, leading to cataract

    PubMed Central

    Lyu, Lei; Whitcomb, Elizabeth A.; Jiang, Shuhong; Chang, Min-Lee; Gu, Yumei; Duncan, Melinda K.; Cvekl, Ales; Wang, Wei-Lin; Limi, Saima; Reneker, Lixing W.; Shang, Fu; Du, Linfang; Taylor, Allen

    2015-01-01

    Failure of lens fiber cell denucleation (LFCD) is associated with congenital cataracts, but the pathobiology awaits elucidation. Recent work has suggested that mechanisms that direct the unidirectional process of LFCD are analogous to the cyclic processes associated with mitosis. We found that lens-specific mutations that elicit an unfolded-protein response (UPR) in vivo accumulate p27(Cdkn1b), show cyclin-dependent kinase (Cdk)-1 inhibition, retain their LFC nuclei, and are cataractous. Although a UPR was not detected in lenses expressing K6W-Ub, they also accumulated p27 and showed failed LFCD. Induction of a UPR in human lens epithelial cells (HLECs) also induced accumulation of p27 associated with decreased levels of S-phase kinase-associated protein (Skp)-2, a ubiquitin ligase that regulates mitosis. These cells also showed decreased lamin A/C phosphorylation and metaphase arrest. The suppression of lamin A/C phosphorylation and metaphase transition induced by the UPR was rescued by knockdown of p27. Taken together, these data indicate that accumulation of p27, whether related to the UPR or not, prevents the phosphorylation of lamin A/C and LFCD in maturing LFCs in vivo, as well as in dividing HLECs. The former leads to cataract and the latter to metaphase arrest. These results suggest that accumulation of p27 is a common mechanism underlying retention of LFC nuclei.—Lei, L., Whitcomb, E. A., Jiang, S., Chang, M.-L., Gu, Y., Duncan, M. K., Cvekl, A., Wang, W.-L., Limi, S., Reneker, L. W., Shang, F., Du, L., Taylor, A. Unfolded protein response–associated stabilization of p27(Cdkn1b) interferes with lens fiber cell denucleation, leading to cataract. PMID:26590164

  20. Antibacterial activity of three newly-synthesized chalcones & synergism with antibiotics against clinical isolates of methicillin-resistant Staphylococcus aureus

    PubMed Central

    Božić, Dragana D.; Milenković, Marina; Ivković, Branka; Cirković, Ivana

    2014-01-01

    Background & objectives: Multidrug-resistance of methicillin-resistant Staphylococcus aureus (MRSA) is a serious therapeutical problem. Chalcones belong to a group of naturally occurring flavonoids, usually found in various plant species, and have potent antibacterial, antiviral and antifungal activities. The goal of this study was to evaluate the antibacterial effect of three newly-synthesized chalcones against clinical isolates of MRSA, and their synergism with β-lactam and non- β-lactam antibiotics. Methods: Antimicrobial activity of the three newly-synthesized chalcones was tested against 19 clinical isolates of MRSA and a laboratory control strain of MRSA (ATCC 43300). The synergism with β-lactams: cefotaxime (CFX), ceftriaxone (CTX), and non-β-lactam antibiotics: ciprofloxacin (CIP), gentamicin (GEN) and trimethoprim/sulphamethoxazole (TMP-SMX) was investigated by checkerboard method. Results: All evaluated compounds showed significant anti-MRSA activity with MIC values from 25-200 μg/ml. Observed synergism with antibiotics demonstrated that chalcones significantly enhanced the efficacy of CIP, GEN and TMP-SMX. Interpretation & conclusions: Our study demonstrated that three newly-synthesized chalcones exhibited significant anti-MRSA effect and synergism with non-β-lactam antibiotics. The most effective compound was 1,3-Bis-(2-hydroxy-phenyl)-propenone. Our results provide useful information for future research of possible application of chalcones in combination with conventional anti-MRSA therapy as promising new antimicrobial agents. PMID:25222788

  1. IFI27, a novel epidermal growth factor-stabilized protein, is functionally involved in proliferation and cell cycling of human epidermal keratinocytes.

    PubMed

    Hsieh, W-L; Huang, Y-H; Wang, T-M; Ming, Y-C; Tsai, C-N; Pang, J-H S

    2015-04-01

    IFI27 is highly expressed in psoriatic lesions but its function has not been known. The present study aimed to explore its role in proliferation of epidermal keratinocytes. IFI27 knockdown and over-expression in keratinocytes were used to compare their proliferation, by MTT assay, apoptosis (by annexin V binding) and cell cycle progression by flow cytometry. Formation of cyclin A/CDK1 complex was examined by a co-immunoprecipitaion method. Anti-proliferation effects of IFI27 were also examined in vivo by topical application of IFI27 siRNA on imiquimod-induced psoriatic lesions, in a mouse model. Epidermal growth factor was demonstrated to increase IFI27 expression by prolonging half-life of IFI27 protein. The IFI27 knockdown in keratinocytes reduced the proliferation rate, but had no effect on apoptosis nor on apoptosis-related genes. Interestingly, IFI27 knockdown resulted in S-phase arrest that was found to be associated with increased Tyr15 phosphorylation of CDK1, reduced CDC25B and reduced formation of cyclin A/CDK1 complex. In addition, IFI27 knockdown was also shown to activate p53 by Ser15 phosphorylation and increase p21 expression. Topical application of IFI27 siRNA on imiquimod-induced psoriatic lesion in a mouse model reduced epidermal thickness, formation of rete ridges and PCNA expression. Our study demonstrates for the first time, that cell function of IFI27 is involved in proliferation of skin keratinocytes both in vitro and in vivo. It suggests that IFI27 might be a suitable target for development of a novel anti-psoriasis therapy. © 2015 John Wiley & Sons Ltd.

  2. Modulation of DNA damage response and induction of apoptosis mediates synergism between doxorubicin and a new imidazopyridine derivative in breast and lung cancer cells.

    PubMed

    El-Awady, Raafat A; Semreen, Mohammad H; Saber-Ayad, Maha M; Cyprian, Farhan; Menon, Varsha; Al-Tel, Taleb H

    2016-01-01

    DNA damage response machinery (DDR) is an attractive target of cancer therapy. Modulation of DDR network may alter the response of cancer cells to DNA damaging anticancer drugs such as doxorubicin. The aim of the present study is to investigate the effects of a newly developed imidazopyridine (IAZP) derivative on the DDR after induction of DNA damage in cancer cells by doxorubicin. Cytotoxicity sulphrhodamine-B assay showed a weak anti-proliferative effect of IAZP alone on six cancer cell lines (MCF7, A549, A549DOX11, HepG2, HeLa and M8) and a normal fibroblast strain. Combination of IAZP with doxorubicin resulted in synergism in lung (A549) and breast (MCF7) cancer cells but neither in the other cancer cell lines nor in normal fibroblasts. Molecular studies revealed that synergism is mediated by modulation of DNA damage response and induction of apoptosis. Using constant-field gel electrophoresis and immunofluorescence detection of γ-H2AX foci, IAZP was shown to inhibit the repair of doxorubicin-induced DNA damage in A549 and MCF7 cells. Immunoblot analysis showed that IAZP suppresses the phosphorylation of the ataxia lelangiectasia and Rad3 related (ATR) protein, which is an important player in the response of cancer cells to chemotherapy-induced DNA damage. Moreover, IAZP augmented the doxorubicin-induced degradation of p21, activation of p53, CDK2, caspase 3/7 and phosphorylation of Rb protein. These effects enhanced doxorubicin-induced apoptosis in both cell lines. Our results indicate that IAZP is a promising agent that may enhance the cytotoxic effects of doxorubicin on some cancer cells through targeting the DDR. It is a preliminary step toward the clinical application of IAZP in combination with anticancer drugs and opens the avenue for the development of compounds targeting the DDR pathway that might improve the therapeutic index of anticancer drugs and enhance their cure rate. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

    PubMed

    Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

    2015-01-01

    Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

  4. Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling*

    PubMed Central

    McGarvey, Jennifer C.; Xiao, Kunhong; Bowman, Shanna L.; Mamonova, Tatyana; Zhang, Qiangmin; Bisello, Alessandro; Sneddon, W. Bruce; Ardura, Juan A.; Jean-Alphonse, Frederic; Vilardaga, Jean-Pierre; Puthenveedu, Manojkumar A.; Friedman, Peter A.

    2016-01-01

    The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor. PMID:27008860

  5. Sudden infant death syndrome: no significant expression of heat-shock proteins (HSP27, HSP70).

    PubMed

    Doberentz, Elke; Führing, Sarah; Madea, Burkhard

    2016-03-01

    In industrialized countries, sudden infant death is the most common cause of death in young children. Although prone sleeping position is a well-known risk factor, hyperthermia might also be important. Pathognomonic findings of premortem hyperthermia do not exist. During stress, including thermal effects, heat-shock protein (HSP) expression increases. This study investigated hyperthermia as a contributing or pathogenic factor for sudden infant death syndrome (SIDS). Immunohistochemical staining for HSP27 and HSP70 in the kidney, heart, and lung from 120 SIDS cases was examined. HSP70 immunostaining was negative in kidney, heart, and lung tissues in all cases and in tissues from the control group. HSP27 staining was positive in the kidney from one case, and was positive in the lungs (respiratory epithelia in 27% of cases; vascular endothelia in 19% of cases) and was negative in the heart. In the control group HSP27 was positive in 8% of renal tubular tissues and in 29% of renal vascular endothelia. Staining for HSP27 in lung tissues was positive in respiratory epithelia in 8% of cases and for vascular endothelia in 29%, whereas tissues from the heart were positive in only 4%. The hypothesis of hyperthermia being a pathogenic factor for SIDS was not supported by immunohistochemical visualization of HSP70 or HSP27.

  6. A phase I dose-escalation study of apatorsen (OGX-427), an antisense inhibitor targeting heat shock protein 27 (Hsp27), in patients with castration-resistant prostate cancer and other advanced cancers.

    PubMed

    Chi, K N; Yu, E Y; Jacobs, C; Bazov, J; Kollmannsberger, C; Higano, C S; Mukherjee, S D; Gleave, M E; Stewart, P S; Hotte, S J

    2016-06-01

    Heat shock protein 27 (Hsp27) is a chaperone protein that regulates cell survival via androgen receptor and other signaling pathways, thereby mediating cancer progression. Apatorsen (OGX-427) is a 2'-methoxyethyl-modified antisense oligonucleotide that inhibits Hsp27 expression. This study evaluated the safety profile and recommended phase II dosing of apatorsen in patients with advanced cancer. Patients with castration-resistant prostate (CRPC), breast, ovary, lung, or bladder cancer were enrolled to this phase I dose-escalation study. Apatorsen was administered i.v. weekly in 21-day cycles following 3 loading doses and over 5 dose levels (200-1000 mg). Apatorsen plasma concentrations, circulating tumor cells (CTCs) and CTC Hsp27 expression, and serum Hsp27 levels were evaluated. Forty-two patients were accrued, of which 52% had CRPC. Patients were heavily pretreated, with 57% having had ≥3 prior chemotherapy regimens. During the loading dose/cycle 1 and overall study period, 93% and 100% of patients (N = 42) experienced treatment-related adverse events, respectively; most were grade 1-2 and included chills, pruritus, flushing, prolonged aPTT, lymphopenia, and anemia. One patient experienced a dose-limiting toxicity at the 600 mg dose level (intracranial hemorrhage in a previously undiagnosed brain metastasis). A maximum tolerated dose was not defined. Apatorsen Cmax increased proportionally with dose. Decreases in tumor markers and declines in CTCs were observed, with a prostate-specific antigen decline >%50% occurring in 10% of patients with CRPC; 29/39 assessable patients (74%) had reductions from ≥5 CTC/7.5 ml at baseline to <5 CTC/7.5 ml post-treatment. Twelve patients had stable measurable disease as best response. Apatorsen was tolerated at the highest dose evaluated (1000 mg). Single-agent activity was suggested by changes in tumor markers, CTC, and stable measurable disease. Phase II studies evaluating apatorsen are underway. NCT00487786. © The

  7. Thrombin Receptor-Activating Protein (TRAP)-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1) from Platelets in DM Patients

    PubMed Central

    Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Matsushima-Nishiwaki, Rie; Akamatsu, Shigeru; Enomoto, Yukiko; Iida, Hiroki; Otsuka, Takanobu; Ogura, Shinji; Iwama, Toru; Kojima, Kumi; Kozawa, Osamu

    2016-01-01

    It is generally known that heat shock protein 27 (HSP27) is phosphorylated through p38 mitogen-activated protein (MAP) kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP) on the release of HSP27 in platelets in type 2 diabetes mellitus (DM) patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78) were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM)-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB) levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC) of small aggregates (9–25 µm) induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25–50 µm), large aggregates (50–70 µm) and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients. PMID:27187380

  8. Thrombin Receptor-Activating Protein (TRAP)-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1) from Platelets in DM Patients.

    PubMed

    Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Matsushima-Nishiwaki, Rie; Akamatsu, Shigeru; Enomoto, Yukiko; Iida, Hiroki; Otsuka, Takanobu; Ogura, Shinji; Iwama, Toru; Kojima, Kumi; Kozawa, Osamu

    2016-05-14

    It is generally known that heat shock protein 27 (HSP27) is phosphorylated through p38 mitogen-activated protein (MAP) kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP) on the release of HSP27 in platelets in type 2 diabetes mellitus (DM) patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78) were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM)-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB) levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC) of small aggregates (9-25 µm) induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25-50 µm), large aggregates (50-70 µm) and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients.

  9. Differential expression and activation of Rab27A in human eosinophils: relationship to blood eosinophilia.

    PubMed

    Coughlin, Jason J; Odemuyiwa, Solomon O; Davidson, Courtney E; Moqbel, Redwan

    2008-08-29

    Eosinophil degranulation is thought to play a pathophysiological role in asthma. Rab27A is a GTP-binding protein that is known to be essential for the degranulation of several leukocyte subsets and thus may be essential for eosinophil granule exocytosis. Here, we show that Rab27A mRNA and protein are expressed in human eosinophils. We have developed a novel assay to assess Rab27A activation and have found a similar activation pattern of this protein upon stimulation of eosinophils, neutrophils and NK cells suggesting a similar function in these cell types. Interestingly, Rab27A expression was elevated in eosinophils from asthmatic donors. Furthermore, eosinophils from eosinophilic donors displayed more rapid Rab27A activation kinetics than those from donors with lower eosinophil counts. Given that elevated blood eosinophil numbers correlate with increased priming of eosinophils, this pattern of Rab27A activation suggests differential protein expression in activated cells may allow eosinophils to degranulate more rapidly upon stimulation.

  10. Role for malonyl coenzyme A:acyl carrier protein transacylase (MCAT) in the growth-inhibitory effect of the calmodulin antagonist trifluoperazine in Mycobacterium bovis BCG.

    PubMed

    Sinha, Indrajit; Dick, Thomas

    2004-06-01

    To determine whether the fatty acid synthesis enzyme malonyl coenzyme A:acyl carrier protein transacylase (MCAT) is involved in the growth-inhibitory effect of trifluoperazine in the tubercle bacillus Mycobacterium bovis BCG. BCG was grown in liquid culture with various concentrations of trifluoperazine and growth was monitored by OD measurement. To determine the effect of trifluoperazine on MCAT protein level, total protein was extracted from BCG cultures and was analysed by 2D gel electrophoresis and western blot. To confirm trifluoperazine-dependent reduction in the MCAT protein level, two BCG strains overexpressing MCAT at a low and high constitutive level were similarly tested. The synergic effect of trifluoperazine and isoniazid was tested at sub-MIC levels in liquid cultures. Trifluoperazine inhibition of growth correlates with reduction in the steady-state level of MCAT protein. Overexpression of MCAT confers resistance to trifluoperazine. Trifluoperazine acts synergically (albeit weakly) with isoniazid and no resistance towards isoniazid alone was observed due to overexpression of MCAT. This suggests MCAT to be a specific target of trifluoperazine. These results indicate MCAT as a target of trifluoperazine and provide an explanation for the inhibitory effect of trifluoperazine on mycobacterial lipid synthesis observed earlier. This makes MCAT a potential target for new antimycobacterials.

  11. Overcoming HSP27-mediated resistance by altered dimerization of HSP27 using small molecules.

    PubMed

    Kim, Jee Hye; Jung, Ye Jin; Choi, Byeol; Lee, Na Lim; Lee, Hae Jun; Kwak, Soo Yeon; Kwon, Youngjoo; Na, Younghwa; Lee, Yun-Sil

    2016-08-16

    Heat shock protein 27 (HSP27, HSPB1) is an anti-apoptotic protein characterized for its tumorigenic and metastatic properties, and now referenced as a major therapeutic target in many types of cancer. The biochemical properties of HSP27 rely on a structural oligomeric and dynamic organization that is important for its chaperone activity. Down-regulation by small interfering RNA or inhibition with a dominant-negative mutant efficiently counteracts the anti-apoptotic and protective properties of HSP27. However, unlike other HSPs such as HSP90 and HSP70, small molecule approaches for neutralization of HSP27 are not well established because of the absence of an ATP binding domain. Previously, we found that a small molecule, zerumbone (ZER), induced altered dimerization of HSP27 by cross linking the cysteine residues required to build a large oligomer, led to sensitization in combination with radiation. In this study, we identified another small molecule, a xanthone compound, more capable of altering dimeric HSP27 than ZER and yielding sensitization in human lung cancer cells when combined with HSP90 inhibitors or standard anticancer modalities such as irradiation and cytotoxic anticancer drugs. Therefore, altered dimerization of HSP27 represents a good strategy for anticancer therapy in HSP27-overexpressing cancer cells.

  12. Overcoming HSP27-mediated resistance by altered dimerization of HSP27 using small molecules

    PubMed Central

    Choi, Byeol; Lee, Na Lim; Lee, Hae Jun; Kwak, Soo Yeon; Kwon, Youngjoo; Na, Younghwa; Lee, Yun-Sil

    2016-01-01

    Heat shock protein 27 (HSP27, HSPB1) is an anti-apoptotic protein characterized for its tumorigenic and metastatic properties, and now referenced as a major therapeutic target in many types of cancer. The biochemical properties of HSP27 rely on a structural oligomeric and dynamic organization that is important for its chaperone activity. Down-regulation by small interfering RNA or inhibition with a dominant-negative mutant efficiently counteracts the anti-apoptotic and protective properties of HSP27. However, unlike other HSPs such as HSP90 and HSP70, small molecule approaches for neutralization of HSP27 are not well established because of the absence of an ATP binding domain. Previously, we found that a small molecule, zerumbone (ZER), induced altered dimerization of HSP27 by cross linking the cysteine residues required to build a large oligomer, led to sensitization in combination with radiation. In this study, we identified another small molecule, a xanthone compound, more capable of altering dimeric HSP27 than ZER and yielding sensitization in human lung cancer cells when combined with HSP90 inhibitors or standard anticancer modalities such as irradiation and cytotoxic anticancer drugs. Therefore, altered dimerization of HSP27 represents a good strategy for anticancer therapy in HSP27-overexpressing cancer cells. PMID:27449291

  13. Investigation of the fatty acid transporter-encoding genes SLC27A3 and SLC27A4 in autism.

    PubMed

    Maekawa, Motoko; Iwayama, Yoshimi; Ohnishi, Tetsuo; Toyoshima, Manabu; Shimamoto, Chie; Hisano, Yasuko; Toyota, Tomoko; Balan, Shabeesh; Matsuzaki, Hideo; Iwata, Yasuhide; Takagai, Shu; Yamada, Kohei; Ota, Motonori; Fukuchi, Satoshi; Okada, Yohei; Akamatsu, Wado; Tsujii, Masatsugu; Kojima, Nobuhiko; Owada, Yuji; Okano, Hideyuki; Mori, Norio; Yoshikawa, Takeo

    2015-11-09

    The solute carrier 27A (SLC27A) gene family encodes fatty acid transport proteins (FATPs) and includes 6 members. During fetal and postnatal periods of development, the growing brain requires a reliable supply of fatty acids. Because autism spectrum disorders (ASD) are now recognized as disorders caused by impaired early brain development, it is possible that functional abnormalities of SLC27A genes may contribute to the pathogenesis of ASD. Here, we confirmed the expression of SLC27A3 and SLC27A4 in human neural stem cells derived from human induced pluripotent stem cells, which suggested their involvement in the developmental stage of the central nervous system. Additionally, we resequenced the SLC27A3 and SLC27A4 genes using 267 ASD patient and 1140 control samples and detected 47 (44 novel and 29 nonsynonymous) and 30 (17 novel and 14 nonsynonymous) variants for the SLC27A3 and SLC27A4, respectively, revealing that they are highly polymorphic with multiple rare variants. The SLC27A4 Ser209 allele was more frequently represented in ASD samples. Furthermore, we showed that a SLC27A4 Ser209 mutant resulted in significantly higher fluorescently-labeled fatty acid uptake into bEnd3 cells, a mouse brain capillary-derived endothelial cell line, compared with SLC27A4 Gly209, suggesting that the functional change may contribute to ASD pathophysiology.

  14. Investigation of the fatty acid transporter-encoding genes SLC27A3 and SLC27A4 in autism

    PubMed Central

    Maekawa, Motoko; Iwayama, Yoshimi; Ohnishi, Tetsuo; Toyoshima, Manabu; Shimamoto, Chie; Hisano, Yasuko; Toyota, Tomoko; Balan, Shabeesh; Matsuzaki, Hideo; Iwata, Yasuhide; Takagai, Shu; Yamada, Kohei; Ota, Motonori; Fukuchi, Satoshi; Okada, Yohei; Akamatsu, Wado; Tsujii, Masatsugu; Kojima, Nobuhiko; Owada, Yuji; Okano, Hideyuki; Mori, Norio; Yoshikawa, Takeo

    2015-01-01

    The solute carrier 27A (SLC27A) gene family encodes fatty acid transport proteins (FATPs) and includes 6 members. During fetal and postnatal periods of development, the growing brain requires a reliable supply of fatty acids. Because autism spectrum disorders (ASD) are now recognized as disorders caused by impaired early brain development, it is possible that functional abnormalities of SLC27A genes may contribute to the pathogenesis of ASD. Here, we confirmed the expression of SLC27A3 and SLC27A4 in human neural stem cells derived from human induced pluripotent stem cells, which suggested their involvement in the developmental stage of the central nervous system. Additionally, we resequenced the SLC27A3 and SLC27A4 genes using 267 ASD patient and 1140 control samples and detected 47 (44 novel and 29 nonsynonymous) and 30 (17 novel and 14 nonsynonymous) variants for the SLC27A3 and SLC27A4, respectively, revealing that they are highly polymorphic with multiple rare variants. The SLC27A4 Ser209 allele was more frequently represented in ASD samples. Furthermore, we showed that a SLC27A4 Ser209 mutant resulted in significantly higher fluorescently-labeled fatty acid uptake into bEnd3 cells, a mouse brain capillary-derived endothelial cell line, compared with SLC27A4 Gly209, suggesting that the functional change may contribute to ASD pathophysiology. PMID:26548558

  15. Silibinin induces hepatic stellate cell cycle arrest via enhancing p53/p27 and inhibiting Akt downstream signaling protein expression.

    PubMed

    Ezhilarasan, Devaraj; Evraerts, Jonathan; Sid, Brice; Calderon, Pedro Buc; Karthikeyan, Sivanesan; Sokal, Etienne; Najimi, Mustapha

    2017-02-01

    Proliferation of hepatic stellate cells (HSCs) plays a pivotal role in the progression of liver fibrosis consequent to chronic liver injury. Silibinin, a flavonoid compound, has been shown to possess anti-fibrogenic effects in animal models of liver fibrosis. This was attributed to an inhibition of cell proliferation of activated HSCs. The present study was to gain insight into the molecular pathways involved in silibinin anti-fibrogenic effect. The study was conducted on LX-2 human stellate cells treated with three concentrations of silibinin (10, 50 and 100 μmol/L) for 24 and 96 hours. At the end of the treatment cell viability and proliferation were evaluated. Protein expression of p27, p21, p53, Akt and phosphorylated-Akt was evaluated by Western blotting analysis and Ki-67 protein expression was by immunocytochemistry. Sirtuin activity was evaluated by chemiluminescence based assay. Silibinin inhibits LX-2 cell proliferation in dose- and time-dependent manner; we showed that silibinin upregulated the protein expressions of p27 and p53. Such regulation was correlated to an inhibition of both downstream Akt and phosphorylated-Akt protein signaling and Ki-67 protein expression. Sirtuin activity also was correlated to silibinin-inhibited proliferation of LX-2 cells. The anti-proliferative effect of silibinin on LX-2 human stellate cells is via the inhibition of the expressions of various cell cycle targets including p27, Akt and sirtuin signaling.

  16. North American Invasion of the Tawny Crazy Ant (Nylanderia fulva) Is Enabled by Pheromonal Synergism from Two Separate Glands.

    PubMed

    Zhang, Qing-He; McDonald, Danny L; Hoover, Doreen R; Aldrich, Jeffrey R; Schneidmiller, Rodney G

    2015-09-01

    A new invader, the "tawny crazy ant", Nylanderia fulva (Hymenoptera: Formicidae; Formicinae), is displacing the red imported fire ant, Solenopsis invicta (Formicidae: Myrmicinae), in the southern U.S., likely through its superior chemical arsenal and communication. Alone, formic acid is unattractive, but this venom (= poison) acid powerfully synergizes attraction of tawny crazy ants to volatiles from the Dufour's gland secretion of N. fulva workers, including the two major components, undecane and 2-tridecanone. The unexpected pheromonal synergism between the Dufour's gland and the venom gland appears to be another key factor, in addition to previously known defensive and detoxification semiochemical features, for the successful invasion and domination of N. fulva in the southern U.S. This synergism is an efficient mechanism enabling N. fulva workers to outcompete Solenopsis and other ant species for food and territory. From a practical standpoint, judicious point-source release formulation of tawny crazy ant volatiles may be pivotal for enhanced attract-and-kill management of this pest.

  17. Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains.

    PubMed

    Van Bortle, Kevin; Ramos, Edward; Takenaka, Naomi; Yang, Jingping; Wahi, Jessica E; Corces, Victor G

    2012-11-01

    Several multiprotein DNA complexes capable of insulator activity have been identified in Drosophila melanogaster, yet only CTCF, a highly conserved zinc finger protein, and the transcription factor TFIIIC have been shown to function in mammals. CTCF is involved in diverse nuclear activities, and recent studies suggest that the proteins with which it associates and the DNA sequences that it targets may underlie these various roles. Here we show that the Drosophila homolog of CTCF (dCTCF) aligns in the genome with other Drosophila insulator proteins such as Suppressor of Hairy wing [SU(HW)] and Boundary Element Associated Factor of 32 kDa (BEAF-32) at the borders of H3K27me3 domains, which are also enriched for associated insulator proteins and additional cofactors. RNAi depletion of dCTCF and combinatorial knockdown of gene expression for other Drosophila insulator proteins leads to a reduction in H3K27me3 levels within repressed domains, suggesting that insulators are important for the maintenance of appropriate repressive chromatin structure in Polycomb (Pc) domains. These results shed new insights into the roles of insulators in chromatin domain organization and support recent models suggesting that insulators underlie interactions important for Pc-mediated repression. We reveal an important relationship between dCTCF and other Drosophila insulator proteins and speculate that vertebrate CTCF may also align with other nuclear proteins to accomplish similar functions.

  18. The EED protein-protein interaction inhibitor A-395 inactivates the PRC2 complex.

    PubMed

    He, Yupeng; Selvaraju, Sujatha; Curtin, Michael L; Jakob, Clarissa G; Zhu, Haizhong; Comess, Kenneth M; Shaw, Bailin; The, Juliana; Lima-Fernandes, Evelyne; Szewczyk, Magdalena M; Cheng, Dong; Klinge, Kelly L; Li, Huan-Qiu; Pliushchev, Marina; Algire, Mikkel A; Maag, David; Guo, Jun; Dietrich, Justin; Panchal, Sanjay C; Petros, Andrew M; Sweis, Ramzi F; Torrent, Maricel; Bigelow, Lance J; Senisterra, Guillermo; Li, Fengling; Kennedy, Steven; Wu, Qin; Osterling, Donald J; Lindley, David J; Gao, Wenqing; Galasinski, Scott; Barsyte-Lovejoy, Dalia; Vedadi, Masoud; Buchanan, Fritz G; Arrowsmith, Cheryl H; Chiang, Gary G; Sun, Chaohong; Pappano, William N

    2017-04-01

    Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.

  19. Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A*

    PubMed Central

    Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

    2016-01-01

    Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase. PMID:27780868

  20. Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A.

    PubMed

    Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

    2016-12-09

    Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Synergism in education: An innovative approach to practice teaching supervision

    NASA Astrophysics Data System (ADS)

    Mereni, Joseph Ibewuike

    1985-12-01

    The problem of effective teacher education is central to the pressing needs of Nigerian education at all levels. Scholars have identified many of them, including the general problems relating to the supervision, guidance, and evaluation of student-practice teaching. The purpose of this essay is briefly to describe an innovative approach undertaken by the Imo State School Board (Nigeria), in collaboration with the Ministry of Education, to resolve some of the problems identified. The underlying assumption is that the student teachers' internship is best conducted with closer collaboration, supervision, and guidance of the teachers. The study employed a theoretical framework synergism in education which integrated earlier studies by both American and Nigerian scholars. The concept of synergism has been defined as the combined healthy action of all `elements' of a system. Application of the theory showed how the State Ministry of Education, the State School Board, the Teacher Training Colleges and the Nigeria Union of Teachers of Imo State collectively resolved in 1980 the problems of inadequate supervisory personnel, high cost of student-teaching internship, and poor student assessment and evaluation. With the synergetic supervisory process, the functions of planning, changing, and decision-making about instructional improvement are shared, with a certain degree of power equalization, among the student teachers, supervisors, co-operating teachers, and the school executives.

  2. A Novel SLC27A4 Splice Acceptor Site Mutation in Great Danes with Ichthyosis.

    PubMed

    Metzger, Julia; Wöhlke, Anne; Mischke, Reinhard; Hoffmann, Annalena; Hewicker-Trautwein, Marion; Küch, Eva-Maria; Naim, Hassan Y; Distl, Ottmar

    2015-01-01

    Ichthyoses are a group of various different types of hereditary disorders affecting skin cornification. They are characterized by hyperkeratoses of different severity levels and are associated with a dry and scaling skin. Genome-wide association analysis of nine affected and 13 unaffected Great Danes revealed a genome-wide significant peak on chromosome 9 at 57-58 Mb in the region of SLC27A4. Sequence analysis of genomic DNA of SLC27A4 revealed the non-synonymous SNV SLC27A4:g.8684G>A in perfect association with ichthyosis-affection in Great Danes. The mutant transcript of SLC27A4 showed an in-frame loss of 54 base pairs in exon 8 probably induced by a new splice acceptor site motif created by the mutated A- allele of the SNV. Genotyping 413 controls from 35 different breeds of dogs and seven wolves revealed that this mutation could not be found in other populations except in Great Danes. Affected dogs revealed high amounts of mutant transcript but only low levels of the wild type transcript. Targeted analyses of SLC27A4 protein from skin tissues of three affected and two unaffected Great Danes indicated a markedly reduced or not detectable wild type and truncated protein levels in affected dogs but a high expression of wild type SLC27A4 protein in unaffected controls. Our data provide evidence of a new splice acceptor site creating SNV that results in a reduction or loss of intact SLC27A4 protein and probably explains the severe skin phenotype in Great Danes. Genetic testing will allow selective breeding to prevent ichthyosis-affected puppies in the future.

  3. Expression and prognostic examination of heat shock proteins (HSP 27, HSP 70, and HSP 90) in medulloblastoma.

    PubMed

    Hauser, Péter; Hanzély, Zoltán; Jakab, Zsuzsanna; Oláh, Lászlóné; Szabó, Erika; Jeney, András; Schuler, Dezso; Fekete, Gyoörgy; Bognár, László; Garami, Miklós

    2006-07-01

    Expression of heat shock proteins (HSPs) is of prognostic significance in several tumor types. HSP expression levels were determined in medulloblastomas and tested whether HSPs expression was associated with prognostic parameters. Expression of antiapoptotic HSP 27, HSP 70, and HSP 90 was investigated by immunohistochemistry, on paraffin-embedded sections from 65 patients. Expression of HSPs was validated on internal vascular controls and by Western blotting analysis. Sample evaluation was based on the estimated percentage of HSP positive tumor cells. For survival analysis Kaplan-Meier method, for statistical analysis chi2 test, univariate analysis, and log rank test were applied. Expression of HSPs varied in medulloblastomas. On the basis of the average expression rate of HSPs, at HSP 27 and HSP 90 with a 10% cut off, and at HSP 70 with a 70% cut off 2 groups were created. The amount of expression of any of the HSP types was not significantly associated with known prognostic factors (age of patient, extent of resection, presence of metastasis) and histologic subtype. After an average follow-up period of 4.30 years, no significant difference was observed in survival depending on the expression of HSP 27 or HSP 70 or HSP 90. The high expression of HSPs indicates that these proteins are potential therapeutic targets.

  4. Intracellular transduction of TAT-Hsp27 fusion protein enhancing cell survival and regeneration capacity of cardiac stem cells in acute myocardial infarction.

    PubMed

    Kim, Hye Jung; Kim, Myoung-Hun; Kim, Jong Tae; Lee, Won-Jin; Kim, Eunjung; Lim, Kwang Suk; Kim, Jang Kyoung; Yang, Young Il; Park, Ki Dong; Kim, Yong-Hee

    2015-10-10

    Myocardial infarction (MI) results in the substantial loss of functional cardiomyocytes, which frequently leads to intractable heart disorders. Cardiac stem cells (CSCs) that retain the capacity to replace all cardiac cells might be a promising strategy for providing a source of new functional cardiomyocytes; however, the poor survival and engraftment of transplanted CSCs in the hostile environment of MI critically mitigate their therapeutic benefits. To capitalize their therapeutic potential, an ex vivo strategy in which CSCs were introduced to the recombinant heat shock protein 27 (Hsp27) through a TAT protein transduction domain for increasing the viability and engraftment in the infarcted myocardium was designed. A recombinant TAT fused Hsp27 (TAT-Hsp27) was able to enter CSCs in a dose-dependent manner. CSCs transduced with TAT-Hsp27 expressed not only endogenous Hsp27 but externally introduced Hsp27, resulting in substantial increase of their anti-oxidative and anti-apoptotic properties via suppressing reactive oxygen species production, the MAPKs signaling pathway, and caspase activation. TAT-Hsp27 enabled CSCs to be protected from apoptotic- and hypoxic-induced cell death during in vitro cardiomyogenic differentiation. In vivo studies demonstrated that CSCs transduced TAT-Hsp27 significantly increased the survival and engraftment in the acutely infarcted myocardium, which is closely related to caspase activity suppression. Finally, CSCs transduced TAT-Hsp27 improved cardiac function and attenuated cardiac remodeling in comparison with non-transduced CSCs. Overall, our approach, which is based on the ex vivo intracellular transduction of TAT-Hsp27 into CSCs before myocardial delivery, might be effective in treating MI. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen

    PubMed Central

    Vijayan, Aneesh; García-Arriaza, Juan; C. Raman, Suresh; Conesa, José Javier; Chichón, Francisco Javier; Santiago, César; Sorzano, Carlos Óscar S.; Carrascosa, José L.; Esteban, Mariano

    2015-01-01

    In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive

  6. Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains

    PubMed Central

    Van Bortle, Kevin; Ramos, Edward; Takenaka, Naomi; Yang, Jingping; Wahi, Jessica E.; Corces, Victor G.

    2012-01-01

    Several multiprotein DNA complexes capable of insulator activity have been identified in Drosophila melanogaster, yet only CTCF, a highly conserved zinc finger protein, and the transcription factor TFIIIC have been shown to function in mammals. CTCF is involved in diverse nuclear activities, and recent studies suggest that the proteins with which it associates and the DNA sequences that it targets may underlie these various roles. Here we show that the Drosophila homolog of CTCF (dCTCF) aligns in the genome with other Drosophila insulator proteins such as Suppressor of Hairy wing [SU(HW)] and Boundary Element Associated Factor of 32 kDa (BEAF-32) at the borders of H3K27me3 domains, which are also enriched for associated insulator proteins and additional cofactors. RNAi depletion of dCTCF and combinatorial knockdown of gene expression for other Drosophila insulator proteins leads to a reduction in H3K27me3 levels within repressed domains, suggesting that insulators are important for the maintenance of appropriate repressive chromatin structure in Polycomb (Pc) domains. These results shed new insights into the roles of insulators in chromatin domain organization and support recent models suggesting that insulators underlie interactions important for Pc-mediated repression. We reveal an important relationship between dCTCF and other Drosophila insulator proteins and speculate that vertebrate CTCF may also align with other nuclear proteins to accomplish similar functions. PMID:22722341

  7. Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat)

    NASA Technical Reports Server (NTRS)

    Cubano, L. A.; Lewis, M. L.

    2001-01-01

    Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription-polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space-flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up-regulate the mRNA for hsp70, although the gene encoding hsp27 is up-regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up-regulation of hsp27 gene expression in microgravity is a new finding.

  8. Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat).

    PubMed

    Cubano, L A; Lewis, M L

    2001-05-01

    Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription-polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space-flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up-regulate the mRNA for hsp70, although the gene encoding hsp27 is up-regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up-regulation of hsp27 gene expression in microgravity is a new finding.

  9. Androgen receptor (AR) inhibitor ErbB3-binding protein-1 (Ebp1) is not targeted by the newly identified AR controlling signaling axis heat-shock protein HSP27 and microRNA miR-1 in prostate cancer cells.

    PubMed

    Stope, Matthias B; Peters, Stefanie; Großebrummel, Hannah; Zimmermann, Uwe; Walther, Reinhard; Burchardt, Martin

    2015-03-01

    Androgen receptor (AR) networks are predominantly involved in prostate cancer (PCa) progression; consequently, factors of AR regulation represent promising targets for PCa therapy. The ErbB3-binding protein 1 (Ebp1) is linked to AR suppression and chemoresistance by so far unknown mechanisms. In this study, an assumed regulation of Ebp1 by the newly identified AR controlling signaling axis heat-shock protein 27 (HSP27)-microRNA-1 (miR-1) was examined. Transfection experiments were carried out overexpressing and knockdown HSP27 and miR-1, respectively, in LNCaP and PC-3 cells. Afterward, HSP27- and miR-1-triggered Ebp1 protein expression was monitored by Western blotting. AR-positive LNCaP cells and AR-negative PC-3 cells possessed diverse basal expression levels of Ebp1. However, subsequent studies revealed no differences in cellular Ebp1 concentrations after modulation of HSP27 and miR-1. Furthermore, docetaxel incubation experiments exhibited no effects on Ebp1 protein synthesis. In PCa, Ebp1 has been described as a regulator of AR functionality and as an effector of PCa therapy resistance. Our data suggest that Ebp1 functionality is independent from heat-shock-protein-regulated progression networks in PCa.

  10. Disorder-function relationships for the cell cycle regulatory proteins p21 and p27.

    PubMed

    Mitrea, Diana M; Yoon, Mi-Kyung; Ou, Li; Kriwacki, Richard W

    2012-04-01

    The classic structure-function paradigm has been challenged by a recently identified class of proteins: intrinsically disordered proteins (IDPs). Despite their lack of stable secondary or tertiary structure, IDPs are prevalent in all forms of life and perform myriad cellular functions, including signaling and regulation. Importantly, disruption of IDP homeostasis is associated with numerous human diseases, including cancer and neurodegeneration. Despite wide recognition of IDPs, the molecular mechanisms underlying their functions are not fully understood. Here we review the structural features and disorder-function relationships for p21 and p27, two cyclin-dependent kinase (Cdk) regulators involved in controlling cell division and fate. Studies of p21 bound to Cdk2/cyclin A revealed that a helix stretching mechanism mediates binding promiscuity. Further, investigations of Tyr88-phosphorylated p27 identified a signaling conduit that controls cell division and is disrupted in certain cancers. These mechanisms rely upon a balance between nascent structure in the free state, induced folding upon binding, and persistent flexibility within functional complexes. Although these disorder-function relationships are likely to be recapitulated in other IDPs, it is also likely that the vocabulary of their mechanisms is much more extensive than is currently understood. Further study of the physical properties of IDPs and elucidation of their links with function are needed to fully understand the mechanistic language of IDPs.

  11. Characterization of the small heat shock protein Hsp27 gene in Chironomus riparius (Diptera) and its expression profile in response to temperature changes and xenobiotic exposures.

    PubMed

    Martínez-Paz, Pedro; Morales, Mónica; Martín, Raquel; Martínez-Guitarte, José Luis; Morcillo, Gloria

    2014-07-01

    Small heat shock proteins constitute the most diverse and least conserved group within the large family of heat shock proteins, which play a crucial role in cell response to environmental insults. Chironomus riparius larvae are widely used in environmental research for testing pollutant toxicity in sediments and freshwater environments. Different genes, such as Hsp70, Hsc70, Hsp90, and Hsp40, have been identified in this species as sensitive biomarkers for xenobiotics, but small Hsps genes remain largely unknown. In this study, the Hsp27 has been characterized in C. riparius and its transcriptional response evaluated under several environmental stimuli. The Hsp27 gene was mapped by FISH on polytene chromosomes at region I-C4 and was found to encode a 195 aa protein, which contains an α-crystallin domain bounded by three conserved regions. This protein shows homology with Drosophila melanogaster HSP27, Ceratitis capitata HSP27, and Sarcophaga crassipalpis HSP25. Real-time reverse transcriptase-polymerase chain reaction analysis showed that heat shock (35 °C) and cadmium dramatically upregulate this gene. Moreover, exposures to triclosan and bisphenol A were able to significantly increase mRNA levels. However, neither nonylphenol nor tributyltin altered Hsp27 gene expression. The transcriptional activity of Hsp27 gene was modulated during cold stress. Interestingly, cold shock (4 °C) significantly reduced Hsp27 transcripts, but this gene was significantly overexpressed during the recovery time at the normal growing temperature. These results show that the Hsp27 gene is sensitive to different environmental stimuli, including endocrine-disrupting pollutants, suggesting its potential as a suitable biomarker for ecotoxicological studies in aquatic systems.

  12. The Candidate Antimalarial Drug MMV665909 Causes Oxygen-Dependent mRNA Mistranslation and Synergizes with Quinoline-Derived Antimalarials

    PubMed Central

    Vallières, Cindy

    2017-01-01

    ABSTRACT To cope with growing resistance to current antimalarials, new drugs with novel modes of action are urgently needed. Molecules targeting protein synthesis appear to be promising candidates. We identified a compound (MMV665909) from the Medicines for Malaria Venture (MMV) Malaria Box of candidate antimalarials that could produce synergistic growth inhibition with the aminoglycoside antibiotic paromomycin, suggesting a possible action of the compound in mRNA mistranslation. This mechanism of action was substantiated with a Saccharomyces cerevisiae model using available reporters of mistranslation and other genetic tools. Mistranslation induced by MMV665909 was oxygen dependent, suggesting a role for reactive oxygen species (ROS). Overexpression of Rli1 (a ROS-sensitive, conserved FeS protein essential in mRNA translation) rescued inhibition by MMV665909, consistent with the drug's action on translation fidelity being mediated through Rli1. The MMV drug also synergized with major quinoline-derived antimalarials which can perturb amino acid availability or promote ROS stress: chloroquine, amodiaquine, and primaquine. The data collectively suggest translation fidelity as a novel target of antimalarial action and support MMV665909 as a promising drug candidate. PMID:28652237

  13. Synergism and antagonism in extracting local anesthetics from aqueous media with mixtures of solvents

    NASA Astrophysics Data System (ADS)

    Sukhanov, P. T.; Chibisova, T. V.; Korenman, Ya. I.

    2014-12-01

    The extraction of local anesthetics from aqueous media with mixtures of solvent is examined and its synergistic and antagonistic effects are determined. Synergism parameters, separation factors, constants for the formation of anesthetic complexes, and solvate numbers are calculated.

  14. Identification of TRIM27 as a novel degradation target of herpes simplex virus 1 ICP0.

    PubMed

    Conwell, Sara E; White, Anne E; Harper, J Wade; Knipe, David M

    2015-01-01

    The herpes simplex virus 1 (HSV-1) immediate early protein ICP0 performs many functions during infection, including transactivation of viral gene expression, suppression of innate immune responses, and modification and eviction of histones from viral chromatin. Although these functions of ICP0 have been characterized, the detailed mechanisms underlying ICP0's complex role during infection warrant further investigation. We thus undertook an unbiased proteomic approach to identifying viral and cellular proteins that interact with ICP0 in the infected cell. Cellular candidates resulting from our analysis included the ubiquitin-specific protease USP7, the transcriptional repressor TRIM27, DNA repair proteins NBN and MRE11A, regulators of apoptosis, including BIRC6, and the proteasome. We also identified two HSV-1 early proteins involved in nucleotide metabolism, UL39 and UL50, as novel candidate interactors of ICP0. Because TRIM27 was the most statistically significant cellular candidate, we investigated the relationship between TRIM27 and ICP0. We observed rapid, ICP0-dependent loss of TRIM27 during HSV-1 infection. TRIM27 protein levels were restored by disrupting the RING domain of ICP0 or by inhibiting the proteasome, arguing that TRIM27 is a novel degradation target of ICP0. A mutant ICP0 lacking E3 ligase activity interacted with endogenous TRIM27 during infection as demonstrated by reciprocal coimmunoprecipitation and supported by immunofluorescence data. Surprisingly, ICP0-null mutant virus yields decreased upon TRIM27 depletion, arguing that TRIM27 has a positive effect on infection despite being targeted for degradation. These results illustrate a complex interaction between TRIM27 and viral infection with potential positive or negative effects of TRIM27 on HSV under different infection conditions. During productive infection, a virus must simultaneously redirect multiple cellular pathways to replicate itself while evading detection by the host's defenses. To

  15. MDM2 antagonists synergize broadly and robustly with compounds targeting fundamental oncogenic signaling pathways

    PubMed Central

    Yu, Dongyin; Lofgren, Julie A.; Osgood, Tao; Robertson, Rebecca; Canon, Jude; Su, Cheng; Jones, Adrie; Zhao, Xiaoning; Deshpande, Chetan; Payton, Marc; Ledell, Jebediah; Hughes, Paul E.; Oliner, Jonathan D.

    2014-01-01

    While MDM2 inhibitors hold great promise as cancer therapeutics, drug resistance will likely limit their efficacy as single agents. To identify drug combinations that might circumvent resistance, we screened for agents that could synergize with MDM2 inhibition in the suppression of cell viability. We observed broad and robust synergy when combining MDM2 antagonists with either MEK or PI3K inhibitors. Synergy was not limited to cell lines harboring MAPK or PI3K pathway mutations, nor did it depend on which node of the PI3K axis was targeted. MDM2 inhibitors also synergized strongly with BH3 mimetics, BCR-ABL antagonists, and HDAC inhibitors. MDM2 inhibitor-mediated synergy with agents targeting these mechanisms was much more prevalent than previously appreciated, implying that clinical translation of these combinations could have far-reaching implications for public health. These findings highlight the importance of combinatorial drug targeting and provide a framework for the rational design of MDM2 inhibitor clinical trials. PMID:24810962

  16. Efficacy of aerosol applications of methoprene and synergized pyrethrin against Tribolium castaneum adults and eggs

    USDA-ARS?s Scientific Manuscript database

    Experiments were performed to determine the efficacy of a single aerosol application of the insecticides methoprene and piperonyl butoxide-synergized pyrethrin, alone or in combination, and the insecticide carrier, Isopar M, against Tribolium castaneum (Herbst), the red flour beetle. The initial tes...

  17. A combination of additives can synergically decrease acrylamide content in gingerbread without compromising sensory quality.

    PubMed

    Komprda, Tomáš; Pridal, Antonin; Mikulíková, Renata; Svoboda, Zdeněk; Cwiková, Olga; Nedomová, Šárka; Sýkora, Vladimír

    2017-02-01

    The present study tested whether replacement of the leavening agent ammonium carbonate by sodium hydrogen carbonate in combination with calcium cation and acidifying agent will synergically decrease acrylamide (AA) content in gingerbread. The type of leavening agent and the presence of Ca 2+ and citric acid accounted for 33.6%, 13.2% and 53.2% of the explained variability of the AA content, respectively (P < 0.01). The AA content in gingerbread produced with (NH 4 ) 2 CO 3 alone was 186.5 µg kg -1 . Irrespective of other tested additives, NaHCO 3 decreased (P < 0.05) AA content to 42% compared to (NH 4 ) 2 CO 3 . Combination of NaHCO 3 + CaCl 2 + citric acid in dough reduced (P < 0.05) AA content below the limit of detection (25 µg kg -1 ). The AA content in gingerbread (y; µg kg -1 ) decreased with an increasing number of additives used (x) according to the equation y = 158.8 - 47.94x (r 2 = 0.42; P < 0.0001). A comprehensive sensory analysis did not indicate any significant deterioration (P > 0.05) in the organoleptic quality of gingerbread produced using calcium cation and citric acid. The present study demonstrates that the combination of additives NaHCO 3 /Ca 2+ /citric acid synergically decreases AA content in gingerbread without compromising the sensory quality. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  18. Synergism between rocuronium and cisatracurium: comparison of the Minto and Greco interaction models.

    PubMed

    Jeon, Soeun; Kwon, Jae Young; Kim, Hae-Kyu; Kim, Tae Kyun

    2016-08-01

    This study was conducted to investigate the pharmacodynamic interaction between rocuronium and cisatracurium using the response surface model, which is not subject to the limitations of traditional isobolographic analysis. One hundred and twenty patients were randomly allocated to receive one of the fifteen predefined combinations of rocuronium and cisatracurium. To study single drugs, cisatracurium 0.2, 0.15, or 0.1 mg/kg or rocuronium 0.8, 0.6 or 0.4 mg/kg doses were administered alone. To study the pharmacodynamic interaction, drugs were applied in three types of combination ratio, i.e., half dose of each drug alone, 75% of each single dose of rocuronium and 25% of each single dose of cisatracurium, and vice versa. Train-of-four (TOF) ratio and T1% (first twitch of the TOF presented as percentage compared to the initial T1) were used as pharmacodynamic endpoints, and the Greco and Minto models were used as surface interaction models. The interaction term α of the Greco model for TOF ratio and T1% measurements showed synergism with values of 0.977 and 1.12, respectively. Application of the Minto model resulted in U50 (θ) values (normalized unit of concentration that produces 50% of the maximal effect in the 0 < θ < 1 region) less than 1 for both TOF ratio and T1% measurements, indicating that rocuronium and cisatracurium exhibit synergism. Response surface modeling of the interaction between rocuronium and cisatracurium, based on considerations of their effects on muscle relaxation as measured by TOF ratio and T1%, indicated that the two drugs show considerable synergism.

  19. Endogenous Noxa Determines the Strong Proapoptotic Synergism of the BH3-Mimetic ABT-737 with Chemotherapeutic Agents in Human Melanoma Cells12

    PubMed Central

    Weber, Arnim; Kirejczyk, Zofia; Potthoff, Stephanie; Ploner, Christian; Häcker, Georg

    2009-01-01

    Human melanoma cells are very resistant to treatment with chemotherapeutic agents, and melanoma shows poor response to chemotherapeutic therapy. We describe a strong synergistic proapoptotic effect of the Bcl-2 family inhibitor ABT-737 and the standard antimelanoma drugs, namely, dacarbazine and fotemustine, and the experimental agent, imiquimod. Experiments with human melanoma cells, keratinocytes, and embryonic fibroblasts showed that all three agents activated the mitochondrial apoptosis pathway. ABT-737 on its own was ineffective in melanoma cells unless Mcl-1 was experimentally downregulated. However, ABT-737 strongly enhanced the proapoptotic activity of the chemotherapeutic drugs. Whereas cell death induction by all three agents involved the activity of both BH3-only proteins, Bim and Noxa, the combination with ABT-737 overcame the requirement for Bim. However, the synergism between ABT-737 and imiquimod or dacarbazine required endogenous Noxa, as demonstrated by experiments with Noxa-specific RNAi. Surprisingly, although Bim was activated, it was unable to replace Noxa. Studies of mitochondrial cytochrome c release using BH3 peptides confirmed that a main effect of dacarbazine, fotemustine, and imiquimod was to neutralize Mcl-1, thereby sensitizing mitochondria to the inhibition of other Bcl-2 family members through ABT-737. ABT-737 is thus a promising agent for combination therapy for human melanoma. Importantly, the efficacy of this therapy depends on endogenous Noxa, and the ability of chemotherapeutic drugs to activate Noxa may be a valuable predictor of their synergism with Bcl-2-targeting drugs. PMID:19412422

  20. Dexamethasone and interleukin-1 potently synergize to stimulate the production of granulocyte colony-stimulating factor in differentiated THP-1 cells.

    PubMed

    Wang, Y; Zhang, J J; Lei, K Y; Pike, J W

    1997-10-29

    The human monocytic leukemic cell line, THP-1, which differentiates toward macrophages in response to phorbol 12-myristate 13-acetate (PMA) was investigated for its ability to produce granulocyte colony-stimulating factor (G-CSF). G-CSF protein was neither produced during PMA-induced differentiation nor in response to dexamethasone (Dex) alone. However, when combined, PMA and Dex synergistically stimulated THP-1 cells to produce G-CSF. The synergistic interaction between PMA and Dex on G-CSF production appeared to be mediated through the production of interleukin-1 (IL-1) since neutralization of IL-1 activity completely inhibited G-CSF production. Further experiments demonstrated that in THP-1 cells pretreated with PMA, Dex potently synergized with IL-1 to stimulate G-CSF production.

  1. Co-administration of recombinant major envelope proteins (rA27L and rH3L) of buffalopox virus provides enhanced immunogenicity and protective efficacy in animal models.

    PubMed

    Kumar, Amit; Yogisharadhya, Revanaiah; Venkatesan, Gnanavel; Bhanuprakash, Veerakyathappa; Pandey, Awadh Bihari; Shivachandra, Sathish Bhadravati

    2017-05-01

    Buffalopox virus (BPXV) and other vaccinia-like viruses (VLVs) are causing an emerging/re-emerging zoonosis affecting buffaloes, cattle and humans in India and other countries. A27L and H3L are immuno-dominant major envelope proteins of intracellular mature virion (IMV) of orthopoxviruses (OPVs) and are highly conserved with an ability to elicit neutralizing antibodies. In the present study, two recombinant proteins namely; rA27L ( 21 S to E 110 ; ∼30 kDa) and rH3L( 1 M to I 280 ; ∼50 kDa) of BPXV-Vij/96 produced from Escherichia coli were used in vaccine formulation. A combined recombinant subunit vaccine comprising rA27L and rH3L antigens (10 μg of each) was used for active immunization of adult mice (20μg/dose/mice) with or without adjuvant (FCA/FIA) by intramuscular route. Immune responses revealed a gradual increase in antigen specific serum IgG as well as neutralizing antibody titers measured by using indirect-ELISA and serum neutralization test (SNT) respectively, which were higher as compared to that elicited by individual antigens. Suckling mice passively administered with combined anti-A27L and anti-H3L sera showed a complete (100%) pre-exposure protection upon challenge with virulent BPXV. Conclusively, this study highlights the potential utility of rA27L and rH3L proteins as safer candidate prophylactic antigens in combined recombinant subunit vaccine for buffalopox as well as passive protective efficacy of combined sera in employing better pre-exposure protection against virulent BPXV. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Tramadol and Tramadol+Caffeine Synergism in the Rat Formalin Test Are Mediated by Central Opioid and Serotonergic Mechanisms

    PubMed Central

    Carrillo-Munguía, Norma; González-Trujano, Ma. Eva; Huerta, Miguel; Trujillo, Xochitl; Díaz-Reval, M. Irene

    2015-01-01

    Different analgesic combinations with caffeine have shown this drug to be capable of increasing the analgesic effect. Many combinations with nonsteroidal anti-inflammatory drugs (NSAIDs) have been carried out, but, in regard to opioids, only combinations with morphine and tramadol have been reported. The antinociceptive synergism mechanism of these combinations is not well understood. The purpose of the present study was to determine the participation of spinal and supraspinal opioidergic and serotonergic systems in the synergic effect of the tramadol+caffeine combination in the rat formalin test. At the supraspinal level, the opioid antagonist, naloxone, completely reversed the effect of the drug combination, whereas ketanserin, a 5-HT2 receptor antagonist, inhibited the effect by 60%; however, ondansetron, a 5-HT3 receptor antagonist, did not alter the combination effect. When the antagonists were intrathecally administered, there was a significant reduction in all tramadol-caffeine combination effects. With respect to tramadol alone, there was significant participation of the opioid system at the supraspinal level, whereas it was the serotonergic system that participated at the spinal level by means of the two receptors studied. In conclusion, the tramadol+caffeine combination synergically activated the opioid and serotonergic systems at the supraspinal level, as well as at the spinal level, to produce the antinociception. PMID:26146627

  3. Expression of HSP27 in Hepatocellular Carcinoma.

    PubMed

    Eto, Daimei; Hisaka, Toru; Horiuchi, Hiroyuki; Uchida, Shinji; Ishikawa, Hiroto; Kawashima, Yusuke; Kinugasa, Tetsushi; Nakashima, Osamu; Yano, Hirohisa; Okuda, Koji; Akagi, Yoshito

    2016-07-01

    Heat-shock protein 27 (HSP27), a low molecular weight stress protein, is recognized as a molecular chaperone. The expression of HSP27 has been detected in some human tumors and while HSP27 is phosphorylated as a reresponse to stress, the function of phosphorylated HSP27 (p-HSP27) is not known. The aim of this study was to investigate what kind of effect expression of HSP27 and p-HSP27 in HCC has on clinicopathological characteristics and prognosis. An immunohistochemical study for HSP27 and p-HSP27 was performed on 194 resected HCC cases. We analyzed the correlation of HSP27 expression with various parameters statistically. There was no correlation between expression of HSP27 and the clinicopathological characteristics and prognosis from the analysis of 194 cases. From the analysis of the hepatitis C virus (HCV)-positive group of 142 cases, those that were p-HSP27-positive had a larger tumor diameter and the portal vein invasion rate was high. The expression of total HSP27 may serve as a new, clinically useful marker of HCC. In addition, the present study suggests that the expression of phosphorylated HSP27 is useful in the screening and grading of HCC occurring in the setting of HCV. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  4. Functional study of miR-27a in human hepatic stellate cells by proteomic analysis: comprehensive view and a role in myogenic tans-differentiation.

    PubMed

    Ji, Yuhua; Zhang, Jinsheng; Wang, Wenwen; Ji, Juling

    2014-01-01

    We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepatic stellate cells (HSCs) activation. To further explore the biological function and underlying mechanisms of miR-27a in HSCs, global protein expression affected by overexpression of miR-27a in HSCs was analyzed by a cleavable isotope-coded affinity tags (cICAT) based comparative proteomic approach. In the present study, 1267 non-redundant proteins were identified with unique accession numbers (score ≥1.3, i.e. confidence ≥95%), among which 1171 were quantified and 149 proteins (12.72%) were differentially expressed with a differential expression ratio of 1.5. We found that up-regulated proteins by miR-27a mainly participate in cell proliferation and myogenesis, while down-regulated proteins were the key enzymes involved in de novo lipid synthesis. The expression of a group of six miR-27a regulated proteins was validated and the function of one miR-27a regulated protein was further validated. The results not only delineated the underlying mechanism of miR-27a in modulating fat metabolism and cell proliferation, but also revealed a novel role of miR-27a in promoting myogenic tans-differentiation during HSCs activation. This study also exemplified proteomics strategy as a powerful tool for the functional study of miRNA.

  5. Synergism between fentanyl and tramadol in tonic inflammatory pain: the orofacial formalin test.

    PubMed

    Miranda, Hugo F; Noriega, Viviana; Zepeda, Ramiro J; Sierralta, Fernando; Prieto, Juan C

    2012-06-01

    Opioids have been used for long time to management of pain, the coadministration of two opioids may induce synergism. The present study was conducted to determine the antinociceptive interaction between the dual mechanism of action of tramadol compared to the main of fentanyl antinociception in the orofacial formalin which represents a model of persistent cutaneous nociception in the region innervated by the trigeminal nerve. The i.p. administration of tramadol and fentanyl induced a dose-dependent antinociception with an ED(50) of 2.97 ± 0.32 mg/kg for phase I and 1.79 ± 0.30 mg/kg for phase II and 0.062 ± 0.0040 mg/kg in phase I and 0.041 ± 0.0039 mg/kg in phase II, respectively. The coadministration of fentanyl with tramadol induced synergism in both phases of the test with an interaction index of 0.343 and 0.163 for phase I and phase II, respectively. This finding could be explained by the more complex pharmacology of tramadol compared to fentanyl.

  6. Antioxidant synergism between tocopherols and ascorbyl palmitate in cooked, minced turkey.

    PubMed

    Bruun-Jensen, L; Skovgaard, I M; Skibsted, L H; Bertelsen, G

    1994-09-01

    In an attempt to reduce oxidative deterioration, including the development of warmed-over flavour (WOF), in cooked, minced turkey meat, a combined strategy using natural antioxidants and modified atmosphere packaging (MAP) was adapted. Tocopherols (200 ppm) each significantly reduced lipid oxidation during 9 days of cold storage (5 degrees C), measured as 2-thiobarbituric-acid-reactive substances (TBARS). Synergism between the two antioxidants was demonstrated as an increasing relative reduction in TBARS values over time when both antioxidants were added, as opposed to the almost constant relative reduction when only one antioxidant was added. Notably, this synergism depended on the availability of O2, and was found to be most significant for atmospheric packaging (21% O2, 79% N2) and most significant towards the end of the storage period. A reduced O2 content in the packages (mixture of O2 and N2, initially: 1.0% O2 or 0.03% O2) had a more pronouced effect on oxidative deterioration than addition of antioxidants. However, during the 9 days of storage, meat balls with the synergistic mixture added, and packed in 1% O2, had TBARS values of 50 compared to 270 mumol malondialdehyde/kg for meat balls without antioxidants packed in 21% O2. This level of reduction in lipid oxidation is comparable with that of meat balls without antioxidants packed in a virtually O2-free atmosphere.

  7. [Synergism inhibition of curcumin combined with cisplatin on T24 bladder carcinoma cells and its related mechanism].

    PubMed

    Zhang, Shao-nan; Yong, Qun; Wu, Xin-li; Liu, Xiao-ping

    2014-11-01

    To investigate the synergism inhibition of curcumin combined with cisplatin on T24 bladder carcinoma cells and the down-regulating effect of curcumin on the Keapl-Nrf2 pathway, a well recognized anti-drug pathway in almost drugged tumor cells. T24 cells were cultured and treated with increasing concentrations of curcumin(5 ,10 and 20 µmol/mL) combined with cisplatin(30 µg/mL) for 24 hours. The inhibitory effects on T24 cells were tested with MTI colorimetric assay. Nuclear Nrf2 and Keapl , cytoplasmic Keapl and two typical phase II enzymes (GSTP1 and NQOl) were checked with Western blotting. The proliferation of T24 cells was significantly inhibited by different concentrations of curcumin combined with cisplatin. After the treatment with different concentrations of curcumin, Nuclear Nrf2 was decreased but Keapl was increased, and GSTP1 and NQO1 were decreased. Synergism inhibition of curcumin combined with cisplatin on T24 bladder carcinoma cells is observed in this research. The Keapl-Nrf2 pathway in T24 cells is down-regulated by curcumin. The expression of typical phase I enzymes (GSTP1 and NQO1) mediated by Nrf2 are decreased by curcumin. The sensitivity of tumor cells to chemotherapeutic drugs is then enhanced. These may be the mechanism of synergism effect of curcumin combined with cisplatin.

  8. Assessment of the synergic effect of immunomodulation on nerve repair using multiparametric magnetic resonance imaging.

    PubMed

    Zheng, Chu-Shan; Zhang, Xiang; Chen, Yue-Yao; Zhang, Fang; Duan, Xiao-Hui; Chen, Mei-Wei; Lu, Lie-Jing; Shen, Jun

    2018-01-01

    The immune system plays a pivotal role in nerve injury. The aim of this study was to determine the role of multiparametric magnetic resonance imaging (MRI) in evaluation of the synergic effect of immunomodulation on nerve regeneration in neurotmesis. Rats with sciatic nerve neurotmesis and surgical repair underwent serial multiparametric MR examinations over an 8-week period after subepineurial microinjection of lipopolysaccharide (LPS) and subsequent subcutaneous injection of FK506 or subepineurial microinjection of LPS or phosphate-buffered saline (PBS) alone. Nerves treated with immunomodulation showed more prominent regeneration than those treated with LPS or PBS alone and more rapid restoration toward normal T2, fractional anisotropy (FA), and radial diffusivity (RD) values than nerves injected with LPS or PBS. Nerves treated with immunomodulation exert synergic beneficial effects on nerve regeneration that can be predicted by T2 measurements and FA and RD values. Muscle Nerve 57: E38-E45, 2018. © 2017 Wiley Periodicals, Inc.

  9. Synergistic growth inhibition of squamous cell carcinoma of the head and neck by erlotinib and epigallocatechin-3-gallate: the role of p53-dependent inhibition of nuclear factor-kappaB.

    PubMed

    Amin, A R M Ruhul; Khuri, Fadlo R; Chen, Zhuo Georgia; Shin, Dong M

    2009-06-01

    We have previously reported that the green tea polyphenol epigallocatechin-3-gallate (EGCG) and the epidermal growth factor receptor-tyrosine kinase inhibitor erlotinib had synergistic growth-inhibitory effects in cell culture and a nude mouse xenograft model of squamous cell carcinoma of the head and neck. However, the mechanism of their antitumor synergism is not fully understood. In the current study, we investigate the mechanism of their synergistic growth-inhibitory effects. The treatment of squamous cell carcinoma of the head and neck cell lines with erlotinib time-dependently increased the expression of cell cycle regulatory proteins p21 and p27 and apoptosis regulatory protein Bim. EGCG alone had very little or no effect on the expression of these proteins among the cell lines. However, simultaneous treatment with EGCG and erlotinib strongly inhibited erlotinib-induced expression of p21 and p27 without affecting the expression of Bim. Moreover, erlotinib increased the expression of p53 protein, the ablation of which by short hairpin RNA strongly inhibited EGCG- and erlotinib-mediated growth inhibition and the expression of p21, p27, and Bim. In addition, combined treatment with erlotinib and EGCG inhibited the protein level of p65 subunit of nuclear factor-kappaB and its transcriptional target Bcl-2, but failed to do so in cells with ablated p53. Taken together, our results, for the first time, suggest that erlotinib treatment activates p53, which plays a critical role in synergistic growth inhibition by erlotinib and EGCG via inhibiting nuclear factor-kappaB signaling pathway. Characterizing the underlying mechanisms of EGCG and erlotinib synergism will provide an important rationale for chemoprevention or treatment trials using this combination.

  10. 78 FR 43889 - Synergizing Efforts in Standards Development for Cellular Therapies and Regenerative Medicine...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-22

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration [Docket No. FDA-2013-N-0001] Synergizing Efforts in Standards Development for Cellular Therapies and Regenerative Medicine Products; Public Workshop AGENCY: Food and Drug Administration, HHS. ACTION: Notice of public workshop. The Food and Drug Administration (FDA), Center for...

  11. Desiccant dusts synergize the effect of Beauveria bassiana (Hyphomycetes: Moniliales) on stored-grain beetles.

    PubMed

    Lord, J C

    2001-04-01

    Diatomaceous earth (DE) is a desiccant insecticide and most efficacious in low humidity. It acts on insect cuticle by absorbing lipids, and perhaps by cuticular abrasion. Beauveria bassiana (Balsamo) Vuillemin, an entomopathogenic fungus, is most efficacious in high humidity and has a complex interaction with cuticular lipids. Interaction between these materials may enhance insect control performance. Assays with stored-grain beetles were conducted with B. bassiana at rates of 11, 33, 100, and 300 mg of conidia per kilogram of grain with and without single rates of DE that killed 10% or less of the target beetles. The assays revealed synergism in effects on adult Rhyzopertha dominica (F.) and Oryzaephilus surinamensis (L.) at all doses. There was statistically significant synergism for adult Cryptolestes ferrugineus (Stephens) and larval R. dominica but at only one B. bassiana rate for each target. Both amorphous silicon dioxide, a sorptive dust, and diamond dust, an abrasive, showed synergistic interaction with B. bassiana on adult R. dominica. These results may provide a basis for a least-toxic approach to control of stored-product beetles and for efficacy-enhancing formulation of entomopathogenic fungi.

  12. The MOF+ Technique: A Significant Synergic Effect Enables High Performance Chromate Removal.

    PubMed

    Luo, Ming Biao; Xiong, Yang Yang; Wu, Hui Qiong; Feng, Xue Feng; Li, Jian Qiang; Luo, Feng

    2017-12-18

    A significant synergic effect between a metal-organic framework (MOF) and Fe 2 SO 4 , the so-called MOF + technique, is exploited for the first time to remove toxic chromate from aqueous solutions. The results show that relative to the pristine MOF samples (no detectable chromate removal), the MOF + method enables super performance, giving a 796 Cr mg g -1 adsorption capacity. The value is almost eight-fold higher than the best value of established MOF adsorbents, and the highest value of all reported porous adsorbents for such use. The adsorption mechanism, unlike the anion-exchange process that dominates chromate removal in all other MOF adsorbents, as unveiled by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM), is due to the surface formation of Fe 0.75 Cr 0.25 (OH) 3 nanospheres on the MOF samples. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Synergism between polyurethane and polydopamine in the synthesis of Ni-Fe alloy monoliths.

    PubMed

    Naresh Kumar, Thangavel; Sivabalan, Santhana; Chandrasekaran, Naveen; Phani, Kanala Lakshminarasimha

    2015-02-04

    Herein, we report the first synthesis of a light-weight macroporous 3-D alloy monolith of Ni-Fe/C using synergism between polydopamine (pDA) and polyurethane (pU); in situ formed polyurethane (pU) enables efficient mixing of pDA (carbon source) and Ni-FeOx resulting in Ni-Fe alloy monoliths at a temperature as low as ∼600 °C. The monolithic Ni-Fe/C exhibits enhanced oxygen evolution activity.

  14. Synergism between NF-kappa B inhibitor, celastrol, and XIAP inhibitor, embelin, in an acute myeloid leukemia cell line, HL-60.

    PubMed

    Pazhang, Yaghub; Jaliani, Hossein Zarei; Imani, Mehdi; Dariushnejad, Hassan

    2016-01-01

    Embelin and celastrol, inhibitors of XIAP and NF-κB proteins respectively, have been derived from natural sources and shown anti-tumor activities against different cancer cell lines. Some interactions have recently been discovered between XIAP and NF-κB pathways, but the effects of these inhibitors in combination have not been investigated yet. We have studied possible synergistic effects of embelin in combination with celastrol, in an acute myeloid leukemia model, HL-60 cell line. Cytotoxicity of embelin and celastrol, separately and in combination, was determined by MTT assay and flow cytometry. Chou-Talalay's method was used to assess the synergistic effect of two components. Immunocytochemistry and western blot analysis of the two tumor marker proteins. (survivin and COX-2) was also performed to investigate downstream effects of two components. Analysis of MTT assay and flow cytometry showed that there is a substantial synergistic effect in some affected fractions of drug-treated HL-60. cells, while in other affected fractions a mild synergism or additive effect was observed. Immunocytochemistry and western blot analysis revealed that the expression of survivin and COX-2 proteins was reduced in treated cells. Embelin and celastrol showed potent antitumor activity and synergistic effects in combination. Therefore targeting XIAP and NF-κB pathways simultaneously can be investigated in more detail to make use of embelin and celastrol as a combination therapy of cancer.

  15. BNGR-A25L and -A27 are two functional G protein-coupled receptors for CAPA periviscerokinin neuropeptides in the silkworm Bombyx mori.

    PubMed

    Shen, Zhangfei; Chen, Yu; Hong, Lingjuan; Cui, Zhenteng; Yang, Huipeng; He, Xiaobai; Shi, Ying; Shi, Liangen; Han, Feng; Zhou, Naiming

    2017-10-06

    CAPA peptides, such as periviscerokinin (PVK), are insect neuropeptides involved in many signaling pathways controlling, for example, metabolism, behavior, and reproduction. They are present in a large number of insects and, together with their cognate receptors, are important for research into approaches for improving insect control. However, the CAPA receptors in the silkworm ( Bombyx mori ) insect model are unknown. Here, we cloned cDNAs of two putative CAPA peptide receptor genes, BNGR-A27 and -A25, from the brain of B. mori larvae. We found that the predicted BNGR-A27 ORF encodes 450 amino acids and that one BNGR-A25 splice variant encodes a full-length isoform (BNGR-A25L) of 418 amino acid residues and another a short isoform (BNGR-A25S) of 341 amino acids with a truncated C-terminal tail. Functional assays indicated that both BNGR-A25L and -A27 are activated by the PVK neuropeptides Bom -CAPA-PVK-1 and -PVK-2, leading to a significant increase in cAMP-response element-controlled luciferase activity and Ca 2+ mobilization in a G q inhibitor-sensitive manner. In contrast, BNGR-A25S was not significantly activated in response to the PVK peptides. Moreover, Bom -CAPA-PVK-1 directly bound to BNGR-A25L and -A27, but not BNGR-A25S. Of note, CAPA-PVK-mediated ERK1/2 phosphorylation and receptor internalization confirmed that BNGR-A25L and -A27 are two canonical receptors for Bombyx CAPA-PVKs. However, BNGR-A25S alone is a nonfunctional receptor but serves as a dominant-negative protein for BNGR-A25L. These results provide evidence that BNGR-A25L and -A27 are two functional G q -coupled receptors for Bombyx CAPA-PVKs, enabling the further elucidation of the endocrinological roles of Bom -CAPA-PVKs and their receptors in insect biology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Denitrification synergized with ANAMMOX for the anaerobic degradation of benzene: performance and microbial community structure.

    PubMed

    Peng, Shuchan; Zhang, Lilan; Zhang, DaiJun; Lu, Peili; Zhang, Xiaoting; He, Qiang

    2017-05-01

    To evaluate the effect of anaerobic ammonium oxidation (ANAMMOX) on benzene degradation under denitrification, a sequencing batch reactor (SBR) under denitrification synergized with ANAMMOX (SBR-DenAna) for benzene degradation was established by inoculating anaerobic ammonium-oxidizing bacteria (AnAOB) into a SBR under denitrification reactor (SBR-Den) for benzene degradation. The average rate of benzene degradation and the maximum first-order kinetic constant in SBR-DenAna were 2.34- and 1.41-fold those in SBR-Den, respectively, indicating that ANAMMOX improved the degradation of benzene under denitrification synergized with ANAMMOX. However, the average rate of benzene degradation decreased by 35% in the denitrification-ANAMMOX synergistic reactor when 10 mg N L -1 NO 2 - was added; the rate recovered once NO 2 - was depleted, indicating that ANAMMOX might detoxify NO 2 - . Results from high-throughput sequencing analysis revealed that Azoarcus within the family Rhodocyclaceae might be associated with benzene degradation in the two SBRs. AnAOB affiliated with the family Candidatus Brocadiaceae were just detected in SBR-DenAna.

  17. Ubiquitination of exposed glycoproteins by SCFFBXO27 directs damaged lysosomes for autophagy

    PubMed Central

    Yoshida, Yukiko; Yasuda, Sayaka; Fujita, Toshiharu; Hamasaki, Maho; Murakami, Arisa; Kawawaki, Junko; Iwai, Kazuhiro; Saeki, Yasushi; Yoshimori, Tamotsu; Matsuda, Noriyuki; Tanaka, Keiji

    2017-01-01

    Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCFFBXO27 ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCFFBXO27 ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy. PMID:28743755

  18. In vitro and in vivo synergism between amoxicillin and clavulanic acid against ampicillin-resistant Haemophilus influenzae type b.

    PubMed Central

    Yogev, R; Melick, C; Kabat, W J

    1981-01-01

    Eight strans of ampicillin-resistant beta-lactamase-producing Haemophilus influenzae type b were studied in vitro for synergy between amoxicillin and clavulanic acid. The minimal inhibitory concentrations for amoxicillin alone were 6.25 to 12.5 microgram/ml, and for clavulanic acid alone they were 12.5 to 25 microgram/ml. However, seven of eight strains were inhibited by a combination of 0.36 microgram of amoxicillin and 0.36 microgram of clavulanic acid per ml. Infant rat models of bacteremia and meningitis were used to test the efficacy of amoxicillin and clavulanic acid alone and in combination upon four strains of ampicillin-resistant H. influenzae. Neither amoxicillin alone (27 animals) nor clavulanic acid alone (20 animals) sterilized the blood or cerebrospinal fluid of the animals. In contrast, 30 of 33 blood cultures and 29 of 33 cerebrospinal fluid cultures were sterile when a combination of the two drugs in the same dosages was used. The observed in vitro and in vivo synergism between amoxicillin and clavulanic acid suggests that the combination may be effective therapy for invasive infections in humans caused by ampicillin-resistant H. influenzae type b. PMID:6973952

  19. Western Spruce Budworm Outbreaks Did Not Increase Fire Risk over the Last Three Centuries: A Dendrochronological Analysis of Inter-Disturbance Synergism

    PubMed Central

    Flower, Aquila; G. Gavin, Daniel; Heyerdahl, Emily K.; Parsons, Russell A.; Cohn, Gregory M.

    2014-01-01

    Insect outbreaks are often assumed to increase the severity or probability of fire occurrence through increased fuel availability, while fires may in turn alter susceptibility of forests to subsequent insect outbreaks through changes in the spatial distribution of suitable host trees. However, little is actually known about the potential synergisms between these natural disturbances. Assessing inter-disturbance synergism is challenging due to the short length of historical records and the confounding influences of land use and climate changes on natural disturbance dynamics. We used dendrochronological methods to reconstruct defoliator outbreaks and fire occurrence at ten sites along a longitudinal transect running from central Oregon to western Montana. We assessed synergism between disturbance types, analyzed long-term changes in disturbance dynamics, and compared these disturbance histories with dendroclimatological moisture availability records to quantify the influence of moisture availability on disturbances. After approximately 1890, fires were largely absent and defoliator outbreaks became longer-lasting, more frequent, and more synchronous at our sites. Fires were more likely to occur during warm-dry years, while outbreaks were most likely to begin near the end of warm-dry periods. Our results show no discernible impact of defoliation events on subsequent fire risk. Any effect from the addition of fuels during defoliation events appears to be too small to detect given the overriding influence of climatic variability. We therefore propose that if there is any relationship between the two disturbances, it is a subtle synergistic relationship wherein climate determines the probability of occurrence of each disturbance type, and each disturbance type damps the severity, but does not alter the probability of occurrence, of the other disturbance type over long time scales. Although both disturbance types may increase in frequency or extent in response to future

  20. Western spruce budworm outbreaks did not increase fire risk over the last three centuries: a dendrochronological analysis of inter-disturbance synergism.

    PubMed

    Flower, Aquila; Gavin, Daniel G; Heyerdahl, Emily K; Parsons, Russell A; Cohn, Gregory M

    2014-01-01

    Insect outbreaks are often assumed to increase the severity or probability of fire occurrence through increased fuel availability, while fires may in turn alter susceptibility of forests to subsequent insect outbreaks through changes in the spatial distribution of suitable host trees. However, little is actually known about the potential synergisms between these natural disturbances. Assessing inter-disturbance synergism is challenging due to the short length of historical records and the confounding influences of land use and climate changes on natural disturbance dynamics. We used dendrochronological methods to reconstruct defoliator outbreaks and fire occurrence at ten sites along a longitudinal transect running from central Oregon to western Montana. We assessed synergism between disturbance types, analyzed long-term changes in disturbance dynamics, and compared these disturbance histories with dendroclimatological moisture availability records to quantify the influence of moisture availability on disturbances. After approximately 1890, fires were largely absent and defoliator outbreaks became longer-lasting, more frequent, and more synchronous at our sites. Fires were more likely to occur during warm-dry years, while outbreaks were most likely to begin near the end of warm-dry periods. Our results show no discernible impact of defoliation events on subsequent fire risk. Any effect from the addition of fuels during defoliation events appears to be too small to detect given the overriding influence of climatic variability. We therefore propose that if there is any relationship between the two disturbances, it is a subtle synergistic relationship wherein climate determines the probability of occurrence of each disturbance type, and each disturbance type damps the severity, but does not alter the probability of occurrence, of the other disturbance type over long time scales. Although both disturbance types may increase in frequency or extent in response to future

  1. A beta-l-Arabinopyranosidase from Streptomyces avermitilis is a novel member of glycoside hydrolase family 27.

    PubMed

    Ichinose, Hitomi; Fujimoto, Zui; Honda, Mariko; Harazono, Koichi; Nishimoto, Yukifumi; Uzura, Atsuko; Kaneko, Satoshi

    2009-09-11

    Arabinogalactan proteins (AGPs) are a family of plant cell surface proteoglycans and are considered to be involved in plant growth and development. Because AGPs are very complex molecules, glycoside hydrolases capable of degrading AGPs are powerful tools for analyses of the AGPs. We previously reported such enzymes from Streptomyces avermitilis. Recently, a beta-l-arabinopyranosidase was purified from the culture supernatant of the bacterium, and its corresponding gene was identified. The primary structure of the protein revealed that the catalytic module was highly similar to that of glycoside hydrolase family 27 (GH27) alpha-d-galactosidases. The recombinant protein was successfully expressed as a secreted 64-kDa protein using a Streptomyces expression system. The specific activity toward p-nitrophenyl-beta-l-arabinopyranoside was 18 micromol of arabinose/min/mg, which was 67 times higher than that toward p- nitrophenyl-alpha-d-galactopyranoside. The enzyme could remove 0.1 and 45% l-arabinose from gum arabic or larch arabinogalactan, respectively. X-ray crystallographic analysis reveals that the protein had a GH27 catalytic domain, an antiparallel beta-domain containing Greek key motifs, another antiparallel beta-domain forming a jellyroll structure, and a carbohydrate-binding module family 13 domain. Comparison of the structure of this protein with that of alpha-d-galactosidase showed a single amino acid substitution (aspartic acid to glutamic acid) in the catalytic pocket of beta-l-arabinopyranosidase, and a space for the hydroxymethyl group on the C-5 carbon of d-galactose bound to alpha-galactosidase was changed in beta-l-arabinopyranosidase. Mutagenesis study revealed that the residue is critical for modulating the enzyme activity. This is the first report in which beta-l-arabinopyranosidase is classified as a new member of the GH27 family.

  2. Evidence of the neuron-restrictive silencer factor (NRSF) interaction with Sp3 and its synergic repression to the mu opioid receptor (MOR) gene

    PubMed Central

    Kim, Chun Sung; Choi, Hack Sun; Hwang, Cheol Kyu; Song, Kyu Young; Lee, Byung-Kwon; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

    2006-01-01

    Previously, we reported that the neuron-restrictive silencer element (NRSE) of mu opioid receptor (MOR) functions as a critical regulator to repress the MOR transcription in specific neuronal cells, depending on neuron-restriction silence factor (NRSF) expression levels [C.S.Kim, C.K.Hwang, H.S.Choi, K.Y.Song, P.Y.Law, L.N.Wei and H.H.Loh (2004) J. Biol. Chem., 279, 46464–46473]. Herein, we identify a conserved GC sequence next to NRSE region in the mouse MOR gene. The inhibition of Sp family factors binding to this GC box by mithramycin A led to a significant increase in the endogenous MOR transcription. In the co-immunoprecipitation experiment, NRSF interacted with the full-length Sp3 factor, but not with Sp1 or two short Sp3 isoforms. The sequence specific and functional binding by Sp3 at this GC box was confirmed by in vitro gel-shift assays using either in vitro translated proteins or nuclear extract, and by in vivo chromatin immunoprecipitation assays. Transient transfection assays showed that Sp3-binding site of the MOR gene is a functionally synergic repressor element with NRSE in NS20Y cells, but not in the NRSF negative PC12 cells. The results suggest that the synergic interaction between NRSF and Sp3 is required to negatively regulate MOR gene transcription and that transcription of MOR gene would be governed by the context of available transcription factors rather than by a master regulator. PMID:17130167

  3. Localization of sweet potato chlorotic stunt virus (SPCSV) in synergic infection with potyviruses in sweet potato.

    PubMed

    Nome, Claudia Fernanda; Nome, Sergio Fernando; Guzmán, Fabiana; Conci, Luis; Laguna, Irma Graciela

    2007-01-01

    Among diseases reported worldwidely for sweet potato (Ipomoea batatas (L) Lam) crop, one of the most frequent is the Sweet potato virus disease (SPVD), caused by sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus (SPFMV) co-infection. In Argentina, there exists the sweet potato chlorotic dwarf (SPCD), a sweet potato disease caused by triple co-infection with SPCSV, SPFMV and sweet potato mild speckling virus (SPMSV). Both diseases cause a synergism between the potyviruses (SPFMV and SPMSV) and the crinivirus (SPCSV). Up to date, studies carried out on the interaction among these three viruses have not described their localization in the infected tissues. In single infections, virions of the crinivirus genus are limited to the phloem while potyviral virions are found in most tissues of the infected plant. The purpose of this work was to localize the heat shock protein 70 homolog (HSP70h), a movement protein for genus crinivirus, of an Argentinean SPCSV isolate in its single infection and in its double and triple co-infection with SPFMV and SPMSV. The localization was made by in situ hybridization (ISH) for electron microscopy (EM) on ultrathin sections of sweet potato cv. Morada INTA infected tissues. The results demonstrated that viral RNA coding HSP70h is restricted to phloem cells during crinivirus single infection, while it was detected outside the phloem in infections combined with the potyviruses involved in chlorotic dwarf disease.

  4. A Targetable Molecular Chaperone Hsp27 Confers Aggressiveness in Hepatocellular Carcinoma.

    PubMed

    Zhang, Yurong; Tao, Xuemei; Jin, Guangzhi; Jin, Haojie; Wang, Ning; Hu, Fangyuan; Luo, Qin; Shu, Huiqun; Zhao, Fangyu; Yao, Ming; Fang, Jingyuan; Cong, Wenming; Qin, Wenxin; Wang, Cun

    2016-01-01

    Heat shock protein 27 (Hsp27) is an ATP-independent molecular chaperone and confers survival advantages and resistance to cancer cells under stress conditions. The effects and molecular mechanisms of Hsp27 in HCC invasion and metastasis are still unclear. In this study, hepatocellular carcinoma (HCC) tissue array (n = 167) was used to investigate the expression and prognostic relevance of Hsp27 in HCC patients. HCC patients with high expression of Hsp27 exhibited poor prognosis. Overexpression of Hsp27 led to the forced invasion of HCC cells, whereas silencing Hsp27 attenuated invasion and metastasis of HCC cells in vitro and in vivo. We revealed that Hsp27 activated Akt signaling, which in turn promoted MMP2 and ITGA7 expression and HCC metastasis. We further observed that targeting Hsp27 using OGX-427 obviously suppressed HCC metastasis in two metastatic models. These findings indicate that Hsp27 is a useful predictive factor for prognosis of HCC and it facilitates HCC metastasis through Akt signaling. Targeting Hsp27 with OGX-427 may represent an attractive therapeutic option for suppressing HCC metastasis.

  5. Meis3 synergizes with Pbx4 and Hoxb1b in promoting hindbrain fates in the zebrafish.

    PubMed

    Vlachakis, N; Choe, S K; Sagerström, C G

    2001-04-01

    Many Hox proteins are thought to require Pbx and Meis co-factors to specify cell identity during embryogenesis. Here we demonstrate that Meis3 synergizes with Pbx4 and Hoxb1b in promoting hindbrain fates in the zebrafish. We find that Hoxb1b and Pbx4 act together to induce ectopic hoxb1a expression in rhombomere 2 of the hindbrain. In contrast, Hoxb1b and Pbx4 acting together with Meis3 induce hoxb1a, hoxb2, krox20 and valentino expression rostrally and cause extensive transformation of forebrain and midbrain fates to hindbrain fates, including differentiation of excess rhombomere 4-specific Mauthner neurons. This synergistic effect requires that Hoxb1b and Meis3 have intact Pbx-interaction domains, suggesting that their in vivo activity is dependent on binding to Pbx4. In the case of Meis3, binding to Pbx4 is also required for nuclear access. Our results are consistent with Hoxb1b and Meis3 interacting with Pbx4 to form complexes that regulate hindbrain development during zebrafish embryogenesis.

  6. Crk synergizes with epidermal growth factor for epithelial invasion and morphogenesis and is required for the met morphogenic program.

    PubMed

    Lamorte, Louie; Rodrigues, Sonia; Naujokas, Monica; Park, Morag

    2002-10-04

    Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor, stimulates cell spreading, cell dispersal, and the inherent morphogenic program of various epithelial cell lines. Although both hepatocyte growth factor and epidermal growth factor (EGF) can activate downstream signaling pathways in Madin-Darby canine kidney epithelial cells, EGF fails to promote the breakdown of cell-cell junctional complexes and initiate an invasive morphogenic program. We have undertaken a strategy to identify signals that synergize with EGF in this process. We provide evidence that the overexpression of the CrkII adapter protein complements EGF-stimulated pathways to induce cell dispersal in two-dimensional cultures and cell invasion and branching morphogenesis in three-dimensional collagen gels. This finding correlates with the ability of CrkII to promote the breakdown of adherens junctions in stable cell lines and the ability of EGF to stimulate enhanced Rac activity in cells overexpressing CrkII. We have previously shown that the Gab1-docking protein is required for branching morphogenesis downstream of the Met receptor. Consistent with a role for CrkII in promoting EGF-dependent branching morphogenesis, the binding of Gab1 to CrkII is required for the branching morphogenic program downstream of Met. Together, our data support a role for the CrkII adapter protein in epithelial invasion and morphogenesis and underscores the importance of considering the synergistic actions of signaling pathways in cancer progression.

  7. Stage and cell-specific expression and intracellular localization of the small heat shock protein Hsp27 during oogenesis and spermatogenesis in the Mediterranean fruit fly, Ceratitis capitata.

    PubMed

    Economou, Katerina; Kotsiliti, Elena; Mintzas, Anastassios C

    2017-01-01

    The cell-specific expression and intracellular distribution of the small heat protein Hsp27 was investigated in the ovaries and testes of the Mediterranean fruit fly, Ceratitis capitata (medfly), under both normal and heat shock conditions. For this study, a gfp-hsp27 strain was used to detect the chimeric protein by confocal microscopy. In unstressed ovaries, the protein was expressed throughout egg development in a stage and cell-specific pattern. In germarium, the protein was detected in the cytoplasm of the somatic cells in both unstressed and heat-shocked ovaries. In the early stages of oogenesis of unstressed ovaries, the protein was mainly located in the perinuclear region of the germ cells and in the cytoplasm of the follicle cells, while in later stages (9-10) it was distributed in the cytoplasm of the germ cells. In late stages (12-14), the protein changed localization pattern and was exclusively associated with the nuclei of the somatic cells. In heat shocked ovaries, the protein was mainly located in the nuclei of the somatic cells throughout egg chamber's development. In unstressed testes, the chimeric protein was detected in the nuclei of primary spermatocytes and in the filamentous structures of spermatid bundles, called actin cones. Interestingly, after a heat shock, the protein presented the same cell-specific localization pattern as in unstressed testes. Furthermore, the protein was also detected in the nuclei of the epithelial cells of the deferent duct, the accessory glands and the ejaculatory bulb. Our data suggest that medfly Hsp27 may have cell-specific functions, especially in the nucleus. Moreover, the association of this protein to actin cones during spermatid individualization, suggests a possible role of the protein in the formation and stabilization of actin cones. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. PnLRR-RLK27, a novel leucine-rich repeats receptor-like protein kinase from the Antarctic moss Pohlia nutans, positively regulates salinity and oxidation-stress tolerance

    PubMed Central

    Wang, Jing; Liu, Shenghao; Li, Chengcheng; Wang, Tailin; Chen, Kaoshan

    2017-01-01

    Leucine-rich repeats receptor-like kinases (LRR-RLKs) play important roles in plant growth and development as well as stress responses. Here, 56 LRR-RLK genes were identified in the Antarctic moss Pohlia nutans transcriptome, which were further classified into 11 subgroups based on their extracellular domain. Of them, PnLRR-RLK27 belongs to the LRR II subgroup and its expression was significantly induced by abiotic stresses. Subcellular localization analysis showed that PnLRR-RLK27 was a plasma membrane protein. The overexpression of PnLRR-RLK27 in Physcomitrella significantly enhanced the salinity and ABA tolerance in their gametophyte growth. Similarly, PnLRR-RLK27 heterologous expression in Arabidopsis increased the salinity and ABA tolerance in their seed germination and early root growth as well as the tolerance to oxidative stress. PnLRR-RLK27 overproduction in these transgenic plants increased the expression of salt stress/ABA-related genes. Furthermore, PnLRR-RLK27 increased the activities of reactive oxygen species (ROS) scavengers and reduced the levels of malondialdehyde (MDA) and ROS. Taken together, these results suggested that PnLRR-RLK27 as a signaling regulator confer abiotic stress response associated with the regulation of the stress- and ABA-mediated signaling network. PMID:28241081

  9. Evidence of significant synergism between antibiotics and the antipsychotic, antimicrobial drug flupenthixol.

    PubMed

    Jeyaseeli, L; Dasgupta, A; Dastidar, S G; Molnar, J; Amaral, L

    2012-06-01

    Previously, the antipsychotic, non-antibiotic compound flupenthixol dihydrochloride (Fp) was shown to exhibit distinct in vitro antibacterial activity against Gram-positive and Gram-negative bacteria and to significantly protect Swiss albino mice challenged with a known mouse virulent salmonella. The present study was designed to ascertain whether this drug could efficiently augment the action of an antibiotic or a non-antibiotic when tested in combination. A total of 12 bacterial strains belonging to various genera were selected for this study and were sensitive to the antibiotics penicillin (Pc), ampicillin, chloramphenicol, tetracycline, streptomycin, gentamicin, erythromycin, ciprofloxacin, and to the non-antibiotics methdilazine, triflupromazine, promethazine, and Fp. Pronounced and statistically significant synergism (p < 0.01) was observed when Fp was combined with Pc following the disc diffusion assay system. With the help of the checkerboard method, the fractional inhibitory concentration (FIC) index of this pair was found to be 0.375, confirming synergism. This pair of Fp+ Pc was then subjected to in vivo experiments in mice challenged with Salmonella enterica serovar Typhimurium NCTC 74. Statistical analysis of the mouse protection test suggested that this combination was highly synergistic (p < 0.001, Chi-squared analysis). Fp also revealed augmentation of its antimicrobial property when combined with streptomycin, gentamicin, ciprofloxacin, and the non-antibiotic methdilazine. The results of this study may provide alternatives for the therapy of problematic infections such as those associated with Salmonella spp.

  10. Type-1-cytokines synergize with oncogene inhibition to induce tumor growth arrest

    PubMed Central

    Acquavella, Nicolas; Clever, David; Yu, Zhiya; Roelke-Parker, Melody; Palmer, Douglas C.; Xi, Liqiang; Pflicke, Holger; Ji, Yun; Gros, Alena; Hanada, Ken-ichi; Goldlust, Ian S.; Mehta, Gautam U.; Klebanoff, Christopher A.; Crompton, Joseph G.; Sukumar, Madhusudhanan; Morrow, James J.; Franco, Zulmarie; Gattinoni, Luca; Liu, Hui; Wang, Ena; Marincola, Francesco; Stroncek, David F.; Lee, Chyi-Chia R.; Raffeld, Mark; Bosenberg, Marcus W.; Roychoudhuri, Rahul; Restifo, Nicholas P.

    2014-01-01

    Both targeted inhibition of oncogenic driver mutations and immune-based therapies show efficacy in treatment of patients with metastatic cancer but responses can be either short-lived or incompletely effective. Oncogene inhibition can augment the efficacy of immune-based therapy but mechanisms by which these two interventions might cooperate are incompletely resolved. Using a novel transplantable BRAFV600E-mutant murine melanoma model (SB-3123), we explore potential mechanisms of synergy between the selective BRAFV600E inhibitor vemurafenib and adoptive cell transfer (ACT)-based immunotherapy. We found that vemurafenib cooperated with ACT to delay melanoma progression without significantly affecting tumor infiltration or effector function of endogenous or adoptively transferred CD8+ T cells as previously observed. Instead, we found that the T-cell cytokines IFNγ and TNFα synergized with vemurafenib to induce cell-cycle arrest of tumor cells in vitro. This combinatorial effect was recapitulated in human melanoma-derived cell lines and was restricted to cancers bearing a BRAFV600E-mutation. Molecular profiling of treated SB-3123 indicated that the provision of vemurafenib promoted the sensitization of SB-3123 to the anti-proliferative effects of T-cell effector cytokines. The unexpected finding that immune cytokines synergize with oncogene inhibitors to induce growth arrest have major implications for understanding cancer biology at the intersection of oncogenic and immune signaling and provides a basis for design of combinatorial therapeutic approaches for patients with metastatic cancer. PMID:25358764

  11. The Induction of Recombinant Protein Bodies in Different Subcellular Compartments Reveals a Cryptic Plastid-Targeting Signal in the 27-kDa γ-Zein Sequence

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

    Hofbauer, Anna; Peters, Jenny; Arcalis, Elsa

    2014-12-11

    Naturally occurring storage proteins such as zeins are used as fusion partners for recombinant proteins because they induce the formation of ectopic storage organelles known as protein bodies (PBs) where the proteins are stabilized by intermolecular interactions and the formation of disulfide bonds. Endogenous PBs are derived from the endoplasmic reticulum (ER). Here, we have used different targeting sequences to determine whether ectopic PBs composed of the N-terminal portion of mature 27 kDa γ-zein added to a fluorescent protein could be induced to form elsewhere in the cell. The addition of a transit peptide for targeting to plastids causes PBmore » formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain. The subcellular localization of the PBs influences their morphology and the solubility of the stored recombinant fusion protein. Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.« less

  12. In vitro synergism of trifluorothymidine and ganciclovir against HSV-1.

    PubMed

    Hobden, Jeffery A; Kumar, Manish; Kaufman, Herbert E; Clement, Christian; Varnell, Emily D; Bhattacharjee, Partha S; Hill, James M

    2011-02-01

    To determine whether trifluorothymidine (TFT) and ganciclovir (GCV) are synergistic against herpes simplex virus type 1 (HSV-1). TFT and GCV activity against 12 strains of HSV-1 (including an acyclovir-resistant strain) was measured by plaque-forming unit (PFU) inhibition. Cellular toxicity was assessed with an MTT dye reduction assay. Synergism was determined by calculating fractional inhibitory concentration (FIC indices) based on PFU reduction. Concentrations of TFT resulting in 50% inhibition of PFUs (IC(50)) of acyclovir-susceptible HSV-1 strains ranged from 3.07 ± 0.36 to 12.52 ± 0.61 μM. GCV IC(50) values ranged from 0.40 ± 0.02 to 1.59 ± 0.14 μM. IC(50) values of TFT and GCV against the acyclovir-resistant strain were 15.40 ± 3.17 and 93.00 ± 9.64 μM, respectively. Concentrations of TFT or GCV resulting in 50% cell cytotoxicity (CC(50)) were 0.99 ± 0.01 and 92.91 ± 8.92 μM, respectively. TFT and GCV combined (10:1) were 10 times more potent against all acyclovir-susceptible HSV-1 strains. For 8 of 12 HSV-1 strains, the IC(50) of TFT and GCV combined was lower than the CC(50) of either drug. For acyclovir-susceptible HSV-1 strains, TFT and GCV combined generated a FIC index of <0.5, suggesting strong synergism between the two drugs. The FIC value for TFT and GCV combined against the acyclovir-resistant HSV-1 strain was 0.84, indicating nonantagonism. TFT and GCV are synergistic against acyclovir-susceptible HSV-1 at concentrations significantly less toxic than if each antiviral were used as a sole agent.

  13. In Vitro Synergism of Trifluorothymidine and Ganciclovir against HSV-1

    PubMed Central

    Hobden, Jeffery A.; Kumar, Manish; Kaufman, Herbert E.; Clement, Christian; Varnell, Emily D.; Bhattacharjee, Partha S.

    2011-01-01

    Purpose. To determine whether trifluorothymidine (TFT) and ganciclovir (GCV) are synergistic against herpes simplex virus type 1 (HSV-1). Methods. TFT and GCV activity against 12 strains of HSV-1 (including an acyclovir-resistant strain) was measured by plaque-forming unit (PFU) inhibition. Cellular toxicity was assessed with an MTT dye reduction assay. Synergism was determined by calculating fractional inhibitory concentration (FIC indices) based on PFU reduction. Results. Concentrations of TFT resulting in 50% inhibition of PFUs (IC50) of acyclovir-susceptible HSV-1 strains ranged from 3.07 ± 0.36 to 12.52 ± 0.61 μM. GCV IC50 values ranged from 0.40 ± 0.02 to 1.59 ± 0.14 μM. IC50 values of TFT and GCV against the acyclovir-resistant strain were 15.40 ± 3.17 and 93.00 ± 9.64 μM, respectively. Concentrations of TFT or GCV resulting in 50% cell cytotoxicity (CC50) were 0.99 ± 0.01 and 92.91 ± 8.92 μM, respectively. TFT and GCV combined (10:1) were 10 times more potent against all acyclovir-susceptible HSV-1 strains. For 8 of 12 HSV-1 strains, the IC50 of TFT and GCV combined was lower than the CC50 of either drug. For acyclovir-susceptible HSV-1 strains, TFT and GCV combined generated a FIC index of <0.5, suggesting strong synergism between the two drugs. The FIC value for TFT and GCV combined against the acyclovir-resistant HSV-1 strain was 0.84, indicating nonantagonism. Conclusions. TFT and GCV are synergistic against acyclovir-susceptible HSV-1 at concentrations significantly less toxic than if each antiviral were used as a sole agent. PMID:20861476

  14. Critical early roles for col27a1a and col27a1b in zebrafish notochord morphogenesis, vertebral mineralization and post-embryonic axial growth.

    PubMed

    Christiansen, Helena E; Lang, Michael R; Pace, James M; Parichy, David M

    2009-12-29

    Fibrillar collagens are well known for their links to human diseases, with which all have been associated except for the two most recently identified fibrillar collagens, type XXIV collagen and type XXVII collagen. To assess functions and potential disease phenotypes of type XXVII collagen, we examined its roles in zebrafish embryonic and post-embryonic development. We identified two type XXVII collagen genes in zebrafish, col27a1a and col27a1b. Both col27a1a and col27a1b were expressed in notochord and cartilage in the embryo and early larva. To determine sites of type XXVII collagen function, col27a1a and col27a1b were knocked down using morpholino antisense oligonucleotides. Knockdown of col27a1a singly or in conjunction with col27a1b resulted in curvature of the notochord at early stages and formation of scoliotic curves as well as dysmorphic vertebrae at later stages. These defects were accompanied by abnormal distributions of cells and protein localization in the notochord, as visualized by transmission electron microscopy, as well as delayed vertebral mineralization as detected histologically. Together, our findings indicate a key role for type XXVII collagen in notochord morphogenesis and axial skeletogenesis and suggest a possible human disease phenotype.

  15. Oligomerization and chaperone-like activity of Drosophila melanogaster small heat shock protein DmHsp27 and three arginine mutants in the alpha-crystallin domain.

    PubMed

    Moutaoufik, Mohamed Taha; Morrow, Geneviève; Maaroufi, Halim; Férard, Céline; Finet, Stéphanie; Tanguay, Robert M

    2017-07-01

    The small Hsp DmHsp27 from Drosophila melanogaster is one of the few small heat shock proteins (sHsps) found within the nucleus. We report that its dimerization is independent of disulfide bond formation and seems to rely on salt bridges. Unlike metazoan sHsps, DmHsp27 forms two populations of oligomers not in equilibrium. Mutations at highly conserved arginine residues in mammalian sHsps have been reported to be associated with protein conformational defects and intracellular aggregation. Independent mutation of three highly conserved arginines (R122, R131, and R135) to glycine in DmHsp27 results in only one population of higher molecular weight form. In vitro, the chaperone-like activity of wild-type DmHsp27 was comparable with that of its two isolated populations and to the single population of the R122G, R131G, and R135G using luciferase as substrate. However, using insulin, the chaperone-like activity of wild-type DmHsp27 was lower than that of R122G and R131G mutants. Altogether, the results characterize wild-type DmHsp27 and its alpha-crystallin domain (ACD) arginine mutants and may give insight into protection mechanism of sHsps.

  16. Western spruce budworm outbreaks did not increase fire risk over the last three centuries: A dendrochronological analysis of inter-disturbance synergism

    Treesearch

    Aquila Flower; Daniel G. Gavin; Emily K. Heyerdahl; Russell A. Parsons; Gregory M. Cohn

    2014-01-01

    Insect outbreaks are often assumed to increase the severity or probability of fire occurrence through increased fuel availability, while fires may in turn alter susceptibility of forests to subsequent insect outbreaks through changes in the spatial distribution of suitable host trees. However, little is actually known about the potential synergisms between these...

  17. Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid Raft-mediated endocytosis negatively regulated by caveolin-1.

    PubMed

    Svensson, Katrin J; Christianson, Helena C; Wittrup, Anders; Bourseau-Guilmain, Erika; Lindqvist, Eva; Svensson, Lena M; Mörgelin, Matthias; Belting, Mattias

    2013-06-14

    The role of exosomes in cancer can be inferred from the observation that they transfer tumor cell derived genetic material and signaling proteins, resulting in e.g. increased tumor angiogenesis and metastasis. However, the membrane transport mechanisms and the signaling events involved in the uptake of these virus-like particles remain ill-defined. We now report that internalization of exosomes derived from glioblastoma (GBM) cells involves nonclassical, lipid raft-dependent endocytosis. Importantly, we show that the lipid raft-associated protein caveolin-1 (CAV1), in analogy with its previously described role in virus uptake, negatively regulates the uptake of exosomes. We find that exosomes induce the phosphorylation of several downstream targets known to associate with lipid rafts as signaling and sorting platforms, such as extracellular signal-regulated kinase-1/2 (ERK1/2) and heat shock protein 27 (HSP27). Interestingly, exosome uptake appears dependent on unperturbed ERK1/2-HSP27 signaling, and ERK1/2 phosphorylation is under negative influence by CAV1 during internalization of exosomes. These findings significantly advance our general understanding of exosome-mediated uptake and offer potential strategies for how this pathway may be targeted through modulation of CAV1 expression and ERK1/2 signaling.

  18. A Targetable Molecular Chaperone Hsp27 Confers Aggressiveness in Hepatocellular Carcinoma

    PubMed Central

    Zhang, Yurong; Tao, Xuemei; Jin, Guangzhi; Jin, Haojie; Wang, Ning; Hu, Fangyuan; Luo, Qin; Shu, Huiqun; Zhao, Fangyu; Yao, Ming; Fang, Jingyuan; Cong, Wenming; Qin, Wenxin; Wang, Cun

    2016-01-01

    Heat shock protein 27 (Hsp27) is an ATP-independent molecular chaperone and confers survival advantages and resistance to cancer cells under stress conditions. The effects and molecular mechanisms of Hsp27 in HCC invasion and metastasis are still unclear. In this study, hepatocellular carcinoma (HCC) tissue array (n = 167) was used to investigate the expression and prognostic relevance of Hsp27 in HCC patients. HCC patients with high expression of Hsp27 exhibited poor prognosis. Overexpression of Hsp27 led to the forced invasion of HCC cells, whereas silencing Hsp27 attenuated invasion and metastasis of HCC cells in vitro and in vivo. We revealed that Hsp27 activated Akt signaling, which in turn promoted MMP2 and ITGA7 expression and HCC metastasis. We further observed that targeting Hsp27 using OGX-427 obviously suppressed HCC metastasis in two metastatic models. These findings indicate that Hsp27 is a useful predictive factor for prognosis of HCC and it facilitates HCC metastasis through Akt signaling. Targeting Hsp27 with OGX-427 may represent an attractive therapeutic option for suppressing HCC metastasis. PMID:26941848

  19. Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing.

    PubMed

    Gamalinda, Michael; Jakovljevic, Jelena; Babiano, Reyes; Talkish, Jason; de la Cruz, Jesús; Woolford, John L

    2013-02-01

    Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2.

  20. Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing

    PubMed Central

    Gamalinda, Michael; Jakovljevic, Jelena; Babiano, Reyes; Talkish, Jason; de la Cruz, Jesús; Woolford, John L.

    2013-01-01

    Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2. PMID:23268442

  1. Plasma Membrane Intrinsic Proteins SlPIP2;1, SlPIP2;7 and SlPIP2;5 Conferring Enhanced Drought Stress Tolerance in Tomato.

    PubMed

    Li, Ren; Wang, Jinfang; Li, Shuangtao; Zhang, Lei; Qi, Chuandong; Weeda, Sarah; Zhao, Bing; Ren, Shuxin; Guo, Yang-Dong

    2016-08-22

    The function of aquaporin (AQP) protein in transporting water is crucial for plants to survive in drought stress. With 47 homologues in tomato (Solanum lycopersicum) were reported, but the individual and integrated functions of aquaporins involved in drought response remains unclear. Here, three plasma membrane intrinsic protein genes, SlPIP2;1, SlPIP2;7 and SlPIP2;5, were identified as candidate aquaporins genes because of highly expressed in tomato roots. Assay on expression in Xenopus oocytes demonstrated that SlPIP2s protein displayed water channel activity and facilitated water transport into the cells. With real-time PCR and in situ hybridization analysis, SlPIP2s were considered to be involved in response to drought treatment. To test its function, transgenic Arabidopsis and tomato lines overexpressing SlPIP2;1, SlPIP2;7 or SlPIP2;5 were generated. Compared with wild type, the over-expression of SlPIP2;1, SlPIP2;7 or SlPIP2;5 transgenic Arabidopsis and tomato plants all showed significantly higher hydraulic conductivity levels and survival rates under both normal and drought conditions. Taken together, this study concludes that aquaporins (SlPIP2;1, SlPIP2;7 and SlPIP2;5) contribute substantially to root water uptake in tomato plants through improving plant water content and maintaining osmotic balance.

  2. Enhanced LPS-induced activation of IL-27 signalling in sarcoidosis.

    PubMed

    Ringkowski, Sabine; Loke, Joshua; Huang, Shuying; Ahmadzai, Hasib; Herth, Felix J F; Thomas, Paul S; Herbert, Cristan

    2016-08-01

    Granulomas in sarcoidosis have recently been described as containing Interleukin (IL)-27, one of the members of the IL-12 family of cytokines, which also includes IL-35. Levels of these cytokines and the IL-27 receptor subunits were hypothesised to differ between patients with sarcoidosis compared to healthy controls in peripheral blood. Using a cross-sectional study design, plasma and peripheral blood mononuclear cells (PBMC) were collected from patients and control subjects. Protein and mRNA (in PBMC) levels for IL-27 and IL-35 (IL27, EBI3, IL12A subunits) as well as IL-27 receptor (IL6ST and IL27RA subunits) were assessed spontaneously and following direct (LPS) and indirect (anti-CD3/28 activation beads) macrophage stimulation using RT- PCR, ELISA and flow cytometry. Following stimulation with LPS, PBMC of patients with sarcoidosis displayed significantly enhanced expression of IL27 and EBI3 mRNA (p = 0.020 and p = 0.037 respectively) compared to PBMCs from healthy controls. There was also significantly enhanced production of IL-27 by PBMC from patients with sarcoidosis compared to healthy controls in response to LPS stimulation (p = 0.027). IL6ST mRNA and IL6ST protein were significantly lower in patients with sarcoidosis (mRNA p = 0.0002; MFI p = 0.0015) whilst IL27RA protein levels were significantly higher in patients with sarcoidosis compared to healthy controls (MFI p < 0.0001). Plasma IL-35 protein levels did not differ between control and sarcoidosis subjects (p = 0.23). These results suggest there may be exaggerated activation of IL-27 signalling in response to LPS in sarcoidosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. GT-094, a NO-NSAID, inhibits colon cancer cell growth by activation of a reactive oxygen species-microRNA-27a: ZBTB10-specificity protein pathway.

    PubMed

    Pathi, Satya S; Jutooru, Indira; Chadalapaka, Gayathri; Sreevalsan, Sandeep; Anand, S; Thatcher, Gregory Rj; Safe, Stephen

    2011-02-01

    Ethyl 2-((2,3-bis(nitrooxy)propyl)disulfanyl)benzoate (GT-094) is a novel nitric oxide (NO) chimera containing an nonsteroidal anti-inflammatory drug (NSAID) and NO moieties and also a disulfide pharmacophore that in itself exhibits cancer chemopreventive activity. In this study, the effects and mechanism of action of GT-094 were investigated in RKO and SW480 colon cancer cells. GT-094 inhibited cell proliferation and induced apoptosis in both cell lines and this was accompanied by decreased mitochondrial membrane potential (MMP) and induction of reactive oxygen species (ROS), and these responses were reversed after cotreatment with the antioxidant glutathione. GT-094 also downregulated genes associated with cell growth [cyclin D1, hepatocyte growth factor receptor (c-Met), epidermal growth factor receptor (EGFR)], survival (bcl-2, survivin), and angiogenesis [VEGF and its receptors (VEGFR1 and VEGFR2)]. Results of previous RNA interference studies in this laboratory has shown that these genes are regulated, in part, by specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 that are overexpressed in colon and other cancer cell lines and not surprisingly, GT-094 also decreased Sp1, Sp3, and Sp4 in colon cancer cells. GT-094-mediated repression of Sp and Sp-regulated gene products was due to downregulation of microRNA-27a (miR-27a) and induction of ZBTB10, an Sp repressor that is regulated by miR-27a in colon cancer cells. Moreover, the effects of GT-094 on Sp1, Sp3, Sp4, miR-27a, and ZBTB10 were also inhibited by glutathione suggesting that the anticancer activity of GT-094 in colon cancer cells is due, in part, to activation of an ROS-miR-27a:ZBTB10-Sp transcription factor pathway.

  4. Protection and synergism by recombinant fowl pox vaccines expressing multiple genes from Marek's disease virus.

    PubMed

    Lee, Lucy E; Witter, R L; Reddy, S M; Wu, P; Yanagida, N; Yoshida, S

    2003-01-01

    Recombinant fowl poxviruses (rFPVs) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B, E, I, H, and UL32 (gB1, gE, gI, gH, and UL32). An additional rFPV was constructed to contain four MDV genes (gB1, gE, gI, and UL32). These rFPVs were evaluated for their ability to protect maternal antibody-positive chickens against challenge with highly virulent MDV isolates. The protection induced by a single rFPV/gB1 (42%) confirmed our previous finding. The protection induced by rFPV/gI (43%), rFPV/gB1UL32 (46%), rFPV/gB1gEgI (72%), and rFPV/gB1gEgIUL32 (70%) contributed to additional knowledge on MDV genes involved in protective immunity. In contrast, the rFPV containing gE, gH, or UL32 did not induce significant protection compared with turkey herpesvirus (HVT). Levels of protection by rFPV/gB1 and rFPV/gl were comparable with that of HVT. Only gB1 and gI conferred synergism in rFPV containing these two genes. Protection by both rFPV/gB1gEgI (72%) and rFPV/gB1gEgIUL32(70%) against Marek's disease was significantly enhanced compared with a single gB1 or gI gene (40%). This protective synergism between gB1 and gI in rFPVs may be the basis for better protection when bivalent vaccines between serotypes 2 and 3 were used. When rFPV/gB1gIgEUL32 + HVT were used as vaccine against Md5 challenge, the protection was significantly enhanced (94%). This synergism between rFPV/gB1gIgEUL32 and HVT indicates additional genes yet to be discovered in HVT may be responsible for the enhancement.

  5. Trait synergisms and the rarity, extirpation, and extinction risk of desert fishes.

    PubMed

    Olden, Julian D; Poff, N LeRoy; Bestgen, Kevin R

    2008-03-01

    Understanding the causes and consequences of species extinctions is a central goal in ecology. Faced with the difficult task of identifying those species with the greatest need for conservation, ecologists have turned to using predictive suites of ecological and life-history traits to provide reasonable estimates of species extinction risk. Previous studies have linked individual traits to extinction risk, yet the nonadditive contribution of multiple traits to the entire extinction process, from species rarity to local extirpation to global extinction, has not been examined. This study asks whether trait synergisms predispose native fishes of the Lower Colorado River Basin (USA) to risk of extinction through their effects on rarity and local extirpation and their vulnerability to different sources of threat. Fish species with "slow" life histories (e.g., large body size, long life, and delayed maturity), minimal parental care to offspring, and specialized feeding behaviors are associated with smaller geographic distribution, greater frequency of local extirpation, and higher perceived extinction risk than that expected by simple additive effects of traits in combination. This supports the notion that trait synergisms increase the susceptibility of native fishes to multiple stages of the extinction process, thus making them prone to the multiple jeopardies resulting from a combination of fewer individuals, narrow environmental tolerances, and long recovery times following environmental change. Given that particular traits, some acting in concert, may differentially predispose native fishes to rarity, extirpation, and extinction, we suggest that management efforts in the Lower Colorado River Basin should be congruent with the life-history requirements of multiple species over large spatial and temporal scales.

  6. Synergic effects of tactolimus and azole antifungal agents against azole-resistant Candida albican strains.

    PubMed

    Maesaki, S; Marichal, P; Hossain, M A; Sanglard, D; Vanden Bossche, H; Kohno, S

    1998-12-01

    We investigated the effects of combining tacrolimus and azole antifungal agents in azole-resistant strains of Candida albicans by comparing the accumulation of [3H]itraconazole. The CDR1-expressing resistant strain C26 accumulated less itraconazole than the CaMDR-expressing resistant strain C40 or the azole-sensitive strain B2630. A CDR1-expressing Saccharomyces cerevisiae mutant, DSY415, showed a marked reduction in the accumulation of both fluconazole and itraconazole. A CaMDR-expressing S. cerevisiae mutant, DSY416, also showed lower accumulation of fluconazole, but not of itraconazole. The addition of sodium azide, an electron-transport chain inhibitor, increased the intracellular accumulation of itraconazole only in the C26 strain, and not in the C40 or B2630 strains. Addition of tacrolimus, an inhibitor of multidrug resistance proteins, resulted in the highest increase in itraconazole accumulation in the C26 strain. The combination of itraconazole and tacrolimus was synergic in azole-resistant C. albicans strains. In the C26 strain, the MIC of itraconazole decreased from >8 to 0.5 mg/L when combined with tacrolimus. Our results showed that two multidrug resistance phenotypes (encoded by the CDR1 and CaMDR genes) in C. albicans have different substrate specificity for azole antifungal agents and that a combination of tacrolimus and azole antifungal agents is effective against azole-resistant strains of C. albicans.

  7. Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses.

    PubMed Central

    Pruss, G; Ge, X; Shi, X M; Carrington, J C; Bowman Vance, V

    1997-01-01

    Synergistic viral diseases of higher plants are caused by the interaction of two independent viruses in the same host and are characterized by dramatic increases in symptoms and in accumulation of one of the coinfecting viruses. In potato virus X (PVX)/potyviral synergism, increased pathogenicity and accumulation of PVX are mediated by the expression of potyviral 5' proximal sequences encoding P1, the helper component proteinase (HC-Pro), and a fraction of P3. Here, we report that the same potyviral sequence (termed P1/HC-Pro) enhances the pathogenicity and accumulation of two other heterologous viruses: cucumber mosaic virus and tobacco mosaic virus. In the case of PVX-potyviral synergism, we show that the expression of the HC-Pro gene product, but not the RNA sequence itself, is sufficient to induce the increase in PVX pathogenicity and that both P1 and P3 coding sequences are dispensable for this aspect of the synergistic interaction. In protoplasts, expression of the potyviral P1/HC-Pro region prolongs the accumulation of PVX (-) strand RNA and transactivates expression of a reporter gene from a PVX subgenomic promoter. Unlike the synergistic enhancement of PVX pathogenicity, which requires only expression of HC-Pro, the enhancement of PVX (-) strand RNA accumulation in protoplasts is significantly greater when the entire P1/HC-Pro sequence is expressed. These results indicate that the potyviral P1/HC-Pro region affects a step in disease development that is common to a broad range of virus infections and suggest a mechanism involving transactivation of viral replication. PMID:9212462

  8. In silico synergism and antagonism of an anti-tumour system intervened by coupling immunotherapy and chemotherapy: a mathematical modelling approach.

    PubMed

    Hu, Wen-Yong; Zhong, Wei-Rong; Wang, Feng-Hua; Li, Li; Shao, Yuan-Zhi

    2012-02-01

    Based on the logistic growth law for a tumour derived from enzymatic dynamics, we address from a physical point of view the phenomena of synergism, additivity and antagonism in an avascular anti-tumour system regulated externally by dual coupling periodic interventions, and propose a theoretical model to simulate the combinational administration of chemotherapy and immunotherapy. The in silico results of our modelling approach reveal that the tumour population density of an anti-tumour system, which is subject to the combinational attack of chemotherapeutical as well as immune intervention, depends on four parameters as below: the therapy intensities D, the coupling intensity I, the coupling coherence R and the phase-shifts Φ between two combinational interventions. In relation to the intensity and nature (synergism, additivity and antagonism) of coupling as well as the phase-shift between two therapeutic interventions, the administration sequence of two periodic interventions makes a difference to the curative efficacy of an anti-tumour system. The isobologram established from our model maintains a considerable consistency with that of the well-established Loewe Additivity model (Tallarida, Pharmacology 319(1):1-7, 2006). Our study discloses the general dynamic feature of an anti-tumour system regulated by two periodic coupling interventions, and the results may serve as a supplement to previous models of drug administration in combination and provide a type of heuristic approach for preclinical pharmacokinetic investigation.

  9. The human ubiquitin-conjugating enzyme Cdc34 controls cellular proliferation through regulation of p27{sup Kip1} protein levels

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

    Butz, Nicole; Ruetz, Stephan; Natt, Francois

    2005-02-15

    Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27{sup Kip1} was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCF{sup Skp2} ubiquitin ligase has been reported to mediate p27{sup Kip1} degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27{sup Kip1}, and prevent cellular proliferation. Elevation of p27{sup Kip1} protein level is found tomore » be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27{sup Kip1} with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCF{sup Skp2} ubiquitin ligase substrate p27{sup Kip1}, but has no concomitant effect on the level of IkB{alpha} and {beta}-catenin, which are known substrates of a closely related SCF ligase.« less

  10. Biofortification of soybean meal: immunological properties of the 27 kDa γ-zein.

    PubMed

    Krishnan, Hari B; Jang, Sungchan; Kim, Won-Seok; Kerley, Monty S; Oliver, Melvin J; Trick, Harold N

    2011-02-23

    Legumes, including soybeans ( Glycine max ), are deficient in sulfur-containing amino acids, which are required for the optimal growth of monogastric animals. This deficiency can be overcome by expressing heterologous proteins rich in sulfur-containing amino acids in soybean seeds. A maize 27 kDa γ-zein, a cysteine-rich protein, has been successfully expressed in several crops including soybean, barley, and alfalfa with the intent to biofortify these crops for animal feed. Previous work has shown that the maize 27 kDa zein can withstand digestion by pepsin and elicit an immunogenic response in young pigs. By use of sera from patients who tested positive by ImmunoCAP assay for elevated IgE to maize proteins, specific IgE binding to the 27 kDa γ-zein is demonstrated. Bioinformatic analysis using the full-length and 80 amino acid sliding window FASTA searches identified significant sequence homology of the 27 kDa γ-zein with several known allergens. Immunoblot analysis using human serum that cross-reacts with maize seed proteins also revealed specific IgE-binding to the 27 kDa γ-zein in soybean seed protein extracts containing the 27 kDa zein. This study demonstrates for the first time the allergenicity potential of the 27 kDa γ-zein and the potential that this protein has to limit livestock performance when used in soybeans that serve as a biofortified feed supplement.

  11. Forced Expression of Heat Shock Protein 27 (Hsp27) Reverses P-Glycoprotein (ABCB1)-mediated Drug Efflux and MDR1 Gene Expression in Adriamycin-resistant Human Breast Cancer Cells*

    PubMed Central

    Kanagasabai, Ragu; Krishnamurthy, Karthikeyan; Druhan, Lawrence J.; Ilangovan, Govindasamy

    2011-01-01

    Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor κB (NF-κB) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-κB (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G2/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-κB-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-κB level will be a potential approach to circumvent drug resistance in breast cancer. PMID:21784846

  12. Forced expression of heat shock protein 27 (Hsp27) reverses P-glycoprotein (ABCB1)-mediated drug efflux and MDR1 gene expression in Adriamycin-resistant human breast cancer cells.

    PubMed

    Kanagasabai, Ragu; Krishnamurthy, Karthikeyan; Druhan, Lawrence J; Ilangovan, Govindasamy

    2011-09-23

    Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor κB (NF-κB) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-κB (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G(2)/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-κB-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-κB level will be a potential approach to circumvent drug resistance in breast cancer.

  13. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

    PubMed Central

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

  14. Extracellular HSP27 acts as a signaling molecule to activate NF-κB in macrophages.

    PubMed

    Salari, Samira; Seibert, Tara; Chen, Yong-Xiang; Hu, Tieqiang; Shi, Chunhua; Zhao, Xiaoling; Cuerrier, Charles M; Raizman, Joshua E; O'Brien, Edward R

    2013-01-01

    Heat shock protein 27 (HSP27) shows attenuated expression in human coronary arteries as the extent of atherosclerosis progresses. In mice, overexpression of HSP27 reduces atherogenesis, yet the precise mechanism(s) are incompletely understood. Inflammation plays a central role in atherogenesis, and of particular interest is the balance of pro- and anti-inflammatory factors produced by macrophages. As nuclear factor-kappa B (NF-κB) is a key immune signaling modulator in atherogenesis, and macrophages are known to secrete HSP27, we sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling in macrophages. Treatment of THP-1 macrophages with rHSP27 resulted in the degradation of an inhibitor of NF-κB, IκBα, nuclear translocation of the NF-κB p65 subunit, and increased NF-κB transcriptional activity. Treatment of THP-1 macrophages with rHSP27 yielded increased expression of a variety of genes, including the pro-inflammatory factors, IL-1β, and TNF-α. However, rHSP27 also increased the expression of the anti-inflammatory factors IL-10 and GM-CSF both at the mRNA and protein levels. Our study suggests that in macrophages, activation of NF-κB signaling by rHSP27 is associated with upregulated expression and secretion of key pro- and anti-inflammatory cytokines. Moreover, we surmise that it is the balance in expression of these mediators and antagonists of inflammation, and hence atherogenesis, that yields a favorable net effect of HSP27 on the vessel wall.

  15. Synergic treatment for monosodium glutamate wastewater by Saccharomyces cerevisiae and Coriolus versicolor.

    PubMed

    Jia, Cuiying; Kang, Ruijuan; Zhang, Yuhui; Cong, Wei; Cai, Zhaoling

    2007-03-01

    Biodegradation and decolorization of monosodium glutamate wastewater were carried out by using an acidophilus yeast strain of Saccharomyces cerevisiae and Coriolus versicolor. For the yeast treatment, the highest COD removal and reducing sugar removal efficiency were 76.6% and 80.2%, respectively. The color removal was only 2%. For C. versicolor treatment, the highest COD removal, color removal and reducing sugar removal efficiencies were 78.7%, 56.5% and 90.9%, respectively. The synergic treatment process, in which the yeast and C. versicolor were successively applied,exhibited great advantage over the individual process.

  16. Antibodies against recombinant heat shock proteins of 60 kDa from enterobacteria in the sera and synovial fluid of HLA-B27 positive ankylosing spondylitis patients.

    PubMed

    Domínguez-López, M L; Ortega-Ortega, Y; Manríquez-Raya, J C; Burgos-Vargas, R; Vega-López, A; García-Latorre, E

    2009-01-01

    To study the association of HLA-B27 with IgG antibodies to different enterobacterial HSP60s in patients with ankylosing spondylitis (AS). IgG antibodies to 60 kDa enterobacterial HSPs were determined by ELISA in paired samples of sera and synovial fluid from 21 HLA-B27+ ankylosing spondylitis (AS) patients; and in sera from 32 HLA-B27+ AS patients, 35 HLA-B27+ healthy relatives of AS patients, and 60 HLA-B27- healthy individuals with no family members with AS. HLA-B27+ patients and healthy individuals showed significantly higher IgG antibody levels to recombinant enterobacterial HSP60s than HLA-B27- healthy controls. The levels of anti-HSP60Sf and anti-HSP60Ec antibodies correlated with disease activity and anti-HSP60Ec antibodies with male gender. No association between enterobacterial HSP60 antibody levels and disease duration was observed. All groups had lower levels of IgG antibodies to rHSP60 from Streptococcus pyogenes (rHSP60 Spy). In paired samples of sera and synovial fluid from B27+ patients, IgG antibodies to enterobacterial HSP60s were detected, but in significantly higher levels in sera than in synovial fluid. The anti-rHSPSpy IgG response in these samples was lower and similar in the three groups. A correlation was found between HLA-B27 and the response to recombinat enterobacterial HSP60s. This response could be associated with disease activitir and gender in some proteins and the presence eof IgG antibodies to these proteins in synovial fluid could be associated with the inflammatory process and initiation of AS.

  17. Spinosad resistance, esterase isoenzymes and temporal synergism in Frankliniella occidentalis (Pergande) in Australia.

    PubMed

    Herron, Grant A; Gunning, Robin V; Cottage, Emma L A; Borzatta, Valerio; Gobbi, Carlotta

    2014-09-01

    Spinosad has been widely used in Australia to control western flower thrips Frankliniella occidentalis (Pergande) but spinosad usefulness is now compromised by resistance. Here we studied a highly spinosad resistant strain of F. occidentalis to explore if esterases had a role in spinosad resistance. Enhanced esterase activity in pressured spinosad-resistant F. occidentalis was confirmed via PAGE electrophoresis and estimated to be approximately three times higher than that in a susceptible strain. Spinosad-esterase inhibition data in the resistant strain, showed a concentration effect with significant esterase-spinosad binding occurring at spinosad concentrations from 6.2× 10(-7) to 1.5× 10(-5) M. Similarly, a spinosad-piperonyl butoxide (PBO) inhibition curve showed a concentration effect, with significant esterase-PBO binding occurring in the resistant strain at PBO concentrations between 3.3× 10(-5) M and 8.4× 10(-4) M. No binding of esterase to spinosad or PBO occurred in the susceptible strain. Results of bioassays in which spinosad resistant F. occidentalis were sprayed with a 4h delayed release formulation of cyclodextrin-complexed spinosad with immediately available PBO demonstrated that spinosad resistance was significantly reduced from 577 to 72-fold. With further development the PBO synergism of spinosad using a delayed release formulation, similar to that used here, may provide effective control for spinosad resistant F. occidentalis. Temporal synergism of spinosad may prove to be effective tactic for the control of spinosad resistant F. occidentalis where the main resistance mechanism involved has been confirmed to be esterase based. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Mutagenic synergism detected between dimethyl sulfate and X-rays but not found between N-methyl-N-nitrosourea and X-rays in the stamen hairs of Tradescantia clone BNL 4430.

    PubMed

    Shima, N; Ichikawa, S

    1995-09-01

    Mutagenic interactions with X-rays of two monofunctional alkylating agents, dimethyl sulfate (DMS) and N-methyl-N-nitrosourea (MNU), were studied in the stamen hairs of Tradescantia clone BNL 4430, a blue/pink heterozygote. The young inflorescence-bearing shoots with roots cultivated in the nutrient solution circulating growth chamber were used as tester plants. Synergism between two different mutagens was judged to have occurred when the mutation frequency observed after applying the two mutagens concurrently was statistically significantly higher than the mutation frequency expected from the additive effects of the two mutagens. Clear synergistic effects in inducing somatic pink mutations were detected with all combinations of doses of DMS and X-rays examined, even in a relatively low X-ray dose range (down to 299 mGy), resembling those confirmed earlier between ethyl methanesulfonate (EMS) and X-rays, but somewhat differing from the synergisms observed earlier between methyl methanesulfonate (MMS) and X-rays. On the other hand, no mutagenic synergism was detected between MNU and X-rays, even in a relatively high X-ray dose range (up to 862 mGy). The presence or absence of mutagenic synergisms of these alkylating agents with X-rays could be related to the action mechanism of each alkylating agent.

  19. Monocyte Proteomics Reveals Involvement of Phosphorylated HSP27 in the Pathogenesis of Osteoporosis.

    PubMed

    Daswani, Bhavna; Gupta, Manoj Kumar; Gavali, Shubhangi; Desai, Meena; Sathe, Gajanan J; Patil, Anushree; Parte, Priyanka; Sirdeshmukh, Ravi; Khatkhatay, M Ikram

    2015-01-01

    Peripheral monocytes, precursors of osteoclasts, have emerged as important candidates for identifying proteins relevant to osteoporosis, a condition characterized by low Bone Mineral Density (BMD) and increased susceptibility for fractures. We employed 4-plex iTRAQ (isobaric tags for relative and absolute quantification) coupled with LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) to identify differentially expressed monocyte proteins from premenopausal and postmenopausal women with low versus high BMD. Of 1801 proteins identified, 45 were differentially abundant in low versus high BMD, with heat shock protein 27 (HSP27) distinctly upregulated in low BMD condition in both premenopausal and postmenopausal categories. Validation in individual samples (n = 80) using intracellular ELISA confirmed that total HSP27 (tHSP27) as well as phosphorylated HSP27 (pHSP27) was elevated in low BMD condition in both categories (P < 0.05). Further, using transwell assays, pHSP27, when placed in the upper chamber, could increase monocyte migration (P < 0.0001) and this was additive in combination with RANKL (receptor activator of NFkB ligand) placed in the lower chamber (P = 0.05). Effect of pHSP27 in monocyte migration towards bone milieu can result in increased osteoclast formation and thus contribute to pathogenesis of osteoporosis. Overall, this study reveals for the first time a novel link between monocyte HSP27 and BMD.

  20. The prohibitin-binding compound fluorizoline induces apoptosis in chronic lymphocytic leukemia cells through the upregulation of NOXA and synergizes with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax

    PubMed Central

    Cosialls, Ana M.; Pomares, Helena; Iglesias-Serret, Daniel; Saura-Esteller, José; Núñez-Vázquez, Sonia; González-Gironès, Diana M.; de la Banda, Esmeralda; Preciado, Sara; Albericio, Fernando; Lavilla, Rodolfo; Pons, Gabriel; González-Barca, Eva M.; Gil, Joan

    2017-01-01

    Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins. In the study herein, the pro-apoptotic effect of fluorizoline was assessed in 34 primary samples from patients with chronic lymphocytic leukemia. Fluorizoline induced apoptosis in chronic lymphocytic leukemia cells at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline irrespective of patients’ clinical or genetic features, whereas normal T lymphocytes were less sensitive. Fluorizoline increased the protein levels of the pro-apoptotic B-cell lymphoma 2 family member NOXA in chronic lymphocytic leukemia cells. Furthermore, fluorizoline synergized with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax to induce apoptosis. These results suggest that targeting prohibitins could be a new therapeutic strategy for chronic lymphocytic leukemia. PMID:28619845

  1. Overexpression of heat shock protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through S-phase arrest and apoptosis

    PubMed Central

    Guo, Yang; Ziesch, Andreas; Hocke, Sandra; Kampmann, Eric; Ochs, Stephanie; De Toni, Enrico N; Göke, Burkhard; Gallmeier, Eike

    2015-01-01

    We previously established a role for HSP27 as a predictive marker for therapeutic response towards gemcitabine in pancreatic cancer. Here, we investigate the underlying mechanisms of HSP27-mediated gemcitabine sensitivity. Utilizing a pancreatic cancer cell model with stable HSP27 overexpression, cell cycle arrest and apoptosis induction were analysed by flow cytometry, nuclear staining, immunoblotting and mitochondrial staining. Drug sensitivity studies were performed by proliferation assays. Hyperthermia was simulated using mild heat shock at 41.8°C. Upon gemcitabine treatment, HSP27-overexpressing cells displayed an early S-phase arrest subsequently followed by a strongly increased sub-G1 fraction. Apoptosis was characterized by PARP-, CASPASE 3-, CASPASE 8-, CASPASE 9- and BIM- activation along with a mitochondrial membrane potential loss. It was reversible through chemical caspase inhibition. Importantly, gemcitabine sensitivity and PARP cleavage were also elicited by heat shock-induced HSP27 overexpression, although to a smaller extent, in a panel of pancreatic cancer cell lines. Finally, HSP27-overexpressing pancreatic cancer cells displayed an increased sensitivity also towards death receptor-targeting agents, suggesting another pro-apoptotic role of HSP27 along the extrinsic apoptosis pathway. Taken together, in contrast to the well-established anti-apoptotic properties of HSP27 in cancer, our study reveals novel pro-apoptotic functions of HSP27—mediated through both the intrinsic and the extrinsic apoptotic pathways—at least in pancreatic cancer cells. HSP27 could represent a predictive marker of therapeutic response towards specific drug classes in pancreatic cancer and provides a novel molecular rationale for current clinical trials applying the combination of gemcitabine with regional hyperthermia in pancreatic cancer patients. PMID:25331547

  2. RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells

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

    Wang, Houcai; Yu, Jing; Zhang, Lixia

    2014-04-18

    Highlights: • RPS27a expression was up-regulated in advanced-phase CML and AL patients. • RPS27a knockdown changed biological property of K562 and K562/G01 cells. • RPS27a knockdown affected Raf/MEK/ERK, P21 and BCL-2 signaling pathways. • RPS27a knockdown may be applicable for new combination therapy in CML patients. - Abstract: Ribosomal protein S27a (RPS27a) could perform extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. The high expression level of RPS27a was reported in solid tumors, and we found that the expression level of RPS27a was up-regulated in advanced-phase chronic myeloid leukemia (CML) and acute leukemia (AL)more » patients. In this study, we explored the function of RPS27a in leukemia cells by using CML cell line K562 cells and its imatinib resistant cell line K562/G01 cells. It was observed that the expression level of RPS27a was high in K562 cells and even higher in K562/G01 cells. Further analysis revealed that RPS27a knockdown by shRNA in both K562 and K562G01 cells inhibited the cell viability, induced cell cycle arrest at S and G2/M phases and increased cell apoptosis induced by imatinib. Combination of shRNA with imatinib treatment could lead to more cleaved PARP and cleaved caspase-3 expression in RPS27a knockdown cells. Further, it was found that phospho-ERK(p-ERK) and BCL-2 were down-regulated and P21 up-regulated in RPS27a knockdown cells. In conclusion, RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells. It appears that drugs targeting RPS27a combining with tyrosine kinase inhibitor (TKI) might represent a novel therapy strategy in TKI resistant CML patients.« less

  3. Synergism studies with binary mixtures of pyrethroid, carbamate and organophosphate insecticides on Frankliniella occidentalis (Pergande).

    PubMed

    Bielza, Pablo; Espinosa, Pedro J; Quinto, Vicente; Abellán, Jaime; Contreras, Josefina

    2007-01-01

    The major mechanism of resistance to most insecticides in Frankliniella occidentalis (Pergande) is metabolic, piperonyl butoxide (PBO) suppressible, mediated by cytochrome-P450 monooxygenases and conferring cross-resistance among insecticide classes. The efficacy of insecticide mixtures of acrinathrin, methiocarb, formetanate and chlorpyrifos was studied by topical exposure in strains of F. occidentalis selected for resistance to each insecticide. The method consisted in combining increasing concentrations of one insecticide with a constant low rate of the second one as synergist. Acrinathrin activity against F. occidentalis was enhanced by carbamate insecticides, methiocarb being a much better synergist than formetanate. Monooxygenase action on the carbamates would prevent degradation of the pyrethroid, hence providing a level of synergism by competitive substrate inhibition. However, the number of insecticides registered for control of F. occidentalis is very limited, and they are needed for antiresistance strategies such as mosaics and rotations. Therefore, a study was made of the synergist effect of other carbamates not used against thrips, such as carbofuran and carbosulfan, against a susceptible strain and a field strain. Neither carbamate showed synergism to acrinathrin in the susceptible strain, but both did in the field strain, carbosulfan being a better synergist than carbofuran. The data obtained indicate that low rates of carbamates could be used as synergists to restore some pyrethroid susceptibility in F. occidentalis. Copyright (c) 2006 Society of Chemical Industry.

  4. The small heat shock protein Hsp27 affects assembly dynamics and structure of keratin intermediate filament networks.

    PubMed

    Kayser, Jona; Haslbeck, Martin; Dempfle, Lisa; Krause, Maike; Grashoff, Carsten; Buchner, Johannes; Herrmann, Harald; Bausch, Andreas R

    2013-10-15

    The mechanical properties of living cells are essential for many processes. They are defined by the cytoskeleton, a composite network of protein fibers. Thus, the precise control of its architecture is of paramount importance. Our knowledge about the molecular and physical mechanisms defining the network structure remains scarce, especially for the intermediate filament cytoskeleton. Here, we investigate the effect of small heat shock proteins on the keratin 8/18 intermediate filament cytoskeleton using a well-controlled model system of reconstituted keratin networks. We demonstrate that Hsp27 severely alters the structure of such networks by changing their assembly dynamics. Furthermore, the C-terminal tail domain of keratin 8 is shown to be essential for this effect. Combining results from fluorescence and electron microscopy with data from analytical ultracentrifugation reveals the crucial role of kinetic trapping in keratin network formation. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  5. In Vivo Suppression of Heat Shock Protein (HSP)27 and HSP70 Accelerates DMBA-Induced Skin Carcinogenesis by Inducing Antigenic Unresponsiveness to the Initiating Carcinogenic Chemical.

    PubMed

    Yusuf, Nabiha; Nasti, Tahseen H; Ahmad, Israr; Chowdhury, Sanim; Mohiuddin, Hasan; Xu, Hui; Athar, Mohammad; Timares, Laura; Elmets, Craig A

    2015-05-15

    Heat shock proteins (HSPs) are constitutively expressed in murine skin. HSP27 is present in the epidermis, and HSP70 can be found in both the epidermis and dermis. The purpose of this study was to investigate the role of these proteins in cutaneous chemical carcinogenesis and to determine whether their effects on cell-mediated immune function were a contributing factor. In vivo inhibition of HSP27 and HSP70 produced a reduction in the T cell-mediated immune response to 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene in C3H/HeN mice and resulted in a state of Ag-specific tolerance. When mice were pretreated with anti-HSP27 and anti-HSP70 Abs in vivo prior to subjecting them to a standard two-stage DMBA/12-O-tetradecanoylphorbol-13-acetate cutaneous carcinogenesis protocol, the percentage of mice with tumors was much greater (p < 0.05) in anti-HSP27- and HSP70-pretreated animals compared with mice pretreated with control Ab. Similar results were obtained when the data were evaluated as the cumulative number of tumors per group. Mice pretreated with HSP27 and HSP70 Abs developed more H-ras mutations and fewer DMBA-specific cytotoxic T lymphocytes. These findings indicate that in mice HSP27 and HSP70 play a key role in the induction of cell-mediated immunity to carcinogenic polyaromatic hydrocarbons. Bolstering the immune response to carcinogenic polyaromatic hydrocarbons may be an effective method for prevention of the tumors that they produce. Copyright © 2015 by The American Association of Immunologists, Inc.

  6. Arabidopsis HIPP27 is a host susceptibility gene for the beet cyst nematode Heterodera schachtii.

    PubMed

    Radakovic, Zoran S; Anjam, Muhammad Shahzad; Escobar, Elizabeth; Chopra, Divykriti; Cabrera, Javier; Silva, Ana Cláudia; Escobar, Carolina; Sobczak, Miroslaw; Grundler, Florian M W; Siddique, Shahid

    2018-02-22

    Sedentary plant-parasitic cyst nematodes are obligate biotrophs that infect the roots of their host plant. Their parasitism is based on the modification of root cells to form a hypermetabolic syncytium from which the nematodes draw their nutrients. The aim of this study was to identify nematode susceptibility genes in Arabidopsis thaliana and to characterize their roles in supporting the parasitism of Heterodera schachtii. By selecting genes that were most strongly upregulated in response to cyst nematode infection, we identified HIPP27 (HEAVY METAL-ASSOCIATED ISOPRENYLATED PLANT PROTEIN 27) as a host susceptibility factor required for beet cyst nematode infection and development. Detailed expression analysis revealed that HIPP27 is a cytoplasmic protein and that HIPP27 is strongly expressed in leaves, young roots and nematode-induced syncytia. Loss-of-function Arabidopsis hipp27 mutants exhibited severely reduced susceptibility to H. schachtii and abnormal starch accumulation in syncytial and peridermal plastids. Our results suggest that HIPP27 is a susceptibility gene in Arabidopsis whose loss of function reduces plant susceptibility to cyst nematode infection without increasing the susceptibility to other pathogens or negatively affecting the plant phenotype. © 2018 UNIVERSITY BONN. MOLECULAR PLANT PATHOLOGY © 2018 BSPP AND JOHN WILEY & SONS LTD.

  7. Overexpression of heat shock protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through S-phase arrest and apoptosis.

    PubMed

    Guo, Yang; Ziesch, Andreas; Hocke, Sandra; Kampmann, Eric; Ochs, Stephanie; De Toni, Enrico N; Göke, Burkhard; Gallmeier, Eike

    2015-02-01

    We previously established a role for HSP27 as a predictive marker for therapeutic response towards gemcitabine in pancreatic cancer. Here, we investigate the underlying mechanisms of HSP27-mediated gemcitabine sensitivity. Utilizing a pancreatic cancer cell model with stable HSP27 overexpression, cell cycle arrest and apoptosis induction were analysed by flow cytometry, nuclear staining, immunoblotting and mitochondrial staining. Drug sensitivity studies were performed by proliferation assays. Hyperthermia was simulated using mild heat shock at 41.8°C. Upon gemcitabine treatment, HSP27-overexpressing cells displayed an early S-phase arrest subsequently followed by a strongly increased sub-G1 fraction. Apoptosis was characterized by PARP-, CASPASE 3-, CASPASE 8-, CASPASE 9- and BIM- activation along with a mitochondrial membrane potential loss. It was reversible through chemical caspase inhibition. Importantly, gemcitabine sensitivity and PARP cleavage were also elicited by heat shock-induced HSP27 overexpression, although to a smaller extent, in a panel of pancreatic cancer cell lines. Finally, HSP27-overexpressing pancreatic cancer cells displayed an increased sensitivity also towards death receptor-targeting agents, suggesting another pro-apoptotic role of HSP27 along the extrinsic apoptosis pathway. Taken together, in contrast to the well-established anti-apoptotic properties of HSP27 in cancer, our study reveals novel pro-apoptotic functions of HSP27-mediated through both the intrinsic and the extrinsic apoptotic pathways-at least in pancreatic cancer cells. HSP27 could represent a predictive marker of therapeutic response towards specific drug classes in pancreatic cancer and provides a novel molecular rationale for current clinical trials applying the combination of gemcitabine with regional hyperthermia in pancreatic cancer patients. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for

  8. miR-27a controls triacylglycerol synthesis in bovine mammary epithelial cells by targeting peroxisome proliferator-activated receptor gamma.

    PubMed

    Tang, K Q; Wang, Y N; Zan, L S; Yang, W C

    2017-05-01

    Growing evidence has revealed that microRNA are central elements in milk fat synthesis in mammary epithelial cells. A negative regulator of adipocyte fat synthesis, miR-27a has been reported to be involved in the regulation of milk fat synthesis in goat mammary epithelial cells; however, the regulatory role of miR-27a in bovine milk fat synthesis remains unclear. In the present study, primary bovine mammary epithelial cells (BMEC) were harvested from mid-lactation cows and cultured in Dulbecco's modified Eagle's medium/F-12 medium with 10% fetal bovine serum, 5 μg/mL of insulin, 1 μg/mL of hydrocortisone, 2 μg/mL of prolactin, 1 μg/mL of progesterone, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. We found that the overexpression of miR-27a significantly suppressed lipid droplet formation and decreased the cellular triacylglycerol (TAG) levels, whereas inhibition of miR-27a resulted in a greater lipid droplet formation and TAG accumulation in BMEC. Meanwhile, overexpression of miR-27a inhibited mRNA expression of peroxisome proliferator-activated receptor gamma (PPARG), CCAAT/enhancer-binding protein beta (C/EBPβ), perilipin 2 (PLIN2), and fatty acid binding protein 3 (FABP3), whereas miR-27a downregulation increased PPARG, C/EBPβ, FABP3, and CCAAT enhancer binding protein alpha (C/EBPα) mRNA expression. Furthermore, Western blot analysis revealed the protein level of PPARG in miR-27a mimic and inhibitor transfection groups to be consistent with the mRNA expression response. Moreover, luciferase reporter assays verified that PPARG was the direct target of miR-27a. In summary, these results indicate that miR-27a has the ability to control TAG synthesis in BMEC via targeting PPARG, suggesting that miR-27a could potentially be used to improve beneficial milk components in dairy cows. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  9. Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori.

    PubMed

    Jeong, Kyoung Yong; Son, Mina; Lee, June Yong; Park, Kyung Hee; Lee, Jae-Hyun; Park, Jung-Won

    2016-01-01

    Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm.

  10. Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori

    PubMed Central

    Son, Mina; Lee, June Yong

    2016-01-01

    Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm. PMID:26770033

  11. Interaction of prostanoid EP3 and TP receptors in guinea-pig isolated aorta: contractile self-synergism of 11-deoxy-16,16-dimethyl PGE2

    PubMed Central

    Jones, RL; Woodward, DF

    2011-01-01

    BACKGROUND AND PURPOSE Surprisingly high contractile activity was reported for 11-deoxy-16,16-dimethyl prostaglandin E2 (DX-DM PGE2) on pig cerebral artery when used as a selective EP3 receptor agonist. This study investigated the selectivity profile of DX-DM PGE2, focusing on the interaction between its EP3 and TP (thromboxane A2-like) agonist activities. EXPERIMENTAL APPROACH Contraction of guinea-pig trachea (EP1 system) and aorta (EP3 and TP systems) was measured in conventional organ baths. KEY RESULTS Strong contraction of guinea-pig aorta to sulprostone and 17-phenyl PGE2 (EP3 agonists) was only seen under priming with a second contractile agent such as phenylephrine, histamine or U-46619 (TP agonist). In contrast, DX-DM PGE2 induced strong contraction, which on the basis of treatment with (DG)-3ap (EP3 antagonist) and/or BMS-180291 (TP antagonist) was attributed to self-synergism arising from co-activation of EP3 and TP receptors. EP3/TP self-synergism also accounted for contraction induced by PGF2α and its analogues (+)-cloprostenol and latanoprost-FA. DX-DM PGE2 also showed significant EP1 agonism on guinea-pig trachea as defined by the EP1 antagonists SC-51322, (ONO)-5-methyl-1 and AH-6809, although AH-6809 exhibited poor specificity at concentrations ≥3 µM. CONCLUSIONS AND IMPLICATIONS EP3/TP self-synergism, as seen with PGE/PGF analogues in this study, may confound EP3 agonist potency comparisons and the characterization of prostanoid receptor systems. The competitive profile of a TP antagonist may be distorted by variation in the silent/overt contraction profile of the EP3 system in different studies. The relevance of self-synergism to in vivo actions of natural prostanoid receptor agonists is discussed. PMID:20955363

  12. PSB27: A thylakoid protein enabling Arabidopsis to adapt to changing light intensity

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

    Hou, Xin; Garcia, Veder J.; Buchanan, Bob B.

    with a larger complex that might be PSII supercomplex. Under objective 2, we have found that FKBP16-2 interacted with PSB27 that was further pursuited and published a research article in PNAS (attached). Under Objective 3, we have identified several mutants of other FKBPs in the thyalkoid lumen that should be further studied if future funding is available. Under Objective 4, we have started to build a network of lumenal proteins that play a number of roles in photosynthesis. For example, the CYP37 and CYP28 are linked to chloroplast signaling to nucleus, critical for controlling plant response to high light and adaptation to climate change. Unfortunately these studies have been terminated due to funding shortage.« less

  13. Antinociceptive synergism of MD-354 and clonidine. Part II. The alpha-adrenoceptor component.

    PubMed

    Young, Shawquia; Vainio, Minna; Scheinin, Mika; Dukat, Małgorzata

    2010-08-01

    Previously, we reported that antinociceptive synergism of a 5-HT(3)/alpha(2)-adrenoceptor ligand MD-354 (m-chlorophenylguanidine) and clonidine combination occurs, in part, through a 5-HT(3) receptor antagonist mechanism. In the present investigation, a possible role for alpha(2)-adrenoceptors was examined. Mechanistic studies using yohimbine (a subtype non-selective alpha(2)-adrenoceptor antagonist), BRL 44408 (a preferential alpha(2A)-adrenoceptor antagonist) and imiloxan (a preferential alpha(2B/C)-adrenoceptor antagonist) on the antinociceptive actions of a MD-354/clonidine combination were conducted. Subcutaneous pre-treatment with all three antagonists inhibited the antinociceptive synergism of MD-354 and clonidine in the mouse tail-flick assay in a dose-dependent manner (AD(50) = 0.33, 2.1, and 0.17 mg/kg, respectively). Enhancement of clonidine antinociception by MD-354 did not potentiate clonidine's locomotor suppressant activity in a mouse locomotor assay. When [ethyl-3H]RS-79948-197 was used as radioligand, MD-354 displayed almost equal affinity to alpha(2A)- and alpha(2B)-adrenoceptors (K(i) = 110 and 220 nM) and showed lower affinity at alpha(2C)-adrenoceptors (K(i) = 4,700 nM). MD-354 had no subtype-selectivity for the alpha(2)-adrenoceptor subtypes as an antagonist in functional [35S]GTPgammaS binding assays. MD-354 was a weak partial agonist at alpha(2A)-adrenoceptors. Overall, in addition to the 5-HT(3) receptor component, the present investigation found MD-354 to be a weak partial alpha(2A)-adrenoceptor agonist that enhances clonidine's thermal antinociceptive actions through an alpha(2)-adrenoceptor-mediated mechanism without augmenting sedation.

  14. Downregulation of Hsp27 (HSPB1) in MCF-7 human breast cancer cells induces upregulation of PTEN.

    PubMed

    Cayado-Gutiérrez, Niubys; Moncalero, Vera L; Rosales, Eliana M; Berón, Walter; Salvatierra, Edgardo E; Alvarez-Olmedo, Daiana; Radrizzani, Martín; Ciocca, Daniel R

    2013-03-01

    Hsp27 (HSPB1) is usually overexpressed in breast cancers affecting the disease outcome and the sensitivity of tumors to chemotherapy and radiotherapy. Hsp27 interacts with other proteins such as β-catenin, histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3. Phosphatase and tensin homologue (PTEN) is a tumor suppressor gene that is deleted in many human tumors. The PI3K/Akt signaling pathway is negatively regulated by PTEN. Hsp27 is described as a key component of the Akt signaling cascade: Akt, BAD, Forkhead transcription factors, Hsp27, mitogen-activated protein kinase kinase-3 and -6. Here, we have examined whether the downregulation of Hsp27 by siHsp27 affects the PTEN levels in the MCF-7 human breast cancer cell line. PTEN was detected with two different antibodies using western blots and immunocytochemistry. p-Akt was also evaluated by western blot. In addition, Hsp27 and PTEN were immunoprecipitated to know whether these proteins interact. Intracellular colocalization studies were carried out by confocal microscopy. A significant reduction in the Hsp27 levels was noted in the siHsp27 transfected cells. These Hsp27 downregulated cells showed a significant increased expression of PTEN. The MW 76 and 55 kDa PTEN forms were upregulated as revealed by two different antibodies. The phosphatase activity of PTEN seems to be active because p-Akt levels were reduced. Hsp27 immunoprecipitation was bringing PTEN and vice versa, these two proteins seem to interact at cytoplasmic level by FRET. Downregulation of Hsp27 stabilized PTEN protein levels. Chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP) levels were not significantly influenced by Hsp27 downregulation. In conclusion, we report a novel function of Hsp27 modulating the PTEN levels in human breast cancer cells suggesting an interaction between these two molecules.

  15. Synergisms between microbial pathogens in plant disease complexes: a growing trend

    PubMed Central

    Lamichhane, Jay Ram; Venturi, Vittorio

    2015-01-01

    Plant diseases are often thought to be caused by one species or even by a specific strain. Microbes in nature, however, mostly occur as part of complex communities and this has been noted since the time of van Leeuwenhoek. Interestingly, most laboratory studies focus on single microbial strains grown in pure culture; we were therefore unaware of possible interspecies and/or inter-kingdom interactions of pathogenic microbes in the wild. In human and animal infections, it is now being recognized that many diseases are the result of multispecies synergistic interactions. This increases the complexity of the disease and has to be taken into consideration in the development of more effective control measures. On the other hand, there are only a few reports of synergistic pathogen–pathogen interactions in plant diseases and the mechanisms of interactions are currently unknown. Here we review some of these reports of synergism between different plant pathogens and their possible implications in crop health. Finally, we briefly highlight the recent technological advances in diagnostics as these are beginning to provide important insights into the microbial communities associated with complex plant diseases. These examples of synergistic interactions of plant pathogens that lead to disease complexes might prove to be more common than expected and understanding the underlying mechanisms might have important implications in plant disease epidemiology and management. PMID:26074945

  16. Synergism between arsenite and proteasome inhibitor MG132 over cell death in myeloid leukaemic cells U937 and the induction of low levels of intracellular superoxide anion

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

    Lombardo, Tomás; Cavaliere, Victoria; Costantino, Susana N.

    Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitt's lymphoma-derived Raji cells, by the Chou–Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O{submore » 2}{sup −}) levels. Our results showed that combined arsenite + MG132 produced low levels of O{sub 2}{sup −} at 6 h and 24 h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80 μM hydrogen peroxide together with arsenite + MG132 changed synergism on cell death to antagonism at all effect levels while increasing O{sub 2}{sup −} levels. Arsenite + hydrogen peroxide also resulted in antagonism with increased O{sub 2}{sup −} levels in U937 cells. In Raji cells, arsenite + MG132 also produced low levels of O{sub 2}{sup −} at 6 h and 24 h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite + CAPE showed high levels of O{sub 2}{sup −} production at 6 h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O{sub 2}{sup −} levels at early time points after exposure. -- Highlights: ► Arsenic combined cytotoxic and anti-proliferative effects by Chou–Talalay method. ► Cytotoxic effect

  17. Probing the Role of Nascent Helicity in p27 Function as a Cell Cycle Regulator

    PubMed Central

    Otieno, Steve; Kriwacki, Richard

    2012-01-01

    p27 regulates the activity of Cdk complexes which are the principal governors of phase transitions during cell division. Members of the p27 family of proteins, which also includes p21 and p57, are called the Cip/Kip cyclin-dependent kinase regulators (CKRs). Interestingly, the Cip/Kip CKRs play critical roles in cell cycle regulation by being intrinsically unstructured, a characteristic contrary to the classical structure-function paradigm. They exhibit nascent helicity which has been localized to a segment referred to as sub-domain LH. The nascent helicity of this sub-domain is conserved and we hypothesize that it is an important determinant of their functional properties. To test this hypothesis, we successfully designed and prepared p27 variants in which domain LH was either more or less helical with respect to the wild-type protein. Thermal denaturation experiments showed that the ternary complexes of the p27 variants bound to Cdk2/Cyclin A were less stable compared to the wild-type complex. Isothermal titration calorimetry experiments showed a decrease in the enthalpy of binding for all the mutants with respect to p27. The free energies of binding varied within a much narrower range. In vitro Cdk2 inhibition assays showed that the p27 variants exhibited disparate inhibitory potencies. Furthermore, when over-expressed in NIH 3T3 mouse fibroblast cells, the less helical p27 variants were less effective in causing cell cycle arrest relative to the wild-type p27. Our results indicate that the nascent helicity of sub-domain LH plays a key role mediating the biological function of p27. PMID:23071750

  18. New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment?

    PubMed

    Coqueret, Olivier

    2003-02-01

    Cell division relies on the activation of cyclins, which bind to cyclin-dependent kinases (CDKs) to induce cell-cycle progression towards S phase and later to initiate mitosis. Since uncontrolled cyclin-dependent kinase activity is often the cause of human cancer, their function is tightly regulated by cell-cycle inhibitors such as the p21 and p27 Cip/Kip proteins. Following anti-mitogenic signals or DNA damage, p21 and p27 bind to cyclin-CDK complexes to inhibit their catalytic activity and induce cell-cycle arrest. Interestingly, recent discoveries suggest that p21 and p27 might have new activities that are unrelated to their function as CDK inhibitors. The identification of new targets of Cip/Kip proteins as well as evidence of Cip/Kip cytoplasmic relocalization have revealed unexpected functions for these proteins in the control of CDK activation, in the regulation of apoptosis and in transcriptional activation. This article discusses recent insights into these possible additional functions of p21 and p27.

  19. Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species

    PubMed Central

    2012-01-01

    Background Streptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. Replication loci of several Streptomyces theta-type plasmids have been reported, but are not characterized in details. Conjugation loci of some Streptomyces rolling-circle-type plasmids are identified and mechanism of conjugal transferring are described. Results We report the detection of a widely distributed Streptomyces strain Y27 and its indigenous plasmid pWTY27 from fourteen plants and four soil samples cross China by both culturing and nonculturing methods. The complete nucleotide sequence of pWTY27 consisted of 14,288 bp. A basic locus for plasmid replication comprised repAB genes and an adjacent iteron sequence, to a long inverted-repeat (ca. 105 bp) of which the RepA protein bound specifically in vitro, suggesting that RepA may recognize a second structure (e.g. a long stem-loop) of the iteron DNA. A plasmid containing the locus propagated in linear mode when the telomeres of a linear plasmid were attached, indicating a bi-directional replication mode for pWTY27. As for rolling-circle plasmids, a single traA gene and a clt sequence (covering 16 bp within traA and its adjacent 159 bp) on pWTY27 were required for plasmid transfer. TraA recognized and bound specifically to the two regions of the clt sequence, one containing all the four DC1 of 7 bp (TGACACC) and one DC2 (CCCGCCC) and most of IC1, and another covering two DC2 and part of IC1, suggesting formation of a high-ordered DNA-protein complex. Conclusions This work (i) isolates a widespread Streptomyces strain Y27 and sequences its indigenous theta-type plasmid pWTY27; (ii) identifies the replication and conjugation loci of pWTY27 and; (iii) characterizes the binding sequences of the RepA and TraA proteins. PMID:23134842

  20. Chaperone Hsp27 Modulates AUF1 Proteolysis and AU-Rich Element-Mediated mRNA Degradation▿

    PubMed Central

    Knapinska, Anna M.; Gratacós, Frances M.; Krause, Christopher D.; Hernandez, Kristina; Jensen, Amber G.; Bradley, Jacquelyn J.; Wu, Xiangyue; Pestka, Sidney; Brewer, Gary

    2011-01-01

    AUF1 is an AU-rich element (ARE)-binding protein that recruits translation initiation factors, molecular chaperones, and mRNA degradation enzymes to the ARE for mRNA destruction. We recently found chaperone Hsp27 to be an AUF1-associated ARE-binding protein required for tumor necrosis factor alpha (TNF-α) mRNA degradation in monocytes. Hsp27 is a multifunctional protein that participates in ubiquitination of proteins for their degradation by proteasomes. A variety of extracellular stimuli promote Hsp27 phosphorylation on three serine residues—Ser15, Ser78, and Ser82—by a number of kinases, including the mitogen-activated protein (MAP) pathway kinases p38 and MK2. Activating either kinase stabilizes ARE mRNAs. Likewise, ectopic expression of phosphomimetic mutant forms of Hsp27 stabilizes reporter ARE mRNAs. Here, we continued to examine the contributions of Hsp27 to mRNA degradation. As AUF1 is ubiquitinated and degraded by proteasomes, we addressed the hypothesis that Hsp27 phosphorylation controls AUF1 levels to modulate ARE mRNA degradation. Indeed, selected phosphomimetic mutants of Hsp27 promote proteolysis of AUF1 in a proteasome-dependent fashion and render ARE mRNAs more stable. Our results suggest that the p38 MAP kinase (MAPK)-MK2–Hsp27 signaling axis may target AUF1 destruction by proteasomes, thereby promoting ARE mRNA stabilization. PMID:21245386

  1. In Vitro Synergism between Azithromycin or Terbinafine and Topical Antimicrobial Agents against Pythium insidiosum

    PubMed Central

    Itaqui, Sabrina R.; Verdi, Camila M.; Tondolo, Juliana S. M.; da Luz, Thaisa S.; Alves, Sydney H.; Santurio, Janio M.

    2016-01-01

    We describe here in vitro activity for the combination of azithromycin or terbinafine and benzalkonium, cetrimide, cetylpyridinium, mupirocin, triclosan, or potassium permanganate. With the exception of potassium permanganate, the remaining antimicrobial drugs were active and had an MIC90 between 2 and 32 μg∕ml. The greatest synergism was observed for the combination of terbinafine and cetrimide (71.4%). In vivo experimental evaluations will clarify the potential of these drugs for the topical treatment of lesions caused by Pythium insidiosum. PMID:27216049

  2. The prohibitin-binding compound fluorizoline induces apoptosis in chronic lymphocytic leukemia cells through the upregulation of NOXA and synergizes with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax.

    PubMed

    Cosialls, Ana M; Pomares, Helena; Iglesias-Serret, Daniel; Saura-Esteller, José; Núñez-Vázquez, Sonia; González-Gironès, Diana M; de la Banda, Esmeralda; Preciado, Sara; Albericio, Fernando; Lavilla, Rodolfo; Pons, Gabriel; González-Barca, Eva M; Gil, Joan

    2017-09-01

    Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins. In the study herein, the pro-apoptotic effect of fluorizoline was assessed in 34 primary samples from patients with chronic lymphocytic leukemia. Fluorizoline induced apoptosis in chronic lymphocytic leukemia cells at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline irrespective of patients' clinical or genetic features, whereas normal T lymphocytes were less sensitive. Fluorizoline increased the protein levels of the pro-apoptotic B-cell lymphoma 2 family member NOXA in chronic lymphocytic leukemia cells. Furthermore, fluorizoline synergized with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax to induce apoptosis. These results suggest that targeting prohibitins could be a new therapeutic strategy for chronic lymphocytic leukemia. Copyright© 2017 Ferrata Storti Foundation.

  3. Increased maternal and fetal cholesterol efflux capacity and placental CYP27A1 expression in preeclampsia.

    PubMed

    Mistry, Hiten D; Kurlak, Lesia O; Mansour, Yosef T; Zurkinden, Line; Mohaupt, Markus G; Escher, Geneviève

    2017-06-01

    Preeclampsia is a pregnancy-specific condition that leads to increased cardiovascular risk in later life. A decrease in cholesterol efflux capacity is linked to CVD. We hypothesized that in preeclampsia there would be a disruption of maternal/fetal plasma to efflux cholesterol, as well as differences in the concentrations of both placental sterol 27-hydroxylase (CYP27A1) and apoA1 binding protein (AIBP). Total, HDL-, and ABCA1-mediated cholesterol effluxes were performed with maternal and fetal plasma from women with preeclampsia and normotensive controls (both n = 17). apoA1 and apoE were quantified by chemiluminescence, and 27-hydroxycholesterol (27-OHC) by GC-MS. Immunohistochemistry was used to determine placental expression/localization of CYP27A1, AIBP, apoA1, apoE, and SRB1. Maternal and fetal total and HDL-mediated cholesterol efflux capacities were increased in preeclampsia (by 10-20%), but ABCA1-mediated efflux was decreased (by 20-35%; P < 0.05). Maternal and fetal apoE concentrations were higher in preeclampsia. Fetal plasma 27-OHC levels were decreased in preeclamptic samples ( P < 0.05). Placental protein expression of both CYP27A1 and AIBP were localized around fetal vessels and significantly increased in preeclampsia ( P = 0.04). Placental 27-OHC concentrations were also raised in preeclampsia ( P < 0.05). Increased HDL-mediated cholesterol efflux capacity and placental CYP27A1/27-OHC could be a rescue mechanism in preeclampsia, to remove cholesterol from cells to limit lipid peroxidation and increase placental angiogenesis. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  4. Intraflagellar transporter protein (IFT27), an IFT25 binding partner, is essential for male fertility and spermiogenesis in mice.

    PubMed

    Zhang, Yong; Liu, Hong; Li, Wei; Zhang, Zhengang; Shang, Xuejun; Zhang, David; Li, Yuhong; Zhang, Shiyang; Liu, Junpin; Hess, Rex A; Pazour, Gregory J; Zhang, Zhibing

    2017-12-01

    Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27: Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survived to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymides contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the "9+2″ axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the

  5. Intraflagellar Transporter Protein (IFT27), an IFT25 binding partner, Is Essential For Male Fertility and Spermiogenesis In Mice

    PubMed Central

    Zhang, Yong; Liu, Hong; Li, Wei; Zhang, Zhengang; Shang, Xuejun; Zhang, David; Li, Yuhong; Zhang, Shiyang; Liu, Junpin; Hess, Rex A; Pazour, Gregory J; Zhang, Zhibing

    2017-01-01

    Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27:Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survive to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymis contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the “9+2” axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the conditional

  6. Overexpression of mutant HSP27 causes axonal neuropathy in mice.

    PubMed

    Lee, Jinho; Jung, Sung-Chul; Joo, Jaesoon; Choi, Yu-Ri; Moon, Hyo Won; Kwak, Geon; Yeo, Ha Kyung; Lee, Ji-Su; Ahn, Hye-Jee; Jung, Namhee; Hwang, Sunhee; Rheey, Jingeun; Woo, So-Youn; Kim, Ji Yon; Hong, Young Bin; Choi, Byung-Ok

    2015-06-19

    Mutations in heat shock 27 kDa protein 1 (HSP27 or HSPB1) cause distal hereditary motor neuropathy (dHMN) or Charcot-Marie-Tooth disease type 2 F (CMT2F) according to unknown factors. Mutant HSP27 proteins affect axonal transport by reducing acetylated tubulin. We generated a transgenic mouse model overexpressing HSP27-S135F mutant protein driven by Cytomegalovirus (CMV) immediate early promoter. The mouse phenotype was similar to dHMN patients in that they exhibit motor neuropathy. To determine the phenotypic aberration of transgenic mice, behavior test, magnetic resonance imaging (MRI), electrophysiological study, and pathology were performed. Rotarod test showed that founder mice exhibited lowered motor performance. MRI also revealed marked fatty infiltration in the anterior and posterior compartments at calf level. Electrophysiologically, compound muscle action potential (CMAP) but not motor nerve conduction velocity (MNCV) was reduced in the transgenic mice. Toluidine staining with semi-thin section of sciatic nerve showed the ratio of large myelinated axon fiber was reduced, which might cause reduced locomotion in the transgenic mice. Electron microscopy also revealed abundant aberrant myelination. Immunohistochemically, neuronal dysfunctions included elevated level of phosphorylated neurofilament and reduced level of acetylated tubulin in the sural nerve of transgenic mice. There was no additional phenotype besides motor neuronal defects. Overexpression of HSP27-S135F protein causes peripheral neuropathy. The mouse model can be applied to future development of therapeutic strategies for dHMN or CMT2F.

  7. PRAK, a novel protein kinase regulated by the p38 MAP kinase.

    PubMed Central

    New, L; Jiang, Y; Zhao, M; Liu, K; Zhu, W; Flood, L J; Kato, Y; Parry, G C; Han, J

    1998-01-01

    We have identified and cloned a novel serine/ threonine kinase, p38-regulated/activated protein kinase (PRAK). PRAK is a 471 amino acid protein with 20-30% sequence identity to the known MAP kinase-regulated protein kinases RSK1/2/3, MNK1/2 and MAPKAP-K2/3. PRAK was found to be expressed in all human tissues and cell lines examined. In HeLa cells, PRAK was activated in response to cellular stress and proinflammatory cytokines. PRAK activity was regulated by p38alpha and p38beta both in vitro and in vivo and Thr182 was shown to be the regulatory phosphorylation site. Activated PRAK in turn phosphorylated small heat shock protein 27 (HSP27) at the physiologically relevant sites. An in-gel kinase assay demonstrated that PRAK is a major stress-activated kinase that can phosphorylate small heat shock protein, suggesting a potential role for PRAK in mediating stress-induced HSP27 phosphorylation in vivo. PMID:9628874

  8. Loss of function mutations in RPL27 and RPS27 identified by whole-exome sequencing in Diamond-Blackfan anaemia.

    PubMed

    Wang, RuNan; Yoshida, Kenichi; Toki, Tsutomu; Sawada, Takafumi; Uechi, Tamayo; Okuno, Yusuke; Sato-Otsubo, Aiko; Kudo, Kazuko; Kamimaki, Isamu; Kanezaki, Rika; Shiraishi, Yuichi; Chiba, Kenichi; Tanaka, Hiroko; Terui, Kiminori; Sato, Tomohiko; Iribe, Yuji; Ohga, Shouichi; Kuramitsu, Madoka; Hamaguchi, Isao; Ohara, Akira; Hara, Junichi; Goi, Kumiko; Matsubara, Kousaku; Koike, Kenichi; Ishiguro, Akira; Okamoto, Yasuhiro; Watanabe, Kenichiro; Kanno, Hitoshi; Kojima, Seiji; Miyano, Satoru; Kenmochi, Naoya; Ogawa, Seishi; Ito, Etsuro

    2015-03-01

    Diamond-Blackfan anaemia is a congenital bone marrow failure syndrome that is characterized by red blood cell aplasia. The disease has been associated with mutations or large deletions in 11 ribosomal protein genes including RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPS29, RPL5, RPL11, RPL26 and RPL35A as well as GATA1 in more than 50% of patients. However, the molecular aetiology of many Diamond-Blackfan anaemia cases remains to be uncovered. To identify new mutations responsible for Diamond-Blackfan anaemia, we performed whole-exome sequencing analysis of 48 patients with no documented mutations/deletions involving known Diamond-Blackfan anaemia genes except for RPS7, RPL26, RPS29 and GATA1. Here, we identified a de novo splicing error mutation in RPL27 and frameshift deletion in RPS27 in sporadic patients with Diamond-Blackfan anaemia. In vitro knockdown of gene expression disturbed pre-ribosomal RNA processing. Zebrafish models of rpl27 and rps27 mutations showed impairments of erythrocyte production and tail and/or brain development. Additional novel mutations were found in eight patients, including RPL3L, RPL6, RPL7L1T, RPL8, RPL13, RPL14, RPL18A and RPL31. In conclusion, we identified novel germline mutations of two ribosomal protein genes responsible for Diamond-Blackfan anaemia, further confirming the concept that mutations in ribosomal protein genes lead to Diamond-Blackfan anaemia. © 2014 John Wiley & Sons Ltd.

  9. Antibacterial activity of epigallocatechin-3-gallate (EGCG) and its synergism with β-lactam antibiotics sensitizing carbapenem-associated multidrug resistant clinical isolates of Acinetobacter baumannii.

    PubMed

    Lee, Spencer; Razqan, Ghaida Saleh Al; Kwon, Dong H

    2017-01-15

    Infections caused by Acinetobacter baumannii were responsive to conventional antibiotic therapy. However, recently, carbapenem-associated multidrug resistant isolates have been reported worldwide and present a major therapeutic challenge. Epigallocatechin-3-Gallate (EGCG) extracted from green tea exhibits antibacterial activity. We evaluated the antibacterial activity of EGCG and possible synergism with antibiotics in carbapenem-associated multidrug resistant A. baumannii. A potential mechanism for synergism was also explored. Seventy clinical isolates of A. baumannii collected from geographically different areas were analyzed by minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EGCG. Checkerboard and time-killing assays were performed to exam the synergism between EGCG and antibiotics. The effects of EGCG on a multidrug efflux pump inhibitor (1-[1-naphthylmethyl] piperazine; NMP) and β-lactamase production were also examined in A. baumannii. Sixty-three of 70 clinical isolates of A. baumannii carried carbapenemase-encoding genes with carbapenem-associated multidrug resistance. Levels of MIC and MBC of EGCG ranged from 64 to 512µg/ml and from 128 to ≥1024µg/ml, respectively among the clinical isolates. MIC 90 and MBC 86 levels were 256µg/ml and 512µg/ml of EGCG, respectively. Subinhibitory concentration of EGCG in combination with all antibiotics tested, including carbapenem, sensitized (MICs fall≤1.0µg/ml) all carbapenem-associated multidrug resistant isolates. Checkerboard and time-killing assays showed synergism between EGCG and meropenem (or carbenicillin) counted as fractional inhibitory concentration of < 0.5 and cell numbers' decrease per ml of >2log10 within 12h, respectively. EGCG significantly increased the effect of NMP but was unrelated to β-lactamase production in A. baumannii, suggesting EGCG may be associated with inhibition of efflux pumps. Overall we suggest that EGCG-antibiotic combinations might

  10. Small molecule inhibitors of ERCC1-XPF protein-protein interaction synergize alkylating agents in cancer cells.

    PubMed

    Jordheim, Lars Petter; Barakat, Khaled H; Heinrich-Balard, Laurence; Matera, Eva-Laure; Cros-Perrial, Emeline; Bouledrak, Karima; El Sabeh, Rana; Perez-Pineiro, Rolando; Wishart, David S; Cohen, Richard; Tuszynski, Jack; Dumontet, Charles

    2013-07-01

    The benefit of cancer chemotherapy based on alkylating agents is limited because of the action of DNA repair enzymes, which mitigate the damage induced by these agents. The interaction between the proteins ERCC1 and XPF involves two major components of the nucleotide excision repair pathway. Here, novel inhibitors of this interaction were identified by virtual screening based on available structures with use of the National Cancer Institute diversity set and a panel of DrugBank small molecules. Subsequently, experimental validation of the in silico screening was undertaken. Top hits were evaluated on A549 and HCT116 cancer cells. In particular, the compound labeled NSC 130813 [4-[(6-chloro-2-methoxy-9-acridinyl)amino]-2-[(4-methyl-1-piperazinyl)methyl

  11. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase

    PubMed Central

    Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. PMID:26867010

  12. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

    PubMed

    Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.

  13. A newly identified protein of Leptospira interrogans mediates binding to laminin.

    PubMed

    Longhi, Mariana T; Oliveira, Tatiane R; Romero, Eliete C; Gonçales, Amane P; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2009-10-01

    Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. The search for novel antigens that could be relevant in host-pathogen interactions is being pursued. These antigens have the potential to elicit several activities, including adhesion. This study focused on a hypothetical predicted lipoprotein of Leptospira, encoded by the gene LIC12895, thought to mediate attachment to extracellular matrix (ECM) components. The gene was cloned and expressed in Escherichia coli BL21 Star (DE3)pLys by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. The capacity of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC12895, named Lsa27 (leptospiral surface adhesin, 27 kDa), bound strongly to laminin in a dose-dependent and saturable fashion. Moreover, Lsa27 was recognized by antibodies from serum samples of confirmed leptospirosis specimens in both the initial and the convalescent phases of the disease. Lsa27 is most likely a surface protein of Leptospira as revealed in liquid-phase immunofluorescence assays with living organisms. Taken together, these data indicate that this newly identified membrane protein is expressed during natural infection and may play a role in mediating adhesion of L. interrogans to its host.

  14. LMO1 Synergizes with MYCN to Promote Neuroblastoma Initiation and Metastasis.

    PubMed

    Zhu, Shizhen; Zhang, Xiaoling; Weichert-Leahey, Nina; Dong, Zhiwei; Zhang, Cheng; Lopez, Gonzalo; Tao, Ting; He, Shuning; Wood, Andrew C; Oldridge, Derek; Ung, Choong Yong; van Ree, Janine H; Khan, Amish; Salazar, Brittany M; Lummertz da Rocha, Edroaldo; Zimmerman, Mark W; Guo, Feng; Cao, Hong; Hou, Xiaonan; Weroha, S John; Perez-Atayde, Antonio R; Neuberg, Donna S; Meves, Alexander; McNiven, Mark A; van Deursen, Jan M; Li, Hu; Maris, John M; Look, A Thomas

    2017-09-11

    A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dβh promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Antitumor activity of a dual cytokine/single-chain antibody fusion protein for simultaneous delivery of GM-CSF and IL-2 to Ep-CAM expressing tumor cells.

    PubMed

    Schanzer, Juergen M; Fichtner, Iduna; Baeuerle, Patrick A; Kufer, Peter

    2006-01-01

    Cytokine targeting to tumor-associated antigens via antibody cytokine fusion proteins has demonstrated potent antitumor activity in numerous animal models and has led to the clinical development of 2 antibody-interleukin-2 (IL-2) fusion proteins. We previously reported on the construction and in vitro properties of a "dual" cytokine fusion protein for simultaneous targeted delivery of human granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-2 to human tumors. The fusion protein is based on a heterodimerized core structure formed by human CH1 and Ckappa domains (heterominibody) with C-terminally fused human cytokines and N-terminally fused single-chain antibody fragments specific for the tumor-associated surface antigen epithelial cell adhesion molecule (Ep-CAM). For testing the antitumor activity in syngeneic mouse xenograft models, we developed "dual cytokine heterominibodies" with murine cytokines (mDCH). mDCH fusion proteins and, as controls, "single cytokine heterominibodies" (SCH) carrying either murine GM-CSF (mGM-CSF) or murine IL-2 (mIL-2) were constructed, of which all retained the specific activities of cytokines and binding to the Ep-CAM antigen on human Ep-CAM transfected mouse colon carcinoma CT26-KSA cells. Over a 5-day treatment course, DCH fusion proteins induced significant inhibition of established pulmonary CT26-KSA metastases in immune-competent Balb/c mice at low daily doses of 1 mug of fusion protein per mouse. However, with the tested dosing schemes, antitumor activity of mDCH was largely independent of cytokine targeting to tumors as demonstrated by a control protein with mutated Ep-CAM binding sites. Single cytokine fusion proteins mSCH-GM-CSF and mSCH-IL-2 showed similar antitumor activity as the dual cytokine fusion protein mDCH, indicating that GM-CSF and IL-2 in one molecule did not significantly synergize in tumor rejection under our experimental conditions. Our results seem to contradict the notion that IL-2 and GM

  16. Synergistic effects of citrulline supplementation and exercise on performance in male rats: evidence for implication of protein and energy metabolisms.

    PubMed

    Goron, Arthur; Lamarche, Frédéric; Cunin, Valérie; Dubouchaud, Hervé; Hourdé, Christophe; Noirez, Philippe; Corne, Christelle; Couturier, Karine; Sève, Michel; Fontaine, Eric; Moinard, Christophe

    2017-04-25

    Background: Exercise and citrulline (CIT) are both regulators of muscle protein metabolism. However, the combination of both has been under-studied yet may have synergistic effects on muscle metabolism and performance. Methods: Three-month-old healthy male rats were randomly assigned to be fed ad libitum for 4 weeks with either a citrulline-enriched diet (1 g·kg -1 ·day -1 ) ( CIT ) or an isonitrogenous standard diet (by addition of nonessential amino acid) ( Ctrl ) and trained (running on treadmill 5 days·week -1 ) ( ex ) or not. Maximal endurance activity and body composition were assessed, and muscle protein metabolism (protein synthesis, proteomic approach) and energy metabolism [energy expenditure, mitochondrial metabolism] were explored. Results: Body composition was affected by exercise but not by CIT supplementation. Endurance training was associated with a higher maximal endurance capacity than sedentary groups ( P <0.001), and running time was 14% higher in the CITex group than the Ctrlex group (139±4 min versus 122±6 min, P <0.05). Both endurance training and CIT supplementation alone increased muscle protein synthesis (by +27% and +33%, respectively, versus Ctrl , P <0.05) with an additive effect (+48% versus Ctrl , P <0.05). Mitochondrial metabolism was modulated by exercise but not directly by CIT supplementation. However, the proteomic approach demonstrated that CIT supplementation was able to affect energy metabolism, probably due to activation of pathways generating acetyl-CoA. Conclusion: CIT supplementation and endurance training in healthy male rats modulates both muscle protein and energy metabolisms, with synergic effects on an array of parameters, including performance and protein synthesis. © 2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  17. Vemurafenib Synergizes with Nutlin-3 to Deplete Survivin and Suppress Melanoma Viability and Tumor Growth

    PubMed Central

    Ji, Zhenyu; Kumar, Raj; Taylor, Michael; Rajadurai, Anpuchchelvi; Marzuka-Alcalá, Alexander; Chen, Y. Erin; Njauw, Ching-Ni Jenny; Flaherty, Keith; Jönsson, Goran; Tsao, Hensin

    2013-01-01

    Background For patients with advanced melanoma, primary and secondary resistance to selective BRAF inhibition remains one of the most critically compelling challenges. One rationale argues that novel biologically-informed strategies are needed to maximally cripple melanoma cells up front before compensatory mechanisms emerge. Since p53 is uncommonly mutated in melanoma, restoration of its function represents an attractive adjunct to selective BRAF inhibition. Experimental Design Thirty-seven BRAF(V600E)-mutated melanoma lines were subjected to synergy studies in vitro using a combination of vemurafenib and nutlin-3 (Nt-3). In addition, cellular responses and in vivo efficacy were also determined. We also analyzed changes in the levels of canonical apoptotic/survival factors in response to vemurafenib. Results Dual targeting of BRAF(V600E) and HDM2 with vemurafenib and Nt-3, respectively, synergistically induced apoptosis and suppressed melanoma viability in vitro and tumor growth in vivo. Suppression of p53 in melanoma cells abrogated Nt-3′s effects fully and vemurafenib’s effects partially. A survey of canonical survival factors revealed that both vemurafenib and Nt-3 independently attenuated levels of the anti-apoptotic protein, survivin. Genetic depletion of survivin reproduces the cytotoxic effects of the combination strategy. Conclusion These results demonstrate preclinical feasibility for overcoming primary vemurafenib resistance by restoring p53 function. Moreover, it identifies survivin as one downstream mediator of the observed synergism and a potential secondary target. PMID:23812671

  18. Doxil Synergizes with Cancer Immunotherapies to Enhance Antitumor Responses in Syngeneic Mouse Models

    PubMed Central

    Rios-Doria, Jonathan; Durham, Nicholas; Wetzel, Leslie; Rothstein, Raymond; Chesebrough, Jon; Holoweckyj, Nicholas; Zhao, Wei; Leow, Ching Ching; Hollingsworth, Robert

    2015-01-01

    Based on the previously described roles of doxorubicin in immunogenic cell death, both doxorubicin and liposomal doxorubicin (Doxil) were evaluated for their ability to boost the antitumor response of different cancer immunotherapies including checkpoint blockers (anti–PD-L1, PD-1, and CTLA-4 mAbs) and TNF receptor agonists (OX40 and GITR ligand fusion proteins) in syngeneic mouse models. In a preventative CT26 mouse tumor model, both doxorubicin and Doxil synergized with anti–PD-1 and CTLA-4 mAbs. Doxil was active when CT26 tumors were grown in immunocompetent mice but not immunocompromised mice, demonstrating that Doxil activity is increased in the presence of a functional immune system. Using established tumors and maximally efficacious doses of Doxil and cancer immunotherapies in either CT26 or MCA205 tumor models, combination groups produced strong synergistic antitumor effects, a larger percentage of complete responders, and increased survival. In vivo pharmacodynamic studies showed that Doxil treatment decreased the percentage of tumor-infiltrating regulatory T cells and, in combination with anti–PD-L1, increased the percentage of tumor-infiltrating CD8+ T cells. In the tumor, Doxil administration increased CD80 expression on mature dendritic cells. CD80 expression was also increased on both monocytic and granulocytic myeloid cells, suggesting that Doxil may induce these tumor-infiltrating cells to elicit a costimulatory phenotype capable of activating an antitumor T-cell response. These results uncover a novel role for Doxil in immunomodulation and support the use of Doxil in combination with checkpoint blockade or TNFR agonists to increase response rates and antitumor activity. PMID:26408258

  19. p27Kip1 regulates alpha-synuclein expression

    PubMed Central

    Gallastegui, Edurne; Domuro, Carla; Serratosa, Joan; Larrieux, Alejandra; Sin, Laura; Martinez, Jonatan; Besson, Arnaud; Morante-Redolat, José Manuel; Orlando, Serena; Aligue, Rosa; Fariñas, Isabel; Pujol, María Jesús; Bachs, Oriol

    2018-01-01

    Alpha-synuclein (α-SYN) is the main component of anomalous protein aggregates (Lewy bodies) that play a crucial role in several neurodegenerative diseases (synucleinopathies) like Parkinson’s disease and multiple system atrophy. However, the mechanisms involved in its transcriptional regulation are poorly understood. We investigated here the role of the cyclin-dependent kinase (Cdk) inhibitor and transcriptional regulator p27Kip1 (p27) in the regulation of α-SYN expression. We observed that selective deletion of p27 by CRISPR/Cas9 technology in neural cells resulted in increased levels of α-SYN. Knock-down of the member of the same family p21Cip1 (p21) also led to increased α-SYN levels, indicating that p27 and p21 collaborate in the repression of α-SYN transcription. We demonstrated that this repression is mediated by the transcription factor E2F4 and the member of the retinoblastoma protein family p130 and that it is dependent of Cdk activity. Chromatin immunoprecipitation analysis revealed specific binding sites for p27, p21 and E2F4 in the proximal α-SYN gene promoter. Finally, luciferase assays revealed a direct action of p27, p21 and E2F4 in α-SYN gene expression. Our findings reveal for the first time a negative regulatory mechanism of α-SYN expression, suggesting a putative role for cell cycle regulators in the etiology of synucleinopathies. PMID:29662651

  20. Growth promotion in pigs by oxytetracycline coincides with down regulation of serum inflammatory parameters and of hibernation-associated protein HP-27.

    PubMed

    Soler, Laura; Miller, Ingrid; Hummel, Karin; Razzazi-Fazeli, Ebrahim; Jessen, Flemming; Escribano, Damian; Niewold, Theo

    2016-05-01

    The growth promoting effect of supplementing animal feed with antibiotics like tetracycline has traditionally been attributed to their antibiotic character. However, more evidence has been accumulated on their direct anti-inflammatory effect during the last two decades. Here we used a pig model to explore the systemic molecular effect of feed supplementation with sub therapeutic levels of oxytetracycline (OTC) by analysis of serum proteome changes. Results showed that OTC promoted growth, coinciding with a significant down regulation of different serum proteins related to inflammation, oxidation and lipid metabolism, confirming the anti-inflammatory mechanism of OTC. Interestingly, apart from the classic acute phase reactants also down regulation was seen of a hibernation associated plasma protein (HP-27), which is to our knowledge the first description in pigs. Although the exact function in non-hibernators is unclear, down regulation of HP-27 could be consistent with increased appetite, which is possibly linked to the anti-inflammatory action of OTC. Given that pigs are good models for human medicine due to their genetic and physiologic resemblance, the present results might also be used for rational intervention in human diseases in which inflammation plays an important role such as obesity, type 2 diabetes and cardiovascular diseases. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Identification and characterization of ART-27, a novel coactivator for the androgen receptor N terminus.

    PubMed

    Markus, Steven M; Taneja, Samir S; Logan, Susan K; Li, Wenhui; Ha, Susan; Hittelman, Adam B; Rogatsky, Inez; Garabedian, Michael J

    2002-02-01

    The androgen receptor (AR) is a ligand-regulated transcription factor that stimulates cell growth and differentiation in androgen-responsive tissues. The AR N terminus contains two activation functions (AF-1a and AF-1b) that are necessary for maximal transcriptional enhancement by the receptor; however, the mechanisms and components regulating AR transcriptional activation are not fully understood. We sought to identify novel factors that interact with the AR N terminus from an androgen-stimulated human prostate cancer cell library using a yeast two-hybrid approach designed to identify proteins that interact with transcriptional activation domains. A 157-amino acid protein termed ART-27 was cloned and shown to interact predominantly with the AR(153-336), containing AF-1a and a part of AF-1b, localize to the nucleus and increase the transcriptional activity of AR when overexpressed in cultured mammalian cells. ART-27 also enhanced the transcriptional activation by AR(153-336) fused to the LexA DNA-binding domain but not other AR N-terminal subdomains, suggesting that ART-27 exerts its effect via an interaction with a defined region of the AR N terminus. ART-27 interacts with AR in nuclear extracts from LNCaP cells in a ligand-independent manner. Interestingly, velocity gradient sedimentation of HeLa nuclear extracts suggests that native ART-27 is part of a multiprotein complex. ART-27 is expressed in a variety of human tissues, including sites of androgen action such as prostate and skeletal muscle, and is conserved throughout evolution. Thus, ART-27 is a novel cofactor that interacts with the AR N terminus and plays a role in facilitating receptor-induced transcriptional activation.

  2. Previous administration of naltrexone did not change synergism between paracetamol and tramadol in mice.

    PubMed

    Miranda, Hugo F; Noriega, Viviana; Prieto, Juan Carlos

    2012-07-01

    In the treatment of acute and chronic pain the most frequently used drugs are nonsteroidal anti-inflammatory drugs (NSAIDs), e.g., paracetamol; opioids, e.g., tramadol, and a group of drugs called coanalgesics or adjuvants (e.g., antidepressants, anticonvulsants). The aim of this work was to determine the nature of the interaction induced by intraperitoneal or intrathecal coadministration of paracetamol and tramadol. The type of interaction was evaluated by means of isobolographic analysis, using the acetic acid writhing test as an algesiometer in mice. In addition, the involvement of opioid receptors in the interaction was studied using naltrexone, a non-selective opioid receptor antagonist. The administration of paracetamol or tramadol induced a dose-dependent antinociceptive activity in the assay. The dose-response curves were characterized by equal efficacy but different potencies, being i.t. paracetamol 11.84 times more potent than i.p. paracetamol, and i.t. tramadol 3.54 times more potent than the i.p. tramadol. The isobolographic analysis indicates a synergistic interaction between the coadministration of i.p. or i.t. paracetamol with tramadol. The interaction index values were similar for the i.p. and i.t. coadministration with values of 0.414 and 0.364, respectively. The different mechanisms of action of paracetamol and tramadol strongly explain the analgesic synergism between them, in agreement with the general theory of drug interaction. This synergic interaction was not modified by the non selective opioid antagonist, naltrexone. This association could be of clinical significance in the treatment of pain with a reduction of doses and adverse effects. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Synergism between tramadol and parecoxib in the orofacial formalin test.

    PubMed

    Isiordia-Espinoza, Mario Alberto; Zapata-Morales, Juan Ramón; Castañeda-Santana, Demian Ismael; de la Rosa-Coronado, Maximiliano; Aragon-Martinez, Othoniel Hugo

    2015-05-01

    The aim of this study was to evaluate the interaction between tramadol and parecoxib in the orofacial formalin test. Tramadol (10, 31.6, 56, and 100 mg/kg ip) or parecoxib (31.6, 56, 100, and 178 mg/kg ip) were administered 10 min before formalin (2.5%) injection into the upper lip to characterize the dose-response curve of each individual drug in the orofacial pain test in mice. Once the dose-response curve of each drug was obtained, an experimental effective dose 50 (ED50 ) value was determined for each drug. The tramadol-parecoxib combination was evaluated in four different groups of animals. The isobolographic analysis and the interaction index were used to evaluate the nature of interaction between both drugs. The isobologram and the interaction index showed increased in the antinociceptive effect of the combination. The tramadol-parecoxib combination produces a synergism in the second phase of the orofacial formalin test. © 2015 Wiley Periodicals, Inc.

  4. Phosphomimics destabilize Hsp27 oligomeric assemblies and enhance chaperone activity.

    PubMed

    Jovcevski, Blagojce; Kelly, Megan A; Rote, Anthea P; Berg, Tracey; Gastall, Heidi Y; Benesch, Justin L P; Aquilina, J Andrew; Ecroyd, Heath

    2015-02-19

    Serine phosphorylation of the mammalian small heat-shock protein Hsp27 at residues 15, 78, and 82 is thought to regulate its structure and chaperone function; however, the site-specific impact has not been established. We used mass spectrometry to assess the combinatorial effect of mutations that mimic phosphorylation upon the oligomeric state of Hsp27. Comprehensive dimerization yielded a relatively uncrowded spectrum, composed solely of even-sized oligomers. Modification at one or two serines decreased the average oligomeric size, while the triple mutant was predominantly a dimer. These changes were reflected in a greater propensity for oligomers to dissociate upon increased modification. The ability of Hsp27 to prevent amorphous or fibrillar aggregation of target proteins was enhanced and correlated with the amount of dissociated species present. We propose that, in vivo, phosphorylation promotes oligomer dissociation, thereby enhancing chaperone activity. Our data support a model in which dimers are the chaperone-active component of Hsp27. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. A U-Rich Element in the 5′ Untranslated Region Is Necessary for the Translation of p27 mRNA

    PubMed Central

    Millard, S. Sean; Vidal, Anxo; Markus, Maurice; Koff, Andrew

    2000-01-01

    Increased translation of p27 mRNA correlates with withdrawal of cells from the cell cycle. This raised the possibility that antimitogenic signals might mediate their effects on p27 expression by altering complexes that formed on p27 mRNA, regulating its translation. In this report, we identify a U-rich sequence in the 5′ untranslated region (5′UTR) of p27 mRNA that is necessary for efficient translation in proliferating and nonproliferating cells. We show that a number of factors bind to the 5′UTR in vitro in a manner dependent on the U-rich element, and their availability in the cytosol is controlled in a growth- and cell cycle-dependent fashion. One of these factors is HuR, a protein previously implicated in mRNA stability, transport, and translation. Another is hnRNP C1 and C2, proteins implicated in mRNA processing and the translation of a specific subset of mRNAs expressed in differentiated cells. In lovastatin-treated MDA468 cells, the mobility of the associated hnRNP C1 and C2 proteins changed, and this correlated with increased p27 expression. Together, these data suggest that the U-rich dependent RNP complex on the 5′UTR may regulate the translation of p27 mRNA and may be a target of antimitogenic signals. PMID:10913178

  6. Expression of heat shock proteins (hsp) 27 and 70 in various organ systems in cases of death due to fire.

    PubMed

    Doberentz, E; Genneper, L; Böker, D; Lignitz, E; Madea, B

    2014-11-01

    The expression of heat shock proteins (hsp) increases in case of variable types of endogenous and exogenous cellular stress, as for example thermal stress. Immunohistochemical staining with hsp antibodies can visualize these stress proteins. Fifty-three cases of death due to heat and a control group of 100 deaths without any antemortem thermic stress were examined regarding hsp27 and hsp70 expression in myocardial, pulmonary, and renal tissues. The results revealed a correlation between hsp expression, survival time, and cause of death. In cases of death due to fire, the expression of hsp is more extensive than in the control group, especially in pulmonary and renal tissues. The immunohistochemical investigation of an hsp expression can support the proof of vitality in cases of death related to fire.

  7. Crucial HSP70 co–chaperone complex unlocks metazoan protein disaggregation

    PubMed Central

    Nillegoda, Nadinath B.; Kirstein, Janine; Szlachcic, Anna; Berynskyy, Mykhaylo; Stank, Antonia; Stengel, Florian; Arnsburg, Kristin; Gao, Xuechao; Scior, Annika; Aebersold, Ruedi; Guilbride, D. Lys; Wade, Rebecca C.; Morimoto, Richard I.; Mayer, Matthias P.; Bukau, Bernd

    2016-01-01

    Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states1,2. Healthy metazoan cells effectively eliminate intracellular protein aggregates3,4, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems5,6, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro4,7. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control. PMID:26245380

  8. The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1 and Cyclin D1 and induces cell cycle exit and neuronal differentiation

    PubMed Central

    Soppa, Ulf; Schumacher, Julian; Florencio Ortiz, Victoria; Pasqualon, Tobias; Tejedor, Francisco J; Becker, Walter

    2014-01-01

    A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G1 phase. Sustained overexpression of DYRK1A induced G0 cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27Kip1 on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27Kip1 Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome. PMID:24806449

  9. Prehension Synergies in the Grasps With Complex Friction Patterns: Local Versus Synergic Effects and the Template Control

    PubMed Central

    Niu, Xun; Latash, Mark L.; Zatsiorsky, Vladimir M.

    2010-01-01

    We studied adjustments of digit forces to changes in the friction. The subjects held a handle statically in a three-digit grasp. The friction under each digit was either high or low, resulting in eight three-element friction sets (such grasps were coined the grasps with complex friction pattern). The total load was also manipulated. It was found that digit forces were adjusted not only to the supported load and local friction, but also to friction at other digits (synergic effects). When friction under a digit was low, its tangential force decreased and the normal force increased (local effects). The synergic effects were directed to maintain the equilibrium of the handle. The relation between the individual digit forces and loads agreed with the triple-product model: fin=ki(2)ki(1)L, where fin is normal force of digit i, L is the load (newtons), ki(1) is a dimensionless coefficient representing sharing the total tangential force among the digits (Σki(1)=1.0), and ki(2) is a coefficient representing the relation between the tangential and normal forces of digit i (the overall friction equivalent, OFE). At each friction set, the central controller selected the grasping template—a three-element array of ki(2)ki(1) products—and then scaled the template with the load magnitude. PMID:17493928

  10. Temozolomide, sirolimus and chloroquine is a new therapeutic combination that synergizes to disrupt lysosomal function and cholesterol homeostasis in GBM cells.

    PubMed

    Hsu, Sanford P C; Kuo, John S; Chiang, Hsin-Chien; Wang, Hsin-Ell; Wang, Yu-Shan; Huang, Cheng-Chung; Huang, Yi-Chun; Chi, Mau-Shin; Mehta, Minesh P; Chi, Kwan-Hwa

    2018-01-23

    Glioblastoma (GBM) cells are characterized by high phagocytosis, lipogenesis, exocytosis activities, low autophagy capacity and high lysosomal demand are necessary for survival and invasion. The lysosome stands at the cross roads of lipid biosynthesis, transporting, sorting between exogenous and endogenous cholesterol. We hypothesized that three already approved drugs, the autophagy inducer, sirolimus (rapamycin, Rapa), the autophagy inhibitor, chloroquine (CQ), and DNA alkylating chemotherapy, temozolomide (TMZ) could synergize against GBM. This repurposed triple therapy combination induced GBM apoptosis in vitro and inhibited GBM xenograft growth in vivo . Cytotoxicity is caused by induction of lysosomal membrane permeabilization and release of hydrolases, and may be rescued by cholesterol supplementation. Triple treatment inhibits lysosomal function, prevents cholesterol extraction from low density lipoprotein (LDL), and causes clumping of lysosome associated membrane protein-1 (LAMP-1) and lipid droplets (LD) accumulation. Co-treatment of the cell lines with inhibitor of caspases and cathepsin B only partially reverse of cytotoxicities, while N-acetyl cysteine (NAC) can be more effective. A combination of reactive oxygen species (ROS) generation from cholesterol depletion are the early event of underling mechanism. Cholesterol repletion abolished the ROS production and reversed the cytotoxicity from QRT treatment. The shortage of free cholesterol destabilizes lysosomal membranes converting aborted autophagy to apoptosis through either direct mitochondria damage or cathepsin B release. This promising anti-GBM triple therapy combination severely decreases mitochondrial function, induces lysosome-dependent apoptotic cell death, and is now poised for further clinical testing and validation.

  11. Temozolomide, sirolimus and chloroquine is a new therapeutic combination that synergizes to disrupt lysosomal function and cholesterol homeostasis in GBM cells

    PubMed Central

    Chiang, Hsin-Chien; Wang, Hsin-Ell; Wang, Yu-Shan; Huang, Cheng-Chung; Huang, Yi-Chun; Chi, Mau-Shin; Mehta, Minesh P.; Chi, Kwan-Hwa

    2018-01-01

    Glioblastoma (GBM) cells are characterized by high phagocytosis, lipogenesis, exocytosis activities, low autophagy capacity and high lysosomal demand are necessary for survival and invasion. The lysosome stands at the cross roads of lipid biosynthesis, transporting, sorting between exogenous and endogenous cholesterol. We hypothesized that three already approved drugs, the autophagy inducer, sirolimus (rapamycin, Rapa), the autophagy inhibitor, chloroquine (CQ), and DNA alkylating chemotherapy, temozolomide (TMZ) could synergize against GBM. This repurposed triple therapy combination induced GBM apoptosis in vitro and inhibited GBM xenograft growth in vivo. Cytotoxicity is caused by induction of lysosomal membrane permeabilization and release of hydrolases, and may be rescued by cholesterol supplementation. Triple treatment inhibits lysosomal function, prevents cholesterol extraction from low density lipoprotein (LDL), and causes clumping of lysosome associated membrane protein-1 (LAMP-1) and lipid droplets (LD) accumulation. Co-treatment of the cell lines with inhibitor of caspases and cathepsin B only partially reverse of cytotoxicities, while N-acetyl cysteine (NAC) can be more effective. A combination of reactive oxygen species (ROS) generation from cholesterol depletion are the early event of underling mechanism. Cholesterol repletion abolished the ROS production and reversed the cytotoxicity from QRT treatment. The shortage of free cholesterol destabilizes lysosomal membranes converting aborted autophagy to apoptosis through either direct mitochondria damage or cathepsin B release. This promising anti-GBM triple therapy combination severely decreases mitochondrial function, induces lysosome-dependent apoptotic cell death, and is now poised for further clinical testing and validation. PMID:29467937

  12. Curcumin synergizes with resveratrol to inhibit colon cancer.

    PubMed

    Majumdar, Adhip P N; Banerjee, Sanjeev; Nautiyal, Jyoti; Patel, Bhaumik B; Patel, Vaishali; Du, Jianhua; Yu, Yingjie; Elliott, Althea A; Levi, Edi; Sarkar, Fazlul H

    2009-01-01

    Development and progression of many malignancies, including colorectal cancer, are associated with activation of multiple signaling pathways. Therefore, inhibition of these signaling pathways with noncytotoxic natural products represents a logical preventive and/or therapeutic approach for colon cancer. Curcumin and resveratrol, both of which inhibit the growth of transformed cells and colon carcinogenesis, were selected to examine whether combining them would be an effective preventive and/or therapeutic strategy for colon cancer. Indeed, the combination of curcumin and resveratrol was found to be more effective in inhibiting growth of p53-positive (wt) and p53-negative colon cancer HCT-116 cells in vitro and in vivo in SCID xenografts of colon cancer HCT-116 (wt) cells than either agent alone. Analysis by Calcusyn software showed synergism between curcumin and resveratrol. The inhibition of tumors in response to curcumin and/or resveratrol was associated with the reduction in proliferation and stimulation of apoptosis accompanied by attenuation of NF-kappaB activity. In vitro studies have further demonstrated that the combinatorial treatment caused a greater inhibition of constitutive activation of EGFR and its family members as well as IGF-1R. Our current data suggest that the combination of curcumin and resveratrol could be an effective preventive/therapeutic strategy for colon cancer.

  13. HSP27 Alleviates Cardiac Aging in Mice via a Mechanism Involving Antioxidation and Mitophagy Activation.

    PubMed

    Lin, Shenglan; Wang, Yana; Zhang, Xiaojin; Kong, Qiuyue; Li, Chuanfu; Li, Yuehua; Ding, Zhengnian; Liu, Li

    2016-01-01

    Aging-induced cardiac dysfunction is a prominent feature of cardiac aging. Heat shock protein 27 (HSP27) protects cardiac function against ischemia or chemical challenge. We hypothesized that HSP27 attenuates cardiac aging. Transgenic (Tg) mice with cardiac-specific expression of the HSP27 gene and wild-type (WT) littermates were employed in the experiments. Echocardiography revealed a significant decline in the cardiac function of old WT mice compared with young WT mice. In striking contrast, the aging-induced impairment of cardiac function was attenuated in old Tg mice compared with old WT mice. Levels of cardiac aging markers were lower in old Tg mouse hearts than in old WT mouse hearts. Less interstitial fibrosis and lower contents of reactive oxygen species and ubiquitin-conjugated proteins were detected in old Tg hearts than in old WT hearts. Furthermore, old Tg hearts demonstrated lower accumulation of LC3-II and p62 than old WT hearts. Levels of Atg13, Vps34, and Rab7 were also higher in old Tg hearts than in old WT hearts. Additionally, old Tg hearts had higher levels of PINK1 and Parkin than old WT hearts, suggesting that mitophagy was activated in old Tg hearts. Taken together, HSP27 alleviated cardiac aging and this action involved antioxidation and mitophagy activation.

  14. Rpl27a mutation in the sooty foot ataxia mouse phenocopies high p53 mouse models

    PubMed Central

    Terzian, Tamara; Dumble, Melissa; Arbab, Farinaz; Thaller, Christina; Donehower, Lawrence A; Lozano, Guillermina; Justice, Monica J; Roop, Dennis R; Box, Neil F

    2013-01-01

    Ribosomal stress is an important, yet poorly understood, mechanism that results in activation of the p53 tumour suppressor. We present a mutation in the ribosomal protein Rpl27a gene (sooty foot ataxia mice), isolated through a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for p53 pathway defects, that shares striking phenotypic similarities with high p53 mouse models, including cerebellar ataxia, pancytopenia and epidermal hyperpigmentation. This phenocopy is rescued in a haploinsufficient p53 background. A detailed examination of the bone marrow in these mice identified reduced numbers of haematopoietic stem cells and a p53-dependent c-Kit down-regulation. These studies suggest that reduced Rpl27a increases p53 activity in vivo, further evident with a delay in tumorigenesis in mutant mice. Taken together, these data demonstrate that Rpl27a plays a crucial role in multiple tissues and that disruption of this ribosomal protein affects both development and transformation. PMID:21674502

  15. Inactivation and unfolding of protein tyrosine phosphatase from Thermus thermophilus HB27 during urea and guanidine hydrochloride denaturation.

    PubMed

    Wang, Yejing; He, Huawei; Liu, Lina; Gao, Chunyan; Xu, Shui; Zhao, Ping; Xia, Qingyou

    2014-01-01

    The effects of urea and guanidine hydrochloride (GdnHCl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase), a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV) circular dichroism (CD), Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS) fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase.

  16. Synergism between hydrogen peroxide and seventeen acids against five agri-food-borne fungi and one yeast strain.

    PubMed

    Martin, H; Maris, P

    2012-12-01

    The objective of this study was to evaluate fungicidal efficacy of hydrogen peroxide administered in combination with 17 mineral and organic acids authorized for use in the food industry. The assays were performed on a 96-well microplate using a microdilution technique based on the checkerboard titration method. The six selected strains (one yeast and five fungi) were reference strains and strains representative of contaminating fungi found in the food industry. Each synergistic hydrogen peroxide/acid combination found after fifteen minutes contact time at 20 °C in distilled water was then tested in conditions simulating four different use conditions. Twelve combinations were synergistic in distilled water, eleven of these remained synergistic with one or more of the four mineral and organic interfering substances selected. Hydrogen peroxide/formic acid combination remained effective against four strains and was never antagonistic against the other two fungi. Combinations with propionic acid and acetic acid stayed synergistic against two strains. Those with oxalic acid and lactic acid kept their synergism only against Candida albicans. No synergism was detected against Penicillium cyclopium. Synergistic combinations of disinfectants were revealed, among them the promising hydrogen peroxide/formic acid combination. A rapid screening method developed in our laboratory for bacteria was adapted to fungi and used to reveal the synergistic potential of disinfectants and/or sanitizers combinations. © 2012 The Society for Applied Microbiology.

  17. Thionin-like peptide from Capsicum annuum fruits: mechanism of action and synergism with fluconazole against Candida species.

    PubMed

    Taveira, Gabriel B; Carvalho, André O; Rodrigues, Rosana; Trindade, Fernanda G; Da Cunha, Maura; Gomes, Valdirene M

    2016-01-27

    Thionins are a family of plant antimicrobial peptides (AMPs), which participate in plant defense system against pathogens. Here we describe some aspects of the CaThi thionin-like action mechanism, previously isolated from Capsicum annuum fruits. Thionin-like peptide was submitted to antimicrobial activity assays against Candida species for IC50 determination and synergism with fluconazole evaluation. Viability and plasma membrane permeabilization assays, induction of intracellular ROS production analysis and CaThi localization in yeast cells were also investigated. CaThi had strong antimicrobial activity against six tested pathogenic Candida species, with IC50 ranging from 10 to 40 μg.mL(-1). CaThi antimicrobial activity on Candida species was candidacidal. Moreover, CaThi caused plasma membrane permeabilization in all yeasts tested and induces oxidative stresses only in Candida tropicalis. CaThi was intracellularly localized in C. albicans and C. tropicalis, however localized in nuclei in C. tropicalis, suggesting a possible nuclear target. CaThi performed synergistically with fluconazole inhibiting all tested yeasts, reaching 100% inhibition in C. parapsilosis. The inhibiting concentrations for the synergic pair ranged from 1.3 to 4.0 times below CaThi IC50 and from zero to 2.0 times below fluconazole IC50. The results reported herein may ultimately contribute to future efforts aiming to employ this plant-derived AMP as a new therapeutic substance against yeasts.

  18. Small heat shock proteins HSP27 (HspB1), αB-crystallin (HspB5) and HSP22 (HspB8) as regulators of cell death.

    PubMed

    Acunzo, Julie; Katsogiannou, Maria; Rocchi, Palma

    2012-10-01

    Hsp27, αB-crystallin and HSP22 are ubiquitous small heat shock proteins (sHsp) whose expression is induced in response to a wide variety of unfavorable physiological and environmental conditions. These sHsp protect cells from otherwise lethal conditions mainly by their involvement in cell death pathways such as necrosis, apoptosis or autophagy. At a molecular level, the mechanisms accounting for sHsp functions in cell death are (1) prevention of denatured proteins aggregation, (2) regulation of caspase activity, (3) regulation of the intracellular redox state, (4) function in actin polymerization and cytoskeleton integrity and (5) proteasome-mediated degradation of selected proteins. In cancer cells, these sHsp are often overexpressed and associated with increased tumorigenicity, cancer cells metastatic potential and resistance to chemotherapy. Altogether, these properties suggest that Hsp27, αB-crystallin and Hsp22 are appropriate targets for modulating cell death pathways. In the present, we briefly review recent reports showing molecular evidence of cell death regulation by these sHsp and co-chaperones. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Intermittent fasting up-regulates Fsp27/Cidec gene expression in white adipose tissue.

    PubMed

    Karbowska, Joanna; Kochan, Zdzislaw

    2012-03-01

    Fat-specific protein of 27 kDa (FSP27) is a novel lipid droplet protein that promotes triacylglycerol storage in white adipose tissue (WAT). The regulation of the Fsp27 gene expression in WAT is largely unknown. We investigated the nutritional regulation of FSP27 in WAT. The effects of intermittent fasting (48 d, eight cycles of 3-d fasting and 3-d refeeding), caloric restriction (48 d), fasting-refeeding (3-d fasting and 3-d refeeding), and fasting (3 d) on mRNA expression of FSP27, peroxisome proliferator-activated receptor γ (PPARγ2), CCAAT/enhancer binding protein α (C/EBPα), and M isoform of carnitine palmitoyltransferase 1 (a positive control for PPARγ activation) in epididymal WAT and on serum triacylglycerol, insulin, and leptin levels were determined in Wistar rats. We also determined the effects of PPARγ activation by rosiglitazone or pioglitazone on FSP27 mRNA levels in primary rat adipocytes. Long-term intermittent fasting, in contrast to other dietary manipulations, significantly up-regulated Fsp27 gene expression in WAT. Moreover, in rats subjected to intermittent fasting, serum insulin levels were elevated; PPARγ2 and C/EBPα mRNA expression in WAT was increased, and there was a positive correlation of Fsp27 gene expression with PPARγ2 and C/EBPα mRNA levels. FSP27 mRNA expression was also increased in adipocytes treated with PPARγ agonists. Our study demonstrates that the transcription of the Fsp27 gene in adipose tissue may be induced in response to nutritional stimuli. Furthermore, PPARγ2, C/EBPα, and insulin may be involved in the nutritional regulation of FSP27. Thus intermittent fasting, despite lower caloric intake, may promote triacylglycerol deposition in WAT by increasing the expression of genes involved in lipid storage, such as Fsp27. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Identification and Characterization of ART-27, a Novel Coactivator for the Androgen Receptor N Terminus

    PubMed Central

    Markus, Steven M.; Taneja, Samir S.; Logan, Susan K.; Li, Wenhui; Ha, Susan; Hittelman, Adam B.; Rogatsky, Inez; Garabedian, Michael J.

    2002-01-01

    The androgen receptor (AR) is a ligand-regulated transcription factor that stimulates cell growth and differentiation in androgen-responsive tissues. The AR N terminus contains two activation functions (AF-1a and AF-1b) that are necessary for maximal transcriptional enhancement by the receptor; however, the mechanisms and components regulating AR transcriptional activation are not fully understood. We sought to identify novel factors that interact with the AR N terminus from an androgen-stimulated human prostate cancer cell library using a yeast two-hybrid approach designed to identify proteins that interact with transcriptional activation domains. A 157-amino acid protein termed ART-27 was cloned and shown to interact predominantly with the AR153–336, containing AF-1a and a part of AF-1b, localize to the nucleus and increase the transcriptional activity of AR when overexpressed in cultured mammalian cells. ART-27 also enhanced the transcriptional activation by AR153–336 fused to the LexA DNA-binding domain but not other AR N-terminal subdomains, suggesting that ART-27 exerts its effect via an interaction with a defined region of the AR N terminus. ART-27 interacts with AR in nuclear extracts from LNCaP cells in a ligand-independent manner. Interestingly, velocity gradient sedimentation of HeLa nuclear extracts suggests that native ART-27 is part of a multiprotein complex. ART-27 is expressed in a variety of human tissues, including sites of androgen action such as prostate and skeletal muscle, and is conserved throughout evolution. Thus, ART-27 is a novel cofactor that interacts with the AR N terminus and plays a role in facilitating receptor-induced transcriptional activation. PMID:11854421

  1. Mixture toxicity in the marine environment: Model development and evidence for synergism at environmental concentrations.

    PubMed

    Deruytter, David; Baert, Jan M; Nevejan, Nancy; De Schamphelaere, Karel A C; Janssen, Colin R

    2017-12-01

    Little is known about the effect of metal mixtures on marine organisms, especially after exposure to environmentally realistic concentrations. This information is, however, required to evaluate the need to include mixtures in future environmental risk assessment procedures. We assessed the effect of copper (Cu)-Nickel (Ni) binary mixtures on Mytilus edulis larval development using a full factorial design that included environmentally relevant metal concentrations and ratios. The reproducibility of the results was assessed by repeating this experiment 5 times. The observed mixture effects were compared with the effects predicted with the concentration addition model. Deviations from the concentration addition model were estimated using a Markov chain Monte-Carlo algorithm. This enabled the accurate estimation of the deviations and their uncertainty. The results demonstrated reproducibly that the type of interaction-synergism or antagonism-mainly depended on the Ni concentration. Antagonism was observed at high Ni concentrations, whereas synergism occurred at Ni concentrations as low as 4.9 μg Ni/L. This low (and realistic) Ni concentration was 1% of the median effective concentration (EC50) of Ni or 57% of the Ni predicted-no-effect concentration (PNEC) in the European Union environmental risk assessment. It is concluded that results from mixture studies should not be extrapolated to concentrations or ratios other than those investigated and that significant mixture interactions can occur at environmentally realistic concentrations. This should be accounted for in (marine) environmental risk assessment of metals. Environ Toxicol Chem 2017;36:3471-3479. © 2017 SETAC. © 2017 SETAC.

  2. The synergic effects of mirror therapy and neuromuscular electrical stimulation for hand function in stroke patients.

    PubMed

    Yun, Gi Jeong; Chun, Min Ho; Park, Ji Young; Kim, Bo Ryun

    2011-06-01

    To investigate the synergic effects of mirror therapy and neuromuscular electrical stimulation (NMES) for hand function in stroke patients. Sixty patients with hemiparesis after stroke were included (41 males and 19 females, average age 63.3 years). Twenty patients had NMES applied and simultaneously underwent mirror therapy. Twenty patients had NMES applied only, and twenty patients underwent mirror therapy only. Each treatment was done five days per week, 30 minutes per day, for three weeks. NMES was applied on the surface of the extensor digitorum communis and extensor pollicis brevis for open-hand motion. Muscle tone, Fugl-Meyer assessment, and power of wrist and hand were evaluated before and after treatment. There were significant improvements in the Fugl-Meyer assessment score in the wrist, hand and coordination, as well as power of wrist and hand in all groups after treatment. The mirror and NMES group showed significant improvements in the Fugl-Meyer scores of hand, wrist, coordination and power of hand extension compared to the other groups. However, the power of hand flexion, wrist flexion, and wrist extension showed no significant differences among the three groups. Muscle tone also showed no significant differences in the three groups. Our results showed that there is a synergic effect of mirror therapy and NMES on hand function. Therefore, a hand rehabilitation strategy combined with NMES and mirror therapy may be more helpful for improving hand function in stroke patients than NMES or mirror therapy only.

  3. Expression, purification and characterization of Der f 27, a new allergen from dermatophagoides farinae

    PubMed Central

    Lin, Jianli; Li, Meng; Liu, Yulin; Jiang, Congli; Wu, Yulan; Wang, Yuanyuan; Gao, Anjian; Liu, Zhigang; Yang, Pingchang; Liu, Xiaoyu

    2015-01-01

    The house dust mite (HDM), Dermatophagoidesfarinae (D. farina), is one of the most important indoor allergen sources and a major elicitor of allergic asthma; itscharacterization is important in the diagnosis and immunotherapy of mite allergen-relevant diseases. This study aims to characterize a novel allergen, the D. farinae-derived serpin (Der f 27). In this study, the total RNA of D. farinae was extracted, and the Der f 27 gene was cloned and expressed. The allergenicity of recombinant Der f 27 protein was determined by enzyme-linked immunosorbent assay, and Western-blotting with the sera of asthma patients, and skin prick test (SPT) in allergic human subjects. A r-Der f 27 allergic asthma mouse model was established. The cloned Der f 27 gene has been presented at the Gene Bank with an accession number of KM009995. The IgE levels of r-Der f 27 in the serum from r-Der f 27 SPT positive allergic patients were 3 folds more than healthy subjects. The Der f 27 SPT positive ratewas 42.1% in 19 DM-SPT positive patients. Airway hyperresponsiveness, serum specific IgE, and levels of interleukin-4 in the spleen cell culture supernatant were significantly increased in allergic asthma mice sensitized to r-Der f 27. In conclusion, Der f 27 is a new subtype of house mite allergen. PMID:26328010

  4. Proteomic identification and purification of seed proteins from native Amazonian species displaying antifungal activity.

    PubMed

    Ramos, Márcio V; Brito, Daniel; Freitas, Cléverson D T; Gonçalves, José Francisco C; Porfirio, Camila T M N; Lobo, Marina D P; Monteiro-Moreira, Ana Cristina O; Souza, Luiz A C; Fernandes, Andreia V

    2018-04-19

    Seeds of native species from the rain forest (Amazon) are source of chitinases and their protein extracts exhibited strong and broad antifungal activity. Numerous plant species native to the Amazon have not yet been chemically studied. Studies of seeds are scarcer, since adversities in accessing study areas and seasonality pose constant hurdles to systematic research. In this study, proteins were extracted from seeds belonging to endemic Amazon species and were investigated for the first time. Proteolytic activity, peptidase inhibitors, and chitinases were identified, but chitinolytic activity predominated. Four proteins were purified through chromatography and identified as lectin and chitinases by MS/MS analyses. The proteins were examined for inhibition of a phytopathogen (Fusarium oxysporum). Analyses by fluorescence microscopy suggested binding of propidium iodide to DNA of fungal spores, revealing that spore integrity was lost when accessed by the proteins. Further structural and functional analyses of defensive proteins belonging to species facing highly complex ecosystems such as Amazonia should be conducted, since these could provide new insights into specificity and synergism involving defense proteins of plants submitted to a very complex ecosystem.

  5. CacyBP/SIP nuclear translocation regulates p27Kip1 stability in gastric cancer cells

    PubMed Central

    Niu, Ying-Lin; Li, Ya-Jun; Wang, Jing-Bo; Lu, Yuan-Yuan; Liu, Zhen-Xiong; Feng, Shan-Shan; Hu, Jian-Guo; Zhai, Hui-Hong

    2016-01-01

    AIM: To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP) nuclear translocation in promoting the proliferation of gastric cancer (GC) cells. METHODS: The effect of CacyBP/SIP nuclear translocation on cell cycle was investigated by cell cycle analysis. Western blot analysis was used to assess the change in expression of cell cycle regulatory proteins and proteasome-mediated degradation of p27Kip1. Co-immunoprecipitation (co-IP) analysis was performed to examine the binding of CacyBP/SIP with Skp1. A CacyBP/SIP truncation mutant which lacked the Skp1 binding site was constructed and fused to a fluorescent protein. Subsequently, the effect on Skp1 binding with the fusion protein was examined by co-IP, while localization of fluorescent fusion protein observed by confocal laser microscopy, and change in p27Kip1 protein expression assessed by Western blot analysis. RESULTS: CacyBP/SIP nuclear translocation induced by gastrin promoted progression of GC cells from G1 phase. However, while CacyBP/SIP nuclear translocation was inhibited using siRNA to suppress CacyBP/SIP expression, cell cycle was clearly inhibited. CacyBP/SIP nuclear translocation significantly decreased the level of cell cycle inhibitor p27Kip1, increased Cyclin E protein expression whereas the levels of Skp1, Skp2, and CDK2 were not affected. Upon inhibition of CacyBP/SIP nuclear translocation, there were no changes in protein levels of p27Kip1 and Cyclin E, while p27Kip1 decrease could be prevented by the proteasome inhibitor MG132. Moreover, CacyBP/SIP was found to bind to Skp1 by immunoprecipitation, an event that was abolished by mutant CacyBP/SIP, which also failed to stimulate p27Kip1 degradation, even though the mutant could still translocate into the nucleus. CONCLUSION: CacyBP/SIP nuclear translocation contributes to the proliferation of GC cells, and CacyBP/SIP exerts this effect, at least in part, by stimulating ubiquitin-mediated degradation of p27

  6. A hidden markov model derived structural alphabet for proteins.

    PubMed

    Camproux, A C; Gautier, R; Tufféry, P

    2004-06-04

    Understanding and predicting protein structures depends on the complexity and the accuracy of the models used to represent them. We have set up a hidden Markov model that discretizes protein backbone conformation as series of overlapping fragments (states) of four residues length. This approach learns simultaneously the geometry of the states and their connections. We obtain, using a statistical criterion, an optimal systematic decomposition of the conformational variability of the protein peptidic chain in 27 states with strong connection logic. This result is stable over different protein sets. Our model fits well the previous knowledge related to protein architecture organisation and seems able to grab some subtle details of protein organisation, such as helix sub-level organisation schemes. Taking into account the dependence between the states results in a description of local protein structure of low complexity. On an average, the model makes use of only 8.3 states among 27 to describe each position of a protein structure. Although we use short fragments, the learning process on entire protein conformations captures the logic of the assembly on a larger scale. Using such a model, the structure of proteins can be reconstructed with an average accuracy close to 1.1A root-mean-square deviation and for a complexity of only 3. Finally, we also observe that sequence specificity increases with the number of states of the structural alphabet. Such models can constitute a very relevant approach to the analysis of protein architecture in particular for protein structure prediction.

  7. Active Hexose-correlated Compound Down-regulates Heat Shock Factor 1, a Transcription Factor for HSP27, in Gemcitabine-resistant Human Pancreatic Cancer Cells.

    PubMed

    Tokunaga, Masayuki; Baron, Byron; Kitagawa, Takao; Tokuda, Kazuhiro; Kuramitsu, Yasuhiro

    2015-11-01

    Active hexose-correlated compound (AHCC) is an extract of a basidiomycete mushroom that enhances the therapeutic effects and reduces the side-effects of chemotherapy. Our previous studies demonstrated that heat-shock protein 27 (HSP27) was involved in gemcitabine-resistance of pancreatic cancer cells and it was down-regulated by AHCC-treatment. However, how AHCC down-regulated HSP27 is unknown. In the present study, we focused on two transcription factors reported to induce HSP27, heat shock factor 1 (HSF1) and high-mobility group box 1 (HMGB1) and investigated the effect of AHCC on their expression. KLM1-R cells were treated with AHCC and the protein expression of HSF1 and HMGB1 were analyzed by western blotting. The protein expression of HSF1 in KLM1-R was down-regulated by AHCC treatment. On the other hand, the protein expression of HMGB1 was not reduced in KLM1-R cells after AHCC treatment. The possibility that AHCC down-regulated HSP27 through down-regulation of the HSF1, was herein shown. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  8. Activity of semisynthetic penicillins and synergism with mecillinam against Bacteroides species.

    PubMed Central

    Trestman, I; Kaye, D; Levison, M E

    1979-01-01

    The minimal inhibitory concentrations (MIC) of six penicillins (ampicillin, carbenicillin, ticarcillin, piperacillin, mezlocillin, and Bay k 4999) against 29 clinical isolates of Bacteriodes spp. (including Bacteroides fragilis, Bacteroides thetaiotaomicron, and Bacteroides vulgatus) were determined by an agar dilution method. Bay k 4999 was most active, followed in descending order by ampicillin, piperacillin, mezlocillin, ticarcillin, and carbenicillin. Mecillinam, a 6 beta-amidino-penicillanic acid, inhibited no strains at 50 micrograms/ml, but when compared with ampicillin, a fourfold or greater increase in MIC for ampicillin (antagonism) was noted in 3 of 29 strains, with no effect on MIC for 26 strains, whereas when combined with carbenicillin, a fourfold or greater decrease in MIC for both antibiotics (synergism) was noted in 12 strains, 4 of which had an MIC of greater than or equal to 250 micrograms/ml for carbenicillin alone. These studies demonstrate the increased activity of some newer semisynthetic penicillins and the potential synergy obtained with mecillinam and carbenicillin against Bacteroides sp. PMID:228593

  9. Sulforaphane activates heat shock response and enhances proteasome activity through up-regulation of Hsp27.

    PubMed

    Gan, Nanqin; Wu, Yu-Chieh; Brunet, Mathilde; Garrido, Carmen; Chung, Fung-Lung; Dai, Chengkai; Mi, Lixin

    2010-11-12

    It is conceivable that stimulating proteasome activity for rapid removal of misfolded and oxidized proteins is a promising strategy to prevent and alleviate aging-related diseases. Sulforaphane (SFN), an effective cancer preventive agent derived from cruciferous vegetables, has been shown to enhance proteasome activities in mammalian cells and to reduce the level of oxidized proteins and amyloid β-induced cytotoxicity. Here, we report that SFN activates heat shock transcription factor 1-mediated heat shock response. Specifically, SFN-induced expression of heat shock protein 27 (Hsp27) underlies SFN-stimulated proteasome activity. SFN-induced proteasome activity was significantly enhanced in Hsp27-overexpressing cells but absent in Hsp27-silenced cells. The role of Hsp27 in regulating proteasome activity was further confirmed in isogenic REG cells, in which SFN-induced proteasome activation was only observed in cells stably overexpressing Hsp27, but not in the Hsp27-free parental cells. Finally, we demonstrated that phosphorylation of Hsp27 is irrelevant to SFN-induced proteasome activation. This study provides a novel mechanism underlying SFN-induced proteasome activity. This is the first report to show that heat shock response by SFN, in addition to the antioxidant response mediated by the Keap1-Nrf2 pathway, may contribute to cytoprotection.

  10. Ibrutinib synergizes with MDM-2 inhibitors in promoting cytotoxicity in B chronic lymphocytic leukemia.

    PubMed

    Voltan, Rebecca; Rimondi, Erika; Melloni, Elisabetta; Rigolin, Gian Matteo; Casciano, Fabio; Arcidiacono, Maria Vittoria; Celeghini, Claudio; Cuneo, Antonio; Zauli, Giorgio; Secchiero, Paola

    2016-10-25

    The aim of this study was to investigate the anti-leukemic activity of the Bruton tyrosine kinase inhibitor Ibrutinib in combination with the small molecule MDM-2 inhibitor Nutlin-3 in preclinical models. The potential efficacy of the Ibrutinib/Nutlin-3 combination was evaluated in vitro in a panel of B leukemic cell lines (EHEB, JVM-2, JVM-3, MEC-1, MEC-2) and in primary B-chronic lymphocytic leukemia (B-CLL) patient samples, by assessing cell viability, cell cycle profile, apoptosis and intracellular pathway modulations. Validation of the combination therapy was assessed in a B leukemic xenograft mouse model. Ibrutinib exhibited variable anti-leukemic activity in vitro and the combination with Nutlin-3 synergistically enhanced the induction of apoptosis independently from the p53 status. Indeed, the Ibrutinib/Nutlin-3 combination was effective in promoting cytotoxicity also in primary B-CLL samples carrying 17p13 deletion and/or TP53 mutations, already in therapy with Ibrutinib. Molecular analyses performed on both B-leukemic cell lines as well as on primary B-CLL samples, while confirming the switch-off of the MAPK and PI3K pro-survival pathways by Ibrutinib, indicated that the synergism of action with Nutlin-3 was independent by p53 pathway and was accompanied by the activation of the DNA damage cascade signaling through the phosphorylation of the histone protein H2A.X. This observation was confirmed also in the JVM-2 B leukemic xenograft mouse model. Taken together, our data emphasize that the Ibrutinib/Nutlin-3 combination merits to be further evaluated as a therapeutic option for B-CLL.

  11. Ibrutinib synergizes with MDM-2 inhibitors in promoting cytotoxicity in B chronic lymphocytic leukemia

    PubMed Central

    Melloni, Elisabetta; Rigolin, Gian Matteo; Casciano, Fabio; Arcidiacono, Maria Vittoria; Celeghini, Claudio; Cuneo, Antonio; Zauli, Giorgio; Secchiero, Paola

    2016-01-01

    Objective The aim of this study was to investigate the anti-leukemic activity of the Bruton tyrosine kinase inhibitor Ibrutinib in combination with the small molecule MDM-2 inhibitor Nutlin-3 in preclinical models. Methods The potential efficacy of the Ibrutinib/Nutlin-3 combination was evaluated in vitro in a panel of B leukemic cell lines (EHEB, JVM-2, JVM-3, MEC-1, MEC-2) and in primary B-chronic lymphocytic leukemia (B-CLL) patient samples, by assessing cell viability, cell cycle profile, apoptosis and intracellular pathway modulations. Validation of the combination therapy was assessed in a B leukemic xenograft mouse model. Results Ibrutinib exhibited variable anti-leukemic activity in vitro and the combination with Nutlin-3 synergistically enhanced the induction of apoptosis independently from the p53 status. Indeed, the Ibrutinib/Nutlin-3 combination was effective in promoting cytotoxicity also in primary B-CLL samples carrying 17p13 deletion and/or TP53 mutations, already in therapy with Ibrutinib. Molecular analyses performed on both B-leukemic cell lines as well as on primary B-CLL samples, while confirming the switch-off of the MAPK and PI3K pro-survival pathways by Ibrutinib, indicated that the synergism of action with Nutlin-3 was independent by p53 pathway and was accompanied by the activation of the DNA damage cascade signaling through the phosphorylation of the histone protein H2A.X. This observation was confirmed also in the JVM-2 B leukemic xenograft mouse model. Conclusions Taken together, our data emphasize that the Ibrutinib/Nutlin-3 combination merits to be further evaluated as a therapeutic option for B-CLL. PMID:27661115

  12. The EED protein–protein interaction inhibitor A-395 inactivates the PRC2 complex

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

    He, Yupeng; Selvaraju, Sujatha; Curtin, Michael L.

    Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed inmore » vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein–protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.« less

  13. Hsp27 (HSPB1) differential expression in normal salivary glands and pleomorphic adenomas and association with an increased Bcl2/Bax ratio.

    PubMed

    Siqueira, Elisa C de; Souza, Fabrício T A; Diniz, Marina G; Gomez, Ricardo S; Gomes, Carolina C

    2015-01-01

    Pleomorphic adenoma (PA) is the most common salivary gland neoplasm. The Hsp27 (HSPB1) is an antiapoptotic protein whose synthesis follows cytotoxic stresses and result in a transient increase in tolerance to subsequent cell injury. Although Hsp27 is expressed in a range of normal tissues and neoplasms, a wide variation in its expression exists among different cells and tissues types. In certain tumours of glandular origin (such as oesophageal adenocarcinomas), the level of Hsp27 is decreased. In the present study, Hsp27 protein levels were evaluated by enzyme-linked immunosorbent assay (ELISA) in a set of 18 fresh PA and 12 normal salivary gland samples. In addition, we tested if Hsp27 protein levels correlated with p53 expression and cell proliferation index, as well as with the transcriptional levels of Bcl-2-associated X protein (BAX), B cell lymphoma 2 (BCL2) and Caspase 3 in PA. We further tested the association between Hsp27 expression and PA tumour size. While all normal salivary gland samples expressed Hsp27 protein, only half of the PA samples expressed it, resulting in a reduced expression of Hsp27 in PA when compared with normal salivary glands (P = 0.003). The expression levels of this protein correlated positively with a higher messenger ribonucleic acid (mRNA) ratio of Bcl2/Bax (R = 0.631; P = 0.01). In conclusion, a decreased Hsp27 protein expression level in PA was found. In addition, Hsp27 levels correlated positively with the Bcl2/Bax mRNA ratio, suggesting an antiapoptotic effect.

  14. Molecular chaperone Hsp27 regulates the Hippo tumor suppressor pathway in cancer.

    PubMed

    Vahid, Sepideh; Thaper, Daksh; Gibson, Kate F; Bishop, Jennifer L; Zoubeidi, Amina

    2016-08-24

    Heat shock protein 27 (Hsp27) is a molecular chaperone highly expressed in aggressive cancers, where it is involved in numerous pro-tumorigenic signaling pathways. Using functional genomics we identified for the first time that Hsp27 regulates the gene signature of transcriptional co-activators YAP and TAZ, which are negatively regulated by the Hippo Tumor Suppressor pathway. The Hippo pathway inactivates YAP by phosphorylating and increasing its cytoplasmic retention with the 14.3.3 proteins. Gain and loss of function experiments in prostate, breast and lung cancer cells showed that Hsp27 knockdown induced YAP phosphorylation and cytoplasmic localization while overexpression of Hsp27 displayed opposite results. Mechanistically, Hsp27 regulates the Hippo pathway by accelerating the proteasomal degradation of ubiquitinated MST1, the core Hippo kinase, resulting in reduced phosphorylation/activity of LATS1 and MOB1, its downstream effectors. Importantly, our in vitro results were supported by data from human tumors; clinically, high expression of Hsp27 in prostate tumors is correlated with increased expression of YAP gene signature and reduced phosphorylation of YAP in lung and invasive breast cancer clinical samples. This study reveals for the first time a link between Hsp27 and the Hippo cascade, providing a novel mechanism of deregulation of this tumor suppressor pathway across multiple cancers.

  15. Heat shock protein 27 overexpression in CHO cells modulates apoptosis pathways and delays activation of caspases to improve recombinant monoclonal antibody titre in fed-batch bioreactors.

    PubMed

    Tan, Janice G L; Lee, Yih Yean; Wang, Tianhua; Yap, Miranda G S; Tan, Tin Wee; Ng, Say Kong

    2015-05-01

    CHO cells are major production hosts for recombinant biologics including the rapidly expanding recombinant monoclonal antibodies (mAbs). Heat shock protein 27 (HSP27) expression was observed to be down-regulated towards the late-exponential and stationary phase of CHO fed-batch bioreactor cultures, whereas HSP27 was found to be highly expressed in human pathological cells and reported to have anti-apoptotic functions. These phenotypes suggest that overexpression of HSP27 is a potential cell line engineering strategy for improving robustness of CHO cells. In this work, HSP27 was stably overexpressed in CHO cells producing recombinant mAb and the effects of HSP27 on cell growth, volumetric production titer and product quality were assessed. Concomitantly, HSP27 anti-apoptosis functions in CHO cells were investigated. Stably transfected clones cultured in fed-batch bioreactors displayed 2.2-fold higher peak viable cell density, delayed loss of culture viability by two days and 2.3-fold increase in mAb titer without affecting the N-glycosylation profile, as compared to clones stably transfected with the vector backbone. Co-immunoprecipitation studies revealed HSP27 interactions with Akt, pro-caspase 3 and Daxx and caspase activity profiling showed delayed increase in caspase 2, 3, 8 and 9 activities. These results suggest that HSP27 modulates apoptosis signaling pathways and delays caspase activities to improve performance of CHO fed-batch bioreactor cultures. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. EDEM1 targets misfolded HLA-B27 dimers for endoplasmic reticulum associated degradation

    PubMed Central

    Guiliano, David B.; Fussell, Helen; Lenart, Izabela; Tsao, Edward; Nesbeth, Darren; Fletcher, Adam J.; Campbell, Elaine C.; Yousaf, Nasim; Williams, Sarah; Santos, Susana; Cameron, Amy; Towers, Greg J.; Kellam, Paul; Hebert, Daniel N.; Gould, Keith; Powis, Simon J.; Antoniou, Antony N.

    2015-01-01

    Objective HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). We wanted to define the role of the UPR induced ER associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers. Methods HeLa cell lines expressing only two copies of a carboxy terminally Sv5 tagged HLA-B27 were generated. The ER stress induced EDEM1 protein was over expressed by transfection and dimer levels monitored by immunoblotting. EDEM1, the UPR associated transcription factor XBP-1, the E3 ubiquitin ligase HRD1, the degradation associated derlin 1 and 2 proteins were inhibited by either short hairpin RNA or dominant negative mutants. The UPR associated ERAD of HLA-B27 was confirmed using ER stress inducing pharamacological agents in kinetic and pulse chase assays. Results We demonstrate that UPR induced machinery can target HLA-B27 dimers, and that dimer formation can be controlled by alterations to expression levels of components of the UPR induced ERAD pathway. HLA-B27 dimers and misfolded MHC class I monomeric molecules were detected bound to EDEM1, with overexpression of EDEM1 inhibiting HLA-B27 dimer formation. EDEM1 inhibition resulted in upregulation of HLA-B27 dimers, whilst UPR induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1 and derlin1/2. Conclusion The UPR ERAD pathway as described here can dispose of HLA-B27 dimers and presents a potential novel therapeutic target for the modulation of HLA-B27 associated inflammatory disease. PMID:25132672

  17. Synergizing vaccinations with therapeutics for measles eradication.

    PubMed

    Plemper, Richard K; Hammond, Anthea L

    2014-02-01

    The measles virus is a major human pathogen responsible for approximately 150,000 deaths annually. The disease is vaccine preventable and eradication of the virus is considered feasible, in principle. However, a herd immunity exceeding 95% is required to prevent sporadic viral outbreaks in a population. Declining disease prevalence, combined with public anxiety over the vaccination's safety, has led to increased vaccine refusal, especially in Europe. This has led to the resurgence of measles in some areas. This article discusses whether synergizing effective measles therapeutics with the measles vaccination could contribute to finally eradicating measles. The authors identify key elements in a desirable drug profile and review current disease management strategies and the state of experimental inhibitor candidates. The authors also evaluate the risk associated with viral escape from inhibition, and consider the potential of measles therapeutics in the management of persistent central nervous system (CNS) viral infection. Finally, the authors contemplate the possible impact of therapeutics in controlling the threat imposed by closely related zoonotic pathogens of the same genus as measles. Efficacious therapeutics used for post-exposure prophylaxis of high-risk social contacts of confirmed index cases may aid measles eradication by closing herd immunity gaps; this is due to vaccine refusal or failure in populations with overall good vaccination coverage. The envisioned primarily prophylactic application of measles therapeutics to a predominantly pediatric and/or adolescent population, dictates the drug profile. It also has to be safe and efficacious, orally available, shelf-stable at ambient temperature and amenable to cost-effective manufacturing.

  18. p27Kip1 localizes to detergent-insoluble microdomains within lymphocyte membranes.

    PubMed Central

    Yaroslavskiy, B. B.; Stolz, D. B.; Watkins, S. C.; Alber, S. M.; Bradbury, N. A.; Steinman, R. A.

    2001-01-01

    BACKGROUND: Low levels of the cyclin-dependent kinase inhibitor p27Kip1 are associated with poor prognosis in cancer. It is unclear whether this is related strictly to p27Kip1-mediated cell cycle inhibition or to other, possibly extranuclear, roles of this protein. In this study, we examined p27Kip1 expression in quiescent and activated lymphocytes. T-cell membranes have been shown to possess sphingolipid and cholesterol-rich microdomains that are insoluble in non-ionic detergents. These "rafts" provide a scaffold for signaling proteins. Signal transduction coincides with coalescence of these microdomains into larger complexes. METHODS: Localization of p27Kip1 was studied by electron and confocal microscopy. Association of p27Kip1 with membrane microdomains in unstimulated and stimulated lymphocytes was determined using Western blots analysis of isolated membranes variably treated with detergents. RESULTS: We demonstrated that p27Kip1 was present in clusters associated with the plasma membrane in normal lymphocytes. The solubility profile of p27Kip1 in isolated membranes indicated that it was localized to raft structures. When lymphocytes were stimulated, however, p27Kip1 was excluded from aggregated raft complexes. CONCLUSIONS: This study identifies, for the first time, the localization of p27 within a membrane microdomain associated with signaling. Because some cell surface signaling complexes lose p27Kip1 upon cellular activation, p27Kip1 may play a functional role in modulating membrane signaling. PMID:11474127

  19. Conifer flavonoid compounds inhibit detoxification enzymes and synergize insecticides.

    PubMed

    Wang, Zhiling; Zhao, Zhong; Cheng, Xiaofei; Liu, Suqi; Wei, Qin; Scott, Ian M

    2016-02-01

    Detoxification by glutathione S-transferases (GSTs) and esterases are important mechanisms associated with insecticide resistance. Discovery of novel GST and esterase inhibitors from phytochemicals could provide potential new insecticide synergists. Conifer tree species contain flavonoids, such as taxifolin, that inhibit in vitro GST activity. The objectives were to test the relative effectiveness of taxifolin as an enzyme inhibitor and as an insecticide synergist in combination with the organophosphorous insecticide, Guthion (50% azinphos-methyl), and the botanical insecticide, pyrethrum, using an insecticide-resistant Colorado potato beetle (CPB) Leptinotarsa decemlineata (Say) strain. Both taxifolin and its isomer, quercetin, increased the mortality of 1(st) instar CPB larvae after 48h when combined with Guthion, but not pyrethrum. Taxifolin had greater in vitro esterase inhibition compared with the commonly used esterase inhibitor, S, S, S-tributyl phosphorotrithioate (DEF). An in vivo esterase and GST inhibition effect after ingestion of taxifolin was measured, however DEF caused a greater suppression of esterase activity. This study demonstrated that flavonoid compounds have both in vitro and in vivo esterase inhibition, which is likely responsible for the insecticide synergism observed in insecticide-resistant CPB. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

  20. Sequence of the amino-terminal region of rat liver ribosomal proteins S4, S6, S8, L6, L7a, L18, L27, L30, L37, L37a, and L39.

    PubMed

    Wittmann-Liebold, B; Geissler, A W; Lin, A; Wool, I G

    1979-01-01

    The sequence of the amino-terminal region of eleven rat liver ribosomal proteins--S4, S6, S8, L6, L7a, L18, L27, L30, L37a, and L39--was determined. The analysis confirmed the homogeneity of the proteins and suggests that they are unique, since no extensive common sequences were found. The N-terminal regions of the rat liver proteins were compared with amino acid sequences in Saccharomyces cerevisiae and in Escherichia coli ribosomal proteins. It seems likely that the proteins L37 from rat liver and Y55 from yeast ribosomes are homologous. It is possible that rat liver L7a or L37a or both are related to S cerevisiae Y44, although the similar sequences are at the amino-terminus of the rat liver proteins and in an internal region of Y44. A number of similarities in the sequences of rat liver and E coli ribosomal proteins have been found; however, it is not yet possible to say whether they connote a common ancestry.

  1. MicroRNA-23a/b and microRNA-27a/b suppress Apaf-1 protein and alleviate hypoxia-induced neuronal apoptosis.

    PubMed

    Chen, Q; Xu, J; Li, L; Li, H; Mao, S; Zhang, F; Zen, K; Zhang, C-Y; Zhang, Q

    2014-03-20

    Expression of apoptotic protease activating factor-1 (Apaf-1) gradually decreases during brain development, and this decrease is likely responsible for the decreased sensitivity of brain tissue to apoptosis. However, the mechanism by which Apaf-1 expression is decreased remains elusive. In the present study, we found that four microRNAs (miR-23a/b and miR-27a/b) of miR-23a-27a-24 and miR-23b-27b-24 clusters play key roles in modulating the expression of Apaf-1. First, we found that miR-23a/b and miR-27a/b suppressed the expression of Apaf-1 in vitro. Interestingly, the expression of the miR-23-27-24 clusters in the mouse cortex gradually increased in a manner that was inversely correlated with the pattern of Apaf-1 expression. Second, hypoxic injuries during fetal distress caused reduced expression of the miR-23b and miR-27b that was inversely correlated with an elevation of Apaf-1 expression during neuronal apoptosis. Third, we made neuronal-specific transgenic mice and found that overexpressing the miR-23b and miR-27b in mouse neurons inhibited the neuronal apoptosis induced by intrauterine hypoxia. In conclusion, our results demonstrate, in central neural system, that miR-23a/b and miR-27a/b are endogenous inhibitory factors of Apaf-1 expression and regulate the sensitivity of neurons to apoptosis. Our findings may also have implications for the potential target role of microRNAs in the treatment of neuronal apoptosis-related diseases.

  2. A Physical Mechanism to Explain the Delivery of Chemical Penetration Enhancers into Skin during Transdermal Sonophoresis - Insight into the Observed Synergism

    PubMed Central

    Polat, Baris E.; Deen, William M.; Langer, Robert; Blankschtein, Daniel

    2011-01-01

    The synergism between low-frequency sonophoresis (LFS) and chemical penetration enhancers (CPEs), especially surfactants, in transdermal enhancement has been investigated extensively since this phenomenon was first observed over a decade ago. In spite of the identifying that the origin of this synergism is the increased penetration and subsequent dispersion of CPEs in the skin in response to LFS treatment, to date, no mechanism has been directly proposed to explain how LFS induces the observed increased transport of CPEs. In this study, we propose a plausible physical mechanism by which the transport of all CPEs is expected to have significantly increased flux into the localized-transport regions (LTRs) of LFS-treated skin. Specifically, the collapse of acoustic cavitation microjets within LTRs induces a convective flux. In addition, because amphiphilic molecules preferentially adsorb onto the gas/water interface of cavitation bubbles, amphiphiles have an additional adsorptive flux. In this sense, the cavitation bubbles effectively act as carriers for amphiphilic molecules, delivering surfactants directly into the skin when they collapse at the skin surface as cavitation microjets. The flux equations derived for CPE delivery into the LTRs and non-LTRs during LFS treatment, compared to that for untreated skin, explain why the transport of all CPEs, and to an even greater extent amphiphilic CPEs, is increased during LFS treatment. The flux model is tested with a non-amphiphilic CPE (propylene glycol) and both nonionic and ionic amphiphilic CPEs (octyl glucoside and sodium lauryl sulfate, respectively), by measuring the flux of each CPE into untreated skin and the LTRs and non-LTRs of LFS-treated skin. The resulting data shows very good agreement with the proposed flux model. PMID:22100440

  3. Functional state of the Hsp27 chaperone as a molecular marker of an unfavorable course of larynx cancer.

    PubMed

    Kaigorodova, Evgeniya V; Zavyalova, Marina V; Bychkov, Vyacheslav A; Perelmuter, Vladimir M; Choynzonov, Evgenii L

    2016-06-24

    The small heat shock protein 27 kDA (Hsp27) acts as an ATP-independent chaperone in protein folding, but is also implicated in architecture of the cytoskeleton, cell migration, metabolism, cell survival, growth/differentiation, mRNA stabilization, and tumor progression. To study the intracellular localization of phosphorylated and non-phosphorylated forms of Hsp27 in squamous cell carcinoma of the larynx (SCCL) and to evaluate their relationship with regional lymphatic metastasis and overall five-year survival. Tumor biopsies of larynx tissue were collected from 50 patients who were between the ages of 30 to 80 years and had a confirmed diagnosis of squamous cell carcinoma of the larynx. Immunohistochemistry was used to determine the intracellular localization of the phosphorylated and non-phosphorylated forms of Hsp27. The study revealed that the Hsp27 chaperone was expressed in both the cytoplasm and the nucleus of tumor cells in SCCL. The biopsies of patients with lymph node metastases showed significantly higher expression of the phosphorylated and unphosphorylated forms of Hsp27 in the nucleus compared to those of patients without lymph node metastases. At the same time, the cytoplasmic expression of Hsp27 in these patients did not differ statistically. Analysis of the overall five-year survival rates showed that negative Hsp27 expression in the nucleus of tumor cells is associated with the survival rate of patients with SCCL. The nuclear expression of phosphorylated and unphosphorylated forms of Hsp27 is a molecular marker of unfavorable squamous cell carcinoma of the larynx associated with lymphogenous metastasis and decreased total five-year survival.

  4. Overexpression of CaWRKY27, a subgroup IIe WRKY transcription factor of Capsicum annuum, positively regulates tobacco resistance to Ralstonia solanacearum infection.

    PubMed

    Dang, Fengfeng; Wang, Yuna; She, Jianju; Lei, Yufen; Liu, Zhiqin; Eulgem, Thomas; Lai, Yan; Lin, Jing; Yu, Lu; Lei, Dan; Guan, Deyi; Li, Xia; Yuan, Qian; He, Shuilin

    2014-03-01

    WRKY proteins are encoded by a large gene family and are linked to many biological processes across a range of plant species. The functions and underlying mechanisms of WRKY proteins have been investigated primarily in model plants such as Arabidopsis and rice. The roles of these transcription factors in non-model plants, including pepper and other Solanaceae, are poorly understood. Here, we characterize the expression and function of a subgroup IIe WRKY protein from pepper (Capsicum annuum), denoted as CaWRKY27. The protein localized to nuclei and activated the transcription of a reporter GUS gene construct driven by the 35S promoter that contained two copies of the W-box in its proximal upstream region. Inoculation of pepper cultivars with Ralstonia solanacearum induced the expression of CaWRKY27 transcript in 76a, a bacterial wilt-resistant pepper cultivar, whereas it downregulated the expression of CaWRKY27 transcript in Gui-1-3, a bacterial wilt-susceptible pepper cultivar. CaWRKY27 transcript levels were also increased by treatments with salicylic acid (SA), methyl jasmonate (MeJA) and ethephon (ETH). Transgenic tobacco plants overexpressing CaWRKY27 exhibited resistance to R. solanacearum infection compared to that of wild-type plants. This resistance was coupled with increased transcript levels in a number of marker genes, including hypersensitive response genes, and SA-, JA- and ET-associated genes. By contrast, virus-induced gene silencing (VIGS) of CaWRKY27 increased the susceptibility of pepper plants to R. solanacearum infection. These results suggest that CaWRKY27 acts as a positive regulator in tobacco resistance responses to R. solanacearum infection through modulation of SA-, JA- and ET-mediated signaling pathways. © 2013 Scandinavian Plant Physiology Society.

  5. Inactivation and Unfolding of Protein Tyrosine Phosphatase from Thermus thermophilus HB27 during Urea and Guanidine Hydrochloride Denaturation

    PubMed Central

    Liu, Lina; Gao, Chunyan; Xu, Shui; Zhao, Ping; Xia, Qingyou

    2014-01-01

    The effects of urea and guanidine hydrochloride (GdnHCl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase), a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV) circular dichroism (CD), Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS) fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase. PMID:25255086

  6. BAG3 Is a Modular, Scaffolding Protein that physically Links Heat Shock Protein 70 (Hsp70) to the Small Heat Shock Proteins.

    PubMed

    Rauch, Jennifer N; Tse, Eric; Freilich, Rebecca; Mok, Sue-Ann; Makley, Leah N; Southworth, Daniel R; Gestwicki, Jason E

    2017-01-06

    Small heat shock proteins (sHsps) are a family of ATP-independent molecular chaperones that are important for binding and stabilizing unfolded proteins. In this task, the sHsps have been proposed to coordinate with ATP-dependent chaperones, including heat shock protein 70 (Hsp70). However, it is not yet clear how these two important components of the chaperone network are linked. We report that the Hsp70 co-chaperone, BAG3, is a modular, scaffolding factor to bring together sHsps and Hsp70s. Using domain deletions and point mutations, we found that BAG3 uses both of its IPV motifs to interact with sHsps, including Hsp27 (HspB1), αB-crystallin (HspB5), Hsp22 (HspB8), and Hsp20 (HspB6). BAG3 does not appear to be a passive scaffolding factor; rather, its binding promoted de-oligomerization of Hsp27, likely by competing for the self-interactions that normally stabilize large oligomers. BAG3 bound to Hsp70 at the same time as Hsp22, Hsp27, or αB-crystallin, suggesting that it might physically bring the chaperone families together into a complex. Indeed, addition of BAG3 coordinated the ability of Hsp22 and Hsp70 to refold denatured luciferase in vitro. Together, these results suggest that BAG3 physically and functionally links Hsp70 and sHsps. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. IFN-gamma synergizes with LPS to induce nitric oxide biosynthesis through glycogen synthase kinase-3-inhibited IL-10.

    PubMed

    Lin, Chiou-Feng; Tsai, Cheng-Chieh; Huang, Wei-Ching; Wang, Chi-Yun; Tseng, Hsiang-Chi; Wang, Yi; Kai, Jui-In; Wang, Szu-Wen; Cheng, Yi-Lin

    2008-10-15

    Interferon-gamma (IFN-gamma) plays a crucial role in innate immunity and inflammation. It causes the synergistic effect on endotoxin lipopolysaccharide (LPS)-stimulated inducible nitric oxide synthase (iNOS)/NO biosynthesis; however, the mechanism remains unclear. In the present study, we investigated the effects of glycogen synthase kinase-3 (GSK-3)-mediated inhibition of anti-inflammatory interleukin-10 (IL-10). We found, in LPS-stimulated macrophages, that IFN-gamma increased iNOS expression and NO production in a time-dependent manner. In addition, ELISA analysis showed the upregulation of tumor necrosis factor-alpha and regulated on activation, normal T expressed and secreted, and the downregulation of IL-10. RT-PCR further showed changes in the IL-10 mRNA level as well. Treating cells with recombinant IL-10 showed a decrease in IFN-gamma/LPS-induced iNOS/NO biosynthesis, whereas anti-IL-10 neutralizing antibodies enhanced this effect, suggesting that IL-10 acts in an anti-inflammatory role. GSK-3-inhibitor treatment blocked IFN-gamma/LPS-induced iNOS/NO biosynthesis but upregulated IL-10 production. Inhibiting GSK-3 using short-interference RNA showed similar results. Additionally, treating cells with anti-IL-10 neutralizing antibodies blocked these effects. We further showed that inhibiting GSK-3 increased phosphorylation of transcription factor cyclic AMP response element binding protein. Inhibiting protein tyrosine kinase Pyk2, an upstream regulator of GSK-3beta, caused inhibition on IFN-gamma/LPS-induced GSK-3beta phosphorylation at tyrosine 216 and iNOS/NO biosynthesis. Taken together, these findings reveal the involvement of GSK-3-inhibited IL-10 on the induction of iNOS/NO biosynthesis by IFN-gamma synergized with LPS. (c) 2008 Wiley-Liss, Inc.

  8. Interleukin-27 is a novel candidate diagnostic biomarker for bacterial infection in critically ill children.

    PubMed

    Wong, Hector R; Cvijanovich, Natalie Z; Hall, Mark; Allen, Geoffrey L; Thomas, Neal J; Freishtat, Robert J; Anas, Nick; Meyer, Keith; Checchia, Paul A; Lin, Richard; Bigham, Michael T; Sen, Anita; Nowak, Jeffrey; Quasney, Michael; Henricksen, Jared W; Chopra, Arun; Banschbach, Sharon; Beckman, Eileen; Harmon, Kelli; Lahni, Patrick; Shanley, Thomas P

    2012-10-29

    Differentiating between sterile inflammation and bacterial infection in critically ill patients with fever and other signs of the systemic inflammatory response syndrome (SIRS) remains a clinical challenge. The objective of our study was to mine an existing genome-wide expression database for the discovery of candidate diagnostic biomarkers to predict the presence of bacterial infection in critically ill children. Genome-wide expression data were compared between patients with SIRS having negative bacterial cultures (n = 21) and patients with sepsis having positive bacterial cultures (n = 60). Differentially expressed genes were subjected to a leave-one-out cross-validation (LOOCV) procedure to predict SIRS or sepsis classes. Serum concentrations of interleukin-27 (IL-27) and procalcitonin (PCT) were compared between 101 patients with SIRS and 130 patients with sepsis. All data represent the first 24 hours of meeting criteria for either SIRS or sepsis. Two hundred twenty one gene probes were differentially regulated between patients with SIRS and patients with sepsis. The LOOCV procedure correctly predicted 86% of the SIRS and sepsis classes, and Epstein-Barr virus-induced gene 3 (EBI3) had the highest predictive strength. Computer-assisted image analyses of gene-expression mosaics were able to predict infection with a specificity of 90% and a positive predictive value of 94%. Because EBI3 is a subunit of the heterodimeric cytokine, IL-27, we tested the ability of serum IL-27 protein concentrations to predict infection. At a cut-point value of ≥5 ng/ml, serum IL-27 protein concentrations predicted infection with a specificity and a positive predictive value of >90%, and the overall performance of IL-27 was generally better than that of PCT. A decision tree combining IL-27 and PCT improved overall predictive capacity compared with that of either biomarker alone. Genome-wide expression analysis has provided the foundation for the identification of IL-27 as a novel

  9. Interleukin-27 is a novel candidate diagnostic biomarker for bacterial infection in critically ill children

    PubMed Central

    2012-01-01

    Introduction Differentiating between sterile inflammation and bacterial infection in critically ill patients with fever and other signs of the systemic inflammatory response syndrome (SIRS) remains a clinical challenge. The objective of our study was to mine an existing genome-wide expression database for the discovery of candidate diagnostic biomarkers to predict the presence of bacterial infection in critically ill children. Methods Genome-wide expression data were compared between patients with SIRS having negative bacterial cultures (n = 21) and patients with sepsis having positive bacterial cultures (n = 60). Differentially expressed genes were subjected to a leave-one-out cross-validation (LOOCV) procedure to predict SIRS or sepsis classes. Serum concentrations of interleukin-27 (IL-27) and procalcitonin (PCT) were compared between 101 patients with SIRS and 130 patients with sepsis. All data represent the first 24 hours of meeting criteria for either SIRS or sepsis. Results Two hundred twenty one gene probes were differentially regulated between patients with SIRS and patients with sepsis. The LOOCV procedure correctly predicted 86% of the SIRS and sepsis classes, and Epstein-Barr virus-induced gene 3 (EBI3) had the highest predictive strength. Computer-assisted image analyses of gene-expression mosaics were able to predict infection with a specificity of 90% and a positive predictive value of 94%. Because EBI3 is a subunit of the heterodimeric cytokine, IL-27, we tested the ability of serum IL-27 protein concentrations to predict infection. At a cut-point value of ≥5 ng/ml, serum IL-27 protein concentrations predicted infection with a specificity and a positive predictive value of >90%, and the overall performance of IL-27 was generally better than that of PCT. A decision tree combining IL-27 and PCT improved overall predictive capacity compared with that of either biomarker alone. Conclusions Genome-wide expression analysis has provided the foundation

  10. Structural deformation upon protein-protein interaction: A structural alphabet approach

    PubMed Central

    Martin, Juliette; Regad, Leslie; Lecornet, Hélène; Camproux, Anne-Claude

    2008-01-01

    Background In a number of protein-protein complexes, the 3D structures of bound and unbound partners significantly differ, supporting the induced fit hypothesis for protein-protein binding. Results In this study, we explore the induced fit modifications on a set of 124 proteins available in both bound and unbound forms, in terms of local structure. The local structure is described thanks to a structural alphabet of 27 structural letters that allows a detailed description of the backbone. Using a control set to distinguish induced fit from experimental error and natural protein flexibility, we show that the fraction of structural letters modified upon binding is significantly greater than in the control set (36% versus 28%). This proportion is even greater in the interface regions (41%). Interface regions preferentially involve coils. Our analysis further reveals that some structural letters in coil are not favored in the interface. We show that certain structural letters in coil are particularly subject to modifications at the interface, and that the severity of structural change also varies. These information are used to derive a structural letter substitution matrix that summarizes the local structural changes observed in our data set. We also illustrate the usefulness of our approach to identify common binding motifs in unrelated proteins. Conclusion Our study provides qualitative information about induced fit. These results could be of help for flexible docking. PMID:18307769

  11. Structural deformation upon protein-protein interaction: a structural alphabet approach.

    PubMed

    Martin, Juliette; Regad, Leslie; Lecornet, Hélène; Camproux, Anne-Claude

    2008-02-28

    In a number of protein-protein complexes, the 3D structures of bound and unbound partners significantly differ, supporting the induced fit hypothesis for protein-protein binding. In this study, we explore the induced fit modifications on a set of 124 proteins available in both bound and unbound forms, in terms of local structure. The local structure is described thanks to a structural alphabet of 27 structural letters that allows a detailed description of the backbone. Using a control set to distinguish induced fit from experimental error and natural protein flexibility, we show that the fraction of structural letters modified upon binding is significantly greater than in the control set (36% versus 28%). This proportion is even greater in the interface regions (41%). Interface regions preferentially involve coils. Our analysis further reveals that some structural letters in coil are not favored in the interface. We show that certain structural letters in coil are particularly subject to modifications at the interface, and that the severity of structural change also varies. These information are used to derive a structural letter substitution matrix that summarizes the local structural changes observed in our data set. We also illustrate the usefulness of our approach to identify common binding motifs in unrelated proteins. Our study provides qualitative information about induced fit. These results could be of help for flexible docking.

  12. Unphosphorylated HSP27 (HSPB1) regulates the translation initiation process via a direct association with eIF4E in osteoblasts.

    PubMed

    Kuroyanagi, Gen; Tokuda, Haruhiko; Yamamoto, Naohiro; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu

    2015-09-01

    Heat-shock protein 27 (HSP27/HSPB1) and its phosphorylation are implicated in multiple physiological and pathophysiological cell functions. Our previous study reported that unphosphorylated HSP27 has an inhibitory role in triiodothyronine (T(3))‑induced osteocalcin (OC) synthesis in osteoblasts. However, the mechanisms behind the HSP27‑mediated effects on osteoblasts remain to be clarified. In the present study, to investigate the exact mechanism of HSP27 and its phosphorylation in osteoblasts, the molecular targets of HSP27 were explored using osteoblast‑like MC3T3‑E1 cells. The levels of OC mRNA induced by T(3) in the HSP27‑overexpressing cells did not show any significant differences compared with those in the control empty vector‑transfected cells. Therefore, the interactions between HSP27 and translational molecules were focused on, including eukaryotic translation initiation factor 4E (eIF4E), eIF4G and 4E‑binding protein 1 (4E‑BP1). The HSP27 protein in the unstimulated cells co‑immunoprecipitated with eIF4E, but not eIF4G or 4E‑BP1. In addition, the association of eIF4E with 4E‑BP1 was observed in the HSP27‑overexpressing cells, as well as in the control cells. Under T(3) stimulation, the binding of eIF4E to eIF4G was markedly attenuated in the HSP27‑overexpressing cells compared with the control cells. In addition, the binding of HSP27 to eIF4E in the unstimulated cells was diminished by the phosphorylation of HSP27. In response to T(3) stimulation, the association of eIF4E with eIF4G in the unphosphorylatable HSP27‑overexpressing cells was markedly reduced compared with the phospho‑mimic HSP27‑overexpressing cells. Taken together, these findings strongly suggest that unphosphorylated HSP27 associates with eIF4E in osteoblasts and suppresses the translation initiation process.

  13. Long (27-nucleotides) small inhibitory RNAs targeting E6 protein eradicate effectively the cervical cancer cells harboring human papilloma virus.

    PubMed

    Cho, Jun Sik; Lee, Shin-Wha; Kim, Yong-Man; Kim, Dongho; Kim, Dae-Yeon; Kim, Young-Tak

    2015-05-01

    This study was to identify small inhibitory RNAs (siRNAs) that are effective in inhibiting growth of cervical cancer cell lines harboring human papilloma virus (HPV) and to examine how siRNAs interact with interferon beta (IFN-β) and thimerosal. The HPV18-positive HeLa and C-4I cell lines were used. Four types of siRNAs were designed according to their target (both E6 and E7 vs. E6 only) and sizes (21- vs. 27-nucleotides); Ex-18E6/21, Ex-18E6/27, Sp-18E6/21, and Sp-18E6/27. Each siRNA-transfected cells were cultured with or without IFN-b and thimerosal and their viability was measured. The viabilities of HPV18-positive tumor cells were reduced by 21- and 27-nucleotide siRNAs in proportion to the siRNA concentrations. Of the two types of siRNAs, the 27-nucleotide siRNA constructs showed greater inhibitory efficacy. Sp-18E6 siRNAs, which selectively downregulates E6 protein only, were more effective than the E6- and E7-targeting Ex-18E6 siRNAs. siRNAs and IFN-β showed the synergistic effect to inhibit HeLa cell survival and the effect was proportional to both siRNA and IFN-β concentrations. Thimerosal in the presence of siRNA exerted a dose-dependent inhibition of C-4I cell survival. Finally, co-treatment with siRNA, IFN-β, and thimerosal induced the most profound decrease in the viability of both cell lines. Long (27-nucleotides) siRNAs targeting E6-E7 mRNAs effectively reduce the viability of HPV18-positive cervical cancer cells and show the synergistic effect in combination with IFN-b and thimerosal. It is necessary to find the rational design of siRNAs and effective co-factors to eradicate particular cervical cancer.

  14. A Newly Isolated Penicillium oxalicum 16 Cellulase with High Efficient Synergism and High Tolerance of Monosaccharide.

    PubMed

    Zhao, Xi-Hua; Wang, Wei; Tong, Bin; Zhang, Su-Ping; Wei, Dong-Zhi

    2016-01-01

    Compared to Trichoderma reesei RUT-C30 cellulase (Trcel), Penicillium oxalicum 16 cellulase (P16cel) from the fermentation supernatant produced a 2-fold higher glucose yield when degrading microcrystalline cellulose (MCC), possessed a 10-fold higher β-glucosidase (BGL) activity, but obtained somewhat lower other cellulase component activities. The optimal temperature and pH of β-1,4-endoglucanase, cellobiohydrolase, and filter paperase from P16cel were 50-60 °C and 4-5, respectively, but those of BGL reached 70 °C and 5. The cellulase cocktail of P16cel and Trcel had a high synergism when solubilizing MCC and generated 1.7-fold and 6.2-fold higher glucose yields than P16cel and Trcel at the same filter paperase loading, respectively. Additional low concentration of fructose enhanced the glucose yield during enzymatic hydrolysis of MCC; however, additional high concentration of monosaccharide (especially glucose) reduced cellulase activities and gave a stronger monosaccharide inhibition on Trcel. These results indicate that P16cel is a more excellent cellulase than Trcel.

  15. Molecular chaperone Hsp27 regulates the Hippo tumor suppressor pathway in cancer

    PubMed Central

    Vahid, Sepideh; Thaper, Daksh; Gibson, Kate F.; Bishop, Jennifer L.; Zoubeidi, Amina

    2016-01-01

    Heat shock protein 27 (Hsp27) is a molecular chaperone highly expressed in aggressive cancers, where it is involved in numerous pro-tumorigenic signaling pathways. Using functional genomics we identified for the first time that Hsp27 regulates the gene signature of transcriptional co-activators YAP and TAZ, which are negatively regulated by the Hippo Tumor Suppressor pathway. The Hippo pathway inactivates YAP by phosphorylating and increasing its cytoplasmic retention with the 14.3.3 proteins. Gain and loss of function experiments in prostate, breast and lung cancer cells showed that Hsp27 knockdown induced YAP phosphorylation and cytoplasmic localization while overexpression of Hsp27 displayed opposite results. Mechanistically, Hsp27 regulates the Hippo pathway by accelerating the proteasomal degradation of ubiquitinated MST1, the core Hippo kinase, resulting in reduced phosphorylation/activity of LATS1 and MOB1, its downstream effectors. Importantly, our in vitro results were supported by data from human tumors; clinically, high expression of Hsp27 in prostate tumors is correlated with increased expression of YAP gene signature and reduced phosphorylation of YAP in lung and invasive breast cancer clinical samples. This study reveals for the first time a link between Hsp27 and the Hippo cascade, providing a novel mechanism of deregulation of this tumor suppressor pathway across multiple cancers. PMID:27555231

  16. Effect of HSP27 on Human Breast Tumor Cell Growth and Motility.

    DTIC Science & Technology

    1997-09-01

    the small heat shock protein, Hsp27 , on growth and motility characteristics of human mammary tumor cell lines. Since Hsp27 regulates actin...microfilament dynamics, we hypothesize that cells expressing high levels of Hsp27 will show increased motility and altered chemotactic properties, in addition to...significantly elevated levels of Hsp27 has proven to be daunting. Down regulation of Hsp27 levels in MCF7 cells using antisense technology has also

  17. Heterodimeric BMP-2/7 Antagonizes the Inhibition of All-Trans Retinoic Acid and Promotes the Osteoblastogenesis

    PubMed Central

    Bi, Wenjuan; Gu, Zhiyuan; Zheng, Yuanna; Zhang, Xiao; Guo, Jing; Wu, Gang

    2013-01-01

    Objectives Hypervitaminosis A and alcoholism can result in a low mineral density and compromised regenerative capacity of bone, thus delaying implant osteointegration. The inhibitory effect of all-trans retinoic acid on osteoblastogenesis is considered to be one of the mechanisms. We hypothesized that heterodimeric bone morphogenetic protein-2/7 could antagonize all-trans retinoic acid and enhance osteoblastogenesis, with an aim to accelerate and enhance bone regeneration and implant osteointegration. Materials and Methods We applied 5 ng/ml or 50 ng/ml bone morphogenetic protein-2/7 to restore the osteoblastogenesis of pre-osteoblasts (MC3T3-E1 cell line) that was inhibited by 1 µM all-trans retinoic acid. We evaluated the efficacy by assessing cell numbers (proliferation), alkaline phosphatase activity (a marker for early differentiation), osteocalcin (a marker for late differentiation), calcium deposition (a marker for final mineralization) and the expression of osteoblastogenic genes (such as Runx2, Collagen Ia, alkaline phosphatase and osteocalcin) at different time points. Results All-trans retinoic acid significantly inhibited the expression of all the tested osteoblastogenic genes and proteins except alkaline phosphatase activity. In the presence of ATRA, 50 ng/ml bone morphogenetic protein-2/7 not only completely restored but also significantly enhanced all the osteoblastogenic genes and proteins. On the 28th day, mineralization was completely inhibited by all-trans retinoic acid. In contrast, 50 ng/ml BMP-2/7 could antagonize ATRA and significantly enhance the mineralization about 2.5 folds in comparison with the control treatment (no ATRA, no BMP2/7). Conclusions Heterodimeric bone morphogenetic protein-2/7 bears a promising application potential to significantly promote bone regeneration and implant osteointegration for the patients with hypervitaminosis A and alcoholism. PMID:24205156

  18. RIPK4 phosphorylates Dishevelled proteins to regulate canonical Wnt signaling

    PubMed Central

    Huang, XiaoDong; McGann, James C.; Liu, Bob Y.; Hannoush, Rami N.; Lill, Jennie R.; Pham, Victoria; Newton, Kim; Kakunda, Michael; Liu, Jinfeng; Yu, Christine; Hymowitz, Sarah G.; Hongo, Jo-Anne; Wynshaw-Boris, Anthony; Polakis, Paul; Harland, Richard M.; Dixit, Vishva M.

    2014-01-01

    Receptor interacting protein kinase 4 (RIPK4) is required for epidermal differentiation (1–4) and is mutated in Bartsocas-Papas syndrome (5, 6). While RIPK4 binds protein kinase C (5, 6), RIPK4 signaling mechanisms are largely unknown. We show that ectopic RIPK4 induces cytosolic β-catenin accumulation and a transcriptional program similar to Wnt3a, whereas kinase-defective or Bartsocas-Papas syndrome RIPK4 mutants do not. Ectopic ripk4 synergized with Wnt family member xwnt8 in Xenopus, whereas ripk4 morpholinos or kinase-defective RIPK4 antagonized Wnt signaling. Mechanistically, RIKP4 interacted constitutively with the Wnt adaptor protein DVL2 and, after Wnt3a stimulation, with the co-receptor LRP6. Phosphorylation of DVL2 at Ser298 and Ser480 by RIPK4 favored canonical Wnt signaling. Growth of a Wnt-dependent N-Tera2 xenograft tumor model was suppressed by RIPK4 knockdown, suggesting that RIPK4 overexpression may contribute to the growth of certain tumor types. PMID:23371553

  19. Cytochrome c conjugated to ZnO-MAA nanoparticles: the study of interaction and influence on protein structure.

    PubMed

    Simšíková, Michaela; Antalík, Marián; Kaňuchová, Mária; Skvarla, Jiří

    2013-08-01

    Nanoparticle-protein conjugates have potential for numerous applications due to the combination of the properties of both components. In this paper we studied the conjugation of horse heart cytochrome c with ZnO nanoparticles modified by mercaptoacetic acid (MAA) which may be a material with great potential in anticancer therapy as a consequence of synergic effect of both components. Cyt c adsorption to the ZnO-MAA NPs surface was studied by UV-vis spectroscopy and by a dynamic light scattering in various pH. The results indicate that the optimal pH for the association of protein with modified nanoparticles is in range 5.8-8.5 where 90-96% of cytochrome c was assembled on ZnO-MAA nanoparticles. The interaction of proteins with nanoparticles often results in denaturation or loss of protein function. Our observations from UV-vis spectroscopy and circular dichroism performed preserved protein structure after the interaction with modified nanoparticles. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Synergism between PKCδ regulators hypericin and rottlerin enhances apoptosis in U87 MG glioma cells after light stimulation.

    PubMed

    Misuth, Matus; Horvath, Denis; Miskovsky, Pavol; Huntosova, Veronika

    2017-06-01

    Gliomas belong to the most infiltrative types of tumors. Photodynamic therapy (PDT) can be applied to regulate glioma cell proliferation. The inhibitors of PKCs (Protein Kinase C) are very promising drugs that can mediate glioma cells apoptosis in PDT. Hypericin is one of PKCs regulators, and thanks to its physicochemical properties it can be used in PDT. Rottlerin is also considered to be the PKCδ inhibitor. Its implementation in PDT may significantly influence glioma cells response to PDT. The viability of U87 MG glioma cells in the presence of rottlerin and hypericin was assessed by MTT assay and flow cytometry in the absence and presence of light. The flow cytometric data were analyzed through Shannon entropy. The oxidative stress and immunocytochemistry of PKCδ and phosphorylated Bcl-2 (the regulators of apoptosis) were observed using fluorescence microscopy. A pretreatment of glioma cells with rottlerin before hypericin induced PDT led to significant increase in apoptosis accompanied by the decrease of intracellular oxidative stress and increase of phosphorylated Bcl-2 in the cytoplasm of U87 MG cells. In conclusion, we assume that the synergism between rottlerin and hypericin leads firstly to activation of rescue mechanisms in the glioma cells, but finally this cooperation triggers apoptosis rather than necrosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Expression of Hsp27 correlated with rat detrusor contraction after acute urinary retention.

    PubMed

    Xiong, Zhiyong; Wang, Yongquan; Gong, Wei; Zhou, Zhansong; Lu, Gensheng

    2013-09-01

    Heat shock protein 27 (Hsp27) can regulate actin cytoskeleton dynamics and contractile protein activation. This study investigates whether Hsp27 expression is related to bladder contractile dysfunction after acute urinary retention (AUR). Female rats were randomized either to AUR by urethral ligation or to normal control group. Bladder and smooth muscle strip contraction at time points from 0 h to 7 days after AUR were estimated by cystometric and organ bath studies. Hsp27 expression in bladder tissue at each time point was detected with immunofluorescence, Western blots, and real-time PCR. Expression of the three phosphorylated forms of Hsp27 was detected by Western blots. Smooth muscle ultrastructure was observed by transmission electron microscopy. Data suggest that maximum detrusor pressure and both carbachol-induced and spontaneous detrusor strip contraction amplitude decreased gradually for the duration from 0 to 6 h, and then increased gradually to near-normal values at 24 h. Treatment of muscle strips with the p38MAK inhibitor, SB203580, inhibited carbachol-induced contractions. Smooth muscle ultrastructure damage was the highest at 6 h after AUR, and then lessened gradually during next 7 days, and ultrastructure was close to normal. Expressions of Hsp27 mRNA and protein and the proteins of the three phosphorylated forms were higher at 0 h, decreased to lower levels up to 6 h, and then gradually increased. Therefore, we conclude that rat bladder contractile function after AUR worsens during 0-6 h, and then gradually recovers. The findings of the current study suggest that Hsp27 modulates bladder smooth muscle contraction after AUR, and that phosphorylation of Hsp27 may be an important pathway modulating actin cytoskeleton dynamics in bladder smooth muscle contraction and reconstruction after injury.

  2. Synergism of herpes simplex virus and tobacco-specific N'-nitrosamines in cell transformation

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

    Park, N.H.; Dokko, H.; Li, S.L.

    1991-03-01

    Previous studies indicate that herpes simplex virus (HSV) enhances the carcinogenic activity of smokeless tobacco and tobacco-related chemical carcinogens in animals. Since tobacco-specific N'-nitrosamines (TSNAs) such as N'-nitrosonornicotine (NNN) and 4-(N-methyl-N'-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are major chemical carcinogens of smokeless tobacco and are known to be responsible for the development of oral cancers in smokeless tobacco users, the combined effects of TSNAs and HSV in cell transformation were investigated. Exposure of cells to NNN or NNK followed by virus infection resulted in a significant enhancement of transformation frequency when compared with that observed with chemical carcinogens or virus alone. This study suggestsmore » that TSNAs and HSV can interact together and show synergism in cell transformation.« less

  3. [Immunohistochemical analysis of cell cycle-regulating protein (p21, p27 and Ki67) expression in endoscopic biopsy samples from patients with gastroesophageal reflux disease].

    PubMed

    Koyama, Shigeki; Nishiyama, Yorihiro; Ishizuka, Izumi

    2007-05-01

    We performed an immunohistochemical analysis of cell cycle-regulating protein (p21, p27 and Ki67) expression in endoscopic biopsy samples from the patients with gastroesophageal reflux disease (GERD) using angled -biopsy forceps. Inflammatory cell accumulation into the lamina propria was detected even in patients with modified Los Angeles (LA) system grades N or M. In grade N or M patients with no changes in the epithelium, the area of p21, p27 and Ki67 positive cells was expanded compared to normal mucosa. The area of p21, p27 and Ki67 positive cells tended to expand upward in the epithelium with GERD severity based on the LA classification grading. These indicate that inflammatory cell infiltration into the lamina propria is initial histological change of GERD.

  4. A chemical proteomics approach reveals Hsp27 as a target for proapoptotic clerodane diterpenes.

    PubMed

    Faiella, Laura; Piaz, Fabrizio Dal; Bisio, Angela; Tosco, Alessandra; De Tommasi, Nunziatina

    2012-10-01

    Clerodane diterpenoids are a class of naturally occurring molecules widely distributed in the Lamiaceae family. Neo-clerodane diterpenoids from Salvia ssp were recently described as compounds inhibiting the proliferation of human cancer cell lines. To gain new insights into molecular mechanism(s) underlying the antitumor potential of this class of compounds, we used a chemical proteomics approach to analyse the cellular interactome of hardwickiic acid (HAA) selected as a representative molecule. HAA was linked to an opportune 1,1'-carbonyldiimidazole modified by 1,12-dodecanediamine and then immobilized on a matrix support. The modified beads were then used as bait for fishing the potential partners of HAA in a U937 cell lysate. We identified heat shock protein 27 (Hsp27), an ATP-independent antiapoptotic chaperone characterized for its tumorigenic and metastatic properties and now referenced as a major therapeutic target in many types of cancer, as a major HAA partner. Here, we also report the study of HAA-Hsp27 interaction by means of a panel of chemical and biological approaches, including surface plasmon resonance measurements limited proteolysis, and biochemical assays. Our data suggest that HAA could provide a potential tool to develop strategies for the discovery of Hsp27 chemical inhibitors.

  5. Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates - A Substudy.

    PubMed

    Hursel, Rick; Martens, Eveline A P; Gonnissen, Hanne K J; Hamer, Henrike M; Senden, Joan M G; van Loon, Luc J C; Westerterp-Plantenga, Margriet S

    2015-01-01

    Based on controlled 36 h experiments a higher dietary protein intake causes a positive protein balance and a negative fat balance. A positive net protein balance may support fat free mass accrual. However, few data are available on the impact of more prolonged changes in habitual protein intake on whole-body protein metabolism and basal muscle protein synthesis rates. To assess changes in whole-body protein turnover and basal muscle protein synthesis rates following 12 weeks of adaptation to a low versus high dietary protein intake. A randomized parallel study was performed in 40 subjects who followed either a high protein (2.4 g protein/kg/d) or low protein (0.4 g protein/kg/d) energy-balanced diet (30/35/35% or 5/60/35% energy from protein/carbohydrate/fat) for a period of 12 weeks. A subgroup of 7 men and 8 women (body mass index: 22.8±2.3 kg/m2, age: 24.3±4.9 y) were selected to evaluate the impact of prolonged adaptation to either a high or low protein intake on whole body protein metabolism and basal muscle protein synthesis rates. After the diet, subjects received continuous infusions with L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosine in an overnight fasted state, with blood samples and muscle biopsies being collected to assess post-absorptive whole-body protein turnover and muscle protein synthesis rates in vivo in humans. After 12 weeks of intervention, whole-body protein balance in the fasted state was more negative in the high protein treatment when compared with the low protein treatment (-4.1±0.5 vs -2.7±0.6 μmol phenylalanine/kg/h;P<0.001). Whole-body protein breakdown (43.0±4.4 vs 37.8±3.8 μmol phenylalanine/kg/h;P<0.03), synthesis (38.9±4.2 vs 35.1±3.6 μmol phenylalanine/kg/h;P<0.01) and phenylalanine hydroxylation rates (4.1±0.6 vs 2.7±0.6 μmol phenylalanine/kg/h;P<0.001) were significantly higher in the high vs low protein group. Basal muscle protein synthesis rates were maintained on a low vs high protein diet (0.042±0.01 vs 0

  6. Development and validation of a general approach to predict and quantify the synergism of anti-cancer drugs using experimental design and artificial neural networks.

    PubMed

    Pivetta, Tiziana; Isaia, Francesco; Trudu, Federica; Pani, Alessandra; Manca, Matteo; Perra, Daniela; Amato, Filippo; Havel, Josef

    2013-10-15

    The combination of two or more drugs using multidrug mixtures is a trend in the treatment of cancer. The goal is to search for a synergistic effect and thereby reduce the required dose and inhibit the development of resistance. An advanced model-free approach for data exploration and analysis, based on artificial neural networks (ANN) and experimental design is proposed to predict and quantify the synergism of drugs. The proposed method non-linearly correlates the concentrations of drugs with the cytotoxicity of the mixture, providing the possibility of choosing the optimal drug combination that gives the maximum synergism. The use of ANN allows for the prediction of the cytotoxicity of each combination of drugs in the chosen concentration interval. The method was validated by preparing and experimentally testing the combinations with the predicted highest synergistic effect. In all cases, the data predicted by the network were experimentally confirmed. The method was applied to several binary mixtures of cisplatin and [Cu(1,10-orthophenanthroline)2(H2O)](ClO4)2, Cu(1,10-orthophenanthroline)(H2O)2(ClO4)2 or [Cu(1,10-orthophenanthroline)2(imidazolidine-2-thione)](ClO4)2. The cytotoxicity of the two drugs, alone and in combination, was determined against human acute T-lymphoblastic leukemia cells (CCRF-CEM). For all systems, a synergistic effect was found for selected combinations. © 2013 Elsevier B.V. All rights reserved.

  7. A novel pathogenic mutation of the CYP27B1 gene in a patient with vitamin D-dependent rickets type 1: a case report.

    PubMed

    Babiker, Amir M I; Al Gadi, Iman; Al-Jurayyan, Nasir A M; Al Nemri, Abdulrahman M H; Al Haboob, Ali Abdu N; Al Boukai, Ahmed Amer; Al Zahrani, Ali; Habib, Hanan Ahmed

    2014-11-05

    Rickets can occur due to Vitamin D deficiency or defects in its metabolism. Three rare genetic types of rickets with different alterations of genes have been reported, including: Vitamin D dependent rickets type 1, Vitamin D dependent rickets type 2 or also known as Vitamin D resistant rickets and 25 hydroxylase deficiency rickets. Vitamin D dependent rickets type 1 is inherited in an autosomal recessive pattern, and is caused by mutations in the CYP27B1 gene encoding the 1α-hydroxylase enzyme. We report here a new mutation in CYP27B1, which lead to Vitamin D dependent rickets type 1. We report on a 13-month-old Arabic Saudi girl with Vitamin D dependent rickets type 1 presented with multiple fractures and classic features of rickets. A whole exome sequencing identified a novel pathogenic missense mutation (CYP27B1:Homozygous c.1510C > T(p.Q504X)) which results in a protein truncating alteration. Both parents are heterozygous carriers of the mutation. Based on data search in Human Gene Mutation Database, 63 CYP27B1 alterations were reported: only 28.6% are protein truncating (5 nonsense, 13 frameshift insertions/deletions, 0 gross deletions), while 61.9% are non-truncating (38 missense, 1 small in-frame insertions/deletion), and 9.5% are possible protein-truncating (5 splice, 1 regulatory). The deleterious effect of this alteration, which was the only mutation detected in the CYP27B1 common gene of Vitamin D dependent rickets type 1 in the proband, and its autosomal recessive inheritance fashion, both support a pathogenic nature of this mutation as the cause of Vitamin D dependent rickets type 1.

  8. JMJ27, an Arabidopsis H3K9 histone demethylase, modulates defense against Pseudomonas syringae and flowering time.

    PubMed

    Dutta, Aditya; Choudhary, Pratibha; Caruana, Julie; Raina, Ramesh

    2017-09-01

    Histone methylation is known to dynamically regulate diverse developmental and physiological processes. Histone methyl marks are written by methyltransferases and erased by demethylases, and result in modification of chromatin structure to repress or activate transcription. However, little is known about how histone methylation may regulate defense mechanisms and flowering time in plants. Here we report characterization of JmjC DOMAIN-CONTAINING PROTEIN 27 (JMJ27), an Arabidopsis JHDM2 (JmjC domain-containing histone demethylase 2) family protein, which modulates defense against pathogens and flowering time. JMJ27 is a nuclear protein containing a zinc-finger motif and a catalytic JmjC domain with conserved Fe(II) and α-ketoglutarate binding sites, and displays H3K9me1/2 demethylase activity both in vitro and in vivo. JMJ27 is induced in response to virulent Pseudomonas syringae pathogens and is required for resistance against these pathogens. JMJ27 is a negative modulator of WRKY25 (a repressor of defense) and a positive modulator of several pathogenesis-related (PR) proteins. Additionally, loss of JMJ27 function leads to early flowering. JMJ27 negatively modulates the major flowering regulator CONSTANS (CO) and positively modulates FLOWERING LOCUS C (FLC). Taken together, our results indicate that JMJ27 functions as a histone demethylase to modulate both physiological (defense) and developmental (flowering time) processes in Arabidopsis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  9. Suppression of HSP27 Restores Retinal Function and Protects Photoreceptors From Apoptosis in a Light-Induced Retinal Degeneration Animal Model.

    PubMed

    Chien, Chih-Cheng; Huang, Chi-Jung; Tien, Lu-Tai; Cheng, Yu-Che; Ke, Chia-Ying; Lee, Yih-Jing

    2017-06-01

    We used a light-induced retinal degeneration animal model to investigate possible roles of heat shock protein 27 (HSP27) in retinal/photoreceptor protection. Sprague-Dawley rats were used for the light-induced retinal degeneration animal model. The histology of eye sections was observed for morphologic changes in the retina. Cell apoptosis was examined in each group using the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and electroretinography was used to evaluate retinal function. Protein and mRNA expression levels of different retinal cell markers were also detected through immunofluorescence staining, Western blotting, and real-time PCR. The thickness of the outer nuclear layer significantly decreased after 7-day light exposure. Moreover, we injected a viral vector for silencing HSP27 expression into the eyes and observed that photoreceptors were better preserved in the HSP27-suppressed (sHSP27) retina 2 weeks after injection. HSP27 suppression also reduced retinal cell apoptosis caused by light exposure. In addition, the loss of retinal function caused by light exposure was reversed on suppressing HSP27 expression. We subsequently found that the expression of the Rho gene and immunofluorescence staining of rhodopsin and arrestin (cell markers for photoreceptors) increased in sHSP27-treated retinas. HSP27 suppression did not affect the survival of ganglion and amacrine cells. Retinal cell apoptosis and functional loss were observed after 7-day light exposure. However, in the following 2 weeks after light exposure, HSP27 suppression may initiate a protective effect for retinal cells, particularly photoreceptors, from light-induced retinal degeneration.

  10. Synergic chemoprevention with dietary carbohydrate restriction and supplementation of AMPK-activating phytochemicals: the role of SIRT1.

    PubMed

    Lee, Jong Doo; Choi, Min-Ah; Ro, Simon Weonsang; Yang, Woo Ick; Cho, Arthur E H; Ju, Hye-Lim; Baek, Sinhwa; Chung, Sook In; Kang, Won Jun; Yun, Mijin; Park, Jeon Han

    2016-01-01

    Calorie restriction or a low-carbohydrate diet (LCD) can increase life span in normal cells while inhibiting carcinogenesis. Various phytochemicals also have calorie restriction-mimetic anticancer properties. We investigated whether an isocaloric carbohydrate-restriction diet and AMP-activated protein kinase (AMPK)-activating phytochemicals induce synergic tumor suppression. We used a mixture of AMPK-activating phytochemical extracts including curcumin, quercetin, catechins, and resveratrol. Survival analysis was carried out in a B16F10 melanoma model fed a control diet (62.14% kcal carbohydrate, 24.65% kcal protein and 13.2% kcal fat), a control diet with multiple phytochemicals (MP), LCD (16.5, 55.2, and 28.3% kcal, respectively), LCD with multiple phytochemicals (LCDmp), a moderate-carbohydrate diet (MCD, 31.9, 62.4, and 5.7% kcal, respectively), or MCD with phytochemicals (MCDmp). Compared with the control group, MP, LCD, or MCD intervention did not produce survival benefit, but LCDmp (22.80±1.58 vs. 28.00±1.64 days, P=0.040) and MCDmp (23.80±1.08 vs. 30.13±2.29 days, P=0.008) increased the median survival time significantly. Suppression of the IGF-1R/PI3K/Akt/mTOR signaling, activation of the AMPK/SIRT1/LKB1pathway, and NF-κB suppression were the critical tumor-suppression mechanisms. In addition, SIRT1 suppressed proliferation of the B16F10 and A375SM cells under a low-glucose condition. Alterations in histone methylation within Pten and FoxO3a were observed after the MCDmp intervention. In the transgenic liver cancer model developed by hydrodynamic transfection of the HrasG12V and shp53, MCDmp and LCDmp interventions induced significant cancer-prevention effects. Microarray analysis showed that PPARα increased with decreased IL-6 and NF-κB within the hepatocytes after an MCDmp intervention. In conclusion, an isocaloric carbohydrate-restriction diet and natural AMPK-activating agents induce synergistic anticancer effects. SIRT1 acts as a

  11. A UBI 31-38 Peptide-coumarin Conjugate: Photophysical Features, Imaging Tracking and Synergism with Amphotericin B Against Cryptococcus.

    PubMed

    Ferreira, Soraya M Z M D; Carneiro, Hellem C; Alves, Rosemeire B; Batista, Ana Carolina S; da Silva Junior, Eufranio N; Dias, Gleiston G; Resende, Jarbas M; Santos, Daniel A; Oliveira, Debora L; Rodrigues, Marcio L; Freitas, Rossimiriam P

    2018-01-01

    Cryptococcosis is a fungal disease of global significance for which new effective treatments are needed. The conjugation of the synthetic antimicrobial peptide fragment UBI 31-38 to a coumarin derivative showed to be an effective approach for the design of a novel anticryptococcal agent. In addition to antifungal activity, the conjugate exhibited intense fluorescence, which could be valuable for mechanistic investigations of this molecule. In this work, we studied the photophysical properties of the conjugate and confocal scanning laser microscopy was used to inspect the distribution of the peptide-coumarin conjugate in Cryptococcus cell. The synergism of this compound with amphotericin B or fluconazole against C. gattii and C. neoformans strains was also investigated. The results indicated that the fluorescent conjugate alone as well as its combination with amphotericin B are promising tools against cryptococcosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. The small heat shock proteins αB-crystallin (HSPB5) and Hsp27 (HSPB1) inhibit the intracellular aggregation of α-synuclein.

    PubMed

    Cox, Dezerae; Ecroyd, Heath

    2017-07-01

    Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. Proteostasis is preserved in the face of stress by a complex network of cellular machinery, including the small heat shock molecular chaperone proteins (sHsps), which act to inhibit the aggregation and deposition of misfolded protein intermediates. Despite this, the pathogenesis of several neurodegenerative diseases has been inextricably linked with the amyloid fibrillar aggregation and deposition of α-synuclein (α-syn). The sHsps are potent inhibitors of α-syn aggregation in vitro. However, the limited availability of a robust, cell-based model of α-syn aggregation has, thus far, restricted evaluation of sHsp efficacy in the cellular context. As such, this work sought to establish a robust model of intracellular α-syn aggregation using Neuro-2a cells. Aggregation of α-syn was found to be sensitive to inhibition of autophagy and the proteasome, resulting in a significant increase in the proportion of cells containing α-syn inclusions. This model was then used to evaluate the capacity of the sHsps, αB-c and Hsp27, to prevent α-syn aggregation in cells. To do so, we used bicistronic expression plasmids to express the sHsps. Unlike traditional fluorescent fusion constructs, these bicistronic expression plasmids enable only individual transfected cells expressing the sHsps (via expression of the fluorescent reporter) to be analysed, but without the need to tag the sHsp, which can affect its oligomeric structure and chaperone activity. Overexpression of both αB-c and Hsp27 significantly reduced the intracellular aggregation of α-syn. Thus, these findings suggest that overexpressing or boosting the activity of sHsps may be a way of preventing amyloid fibrillar aggregation of α-syn in the context of neurodegenerative disease.

  13. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies.

    PubMed

    Chou, Ting-Chao

    2006-09-01

    The median-effect equation derived from the mass-action law principle at equilibrium-steady state via mathematical induction and deduction for different reaction sequences and mechanisms and different types of inhibition has been shown to be the unified theory for the Michaelis-Menten equation, Hill equation, Henderson-Hasselbalch equation, and Scatchard equation. It is shown that dose and effect are interchangeable via defined parameters. This general equation for the single drug effect has been extended to the multiple drug effect equation for n drugs. These equations provide the theoretical basis for the combination index (CI)-isobologram equation that allows quantitative determination of drug interactions, where CI < 1, = 1, and > 1 indicate synergism, additive effect, and antagonism, respectively. Based on these algorithms, computer software has been developed to allow automated simulation of synergism and antagonism at all dose or effect levels. It displays the dose-effect curve, median-effect plot, combination index plot, isobologram, dose-reduction index plot, and polygonogram for in vitro or in vivo studies. This theoretical development, experimental design, and computerized data analysis have facilitated dose-effect analysis for single drug evaluation or carcinogen and radiation risk assessment, as well as for drug or other entity combinations in a vast field of disciplines of biomedical sciences. In this review, selected examples of applications are given, and step-by-step examples of experimental designs and real data analysis are also illustrated. The merging of the mass-action law principle with mathematical induction-deduction has been proven to be a unique and effective scientific method for general theory development. The median-effect principle and its mass-action law based computer software are gaining increased applications in biomedical sciences, from how to effectively evaluate a single compound or entity to how to beneficially use multiple

  14. Lovastatin induces apoptosis of ovarian cancer cells and synergizes with doxorubicin: potential therapeutic relevance.

    PubMed

    Martirosyan, Anna; Clendening, James W; Goard, Carolyn A; Penn, Linda Z

    2010-03-18

    Ovarian carcinoma is a rarely curable disease, for which new treatment options are required. As agents that block HMG-CoA reductase and the mevalonate pathway, the statin family of drugs are used in the treatment of hypercholesterolemia and have been shown to trigger apoptosis in a tumor-specific manner. Recent clinical trials show that the addition of statins to traditional chemotherapeutic strategies can increase efficacy of targeting statin-sensitive tumors. Our goal was to assess statin-induced apoptosis of ovarian cancer cells, either alone or in combination with chemotherapeutics, and then determine these mechanisms of action. The effect of lovastatin on ovarian cancer cell lines was evaluated alone and in combination with cisplatin and doxorubicin using several assays (MTT, TUNEL, fixed PI, PARP cleavage) and synergy determined by evaluating the combination index. The mechanisms of action were evaluated using functional, molecular, and pharmacologic approaches. We demonstrate that lovastatin induces apoptosis of ovarian cancer cells in a p53-independent manner and synergizes with doxorubicin, a chemotherapeutic agent used to treat recurrent cases of ovarian cancer. Lovastatin drives ovarian tumor cell death by two mechanisms: first, by blocking HMG-CoA reductase activity, and second, by sensitizing multi-drug resistant cells to doxorubicin by a novel mevalonate-independent mechanism. This inhibition of drug transport, likely through inhibition of P-glycoprotein, potentiates both DNA damage and tumor cell apoptosis. The results of this research provide pre-clinical data to warrant further evaluation of statins as potential anti-cancer agents to treat ovarian carcinoma. Many statins are inexpensive, off-patent generic drugs that are immediately available for use as anti-cancer agents. We provide evidence that lovastatin triggers apoptosis of ovarian cancer cells as a single agent by a mevalonate-dependent mechanism. Moreover, we also show lovastatin synergizes

  15. Self-Assembled Coacervates of Chitosan and an Insect Cuticle Protein Containing a Rebers-Riddiford Motif.

    PubMed

    Vaclaw, M Coleman; Sprouse, Patricia A; Dittmer, Neal T; Ghazvini, Saba; Middaugh, C Russell; Kanost, Michael R; Gehrke, Stevin H; Dhar, Prajnaparamita

    2018-05-09

    The interactions among biomacromolecules within insect cuticle may offer new motifs for biomimetic material design. CPR27 is an abundant protein in the rigid cuticle of the elytron from Tribolium castaneum. CPR27 contains the Rebers-Riddiford (RR) motif, which is hypothesized to bind chitin. In this study, active magnetic microrheology coupled with microscopy and protein particle analysis techniques were used to correlate alterations in the viscosity of chitosan solutions with changes in solution microstructure. Addition of CPR27 to chitosan solutions led to a 3-fold drop in viscosity. This change was accompanied by the presence of micrometer-sized coacervate particles in solution. Coacervate formation had a strong dependence on chitosan concentration. Analysis showed the existence of a critical CPR27 concentration beyond which a significant increase in particle count was observed. These effects were not observed when a non-RR cuticular protein, CP30, was tested, providing evidence of a structure-function relationship related to the RR motif.

  16. Synthesis and Anticancer Mechanism Investigation of Dual Hsp27 and Tubulin Inhibitors

    PubMed Central

    Zhong, Bo; Chennamaneni, Snigdha; Lama, Rati; Yi, Xin; Geldenhuys, Werner J.; Pink, John J.; Dowlati, Afshin; Xu, Yan; Zhou, Aimin; Su, Bin

    2013-01-01

    Heat shock protein 27 (Hsp27) is a chaperone protein, and its expression is increased in response to various stress stimuli including anticancer chemotherapy, which allows the cells to survive and causes drug resistance. We previously identified lead compounds that bound to Hsp27 and tubulin via proteomic approaches. Systematic ligand based optimization in the current study significantly increased the cell growth inhibition and apoptosis inducing activities of the compounds. Compared to the lead compounds, one of the new derivatives exhibited much better potency to inhibit tubulin polymerization but a decreased activity to inhibit Hsp27 chaperone function, suggesting that the structural modification dissected the dual targeting effects of the compound. The most potent compounds 20 and 22 exhibited strong cell proliferation inhibitory activities at subnanomolar concentration against 60 human cancer cell lines conducted by Developmental Therapeutic Program at the National Cancer Institute and represented promising candidates for anticancer drug development. PMID:23767669

  17. Epidermal growth factor/heat shock protein 27 pathway regulates vasculogenic mimicry activity of breast cancer stem/progenitor cells.

    PubMed

    Lee, Che-Hsin; Wu, Yu-Ting; Hsieh, Hung-Chun; Yu, Yun; Yu, Alice L; Chang, Wen-Wei

    2014-09-01

    Tumor vascularization, which is mainly contributed by angiogenesis and vascularization, is necessary for tumor maintenance and progression. Vasculogenic mimicry (VM), vascular-like channels which are lack of the involvement of endothelial cells, has been observed in aggressive cancers and also involves in tumor vascularization. Breast cancer stem/progenitor cells (BCSCs) have been identified as a subpopulation of breast cancer cells with markers of CD24(-)CD44(+), high aldehyde dehydrogenase activity (ALDH(+)) or could be enriched by mammosphere cultivation. These cells have been proven to be associated with tumor vascularization. Here we investigated the molecular mechanisms in VM activity of BCSCs. By periodic acid-Schiff or hematoxylin-eosin stain, we found that there were VM structures in two xenografted human breast cancer tissues established from CD24(-)CD44(+) or ALDH(+) cells. Only ALDH(+) or mammosphere-forming BCSCs could form tube structures on matrigel-coated surface as similar as microvascular endothelial cells. Inhibition of the phosphorylation of epidermal growth factor receptor (EGFR) by gefitinib or knockdown of EGFR by lentiviral shRNA abolished the in vitro VM activity of BCSCs. By quercetin treatment, a plant flavonoid compound which is known to suppress heat shock proteins, or siRNA-mediated gene silencing, both Hsp27 expression and VM capability of BCSCs were suppressed. Forced expression of phosphor-mimic form of Hsp27 in ALDH(+) BCSCs could overcome the inhibitory effect of gefitinib. In conclusion, our data demonstrate that VM activity of BCSCs is mediated by EGF/Hsp27 signaling and targeting this pathway may benefit to breast cancer therapy. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  18. Tachyphylaxis to PACAP-27 after inhibition of NO synthesis: a loss of adenylate cyclase activation

    NASA Technical Reports Server (NTRS)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The vasodilator effects of pituitary adenylate cyclase activating polypeptide (PACAP-27) are subject to tachyphylaxis in rats treated with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). This study examined whether this tachyphylaxis is due to the loss of vasodilator potency of cAMP generated by activation of the G(s) protein-coupled PACAP receptors. Five successive treatments with PACAP-27 (2 nmol/kg iv) produced pronounced vasodilator responses in saline-treated rats that were not subject to tachyphylaxis. The first injection of PACAP-27 (2 nmol/kg iv) in L-NAME (50 micromol/kg iv)-treated rats produced vasodilator responses of similar magnitude to those in saline-treated rats, whereas four subsequent injections produced progressively and markedly smaller responses. The hemodynamic effects of the membrane-permeable cAMP analog 8-(4-chlorophenylthiol)-cAMP (8-CPT-cAMP; 5-15 micromol/kg iv) were similar in L-NAME-treated rats and in L-NAME-treated rats that had received the five injections of PACAP-27. In addition, five injections of 8-CPT-cAMP (10 micromol/kg iv) produced pronounced vasodilator responses in saline- and L-NAME-treated rats that were not subject to the development of tachyphylaxis. These results suggest that a loss of biological potency of cAMP is not responsible for tachyphylaxis to PACAP-27 in L-NAME-treated rats. This tachyphylaxis may be due to the inability of the G(s) protein-coupled PACAP receptor to activate adenylate cyclase.

  19. The metazoan protein disaggregase and amyloid depolymerase system: Hsp110, Hsp70, Hsp40, and small heat shock proteins.

    PubMed

    Torrente, Mariana P; Shorter, James

    2013-01-01

    A baffling aspect of metazoan proteostasis is the lack of an Hsp104 ortholog that rapidly disaggregates and reactivates misfolded polypeptides trapped in stress induced disordered aggregates, preamyloid oligomers, or amyloid fibrils. By contrast, in bacteria, protozoa, chromista, fungi, and plants, Hsp104 orthologs are highly conserved and confer huge selective advantages in stress tolerance. Moreover, in fungi, the amyloid remodeling activity of Hsp104 has enabled deployment of prions for various beneficial modalities. Thus, a longstanding conundrum has remained unanswered: how do metazoan cells renature aggregated proteins or resolve amyloid fibrils without Hsp104? Here, we highlight recent advances that unveil the metazoan protein-disaggregase machinery, comprising Hsp110, Hsp70, and Hsp40, which synergize to dissolve disordered aggregates, but are unable to rapidly solubilize stable amyloid fibrils. However, Hsp110, Hsp70, and Hsp40 exploit the slow monomer exchange dynamics of amyloid, and can slowly depolymerize amyloid fibrils from their ends in a manner that is stimulated by small heat shock proteins. Upregulation of this system could have key therapeutic applications in various protein-misfolding disorders. Intriguingly, yeast Hsp104 can interface with metazoan Hsp110, Hsp70, and Hsp40 to rapidly eliminate disease associated amyloid. Thus, metazoan proteostasis is receptive to augmentation with exogenous disaggregases, which opens a number of therapeutic opportunities.

  20. Evaluation of the synergistic effects of milk proteins in a rapid viscosity analyzer.

    PubMed

    Stephani, Rodrigo; Borges de Souza, Alisson; Leal de Oliveira, Marcone Augusto; Perrone, Ítalo Tuler; Fernandes de Carvalho, Antônio; Cappa de Oliveira, Luiz Fernando

    2015-12-01

    Protein systems (PS) are routinely used by companies from Brazil and around the globe to improve the texture, yield, and palatability of processed foods. Understanding the synergistic behavior among the different protein structures of these systems during thermal treatment under the influence of pH can help to better define optimum conditions for products and processes. The interpretation of the reactions and interactions that occur simultaneously among the protein constituents of these systems as dispersions during thermal processing is still a major challenge. Here, using a rapid viscosity analyzer, we observed the rheological changes in the startup viscosities of 5 PS obtained by combining varying proportions of milk protein concentrate and whey protein concentrate under different conditions of pH (5.0, 6.5, and 7.0) and heat processing (85°C/15min and 95°C/5min). The solutions were standardized to 25% of total solids and 17% of protein. Ten analytical parameters were used to characterize each of the startup-viscosity ramps for 35 experiments conducted in a 2×3 × 5 mixed planning matrix, using principal component analysis to interpret behavioral similarities. The study showed the clear influence of pH 5.5 in the elevation of the initial temperature of the PS startup viscosity by at least 5°C, as well as the effect of different milk protein concentrate:whey protein concentrate ratios above 15:85 at pH 7.0 on the viscographic profile curves. These results suggested that the primary agent driving the changes was the synergism among the reactions and interactions of casein with whey proteins during processing. This study reinforces the importance of the rapid viscosity analyzer as an analytical tool for the simulation of industrial processes involving PS, and the use of the startup viscosity ramp as a means of interpreting the interactions of system components with respect to changes related to the treatment temperature. Copyright © 2015 American Dairy Science

  1. The Functional Landscape of Hsp27 Reveals New Cellular Processes such as DNA Repair and Alternative Splicing and Proposes Novel Anticancer Targets*

    PubMed Central

    Katsogiannou, Maria; Andrieu, Claudia; Baylot, Virginie; Baudot, Anaïs; Dusetti, Nelson J.; Gayet, Odile; Finetti, Pascal; Garrido, Carmen; Birnbaum, Daniel; Bertucci, François; Brun, Christine; Rocchi, Palma

    2014-01-01

    Previously, we identified the stress-induced chaperone, Hsp27, as highly overexpressed in castration-resistant prostate cancer and developed an Hsp27 inhibitor (OGX-427) currently tested in phase I/II clinical trials as a chemosensitizing agent in different cancers. To better understand the Hsp27 poorly-defined cytoprotective functions in cancers and increase the OGX-427 pharmacological safety, we established the Hsp27-protein interaction network using a yeast two-hybrid approach and identified 226 interaction partners. As an example, we showed that targeting Hsp27 interaction with TCTP, a partner protein identified in our screen increases therapy sensitivity, opening a new promising field of research for therapeutic approaches that could decrease or abolish toxicity for normal cells. Results of an in-depth bioinformatics network analysis allying the Hsp27 interaction map into the human interactome underlined the multifunctional character of this protein. We identified interactions of Hsp27 with proteins involved in eight well known functions previously related to Hsp27 and uncovered 17 potential new ones, such as DNA repair and RNA splicing. Validation of Hsp27 involvement in both processes in human prostate cancer cells supports our system biology-predicted functions and provides new insights into Hsp27 roles in cancer cells. PMID:25277244

  2. Quinone-induced protein handling changes: Implications for major protein handling systems in quinone-mediated toxicity

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

    Xiong, Rui; Siegel, David; Ross, David, E-mail: david.ross@ucdenver.edu

    2014-10-15

    Para-quinones such as 1,4-Benzoquinone (BQ) and menadione (MD) and ortho-quinones including the oxidation products of catecholamines, are derived from xenobiotics as well as endogenous molecules. The effects of quinones on major protein handling systems in cells; the 20/26S proteasome, the ER stress response, autophagy, chaperone proteins and aggresome formation, have not been investigated in a systematic manner. Both BQ and aminochrome (AC) inhibited proteasomal activity and activated the ER stress response and autophagy in rat dopaminergic N27 cells. AC also induced aggresome formation while MD had little effect on any protein handling systems in N27 cells. The effect of NQO1more » on quinone induced protein handling changes and toxicity was examined using N27 cells stably transfected with NQO1 to generate an isogenic NQO1-overexpressing line. NQO1 protected against BQ–induced apoptosis but led to a potentiation of AC- and MD-induced apoptosis. Modulation of quinone-induced apoptosis in N27 and NQO1-overexpressing cells correlated only with changes in the ER stress response and not with changes in other protein handling systems. These data suggested that NQO1 modulated the ER stress response to potentiate toxicity of AC and MD, but protected against BQ toxicity. We further demonstrated that NQO1 mediated reduction to unstable hydroquinones and subsequent redox cycling was important for the activation of the ER stress response and toxicity for both AC and MD. In summary, our data demonstrate that quinone-specific changes in protein handling are evident in N27 cells and the induction of the ER stress response is associated with quinone-mediated toxicity. - Highlights: • Unstable hydroquinones contributed to quinone-induced ER stress and toxicity.« less

  3. HSP27 phosphorylation modulates TRAIL-induced activation of Src-Akt/ERK signaling through interaction with β-arrestin2.

    PubMed

    Qi, Shimei; Xin, Yinqiang; Qi, Zhilin; Xu, Yimiao; Diao, Ying; Lan, Lei; Luo, Lan; Yin, Zhimin

    2014-03-01

    Heat shock protein 27 (HSP27) regulates critical cellular functions such as development, differentiation, cell growth and apoptosis. A variety of stimuli induce the phosphorylation of HSP27, which affects its cellular functions. However, most previous studies focused on the role of HSP27 protein itself in apoptosis, the particular role of its phosphorylation state in signaling transduction remains largely unclear. In the present study, we reported that HSP27 phosphorylation modulated TRAIL-triggered pro-survival signaling transduction. In HeLa cells, suppression of HSP27 phosphorylation by specific inhibitor KRIBB3 or MAPKAPK2 (MK2) knockdown and by overexpression of non-phosphorylatable HSP27(3A) mutant demonstrated that hindered HSP27 phosphorylation enhanced the TRAIL-induced apoptosis. In addition, reduced HSP27 phosphorylation by KRIBB3 treatment or MK2 knockdown attenuated the TRAIL-induced activation of Akt and ERK survival signaling through suppressing the phosphorylation of Src. By overexpression of HSP27(15A) or HSP27(78/82A) phosphorylation mutant, we further showed that phosphorylation of HSP27 at serine 78/82 residues was essential to TRAIL-triggered Src-Akt/ERK signaling transduction. Co-immunoprecipitation and confocal microscopy showed that HSP27 interacted with Src and scaffolding protein β-arrestin2 in response of TRAIL stimulation and suppression of HSP27 phosphorylation apparently disrupted the TRAIL-induced interaction of HSP27 and Src or interaction of HSP27 and β-arrestin2. We further demonstrated that β-arrestin2 mediated HSP27 action on TRAIL-induced Src activation, which was achieved by recruiting signaling complex of HSP27/β-arrestin2/Src in response to TRAIL. Taken together, our study revealed that HSP27 phosphorylation modulates TRAIL-triggered activation of Src-Akt/ERK pro-survival signaling via interacting with β-arrestin2 in HeLa cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Pro-inflammatory cytokines downregulate Hsp27 and cause apoptosis of human retinal capillary endothelial cells.

    PubMed

    Nahomi, Rooban B; Palmer, Allison; Green, Katelyn M; Fort, Patrice E; Nagaraj, Ram H

    2014-02-01

    The formation of acellular capillaries in the retina, a hallmark feature of diabetic retinopathy, is caused by apoptosis of endothelial cells and pericytes. The biochemical mechanism of such apoptosis remains unclear. Small heat shock proteins play an important role in the regulation of apoptosis. In the diabetic retina, pro-inflammatory cytokines are upregulated. In this study, we investigated the effects of pro-inflammatory cytokines on small heat shock protein 27 (Hsp27) in human retinal endothelial cells (HREC). In HREC cultured in the presence of cytokine mixtures (CM), a significant downregulation of Hsp27 at the protein and mRNA level occurred, with no effect on HSF-1, the transcription factor for Hsp27. The presence of high glucose (25mM) amplified the effects of cytokines on Hsp27. CM activated indoleamine 2,3-dioxygenase (IDO) and enhanced the production of kynurenine and ROS. An inhibitor of IDO, 1-methyl tryptophan (MT), inhibited the effects of CM on Hsp27. CM also upregulated NOS2 and, consequently, nitric oxide (NO). A NOS inhibitor, L-NAME, and a ROS scavenger blocked the CM-mediated Hsp27 downregulation. While a NO donor in the culture medium did not decrease the Hsp27 content, a peroxynitrite donor and exogenous peroxynitrite did. The cytokines and high glucose-induced apoptosis of HREC were inhibited by MT and L-NAME. Downregulation of Hsp27 by a siRNA treatment promoted apoptosis in HREC. Together, these data suggest that pro-inflammatory cytokines induce the formation of ROS and NO, which, through the formation of peroxynitrite, reduce the Hsp27 content and bring about apoptosis of retinal capillary endothelial cells. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. A Tick Antivirulence Protein Potentiates Antibiotics against Staphylococcus aureus

    PubMed Central

    Abraham, Nabil M.; Liu, Lei; Jutras, Brandon L.; Murfin, Kristen; Acar, Ali; Yarovinsky, Timur O.; Sutton, Erica; Heisig, Martin; Jacobs-Wagner, Christine

    2017-01-01

    ABSTRACT New strategies are needed to combat antibiotic resistance, especially against pathogens such as methicillin-resistant Staphylococcus aureus. A tick antifreeze glycoprotein, IAFGP, possesses potent antibiofilm properties against a variety of clinical pathogens, including S. aureus. Synergy between IAFGP, or a peptide (P1) representative of a repeat region of the protein, with different antibiotics was assessed in vitro. Antibiotics that synergized with either IAFPG or P1 were further evaluated in vivo using vertebrate and invertebrate infection models. IAFGP readily enhanced the efficacy of antibiotics against S. aureus. Synergy with daptomycin, an antibiotic used to treat methicillin-resistant S. aureus, was observed in vitro and in vivo using iafgp-transgenic mice and flies. Furthermore, synergy with ciprofloxacin or gentamicin, antibiotics not generally used to treat S. aureus, was also perceived. The combined effect of the antibiotic and IAFGP was associated with improved permeation of the antibiotic into the cell. Our results highlight that synergy of IAFGP with antibiotics traditionally used to treat this pathogen, and enhancement of the potency of antibiotics not commonly used against this microbe, can provide novel alternative therapeutic strategies to combat bacterial infections. PMID:28438938

  6. Silk-fibronectin protein alloy fibres support cell adhesion and viability as a high strength, matrix fibre analogue

    NASA Astrophysics Data System (ADS)

    Jacobsen, Matthew M.; Li, David; Gyune Rim, Nae; Backman, Daniel; Smith, Michael L.; Wong, Joyce Y.

    2017-04-01

    Silk is a natural polymer with broad utility in biomedical applications because it exhibits general biocompatibility and high tensile material properties. While mechanical integrity is important for most biomaterial applications, proper function and integration also requires biomaterial incorporation into complex surrounding tissues for many physiologically relevant processes such as wound healing. In this study, we spin silk fibroin into a protein alloy fibre with whole fibronectin using wet spinning approaches in order to synergize their respective strength and cell interaction capabilities. Results demonstrate that silk fibroin alone is a poor adhesive surface for fibroblasts, endothelial cells, and vascular smooth muscle cells in the absence of serum. However, significantly improved cell attachment is observed to silk-fibronectin alloy fibres without serum present while not compromising the fibres’ mechanical integrity. Additionally, cell viability is improved up to six fold on alloy fibres when serum is present while migration and spreading generally increase as well. These findings demonstrate the utility of composite protein alloys as inexpensive and effective means to create durable, biologically active biomaterials.

  7. SUMOylation of HSP27 by small ubiquitin-like modifier 2/3 promotes proliferation and invasion of hepatocellular carcinoma cells.

    PubMed

    Ge, Haize; Du, Juan; Xu, Jingman; Meng, Xiangliang; Tian, Jinchuan; Yang, Jie; Liang, Huimin

    2017-08-03

    Primary hepatocellular carcinoma (PHC) is a major health problem worldwide and is one of the 10 most commonly diagnosed cancers in China. Heat shock protein 27 (HSP27) were found to be overexpressed in a wide range of malignancies including PHC, however, post-translational modification of HSP27 still needs exploration in PHC. Recently, SUMOylation, an important post-translational modification associating with the development of many kinds of cancers has been intensively studied. In the current study, mRNA and protein level of HSP27 in archived tumor samples representing various pathological characteristics of PHC were examined, and modification of HSP27 by SUMO2/3 was investigated. HSP27 were expressed abundantly in patients' tumor tissues, and found to be associated with pathological progression. Besides, HSP27 was also elevated significantly in liver cancer cell lines Huh7 and HepG2 compared with human hepatocyte cells L02. Furthermore, knockdown of HSP27 was found to be associated with the decreased proliferation and invasion ability in Huh7 and HepG2 cells. Immunofluorescence assay showed that HSP27 and SUMO2/3 were co-localized in the subcellular, and co-immunoprecipitation verified the interaction between HSP27 and SUMO2/3. Overexpression of SUMO2/3 upregulated the HSP27 protein level and promotes Huh7 and HepG2 cell proliferation and invasion, and vice versa when the SUMO2/3 was knockdown. Taken together, increased protein level of HSP27 through SUMO2/3-mediated SUMOylation plays crucial roles in the progression of PHC, and this finding may shed light on developing potential therapeutic targets for PHC.

  8. Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates – A Substudy

    PubMed Central

    Hursel, Rick; Martens, Eveline A. P.; Gonnissen, Hanne K. J.; Hamer, Henrike M.; Senden, Joan M. G.; van Loon, Luc J. C.; Westerterp-Plantenga, Margriet S.

    2015-01-01

    Background Based on controlled 36 h experiments a higher dietary protein intake causes a positive protein balance and a negative fat balance. A positive net protein balance may support fat free mass accrual. However, few data are available on the impact of more prolonged changes in habitual protein intake on whole-body protein metabolism and basal muscle protein synthesis rates. Objective To assess changes in whole-body protein turnover and basal muscle protein synthesis rates following 12 weeks of adaptation to a low versus high dietary protein intake. Methods A randomized parallel study was performed in 40 subjects who followed either a high protein (2.4 g protein/kg/d) or low protein (0.4 g protein/kg/d) energy-balanced diet (30/35/35% or 5/60/35% energy from protein/carbohydrate/fat) for a period of 12 weeks. A subgroup of 7 men and 8 women (body mass index: 22.8±2.3 kg/m2, age: 24.3±4.9 y) were selected to evaluate the impact of prolonged adaptation to either a high or low protein intake on whole body protein metabolism and basal muscle protein synthesis rates. After the diet, subjects received continuous infusions with L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosine in an overnight fasted state, with blood samples and muscle biopsies being collected to assess post-absorptive whole-body protein turnover and muscle protein synthesis rates in vivo in humans. Results After 12 weeks of intervention, whole-body protein balance in the fasted state was more negative in the high protein treatment when compared with the low protein treatment (-4.1±0.5 vs -2.7±0.6 μmol phenylalanine/kg/h;P<0.001). Whole-body protein breakdown (43.0±4.4 vs 37.8±3.8 μmol phenylalanine/kg/h;P<0.03), synthesis (38.9±4.2 vs 35.1±3.6 μmol phenylalanine/kg/h;P<0.01) and phenylalanine hydroxylation rates (4.1±0.6 vs 2.7±0.6 μmol phenylalanine/kg/h;P<0.001) were significantly higher in the high vs low protein group. Basal muscle protein synthesis rates were maintained on a low

  9. Evidence of cue synergism in termite corpse response behavior

    NASA Astrophysics Data System (ADS)

    Ulyshen, Michael D.; Shelton, Thomas G.

    2012-02-01

    Subterranean termites of the genus Reticulitermes are known to build walls and tubes and move considerable amounts of soil into wood but the causes of this behavior remain largely unexplored. In laboratory assays, we tested the hypothesis that Reticulitermes virginicus (Banks) would carry more sand into wooden blocks containing corpses compared to corpse-free controls. We further predicted that the corpses of predatory ants would elicit a stronger response than those of a benign beetle species or nestmates. As hypothesized, significantly more sand was carried into blocks containing corpses and this material was typically used to build partitions separating the dead from the rest of the colony. Contrary to expectations, however, this behavior did not vary among corpse types. We then tested the hypothesis that oleic acid, an unsaturated fatty acid released during arthropod decay and used by ants and other arthropod taxa in corpse recognition, would induce a similar building response in R. virginicus. To additionally determine the role of foreign objects in giving rise to this behavior, the experiment was carried out with and without imitation corpses (i.e., small glass beads). As predicted, oleic acid induced building (a tenfold increase) but only when applied to beads, suggesting strong synergism between tactile and chemical cues. Oleic acid also significantly reduced the amount of wood consumed by R. virginicus and may possess useful repellent properties.

  10. Hsp27 promotes ABCA1 expression and cholesterol efflux through the PI3K/PKCζ/Sp1 pathway in THP-1 macrophages.

    PubMed

    Kuang, Hai-Jun; Zhao, Guo-Jun; Chen, Wu-Jun; Zhang, Min; Zeng, Gao-Feng; Zheng, Xi-Long; Tang, Chao-Ke

    2017-09-05

    Heat shock protein 27 (Hsp27) is a putative biomarker and therapeutic target in atherosclerosis. This study was to explore the potential mechanisms underlying Hsp27 effects on ATP-binding cassette transporter A1 (ABCA1) expression and cellular cholesterol efflux. THP-1 macrophage-derived foam cells were infected with adenovirus to express wild-type Hsp27, hyper-phosphorylated Hsp27 mimic (3D Hsp27), antisense Hsp27 or hypo-phosphorylated Hsp27 mimic (3A Hsp27). Wild-type and 3D Hsp27 were found to up-regulate ABCA1 mRNA and protein expression and increase cholesterol efflux from cells. Expression of antisense or 3A Hsp27 suppressed the expression of ABCA1 and cholesterol efflux. Furthermore, over-expression of wild-type and 3D Hsp27 significantly increased the levels of phosphorylated specificity protein 1 (Sp1), protein kinase C ζ (PKCζ) and phosphatidylinositol 3-kinase (PI3K). In addition, the up-regulation of ABCA1 expression and cholesterol efflux induced by 3D Hsp27 was suppressed by inhibition of Sp1, PKCζ and PI3K with specific kinase inhibitors. Taken together, our results revealed that Hsp27 may up-regulate the expression of ABCA1 and promotes cholesterol efflux through activation of the PI3K/PKCζ/Sp1 signal pathway in THP-1 macrophage-derived foam cells. Our findings may partly explain the mechanisms underlying the anti-atherogenic effect of Hsp27. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Synergism between Enantiomers Creates Species-Specific Pheromone Blends and Minimizes Cross-Attraction for Two Species of Cerambycid Beetles.

    PubMed

    Meier, Linnea R; Zou, Yunfan; Millar, Jocelyn G; Mongold-Diers, Judith A; Hanks, Lawrence M

    2016-11-01

    Research over the last decade has revealed extensive parsimony among pheromones within the large insect family Cerambycidae, with males of many species producing the same, or very similar aggregation pheromones. Among some species in the subfamily Cerambycinae, interspecific attraction is minimized by temporal segregation, and/or by minor pheromone components that synergize attraction of conspecifics or inhibit attraction of heterospecifics. Less is known about pheromone-based mechanisms of reproductive isolation among species in the largest subfamily, the Lamiinae. Here, we present evidence that the pheromone systems of two sympatric lamiine species consist of synergistic blends of enantiomers of (E)-6,10-dimethyl-5,9-undecadien-2-ol (fuscumol) and the structurally related (E)-6,10-dimethyl-5,9-undecadien-2-yl acetate (fuscumol acetate), as a mechanism by which species-specific blends of pheromone components can minimize interspecific attraction. Male Astylidius parvus (LeConte) were found to produce (R)- and (S)-fuscumol + (R)-fuscumol acetate + geranylacetone, whereas males of Lepturges angulatus (LeConte) produced (R)- and (S)-fuscumol acetate + geranylacetone. Field experiments confirmed that adult beetles were attracted only by their species-specific blend of the enantiomers of fuscumol and fuscumol acetate, respectively, and not to the individual enantiomers. Because other lamiine species are known to produce single enantiomers or blends of enantiomers of fuscumol and/or fuscumol acetate, synergism between enantiomers, or inhibition by enantiomers, may be a widespread mechanism for forming species-specific pheromone blends in this subfamily.

  12. Hsp27 binding to the 3′UTR of bim mRNA prevents neuronal death during oxidative stress–induced injury: a novel cytoprotective mechanism

    PubMed Central

    Dávila, David; Jiménez-Mateos, Eva M.; Mooney, Claire M.; Velasco, Guillermo; Henshall, David C.; Prehn, Jochen H. M.

    2014-01-01

    Neurons face a changeable microenvironment and therefore need mechanisms that allow rapid switch on/off of their cytoprotective and apoptosis-inducing signaling pathways. Cellular mechanisms that control apoptosis activation include the regulation of pro/antiapoptotic mRNAs through their 3′-untranslated region (UTR). This region holds binding elements for RNA-binding proteins, which can control mRNA translation. Here we demonstrate that heat shock protein 27 (Hsp27) prevents oxidative stress–induced cell death in cerebellar granule neurons by specific regulation of the mRNA for the proapoptotic BH3-only protein, Bim. Hsp27 depletion induced by oxidative stress using hydrogen peroxide (H2O2) correlated with bim gene activation and subsequent neuronal death, whereas enhanced Hsp27 expression prevented these. This effect could not be explained by proteasomal degradation of Bim or bim promoter inhibition; however, it was associated with a specific increase in the levels of bim mRNA and with its binding to Hsp27. Finally, we determined that enhanced Hsp27 expression in neurons exposed to H2O2 or glutamate prevented the translation of a reporter plasmid where bim-3′UTR mRNA sequence was cloned downstream of a luciferase gene. These results suggest that repression of bim mRNA translation through binding to the 3′UTR constitutes a novel cytoprotective mechanism of Hsp27 during stress in neurons. PMID:25187648

  13. Studies of an Androgen-Binding Protein Knockout Corroborate a Role for Salivary ABP in Mouse Communication

    PubMed Central

    Chung, Amanda G.; Belone, Phillip M.; Bímová, Barbora Vošlajerová; Karn, Robert C.; Laukaitis, Christina M.

    2017-01-01

    The house mouse Androgen-binding protein (Abp) gene family is comprised of 64 paralogs, 30 Abpa and 34 Abpbg, encoding the alpha (ABPA) and beta-gamma (ABPBG) protein subunits that are disulfide-bridged to form dimers in secretions. Only 14 Abp genes are expressed in distinct patterns in the lacrimal (11) and submandibular glands (3). We created a knockout mouse line lacking two of the three genes expressed in submandibular glands, Abpa27 and Abpbg27, by replacing them with the neomycin resistance gene. The knockout genotype (−/−) showed no Abpa27 or Abpbg27 transcripts in submandibular gland complementary DNA (cDNA) libraries and there was a concomitant lack of protein expression of ABPA27 and ABPBG27 in the −/− genotype saliva, shown by elimination of these two proteins from the saliva proteome and the loss of cross-reactive material in the acinar cells of the submandibular glands. We also observed a decrease in BG26 protein in the −/− animals, suggesting monomer instability. Overall, we observed no major phenotypic changes in the −/− genotype, compared with their +/+ and +/− siblings raised in a laboratory setting, including normal growth curves, tissue histology, fecundity, and longevity. The only difference is that male and female C57BL/6 mice preferred saliva of the opposite sex containing ABP statistically significantly more than saliva of the opposite sex without ABP in a Y-maze test. These results show for the first time that mice can sense the presence of ABP between saliva targets with and without ABPs, and that they spend more time investigating the target containing ABP. PMID:28159752

  14. Studies of an Androgen-Binding Protein Knockout Corroborate a Role for Salivary ABP in Mouse Communication.

    PubMed

    Chung, Amanda G; Belone, Phillip M; Bímová, Barbora Vošlajerová; Karn, Robert C; Laukaitis, Christina M

    2017-04-01

    The house mouse Androgen-binding protein ( Abp ) gene family is comprised of 64 paralogs, 30 Abpa and 34 Abpbg , encoding the alpha (ABPA) and beta-gamma (ABPBG) protein subunits that are disulfide-bridged to form dimers in secretions. Only 14 Abp genes are expressed in distinct patterns in the lacrimal (11) and submandibular glands (3). We created a knockout mouse line lacking two of the three genes expressed in submandibular glands, Abpa27 and Abpbg27 , by replacing them with the neomycin resistance gene. The knockout genotype (-/-) showed no Abpa27 or Abpbg27 transcripts in submandibular gland complementary DNA (cDNA) libraries and there was a concomitant lack of protein expression of ABPA27 and ABPBG27 in the -/- genotype saliva, shown by elimination of these two proteins from the saliva proteome and the loss of cross-reactive material in the acinar cells of the submandibular glands. We also observed a decrease in BG26 protein in the -/- animals, suggesting monomer instability. Overall, we observed no major phenotypic changes in the -/- genotype, compared with their +/+ and +/- siblings raised in a laboratory setting, including normal growth curves, tissue histology, fecundity, and longevity. The only difference is that male and female C57BL/6 mice preferred saliva of the opposite sex containing ABP statistically significantly more than saliva of the opposite sex without ABP in a Y-maze test. These results show for the first time that mice can sense the presence of ABP between saliva targets with and without ABPs, and that they spend more time investigating the target containing ABP. Copyright © 2017 by the Genetics Society of America.

  15. Ras-Association Domain of Sorting Nexin 27 Is Critical for Regulating Expression of GIRK Potassium Channels

    PubMed Central

    Bodhinathan, Karthik; Taura, Jaume J.; Taylor, Natalie M.; Nettleton, Margaret Y.; Ciruela, Francisco; Slesinger, Paul A.

    2013-01-01

    G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-ΔRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability. PMID:23536889

  16. Salinity-mediated transcriptional and post-translational regulation of the Arabidopsis aquaporin PIP2;7.

    PubMed

    Pou, Alicia; Jeanguenin, Linda; Milhiet, Thomas; Batoko, Henri; Chaumont, François; Hachez, Charles

    2016-12-01

    Salt stress triggers a simultaneous transcriptional repression and aquaporin internalization to modify root cell water conductivity. Plasma membrane intrinsic proteins (PIPs) are involved in the adjustment of plant water balance in response to changing environmental conditions. In this study, Arabidopsis wild-type (Col-0) and transgenic lines overexpressing PIP2;7 were used to investigate and compare their response to salt stress. Hydraulic conductivity measurements using a high-pressure flowmeter (HPFM) revealed that overexpression of PIP2;7 induced a sixfold increase in root hydraulic conductivity of four week-old Arabidopsis thaliana plants compared to WT. Exposure to a high salt stress (150 mM NaCl) triggered a rapid repression of overall aquaporin activity in both genotypes. Response to salt stress was also investigated in 8 day-old seedlings. Exposure to salt led to a repression of PIP2;7 promoter activity and a significant decrease in PIP2;7 mRNA abundance within 2 h. Concomitantly, a rapid internalization of fluorescently-tagged PIP2;7 proteins was observed but removal from the cell membrane was not accompanied by further degradation of the protein within 4 h of exposure to salinity stress. These data suggest that PIP transcriptional repression and channel internalization act in concert during salt stress conditions to modulate aquaporin activity, thereby significantly altering the plant hydraulic parameters in the short term.

  17. Proteomic profiling reveals that resveratrol inhibits HSP27 expression and sensitizes breast cancer cells to doxorubicin therapy.

    PubMed

    Díaz-Chávez, José; Fonseca-Sánchez, Miguel A; Arechaga-Ocampo, Elena; Flores-Pérez, Ali; Palacios-Rodríguez, Yadira; Domínguez-Gómez, Guadalupe; Marchat, Laurence A; Fuentes-Mera, Lizeth; Mendoza-Hernández, Guillermo; Gariglio, Patricio; López-Camarillo, César

    2013-01-01

    The use of chemopreventive natural compounds represents a promising strategy in the search for novel therapeutic agents in cancer. Resveratrol (3,4',5-trans-trihydroxystilbilene) is a dietary polyphenol found in fruits, vegetables and medicinal plants that exhibits chemopreventive and antitumor effects. In this study, we searched for modulated proteins with preventive or therapeutic potential in MCF-7 breast cancer cells exposed to resveratrol. Using two-dimensional electrophoresis we found significant changes (FC >2.0; p≤0.05) in the expression of 16 proteins in resveratrol-treated MCF-7 cells. Six down-regulated proteins were identified by tandem mass spectrometry (ESI-MS/MS) as heat shock protein 27 (HSP27), translationally-controlled tumor protein, peroxiredoxin-6, stress-induced-phosphoprotein-1, pyridoxine-5'-phosphate oxidase-1 and hypoxanthine-guanine phosphoribosyl transferase; whereas one up-regulated protein was identified as triosephosphate isomerase. Particularly, HSP27 overexpression has been associated to apoptosis inhibition and resistance of human cancer cells to therapy. Consistently, we demonstrated that resveratrol induces apoptosis in MCF-7 cells. Apoptosis was associated with a significant increase in mitochondrial permeability transition, cytochrome c release in cytoplasm, and caspases -3 and -9 independent cell death. Then, we evaluated the chemosensitization effect of increasing concentrations of resveratrol in combination with doxorubicin anti-neoplastic agent in vitro. We found that resveratrol effectively sensitize MCF-7 cells to cytotoxic therapy. Next, we evaluated the relevance of HSP27 targeted inhibition in therapy effectiveness. Results evidenced that HSP27 inhibition using RNA interference enhances the cytotoxicity of doxorubicin. In conclusion, our data indicate that resveratrol may improve the therapeutic effects of doxorubicin in part by cell death induction. We propose that potential modulation of HSP27 levels using natural

  18. Phene Synergism between Root Hair Length and Basal Root Growth Angle for Phosphorus Acquisition1[OPEN

    PubMed Central

    Miguel, Magalhaes Amade

    2015-01-01

    Shallow basal root growth angle (BRGA) increases phosphorus acquisition efficiency by enhancing topsoil foraging because in most soils, phosphorus is concentrated in the topsoil. Root hair length and density (RHL/D) increase phosphorus acquisition by expanding the soil volume subject to phosphorus depletion through diffusion. We hypothesized that shallow BRGA and large RHL/D are synergetic for phosphorus acquisition, meaning that their combined effect is greater than the sum of their individual effects. To evaluate this hypothesis, phosphorus acquisition in the field in Mozambique was compared among recombinant inbred lines of common bean (Phaseolus vulgaris) having four distinct root phenotypes: long root hairs and shallow basal roots, long root hairs and deep basal roots, short root hairs and shallow basal roots, and short root hairs and deep basal roots. The results revealed substantial synergism between BRGA and RHL/D. Compared with short-haired, deep-rooted phenotypes, long root hairs increased shoot biomass under phosphorus stress by 89%, while shallow roots increased shoot biomass by 58%. Genotypes with both long root hairs and shallow roots had 298% greater biomass accumulation than short-haired, deep-rooted phenotypes. Therefore, the utility of shallow basal roots and long root hairs for phosphorus acquisition in combination is twice as large as their additive effects. We conclude that the anatomical phene of long, dense root hairs and the architectural phene of shallower basal root growth are synergetic for phosphorus acquisition. Phene synergism may be common in plant biology and can have substantial importance for plant fitness, as shown here. PMID:25699587

  19. miR-27b Represses Migration of Mouse MSCs to Burned Margins and Prolongs Wound Repair through Silencing SDF-1a

    PubMed Central

    Chen, Ling; Peng, Xi; Chen, Jian; Hu, Jiong-Yu; Teng, Miao; Liang, Guang-Ping

    2013-01-01

    Background Interactions between stromal cell-derived factor-1α (SDF-1α) and its cognate receptor CXCR4 are crucial for the recruitment of mesenchymal stem cells (MSCs) from bone marrow (BM) reservoirs to damaged tissues for repair during alarm situations. MicroRNAs are differentially expressed in stem cell niches, suggesting a specialized role in stem cell regulation. Here, we gain insight into the molecular mechanisms involved in regulating SDF-1α. Methods MSCs from green fluorescent protein transgenic male mice were transfused to irradiated recipient female C57BL/6 mice, and skin burn model of bone marrow-chimeric mice were constructed. Six miRNAs with differential expression in burned murine skin tissue compared to normal skin tissue were identified using microarrays and bioinformatics. The expression of miR-27b and SDF-1α was examined in burned murine skin tissue using quantitative real-time PCR (qPCR) and immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA). The Correlation of miR-27b and SDF-1α expression was analyzed by Pearson analysis Correlation. miRNAs suppressed SDF-1α protein expression by binding directly to its 3′UTR using western blot and luciferase reporter assay. The importance of miRNAs in MSCs chemotaxis was further estimated by decreasing SDF-1α in vivo and in vitro. Results miR-23a, miR-27a and miR-27b expression was significantly lower in the burned skin than in the normal skin (p<0.05). We also found that several miRNAs suppressed SDF-1α protein expression, while just miR-27a and miR-27b directly bound to the SDF-1α 3′UTR. Moreover, the forced over-expression of miR-27a and miR-27b significantly reduced the directional migration of mMSCs in vitro. However, only miR-27b in burn wound margins significantly inhibited the mobilization of MSCs to the epidermis. Conclusion miR-27b may be a unique signature of the stem cell niche in burned mouse skin and can suppress the directional migration of mMSCs by targeting SDF

  20. 27 CFR 6.27 - Proprietary interest.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Proprietary interest. 6.27 Section 6.27 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS âTIED-HOUSEâ Unlawful Inducements Interest in Retail License § 6.27 Proprietary interest. (a) Complete ownership....

  1. 27 CFR 6.27 - Proprietary interest.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Proprietary interest. 6.27 Section 6.27 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS âTIED-HOUSEâ Unlawful Inducements Interest in Retail License § 6.27 Proprietary interest. (a) Complete ownership....

  2. HSP27 as a biomarker for predicting skin irritation in human skin and reconstructed organotypic skin model.

    PubMed

    Chen, Hongxia; Li, Shuhua; Meng, Tian; Zhang, Lei; Dai, Taoli; Xiang, Qi; Su, Zhijian; Zhang, Qihao; Huang, Yadong

    2014-04-21

    In vitro alternative tests aiming at replacing the traditional animal test for predicting the irritant potential of chemicals have been developed, but the assessing parameters or endpoints are still not sufficient. To discover novel endpoints for skin irritation responses, 2DE-based proteomics was used to analyze the protein expression in human skin exposed to sodium lauryl sulfate (SLS) following the test protocol of the European Centre for the Validation of Alternative Methods (ECVAM) in the present study. HSP27 was up-regulated most significantly among the eight identified proteins, consistent with our previous reports. Acid and basic chemicals were applied on human skin for further validation and results showed that the up-regulated expression of HSP27 was induced in 24h after the exposure. Skin-equivalent constructed with fibroblasts, basement membrane and keratinocytes was used to investigate the potential of HSP27 as a biomarker or additional endpoint for the hazard assessment of skin irritation. Our skin-equivalent (Reconstructed Organotypic Skin Model, ROSM) had excellent epidermal differentiation and was suitable for the skin irritation test. HSP27 also displayed an up-regulated expression in the ROSM in 24h after the irritants exposure for 15min. All these results suggest that HSP27 may represent a potential marker or additional endpoint for the hazard assessment of skin irritation caused by chemical products. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. The functional landscape of Hsp27 reveals new cellular processes such as DNA repair and alternative splicing and proposes novel anticancer targets.

    PubMed

    Katsogiannou, Maria; Andrieu, Claudia; Baylot, Virginie; Baudot, Anaïs; Dusetti, Nelson J; Gayet, Odile; Finetti, Pascal; Garrido, Carmen; Birnbaum, Daniel; Bertucci, François; Brun, Christine; Rocchi, Palma

    2014-12-01

    Previously, we identified the stress-induced chaperone, Hsp27, as highly overexpressed in castration-resistant prostate cancer and developed an Hsp27 inhibitor (OGX-427) currently tested in phase I/II clinical trials as a chemosensitizing agent in different cancers. To better understand the Hsp27 poorly-defined cytoprotective functions in cancers and increase the OGX-427 pharmacological safety, we established the Hsp27-protein interaction network using a yeast two-hybrid approach and identified 226 interaction partners. As an example, we showed that targeting Hsp27 interaction with TCTP, a partner protein identified in our screen increases therapy sensitivity, opening a new promising field of research for therapeutic approaches that could decrease or abolish toxicity for normal cells. Results of an in-depth bioinformatics network analysis allying the Hsp27 interaction map into the human interactome underlined the multifunctional character of this protein. We identified interactions of Hsp27 with proteins involved in eight well known functions previously related to Hsp27 and uncovered 17 potential new ones, such as DNA repair and RNA splicing. Validation of Hsp27 involvement in both processes in human prostate cancer cells supports our system biology-predicted functions and provides new insights into Hsp27 roles in cancer cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. 27 CFR 7.27 - Net contents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Net contents. 7.27 Section 7.27 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL LABELING AND ADVERTISING OF MALT BEVERAGES Labeling Requirements for Malt Beverages § 7.27 Net contents. (a) Net contents shall...

  5. 27 CFR 7.27 - Net contents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Net contents. 7.27 Section 7.27 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL LABELING AND ADVERTISING OF MALT BEVERAGES Labeling Requirements for Malt Beverages § 7.27 Net contents. (a) Net contents shall...

  6. 27 CFR 7.27 - Net contents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Net contents. 7.27 Section 7.27 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS LABELING AND ADVERTISING OF MALT BEVERAGES Labeling Requirements for Malt Beverages § 7.27 Net contents. (a) Net contents shall...

  7. 27 CFR 7.27 - Net contents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Net contents. 7.27 Section 7.27 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS LABELING AND ADVERTISING OF MALT BEVERAGES Labeling Requirements for Malt Beverages § 7.27 Net contents. (a) Net contents shall...

  8. 27 CFR 7.27 - Net contents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Net contents. 7.27 Section 7.27 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS LABELING AND ADVERTISING OF MALT BEVERAGES Labeling Requirements for Malt Beverages § 7.27 Net contents. (a) Net contents shall...

  9. 27 CFR 27.42 - Wines.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Wines. 27.42 Section 27.42... TREASURY LIQUORS IMPORTATION OF DISTILLED SPIRITS, WINES, AND BEER Tax On Imported Distilled Spirits, Wines, and Beer Wines § 27.42 Wines. All wines (including imitation, substandard, or artificial wine, and...

  10. EZH2-mediated H3K27 trimethylation mediates neurodegeneration in ataxia-telangiectasia

    PubMed Central

    Li, Jiali; Hart, Ronald P.; Mallimo, Elyse M.; Swerdel, Mavis R.; Kusnecov, Alexander; Herrup, Karl

    2014-01-01

    The symptoms of ataxia-telangiectasia (A-T) include a progressive neurodegeneration caused by ATM protein deficiency. We previously found that nuclear accumulation of histone deacetylase-4, HDAC4, contributes to this degeneration; we now report that increased histone H3K27 trimethylation (H3K27me3) mediated by polycomb repressive complex 2 (PRC2) also plays an important role in the A-T phenotype. Enhancer of zeste homolog 2 (EZH2), a core catalytic component of PRC2, is a new ATM kinase target, and ATM-mediated S734 phosphorylation of EZH2 reduces protein stability. Thus, PRC2 formation is elevated along with H3K27me3in ATM deficiency. ChIP-sequencing shows a significant increase in H3K27me3 ‘marks’ and a dramatic shift in their location. The change of H3K27me3 chromatin-binding pattern is directly related to cell cycle re-entry and cell death of ATM-deficient neurons. Lentiviral knockdown of EZH2 rescues Purkinje cell degeneration and behavioral abnormalities in Atm−/− mice, demonstrating that EZH2 hyperactivity is another key factor in A-T neurodegeneration. PMID:24162653

  11. 27 CFR 27.59 - Wines.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Wines. 27.59 Section 27.59... TREASURY LIQUORS IMPORTATION OF DISTILLED SPIRITS, WINES, AND BEER General Requirements Marking and Labeling of Wines and Beer § 27.59 Wines. All imported wines containing not less than 7 percent and not...

  12. 27 CFR 27.60 - Beer.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Beer. 27.60 Section 27.60... TREASURY ALCOHOL IMPORTATION OF DISTILLED SPIRITS, WINES, AND BEER General Requirements Marking and Labeling of Wines and Beer § 27.60 Beer. All imported beer is required to be released from customs custody...

  13. 27 CFR 27.60 - Beer.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Beer. 27.60 Section 27.60... TREASURY LIQUORS IMPORTATION OF DISTILLED SPIRITS, WINES, AND BEER General Requirements Marking and Labeling of Wines and Beer § 27.60 Beer. All imported beer is required to be released from customs custody...

  14. 27 CFR 27.60 - Beer.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Beer. 27.60 Section 27.60... TREASURY LIQUORS IMPORTATION OF DISTILLED SPIRITS, WINES, AND BEER General Requirements Marking and Labeling of Wines and Beer § 27.60 Beer. All imported beer is required to be released from customs custody...

  15. 27 CFR 27.60 - Beer.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Beer. 27.60 Section 27.60... TREASURY ALCOHOL IMPORTATION OF DISTILLED SPIRITS, WINES, AND BEER General Requirements Marking and Labeling of Wines and Beer § 27.60 Beer. All imported beer is required to be released from customs custody...

  16. 27 CFR 27.60 - Beer.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Beer. 27.60 Section 27.60... TREASURY LIQUORS IMPORTATION OF DISTILLED SPIRITS, WINES, AND BEER General Requirements Marking and Labeling of Wines and Beer § 27.60 Beer. All imported beer is required to be released from customs custody...

  17. Small heat shock proteins protect against {alpha}-synuclein-induced toxicity and aggregation

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

    Outeiro, Tiago Fleming; Klucken, Jochen; Strathearn, Katherine E.

    Protein misfolding and inclusion formation are common events in neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD) or Huntington's disease (HD). {alpha}-Synuclein (aSyn) is the main protein component of inclusions called Lewy bodies (LB) which are pathognomic of PD, Dementia with Lewy bodies (DLB), and other diseases collectively known as LB diseases. Heat shock proteins (HSPs) are one class of the cellular quality control system that mediate protein folding, remodeling, and even disaggregation. Here, we investigated the role of the small heat shock proteins Hsp27 and {alpha}B-crystallin, in LB diseases. We demonstrate, via quantitative PCR, that Hsp27 messengermore » RNA levels are {approx}2-3-fold higher in DLB cases compared to control. We also show a corresponding increase in Hsp27 protein levels. Furthermore, we found that Hsp27 reduces aSyn-induced toxicity by {approx}80% in a culture model while {alpha}B-crystallin reduces toxicity by {approx}20%. In addition, intracellular inclusions were immunopositive for endogenous Hsp27, and overexpression of this protein reduced aSyn aggregation in a cell culture model.« less

  18. Essential role of the NH2-terminal WD/EPF motif in the phosphorylation-activated protective function of mammalian Hsp27.

    PubMed

    Thériault, Jimmy R; Lambert, Herman; Chávez-Zobel, Aura T; Charest, Gabriel; Lavigne, Pierre; Landry, Jacques

    2004-05-28

    Hsp27 is expressed at high levels after mild heat shock and contributes to making cells extremely resistant to subsequent treatments. The activity of the protein is regulated at the transcriptional level, but also by phosphorylation, which occurs rapidly during stress and is responsible for causing the dissociation of large 700-kDa Hsp27 oligomers into dimers. We investigated the mechanism by which phosphorylation and oligomerization modulate the protective activity of Chinese hamster Hsp27. In contrast to oligomer dissociation, which only required Ser90 phosphorylation, activation of Hsp27 thermoprotective activity required the phosphorylation of both Ser90 and Ser15. Replacement of Ser90 by Ala90, which prevented the dissociation of the oligomer upon stress, did cause a severe defect in the protective activity. Dissociation was, however, not a sufficient condition to activate the protein because replacement of Ser15 by Ala15, which caused little effect in the oligomeric organization of the protein, also yielded an inactive protein. Analyzes of mutants with short deletions in the NH2 terminus identified the Hsp27 WD/EPF or PF-rich domain as essential for protection, maintenance of the oligomeric structure, and in vitro chaperone activity of the protein. In light of a three-dimensional model of Hsp27 based on the crystallographic structure of wheat Hsp16.9, we propose that the conserved WD/EPF motif of mammalian Hsp27 mediates important intramolecular interactions with hydrophic surfaces of the alpha-crystallin domain of the protein. These interactions are destabilized by Ser90 phosphorylation, making the motif free to interact with heterologous molecular targets upon the additional phosphorylation of the nearby Ser15.

  19. HSP27 Inhibits Homocysteine-Induced Endothelial Apoptosis by Modulation of ROS Production and Mitochondrial Caspase-Dependent Apoptotic Pathway.

    PubMed

    Tian, Xin; Zhao, Lei; Song, Xianjing; Yan, Youyou; Liu, Ning; Li, Tianyi; Yan, Bingdi; Liu, Bin

    2016-01-01

    Objectives. Elevated plasma homocysteine (Hcy) could lead to endothelial dysfunction and is viewed as an independent risk factor for atherosclerosis. Heat shock protein 27 (HSP27), a small heat shock protein, is reported to exert protective effect against atherosclerosis. This study aims to investigate the protective effect of HSP27 against Hcy-induced endothelial cell apoptosis in human umbilical vein endothelial cells (HUVECs) and to determine the underlying mechanisms. Methods. Apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) of normal or HSP27-overexpressing HUVECs in the presence of Hcy were analyzed by flow cytometry. The mRNA and protein expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Results. We found that Hcy could induce cell apoptosis with corresponding decrease of nitric oxide (NO) level, increase of endothelin-1 (ET-1), intracellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1) levels, elevation of ROS, and dissipation of MMP. In addition, HSP27 could protect the cell against Hcy-induced apoptosis and inhibit the effect of Hcy on HUVECs. Furthermore, HSP27 could increase the ratio of Bcl-2/Bax and inhibit caspase-3 activity. Conclusions. Therefore, we concluded that HSP27 played a protective role against Hcy-induced endothelial apoptosis through modulation of ROS production and the mitochondrial caspase-dependent apoptotic pathway.

  20. Bifurcation-based approach reveals synergism and optimal combinatorial perturbation.

    PubMed

    Liu, Yanwei; Li, Shanshan; Liu, Zengrong; Wang, Ruiqi

    2016-06-01

    Cells accomplish the process of fate decisions and form terminal lineages through a series of binary choices in which cells switch stable states from one branch to another as the interacting strengths of regulatory factors continuously vary. Various combinatorial effects may occur because almost all regulatory processes are managed in a combinatorial fashion. Combinatorial regulation is crucial for cell fate decisions because it may effectively integrate many different signaling pathways to meet the higher regulation demand during cell development. However, whether the contribution of combinatorial regulation to the state transition is better than that of a single one and if so, what the optimal combination strategy is, seem to be significant issue from the point of view of both biology and mathematics. Using the approaches of combinatorial perturbations and bifurcation analysis, we provide a general framework for the quantitative analysis of synergism in molecular networks. Different from the known methods, the bifurcation-based approach depends only on stable state responses to stimuli because the state transition induced by combinatorial perturbations occurs between stable states. More importantly, an optimal combinatorial perturbation strategy can be determined by investigating the relationship between the bifurcation curve of a synergistic perturbation pair and the level set of a specific objective function. The approach is applied to two models, i.e., a theoretical multistable decision model and a biologically realistic CREB model, to show its validity, although the approach holds for a general class of biological systems.

  1. 27 CFR 27.133 - General requirements.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false General requirements. 27.133 Section 27.133 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU... Reports Record and Report of Imported Liquors § 27.133 General requirements. Except as provided in § 27...

  2. Structural and dynamic features of HLA-B27 subtypes.

    PubMed

    Uchanska-Ziegler, Barbara; Ziegler, Andreas; Schmieder, Peter

    2013-07-01

    The differential association of HLA-B27 subtypes with ankylosing spondylitis provides the rationale for a comparative investigation of these proteins. Results from the last 2 years of research on minimally distinct HLA-B27 subtypes, primarily using biochemical and biophysical techniques, are presented and discussed. We summarize evidence that micropolymorphisms within the molecules' peptide-binding groove influence wide-ranging biochemical, biophysical and antigenic properties of HLA-B27 molecules, and suggest that distinct, subtype and peptide-dependent dynamics of peptide - heavy chain - β(2)-microglobulin heterotrimers could be instrumental for an understanding of the initiation of disease processes that are connected with certain HLA-B27 subtypes. The results indicate that mAbs that bind only to structurally distinguishable subsets of HLA-B27 molecules as well as techniques that assess the flexibility of these antigens may hold the key to comprehend molecular events contributing to the initial stages of disease pathogenesis in spondyloarthropathies.

  3. Anaphase-promoting complex/cyclosome protein Cdc27 is a target for curcumin-induced cell cycle arrest and apoptosis.

    PubMed

    Lee, Seung Joon; Langhans, Sigrid A

    2012-01-26

    Curcumin (diferuloylmethane), the yellow pigment in the Asian spice turmeric, is a hydrophobic polyphenol from the rhizome of Curcuma longa. Because of its chemopreventive and chemotherapeutic potential with no discernable side effects, it has become one of the major natural agents being developed for cancer therapy. Accumulating evidence suggests that curcumin induces cell death through activation of apoptotic pathways and inhibition of cell growth and proliferation. The mitotic checkpoint, or spindle assembly checkpoint (SAC), is the major cell cycle control mechanism to delay the onset of anaphase during mitosis. One of the key regulators of the SAC is the anaphase promoting complex/cyclosome (APC/C) which ubiquitinates cyclin B and securin and targets them for proteolysis. Because APC/C not only ensures cell cycle arrest upon spindle disruption but also promotes cell death in response to prolonged mitotic arrest, it has become an attractive drug target in cancer therapy. Cell cycle profiles were determined in control and curcumin-treated medulloblastoma and various other cancer cell lines. Pull-down assays were used to confirm curcumin binding. APC/C activity was determined using an in vitro APC activity assay. We identified Cdc27/APC3, a component of the APC/C, as a novel molecular target of curcumin and showed that curcumin binds to and crosslinks Cdc27 to affect APC/C function. We further provide evidence that curcumin preferably induces apoptosis in cells expressing phosphorylated Cdc27 usually found in highly proliferating cells. We report that curcumin directly targets the SAC to induce apoptosis preferably in cells with high levels of phosphorylated Cdc27. Our studies provide a possible molecular mechanism why curcumin induces apoptosis preferentially in cancer cells and suggest that phosphorylation of Cdc27 could be used as a biomarker to predict the therapeutic response of cancer cells to curcumin.

  4. Anaphase-promoting complex/cyclosome protein Cdc27 is a target for curcumin-induced cell cycle arrest and apoptosis

    PubMed Central

    2012-01-01

    Background Curcumin (diferuloylmethane), the yellow pigment in the Asian spice turmeric, is a hydrophobic polyphenol from the rhizome of Curcuma longa. Because of its chemopreventive and chemotherapeutic potential with no discernable side effects, it has become one of the major natural agents being developed for cancer therapy. Accumulating evidence suggests that curcumin induces cell death through activation of apoptotic pathways and inhibition of cell growth and proliferation. The mitotic checkpoint, or spindle assembly checkpoint (SAC), is the major cell cycle control mechanism to delay the onset of anaphase during mitosis. One of the key regulators of the SAC is the anaphase promoting complex/cyclosome (APC/C) which ubiquitinates cyclin B and securin and targets them for proteolysis. Because APC/C not only ensures cell cycle arrest upon spindle disruption but also promotes cell death in response to prolonged mitotic arrest, it has become an attractive drug target in cancer therapy. Methods Cell cycle profiles were determined in control and curcumin-treated medulloblastoma and various other cancer cell lines. Pull-down assays were used to confirm curcumin binding. APC/C activity was determined using an in vitro APC activity assay. Results We identified Cdc27/APC3, a component of the APC/C, as a novel molecular target of curcumin and showed that curcumin binds to and crosslinks Cdc27 to affect APC/C function. We further provide evidence that curcumin preferably induces apoptosis in cells expressing phosphorylated Cdc27 usually found in highly proliferating cells. Conclusions We report that curcumin directly targets the SAC to induce apoptosis preferably in cells with high levels of phosphorylated Cdc27. Our studies provide a possible molecular mechanism why curcumin induces apoptosis preferentially in cancer cells and suggest that phosphorylation of Cdc27 could be used as a biomarker to predict the therapeutic response of cancer cells to curcumin. PMID:22280307

  5. Therapeutic silencing of FSP27 reduces the progression of atherosclerosis in Ldlr-/- mice.

    PubMed

    Rajamoorthi, Ananthi; Lee, Richard G; Baldán, Ángel

    2018-05-24

    Obesity, hepatosteatosis, and hypertriglyceridemia are components of the metabolic syndrome and independent risk factors for cardiovascular disease. The lipid droplet-associated protein CIDEC (cell death-inducing DFFA-like effector C), known in mice as FSP27 (fat-specific protein 27), plays a key role in maintaining triacylglyceride (TAG) homeostasis in adipose tissue and liver, and controls circulating TAG levels in mice. Importantly, mutations and SNPs in CIDEC are associated with dyslipidemia and altered metabolic function in humans. Here we tested whether systemic silencing of Fsp27 using antisense oligonucleotides (ASOs) was atheroprotective in LDL receptor knock-out (Ldlr -/- ) mice. Atheroprone Ldlr -/- mice were fed a high-fat, high-cholesterol diet for 12 weeks while simultaneously dosed with saline, ASO-ctrl, or ASO-Fsp27. Data show that, compared to control treatments, silencing Fsp27 significantly reduced body weight gain and visceral adiposity, prevented diet-induced hypertriglyceridemia, and reduced atherosclerotic lesion size both in en face aortas and in the aortic root. Our findings suggest that therapeutic silencing of Fsp27 with ASOs may be beneficial in the prevention and management of atherogenic disease in patients with metabolic syndrome. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  6. ATXR5 and ATXR6 are novel H3K27 monomethyltransferases required for chromatin structure and gene silencing

    PubMed Central

    Jacob, Yannick; Feng, Suhua; LeBlanc, Chantal A.; Bernatavichute, Yana V.; Stroud, Hume; Cokus, Shawn; Johnson, Lianna M.; Pellegrini, Matteo; Jacobsen, Steven E.; Michaels, Scott D.

    2009-01-01

    Constitutive heterochromatin in Arabidopsis thaliana is marked by repressive chromatin modifications including DNA methylation, histone H3 dimethylation at lysine 9 (H3K9me2), and monomethylation at lysine 27 (H3K27me1). The enzymes catalyzing DNA methylation and H3K9me2 have been identified and mutations in these proteins lead to the reactivation of silenced heterochromatic elements. The enzymes responsible for heterochromatic H3K27me1, in contrast, remain unknown. Here we show that the divergent SET-domain proteins ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6 exhibit H3K27 monomethyltransferase activity and double mutants have reduced H3K27me1 in vivo and show partial heterochromatin decondensation. Mutations in atxr5 and atxr6 also lead to transcriptional activation of repressed heterochromatic elements. Interestingly, H3K9me2 and DNA methylation are unaffected in the double mutant. These results indicate that ATXR5 and ATXR6 form a novel class of H3K27 methyltransferases and that H3K27me1 represents a new pathway required for transcriptional repression in Arabidopsis. PMID:19503079

  7. 47 CFR 27.61-27.62 - [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 2 2011-10-01 2011-10-01 false [Reserved] 27.61-27.62 Section 27.61-27.62 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES Technical Standards §§ 27.61-27.62 [Reserved] ...

  8. 47 CFR 27.61-27.62 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false [Reserved] 27.61-27.62 Section 27.61-27.62 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES Technical Standards §§ 27.61-27.62 [Reserved] ...

  9. Caffeine synergizes with another coffee component to increase plasma GCSF: linkage to cognitive benefits in Alzheimer's mice.

    PubMed

    Cao, Chuanhai; Wang, Li; Lin, Xiaoyang; Mamcarz, Malgorzata; Zhang, Chi; Bai, Ge; Nong, Jasson; Sussman, Sam; Arendash, Gary

    2011-01-01

    Retrospective and prospective epidemiologic studies suggest that enhanced coffee/caffeine intake during aging reduces risk of Alzheimer's disease (AD). Underscoring this premise, our studies in AD transgenic mice show that long-term caffeine administration protects against cognitive impairment and reduces brain amyloid-β levels/deposition through suppression of both β- and γ-secretase. Because coffee contains many constituents in addition to caffeine that may provide cognitive benefits against AD, we examined effects of caffeinated and decaffeinated coffee on plasma cytokines, comparing their effects to caffeine alone. In both AβPPsw+PS1 transgenic mice and non-transgenic littermates, acute i.p. treatment with caffeinated coffee greatly and specifically increased plasma levels of granulocyte-colony stimulating factor (GCSF), IL-10, and IL-6. Neither caffeine solution alone (which provided high plasma caffeine levels) or decaffeinated coffee provided this effect, indicating that caffeine synergized with some as yet unidentified component of coffee to selectively elevate these three plasma cytokines. The increase in GCSF is particularly important because long-term treatment with coffee (but not decaffeinated coffee) enhanced working memory in a fashion that was associated only with increased plasma GCSF levels among all cytokines. Since we have previously reported that long-term GCSF treatment enhances cognitive performance in AD mice through three possible mechanisms (e.g., recruitment of microglia from bone marrow, synaptogenesis, and neurogenesis), the same mechanisms could be complimentary to caffeine's established ability to suppress Aβ production. We conclude that coffee may be the best source of caffeine to protect against AD because of a component in coffee that synergizes with caffeine to enhance plasma GCSF levels, resulting in multiple therapeutic actions against AD.

  10. Dual pharmacological inhibition of glutathione and thioredoxin systems synergizes to kill colorectal carcinoma stem cells.

    PubMed

    Tanaka, Genki; Inoue, Ken-Ichi; Shimizu, Takayuki; Akimoto, Kazumi; Kubota, Keiichi

    2016-09-01

    NRF2 stabilizes redox potential through genes for glutathione and thioredoxin antioxidant systems. Whether blockade of glutathione and thioredoxin is useful in eliminating cancer stem cells remain unknown. We used xenografts derived from colorectal carcinoma patients to investigate the pharmacological inhibition of glutathione and thioredoxin systems. Higher expression of five glutathione S-transferase isoforms (GSTA1, A2, M4, O2, and P1) was observed in xenograft-derived spheroids than in fibroblasts. Piperlongumine (2.5-10 μmol/L) and auranofin (0.25-4 μmol/L) were used to inhibit glutathione S-transferase π and thioredoxin reductase, respectively. Piperlongumine or auranofin alone up-regulated the expression of NRF2 target genes, but not TP53 targets. While piperlongumine showed modest cancer-specific cell killing (IC50 difference between cancer spheroids and fibroblasts: P = 0.052), auranofin appeared more toxic to fibroblasts (IC50 difference between cancer spheroids and fibroblasts: P = 0.002). The synergism of dual inhibition was evaluated by determining the Combination Index, based on the number of surviving cells with combination treatments. Molar ratios indicated synergism in cancer spheroids, but not in fibroblasts: (auranofin:piperlongumine) = 2:5, 1:5, 1:10, and 1:20. Cancer-specific cell killing was achieved at the following drug concentrations (auranofin:piperlongumine): 0.25:2.5 μmol/L, 0.5:2.5 μmol/L, or 0.25:5 μmol/L. The dual inhibition successfully decreased CD44v9 surface presentation and delayed tumor emergence in nude mouse. However, a small subpopulation persistently survived and accumulated phosphorylated histone H2A. Such "persisters" still retained lesser but significant tumorigenicity. Thus, dual inhibition of glutathione S-transferase π and thioredoxin reductase could be a feasible option for decreasing the tumor mass and CD44v9-positive fraction by disrupting redox regulation. © 2016 The Authors. Cancer Medicine

  11. Home cooking and ingredient synergism improve lycopene isomer production in Sofrito.

    PubMed

    Rinaldi de Alvarenga, José Fernando; Tran, Camilla; Hurtado-Barroso, Sara; Martinez-Huélamo, Miriam; Illan, Montserrat; Lamuela-Raventos, Rosa M

    2017-09-01

    There has been increasing interest in tomato products rich in lycopene Z-isomers since these carotenoids present greater bioavailability and antioxidant capacity than the all-E lycopene form. Intrinsic food properties as well as processing and the interaction between dietary components can all influence the content, type and bioavailability of carotenoids. The aim of this study was to evaluate whether carotenoid content and isomerization in tomato-based Mediterranean sofrito is affected by the process of home cooking and the presence of other ingredients such as extra virgin olive oil, onion and garlic. We used a full factorial design to clarify the contribution of each ingredient to the carotenoid composition of sofrito and to determine whether this can be improved by the cooking time and ingredient synergism. Cooking time and onion content were associated with a higher production of 5-Z-lycopene, 9-Z-lycopene and 13-Z-lycopene in sofrito. Onion proved to be the most interesting ingredient in the sofrito formulation due to their enhancing effect on lycopene isomerization. The use of onion combined with an adequate processing time may improve the bioavailability of lycopene in tomato products. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. 27 CFR 6.27 - Proprietary interest.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Proprietary interest. 6.27... OF THE TREASURY ALCOHOL âTIED-HOUSEâ Unlawful Inducements Interest in Retail License § 6.27 Proprietary interest. (a) Complete ownership. Outright ownership of a retail business by an industry member is...

  13. 27 CFR 6.27 - Proprietary interest.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Proprietary interest. 6.27... OF THE TREASURY LIQUORS âTIED-HOUSEâ Unlawful Inducements Interest in Retail License § 6.27 Proprietary interest. (a) Complete ownership. Outright ownership of a retail business by an industry member is...

  14. Effect of HSP27 on Human Breast Tumor Cell Growth and Motility

    DTIC Science & Technology

    1999-08-01

    the small stress protein, HSP27 , on growth and motility characteristics of normal and tumor-derived human mammary cell lines. We hypothesized that...cells overexpressing HSP27 would show increased motility, altered chemotactic properties, increased resistance to heat killing and to certain drugs...Donna has prepared and studied 19 clonal MDA23 1 breast tumor cell lines that overexpress human HSP27 , and determined that, while heat resistance is

  15. 47 CFR 27.322-27.325 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false [Reserved] 27.322-27.325 Section 27.322-27.325 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES Application, Licensing, and Processing Rules for WCS §§ 27.322-27.325 [Reserved] ...

  16. 47 CFR 27.304-27.307 - [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 2 2011-10-01 2011-10-01 false [Reserved] 27.304-27.307 Section 27.304-27.307 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES Application, Licensing, and Processing Rules for WCS §§ 27.304-27.307 [Reserved] ...

  17. 47 CFR 27.310-27.320 - [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 2 2011-10-01 2011-10-01 false [Reserved] 27.310-27.320 Section 27.310-27.320 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES Application, Licensing, and Processing Rules for WCS §§ 27.310-27.320 [Reserved] ...

  18. 47 CFR 27.322-27.325 - [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 2 2011-10-01 2011-10-01 false [Reserved] 27.322-27.325 Section 27.322-27.325 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES Application, Licensing, and Processing Rules for WCS §§ 27.322-27.325 [Reserved] ...

  19. 47 CFR 27.310-27.320 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false [Reserved] 27.310-27.320 Section 27.310-27.320 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES Application, Licensing, and Processing Rules for WCS §§ 27.310-27.320 [Reserved] ...

  20. 47 CFR 27.304-27.307 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false [Reserved] 27.304-27.307 Section 27.304-27.307 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES Application, Licensing, and Processing Rules for WCS §§ 27.304-27.307 [Reserved] ...

  1. HSP27 Protects the Blood-Brain Barrier Against Ischemia-Induced Loss of Integrity

    PubMed Central

    Leak, Rehana K.; Zhang, Lili; Stetler, R. Anne; Weng, Zhongfang; Li, Peiying; Atkins, G. Brandon; Gao, Yanqin; Chen, Jun

    2014-01-01

    Loss of integrity of the blood-brain barrier (BBB) in stroke victims initiates a devastating cascade of events including extravasation of blood-borne molecules, water, and inflammatory cells deep into brain parenchyma. Thus, it is important to identify mechanisms by which BBB integrity can be maintained in the face of ischemic injury in experimental stroke. We previously demonstrated that the phylogenetically conserved small heat shock protein 27 (HSP27) protects against transient middle cerebral artery occlusion (tMCAO). Here we show that HSP27 transgenic overexpression also maintains the integrity of the BBB in mice subjected to tMCAO. Extravasation of endogenous IgG antibodies and exogenous FITC-albumin into the brain following tMCAO was reduced in transgenic mice, as was total brain water content. HSP27 overexpression abolished the appearance of TUNEL-positive profiles in microvessel walls. Transgenics also exhibited less loss of microvessel proteins following tMCAO. Notably, primary endothelial cell cultures were rescued from oxygen-glucose deprivation (OGD) by lentiviral HSP27 overexpression according to four viability assays, supporting a direct effect on this cell type. Finally, HSP27 overexpression reduced the appearance of neutrophils in the brain and inhibited the secretion of five cytokines. These findings reveal a novel role for HSP27 in attenuating ischemia/reperfusion injury - the maintenance of BBB integrity. Endogenous upregulation of HSP27 after ischemia in wild-type animals may exert similar protective functions and warrants further investigation. Exogenous enhancement of HSP27 by rational drug design may lead to future therapies against a host of injuries, including but not limited to a harmful breach in brain vasculature. PMID:23469858

  2. Characterization of a proteasome and TAP-independent presentation of intracellular epitopes by HLA-B27 molecules.

    PubMed

    Magnacca, Adriana; Persiconi, Irene; Nurzia, Elisa; Caristi, Silvana; Meloni, Francesca; Barnaba, Vincenzo; Paladini, Fabiana; Raimondo, Domenico; Fiorillo, Maria Teresa; Sorrentino, Rosa

    2012-08-31

    Nascent HLA-class I molecules are stabilized by proteasome-derived peptides in the ER and the new complexes proceed to the cell surface through the post-ER vesicles. It has been shown, however, that less stable complexes can exchange peptides in the Trans Golgi Network (TGN). HLA-B27 are the most studied HLA-class I molecules due to their association with Ankylosing Spondylitis (AS). Chimeric proteins driven by TAT of HIV have been exploited by us to deliver viral epitopes, whose cross-presentation by the HLA-B27 molecules was proteasome and TAP-independent and not restricted to Antigen-Presenting Cells (APC). Here, using these chimeric proteins as epitope suppliers, we compared with each other and with the HLA-A2 molecules, the two HLA-B*2705 and B*2709 alleles differing at residue 116 (D116H) and differentially associated with AS. We found that the antigen presentation by the two HLA-B27 molecules was proteasome-, TAP-, and APC-independent whereas the presentation by the HLA-A2 molecules required proteasome, TAP and professional APC. Assuming that such difference could be due to the unpaired, highly reactive Cys-67 distinguishing the HLA-B27 molecules, C67S mutants in HLA-B*2705 and B*2709 and V67C mutant in HLA-A*0201 were also analyzed. The results showed that this mutation did not influence the HLA-A2-restricted antigen presentation while it drastically affected the HLA-B27-restricted presentation with, however, remarkable differences between B*2705 and B*2709. The data, together with the occurrence on the cell surface of unfolded molecules in the case of C67S-B*2705 mutant but not in that of C67S-B*2709 mutant, indicates that Cys-67 has a more critical role in stabilizing the B*2705 rather than the B*2709 complexes.

  3. Determination of Protein by Fluorescence Enhancement of Curcumin in Lanthanum-Curcumin-Sodium Dodecyl Benzene Sulfonate-Protein System

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

    Wang, Feng; Huang, Wei; Zhang, Yunfeng

    2011-01-01

    We found that the fluorescence intensity of the lanthanum (La(3+))-curcumin (CU) complex can be highly enhanced by proteins in the presence of sodium dodecyl benzene sulphonate (SDBS). Based on this finding, a new fluorimetric method for the determination of protein was developed. Under optimized conditions, the enhanced intensities of fluorescence are quantitatively in proportion to the concentrations of proteins in the range 0.0080-20.0 g mL(-1) for bovine serum albumin (BSA) and 0.00080-20.0 g mL(-1) for human serum albumin (HSA) with excitation of 425 nm, and 0.00020-20.0 g mL(-1) for bovine serum albumin (BSA) and 0.00080-20.0 g mL(-1)for human serum albuminmore » (HSA) with excitation of 280 nm, while corresponding qualitative detection limits (S/N 3) are as low as 5.368, 0.573, 0.049, 0.562 g mL(-1), respectively. Study on reaction mechanism reveals that proteins can bind with La(3+), CU and SDBS through self-assembling function with electrostatic attraction, hydrogen bonding, hydrophobic interaction and van der Waals forces, etc. The proteins form a supermolecular association with multilayer structure, in which La(3+)-CU is clamped between BSA and SDBS. The unique high fluorescence enhancement of CU is resulted through synergic effects of favorable hydrophobic microenvironment provided by BSA and SDBS, and efficient intermolecular energy transfer among BSA, SDBS and CU. In energy transfer process, La(3+) plays a crucial role because it not only shortens the distance between SDBS and CU, but also acts as a "bridge" for transferring the energy from BSA to CU.« less

  4. Phosphorylation and Subcellular Localization of p27Kip1 Regulated by Hydrogen Peroxide Modulation in Cancer Cells

    PubMed Central

    Ibañez, Irene L.; Bracalente, Candelaria; Notcovich, Cintia; Tropper, Ivanna; Molinari, Beatriz L.; Policastro, Lucía L.; Durán, Hebe

    2012-01-01

    The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H2O2) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H2O2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H2O2 (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H2O2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27Kip1. PMID

  5. Cigarette Smoke-Related Hydroquinone Induces Filamentous Actin Reorganization and Heat Shock Protein 27 Phosphorylation through p38 and Extracellular Signal-Regulated Kinase 1/2 in Retinal Pigment Epithelium

    PubMed Central

    Pons, Marianne; Cousins, Scott W.; Csaky, Karl G.; Striker, Gary; Marin-Castaño, Maria E.

    2010-01-01

    Retinal pigment epithelium (RPE)-derived membranous debris named blebs, may accumulate and contribute to sub-RPE deposit formation, which is the earliest sign of age-related macular degeneration (AMD). Oxidative injury to the RPE might play a significant role in AMD. However, the underlying mechanisms are unknown. We previously reported that hydroquinone (HQ), a major pro-oxidant in cigarette smoke, foodstuff, and atmospheric pollutants, induces actin rearrangement and membrane blebbing in RPE cells as well as sub-RPE deposits in mice. Here, we show for the first time that phosphorylated Heat shock protein 27 (Hsp27), a key regulator of actin filaments dynamics, is up-regulated in RPE from patients with AMD. Also, HQ-induced nonlethal oxidative injury led to Hsp27mRNA up-regulation, dimer formation, and Hsp27 phosphorylation in ARPE-19 cells. Furthermore, we found that a cross talk between p38 and extracellular signal-regulated kinase (ERK) mediates HQ-induced Hsp27 phosphorylation and actin aggregate formation, revealing ERK as a novel upstream mediator of Hsp27 phosphorylation. Finally, we demonstrated that Hsp25, p38, and ERK phosphorylation are increased in aging C57BL/6 mice chronically exposed to HQ, whereas Hsp25 expression is decreased. Our data suggest that phosphorylated Hsp27 might be a key mediator in AMD and HQ-induced oxidative injury to the RPE, which may provide helpful insights into the early cellular events associated with actin reorganization and bleb formation involved in sub-RPE deposits formation relevant to the pathogenesis of AMD. PMID:20651235

  6. A687V EZH2 is a driver of histone H3 lysine 27 (H3K27) hypertrimethylation.

    PubMed

    Ott, Heidi M; Graves, Alan P; Pappalardi, Melissa B; Huddleston, Michael; Halsey, Wendy S; Hughes, Ashley M; Groy, Arthur; Dul, Edward; Jiang, Yong; Bai, Yuchen; Annan, Roland; Verma, Sharad K; Knight, Steven D; Kruger, Ryan G; Dhanak, Dashyant; Schwartz, Benjamin; Tummino, Peter J; Creasy, Caretha L; McCabe, Michael T

    2014-12-01

    The EZH2 methyltransferase silences gene expression through methylation of histone H3 on lysine 27 (H3K27). Recently, EZH2 mutations have been reported at Y641, A677, and A687 in non-Hodgkin lymphoma. Although the Y641F/N/S/H/C and A677G mutations exhibit clearly increased activity with substrates dimethylated at lysine 27 (H3K27me2), the A687V mutant has been shown to prefer a monomethylated lysine 27 (H3K27me1) with little gain of activity toward H3K27me2. Herein, we demonstrate that despite this unique substrate preference, A687V EZH2 still drives increased H3K27me3 when transiently expressed in cells. However, unlike the previously described mutants that dramatically deplete global H3K27me2 levels, A687V EZH2 retains normal levels of H3K27me2. Sequencing of B-cell-derived cancer cell lines identified an acute lymphoblastic leukemia cell line harboring this mutation. Similar to exogenous expression of A687V EZH2, this cell line exhibited elevated H3K27me3 while possessing H3K27me2 levels higher than Y641- or A677-mutant lines. Treatment of A687V EZH2-mutant cells with GSK126, a selective EZH2 inhibitor, was associated with a global decrease in H3K27me3, robust gene activation, caspase activation, and decreased proliferation. Structural modeling of the A687V EZH2 active site suggests that the increased catalytic activity with H3K27me1 may be due to a weakened interaction with an active site water molecule that must be displaced for dimethylation to occur. These findings suggest that A687V EZH2 likely increases global H3K27me3 indirectly through increased catalytic activity with H3K27me1 and cells harboring this mutation are highly dependent on EZH2 activity for their survival. ©2014 American Association for Cancer Research.

  7. Hsp27 binding to the 3'UTR of bim mRNA prevents neuronal death during oxidative stress-induced injury: a novel cytoprotective mechanism.

    PubMed

    Dávila, David; Jiménez-Mateos, Eva M; Mooney, Claire M; Velasco, Guillermo; Henshall, David C; Prehn, Jochen H M

    2014-11-01

    Neurons face a changeable microenvironment and therefore need mechanisms that allow rapid switch on/off of their cytoprotective and apoptosis-inducing signaling pathways. Cellular mechanisms that control apoptosis activation include the regulation of pro/antiapoptotic mRNAs through their 3'-untranslated region (UTR). This region holds binding elements for RNA-binding proteins, which can control mRNA translation. Here we demonstrate that heat shock protein 27 (Hsp27) prevents oxidative stress-induced cell death in cerebellar granule neurons by specific regulation of the mRNA for the proapoptotic BH3-only protein, Bim. Hsp27 depletion induced by oxidative stress using hydrogen peroxide (H2O2) correlated with bim gene activation and subsequent neuronal death, whereas enhanced Hsp27 expression prevented these. This effect could not be explained by proteasomal degradation of Bim or bim promoter inhibition; however, it was associated with a specific increase in the levels of bim mRNA and with its binding to Hsp27. Finally, we determined that enhanced Hsp27 expression in neurons exposed to H2O2 or glutamate prevented the translation of a reporter plasmid where bim-3'UTR mRNA sequence was cloned downstream of a luciferase gene. These results suggest that repression of bim mRNA translation through binding to the 3'UTR constitutes a novel cytoprotective mechanism of Hsp27 during stress in neurons. © 2014 Dávila et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  8. New Frontiers in NanoBiotechnology: Monitoring the Protein Function With Single Protein Resolution

    DTIC Science & Technology

    2005-03-29

    Protein (GFP) is a spontaneously fluorescent polypeptide of 27 kD from the jellyfish Aequorea victoria that absorbs UV-blue light and emits in the...will have vast applications in science. Relationship between structure and optical properties in Green Fluorescent Proteins : A quantum mechanical study...RESEARCH AND DEVELOPMENT Invited talks Folding, stability and fluorescence efficiency of the Green and Red Fluorescent Proteins Saverio Alberti Lab.

  9. Synergic chemoprevention with dietary carbohydrate restriction and supplementation of AMPK-activating phytochemicals: the role of SIRT1

    PubMed Central

    Choi, Min-Ah; Ro, Simon Weonsang; Yang, Woo Ick; Cho, Arthur E.H.; Ju, Hye-Lim; Baek, Sinhwa; Chung, Sook In; Kang, Won Jun; Yun, Mijin; Park, Jeon Han

    2016-01-01

    Calorie restriction or a low-carbohydrate diet (LCD) can increase life span in normal cells while inhibiting carcinogenesis. Various phytochemicals also have calorie restriction-mimetic anticancer properties. We investigated whether an isocaloric carbohydrate-restriction diet and AMP-activated protein kinase (AMPK)-activating phytochemicals induce synergic tumor suppression. We used a mixture of AMPK-activating phytochemical extracts including curcumin, quercetin, catechins, and resveratrol. Survival analysis was carried out in a B16F10 melanoma model fed a control diet (62.14% kcal carbohydrate, 24.65% kcal protein and 13.2% kcal fat), a control diet with multiple phytochemicals (MP), LCD (16.5, 55.2, and 28.3% kcal, respectively), LCD with multiple phytochemicals (LCDmp), a moderate-carbohydrate diet (MCD, 31.9, 62.4, and 5.7% kcal, respectively), or MCD with phytochemicals (MCDmp). Compared with the control group, MP, LCD, or MCD intervention did not produce survival benefit, but LCDmp (22.80±1.58 vs. 28.00±1.64 days, P=0.040) and MCDmp (23.80±1.08 vs. 30.13±2.29 days, P=0.008) increased the median survival time significantly. Suppression of the IGF-1R/PI3K/Akt/mTOR signaling, activation of the AMPK/SIRT1/LKB1pathway, and NF-κB suppression were the critical tumor-suppression mechanisms. In addition, SIRT1 suppressed proliferation of the B16F10 and A375SM cells under a low-glucose condition. Alterations in histone methylation within Pten and FoxO3a were observed after the MCDmp intervention. In the transgenic liver cancer model developed by hydrodynamic transfection of the HrasG12V and shp53, MCDmp and LCDmp interventions induced significant cancer-prevention effects. Microarray analysis showed that PPARα increased with decreased IL-6 and NF-κB within the hepatocytes after an MCDmp intervention. In conclusion, an isocaloric carbohydrate-restriction diet and natural AMPK-activating agents induce synergistic anticancer effects. SIRT1 acts as a

  10. Additive effect of rPb27 immunization and chemotherapy in experimental paracoccidioidomycosis.

    PubMed

    Fernandes, Viviane C; Martins, Estefânia M N; Boeloni, Jankerle N; Coitinho, Juliana B; Serakides, Rogéria; Goes, Alfredo M

    2011-03-10

    Paracoccidioidomycosis, PCM, the major systemic mycosis in Latin America, is caused by the termally dimorphic fungus Paracoccidioides brasiliensis and requires extended periods of chemotherapy with a significant frequency of relapsing disease. The search for new alternatives of treatment is necessary. rPb27 is an antigenic protein from P. brasiliensis that already showed a significant protective activity as a vaccine for PCM in experimental models. The cDNA of rPb27 was subcloned into a pET-DEST 42 plasmid, expressed in E. coli with a his-tag and purified by affinity chromatography. Immunization with this recombinant protein and chemotherapy were used together in an attempt to improve treatment of PCM. For this, BALB/c mice were challenged with pathogenic P. brasiliensis strain and after immunized with rPb27, in the presence of Corynebacterium parvum and Al(OH)(3), some groups were also treated with fluconazole. After 40 days of treatment, the combined drug/rPb27 administration controlled PCM in the liver and spleen, with long lasting protection, and largely preserved tissues structures of these organs. Additionally, in the lungs after 40 days of treatment there was a significant reduction in the fungal load and size of lesions. At the same time, the levels of TNF-α were higher than infected-only mice. Moreover, significant levels of anti-rPb27 specific IgG1, IgG2a and IgG2b isotypes were detected in the sera of mice immunized with rPb27 fluconazole treated or not. These results showed an additive protective effect of rPb27 immunization and chemotherapy, suggesting that an rPb27-based vaccine can be used to enhance PCM antifungal treatment.

  11. Synergizing 13C Metabolic Flux Analysis and Metabolic Engineering for Biochemical Production.

    PubMed

    Guo, Weihua; Sheng, Jiayuan; Feng, Xueyang

    Metabolic engineering of industrial microorganisms to produce chemicals, fuels, and drugs has attracted increasing interest as it provides an environment-friendly and renewable route that does not depend on depleting petroleum sources. However, the microbial metabolism is so complex that metabolic engineering efforts often have difficulty in achieving a satisfactory yield, titer, or productivity of the target chemical. To overcome this challenge, 13 C Metabolic Flux Analysis ( 13 C-MFA) has been developed to investigate rigorously the cell metabolism and quantify the carbon flux distribution in central metabolic pathways. In the past decade, 13 C-MFA has been widely used in academic labs and the biotechnology industry to pinpoint the key issues related to microbial-based chemical production and to guide the development of the appropriate metabolic engineering strategies for improving the biochemical production. In this chapter we introduce the basics of 13 C-MFA and illustrate how 13 C-MFA has been applied to synergize with metabolic engineering to identify and tackle the rate-limiting steps in biochemical production.

  12. 27 CFR 4.27 - Vintage wine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Vintage wine. 4.27 Section... THE TREASURY LIQUORS LABELING AND ADVERTISING OF WINE Standards of Identity for Wine § 4.27 Vintage wine. (a) General. Vintage wine is wine labeled with the year of harvest of the grapes and made in...

  13. Relevance of anti-inflammatory and antioxidant activities of exemestane and synergism with sulforaphane for disease prevention

    PubMed Central

    Liu, Hua; Talalay, Paul

    2013-01-01

    Exemestane (6-methyleneandrosta-1,4-diene-3,17-dione) is a synthetic steroidal inhibitor of the aromatase reaction that catalyzes the terminal and rate-limiting step of the biosynthesis of estrogens. It is active clinically in preventing, delaying progression of, and treating mammary cancers, many of which are estrogen receptor-positive. A striking feature of the structure of exemestane is an extended system of conjugated Michael reaction functions, which is also characteristic of inducers of a broad network of chemoprotective genes regulated by the Keap1 (Kelch-like ECA-associated protein)/Nrf2 (nuclear factor E2-related factor 2)/ARE (antioxidant response element) signaling system. These genes are largely involved in xenobiotic metabolism and antioxidative and anti-inflammatory protection, as well as the synthesis and reduction of glutathione. We show here that exemestane transcriptionally activates NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1), typical chemoprotective gene products, in a wide variety of mouse, rat, and human cells. It protects several cell lines against oxidative toxicity of tert-butyl hydroperoxide and 4-hydroxynonenal, against free radical damage arising from hypoxia–reoxygenation, and against UVA radiation damage. Exemestane also inhibits the inflammatory increases in inducible nitric oxide synthase (iNOS) in mouse macrophages exposed to LPS (lipopolysaccharide), thereby resembling the isothiocyanate sulforaphane derived from broccoli. Remarkably, combinations of exemestane and sulforaphane act highly synergistically, and this property is also displayed by several other phytochemicals. Thus, exemestane has a wide range of previously unrecognized protective activities, probably unrelated to aromatase inhibition. Its potential for reducing the risk, not only of breast cancer, but also of other chronic diseases that arise from inflammation, oxidative stress, and DNA-damaging electrophiles, requires exploration

  14. Relevance of anti-inflammatory and antioxidant activities of exemestane and synergism with sulforaphane for disease prevention.

    PubMed

    Liu, Hua; Talalay, Paul

    2013-11-19

    Exemestane (6-methyleneandrosta-1,4-diene-3,17-dione) is a synthetic steroidal inhibitor of the aromatase reaction that catalyzes the terminal and rate-limiting step of the biosynthesis of estrogens. It is active clinically in preventing, delaying progression of, and treating mammary cancers, many of which are estrogen receptor-positive. A striking feature of the structure of exemestane is an extended system of conjugated Michael reaction functions, which is also characteristic of inducers of a broad network of chemoprotective genes regulated by the Keap1 (Kelch-like ECA-associated protein)/Nrf2 (nuclear factor E2-related factor 2)/ARE (antioxidant response element) signaling system. These genes are largely involved in xenobiotic metabolism and antioxidative and anti-inflammatory protection, as well as the synthesis and reduction of glutathione. We show here that exemestane transcriptionally activates NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1), typical chemoprotective gene products, in a wide variety of mouse, rat, and human cells. It protects several cell lines against oxidative toxicity of tert-butyl hydroperoxide and 4-hydroxynonenal, against free radical damage arising from hypoxia-reoxygenation, and against UVA radiation damage. Exemestane also inhibits the inflammatory increases in inducible nitric oxide synthase (iNOS) in mouse macrophages exposed to LPS (lipopolysaccharide), thereby resembling the isothiocyanate sulforaphane derived from broccoli. Remarkably, combinations of exemestane and sulforaphane act highly synergistically, and this property is also displayed by several other phytochemicals. Thus, exemestane has a wide range of previously unrecognized protective activities, probably unrelated to aromatase inhibition. Its potential for reducing the risk, not only of breast cancer, but also of other chronic diseases that arise from inflammation, oxidative stress, and DNA-damaging electrophiles, requires exploration, particularly

  15. Combined metabolic and transcriptional profiling identifies pentose phosphate pathway activation by HSP27 phosphorylation during cerebral ischemia.

    PubMed

    Imahori, Taichiro; Hosoda, Kohkichi; Nakai, Tomoaki; Yamamoto, Yusuke; Irino, Yasuhiro; Shinohara, Masakazu; Sato, Naoko; Sasayama, Takashi; Tanaka, Kazuhiro; Nagashima, Hiroaki; Kohta, Masaaki; Kohmura, Eiji

    2017-05-04

    The metabolic pathophysiology underlying ischemic stroke remains poorly understood. To gain insight into these mechanisms, we performed a comparative metabolic and transcriptional analysis of the effects of cerebral ischemia on the metabolism of the cerebral cortex using middle cerebral artery occlusion (MCAO) rat model. Metabolic profiling by gas-chromatography/mass-spectrometry analysis showed clear separation between the ischemia and control group. The decreases of fructose 6-phosphate and ribulose 5-phosphate suggested enhancement of the pentose phosphate pathway (PPP) during cerebral ischemia (120-min MCAO) without reperfusion. Transcriptional profiling by microarray hybridization indicated that the Toll-like receptor and mitogen-activated protein kinase (MAPK) signaling pathways were upregulated during cerebral ischemia without reperfusion. In relation to the PPP, upregulation of heat shock protein 27 (HSP27) was observed in the MAPK signaling pathway and was confirmed through real-time polymerase chain reaction. Immunoblotting showed a slight increase in HSP27 protein expression and a marked increase in HSP27 phosphorylation at serine 85 after 60-min and 120-min MCAO without reperfusion. Corresponding upregulation of glucose 6-phosphate dehydrogenase (G6PD) activity and an increase in the NADPH/NAD + ratio were also observed after 120-min MCAO. Furthermore, intracerebroventricular injection of ataxia telangiectasia mutated (ATM) kinase inhibitor (KU-55933) significantly reduced HSP27 phosphorylation and G6PD upregulation after MCAO, but that of protein kinase D inhibitor (CID755673) did not affect HSP27 phosphorylation. Consequently, G6PD activation via ischemia-induced HSP27 phosphorylation by ATM kinase may be part of an endogenous antioxidant defense neuroprotection mechanism during the earliest stages of ischemia. These findings have important therapeutic implications for the treatment of stroke. Copyright © 2017 IBRO. Published by Elsevier Ltd. All

  16. The flavonoid tangeretin activates the unfolded protein response and synergizes with imatinib in the erythroleukemia cell line K562.

    PubMed

    Lust, Sofie; Vanhoecke, Barbara; Van Gele, Mireille; Philippé, Jan; Bracke, Marc; Offner, Fritz

    2010-06-01

    We explored the mechanism of cell death of the polymethoxyflavone tangeretin (TAN) in K562 breakpoint cluster region-abelson murine leukemia (Bcr-Abl+) cells. Flow cytometric analysis showed that TAN arrested the cells in the G(2)/M phase and stimulated an accumulation of the cells in the sub-G(0) phase. TAN-induced cell death was evidenced by poly(ADP)-ribose polymerase cleavage, DNA laddering fragmentation, activation of the caspase cascade and downregulation of the antiapoptotic proteins Mcl-1 and Bcl-x(L). Pretreatment with the pancaspase inhibitor Z-VAD-FMK_blocked caspase activation and cell cycle arrest but did not inhibit apoptosis which suggest that other cell killing mechanisms like endoplasmic reticulum (ER)-associated cell death pathways could be involved. We demonstrated that TAN-induced apoptosis was preceded by a rapid activation of the proapoptotic arm of the unfolded protein response, namely PKR-like ER kinase. This was accompanied by enhanced levels of glucose-regulated protein of 78 kDa and of spliced X-box binding protein 1. Furthermore, TAN sensitized K562 cells to the cell killing effects of imatinib via an apoptotic mechanism. In conclusion, our results suggest that TAN is able to induce apoptosis in Bcr-Abl+ cells via cell cycle arrest and the induction of the unfolded protein response, and has synergistic cytotoxicity with imatinib.

  17. Ubiquitin--conserved protein or selfish gene?

    PubMed

    Catic, André; Ploegh, Hidde L

    2005-11-01

    The posttranslational modifier ubiquitin is encoded by a multigene family containing three primary members, which yield the precursor protein polyubiquitin and two ubiquitin moieties, Ub(L40) and Ub(S27), that are fused to the ribosomal proteins L40 and S27, respectively. The gene encoding polyubiquitin is highly conserved and, until now, those encoding Ub(L40) and Ub(S27) have been generally considered to be equally invariant. The evolution of the ribosomal ubiquitin moieties is, however, proving to be more dynamic. It seems that the genes encoding Ub(L40) and Ub(S27) are actively maintained by homologous recombination with the invariant polyubiquitin locus. Failure to recombine leads to deterioration of the sequence of the ribosomal ubiquitin moieties in several phyla, although this deterioration is evidently constrained by the structural requirements of the ubiquitin fold. Only a few amino acids in ubiquitin are vital for its function, and we propose that conservation of all three ubiquitin genes is driven not only by functional properties of the ubiquitin protein, but also by the propensity of the polyubiquitin locus to act as a 'selfish gene'.

  18. sugE: A gene involved in tributyltin (TBT) resistance of Aeromonas molluscorum Av27.

    PubMed

    Cruz, Andreia; Micaelo, Nuno; Félix, Vitor; Song, Jun-Young; Kitamura, Shin-Ichi; Suzuki, Satoru; Mendo, Sónia

    2013-01-01

    The mechanism of bacterial resistance to tributyltin (TBT) is still unclear. The results herein presented contribute to clarify that mechanism in the TBT-resistant bacterium Aeromonas molluscorum Av27. We have identified and cloned a new gene that is involved in TBT resistance in this strain. The gene is highly homologous (84%) to the Aeromonas hydrophila-sugE gene belonging to the small multidrug resistance gene family (SMR), which includes genes involved in the transport of lipophilic drugs. In Av27, expression of the Av27-sugE was observed at the early logarithmic growth phase in the presence of a high TBT concentration (500 μM), thus suggesting the contribution of this gene for TBT resistance. E. coli cells transformed with Av27-sugE become resistant to ethidium bromide (EtBr), chloramphenicol (CP) and tetracycline (TE), besides TBT. According to the Moriguchi logP (miLogP) values, EtBr, CP and TE have similar properties and are substrates for the sugE-efflux system. Despite the different miLogP of TBT, E. coli cells transformed with Av27-sugE become resistant to this compound. So it seems that TBT is also a substrate for the SugE protein. The modelling studies performed also support this hypothesis. The data herein presented clearly indicate that sugE is involved in TBT resistance of this bacterium.

  19. Investigation of surfactant/cosurfactant synergism impact on ibuprofen solubilization capacity and drug release characteristics of nonionic microemulsions.

    PubMed

    Djekic, Ljiljana; Primorac, Marija; Filipic, Slavica; Agbaba, Danica

    2012-08-20

    The current study investigates the performances of the multicomponent mixtures of nonionic surfactants regarding the microemulsion stabilisation, drug solubilization and in vitro drug release kinetic. The primary surfactant was PEG-8 caprylic/capric glycerides (Labrasol). The cosurfactants were commercially available mixtures of octoxynol-12 and polysorbate 20 without or with the addition of PEG-40 hydrogenated castor oil (Solubilisant gamma 2421 and Solubilisant gamma 2429, respectively). The oil phase of microemulsions was isopropyl myristate. Phase behaviour study of the pseudo-ternary systems Labrasol/cosurfactant/oil/water at surfactant-to-cosurfactant weight ratios (K(m)) 40:60, 50:50 and 60:40, revealed a strong synergism in the investigated tensides mixtures for stabilisation of microemulsions containing up to 80% (w/w) of water phase at surfactant +cosurfactant-to-oil weight ratio (SCoS/O) 90:10. Solubilization of a model drug ibuprofen in concentration common for topical application (5%, w/w) was achieved at the water contents below 50% (w/w). Drug free and ibuprofen-loaded microemulsions M1-M6, containing 45% (w/w) of water phase, were prepared and characterized by polarized light microscopy, conductivity, pH, rheological and droplet size measurements. In vitro ibuprofen release kinetics from the microemulsions was investigated using paddle-over-enhancer cell method and compared with the commercial 5% (w/w) ibuprofen hydrogel product (Deep Relief, Mentholatum Company Ltd., USA). The investigated microemulsions were isotropic, low viscous Bingham-type liquids with the pH value (4.70-6.61) suitable for topical application. The different efficiency of the tensides mixtures for microemulsion stabilisation was observed, depending on the cosurfactant type and K(m) value. Solubilisant gamma 2429 as well as higher K(m) (i.e., lower relative content of the cosurfactant) provided higher surfactant/cosurfactant synergism. The drug molecules were predominantly

  20. Potential use of TIA-1, MFF, microRNA-200a-3p, and microRNA-27 as a novel marker for hepatocellular carcinoma.

    PubMed

    Tak, Hyosun; Kang, Hoin; Ji, Eunbyul; Hong, Youlim; Kim, Wook; Lee, Eun Kyung

    2018-03-18

    Precise and early diagnosis is critical to improve the survival rate of hepatocellular carcinoma (HCC) patients. Although several genetic and protein markers have been developed and are currently used for diagnosis, prognosis, risk stratification, and therapeutic monitoring, application of these markers still needs to be improved for better specificity and efficacy. In this study, we investigated the relative expression of mitochondrial dynamics-regulating factors including T-cell intercellular antigen protein-1 (TIA-1), mitochondrial fission factor (MFF), microRNA (miR)-200a-3p, and miR-27a/b in the liver tissues from HCC patients. The expressions of TIA-1 and MFF were augmented in the cancerous liver tissues compared to the corresponding non-tumor tissues at mRNA and protein level, while the levels of miR-200a-3p and miR-27a/b were relatively lower in the cancerous liver tissues. In addition, high levels of TIA-1 and MFF mRNA were related to the poor survival rate of HCC patients. Our results indicated that the expressions of TIA-1, MFF, miR-200a-3p, and miR-27a/b in the cancerous liver tissues differed to these in non-cancerous tissues of HCC patients, demonstrating that these gene expressions could be potential markers for the diagnosis and prognosis of HCC. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Hepatitis C virus inhibitor synergism suggests multistep interactions between heat-shock protein 90 and hepatitis C virus replication

    PubMed Central

    Kubota, Naoko; Nomoto, Masataka; Hwang, Gi-Wook; Watanabe, Toshihiko; Kohara, Michinori; Wakita, Takaji; Naganuma, Akira; Kuge, Shusuke

    2016-01-01

    AIM: To address the effect of heat-shock protein 90 (HSP90) inhibitors on the release of the hepatitis C virus (HCV), a cell culture-derived HCV (JFH1/HCVcc) from Huh-7 cells was examined. METHODS: We quantified both the intracellular and extracellular (culture medium) levels of the components (RNA and core) of JFH-1/HCVcc. The intracellular HCV RNA and core levels were determined after the JFH1/HCVcc-infected Huh-7 cells were treated with radicicol for 36 h. The extracellular HCV RNA and core protein levels were determined from the medium of the last 24 h of radicicol treatment. To determine the possible role of the HSP90 inhibitor in HCV release, we examined the effect of a combined application of low doses of the HSP90 inhibitor radicicol and the RNA replication inhibitors cyclosporin A (CsA) or interferon. Finally, we statistically examined the combined effect of radicicol and CsA using the combination index (CI) and graphical representation proposed by Chou and Talalay. RESULTS: We found that the HSP90 inhibitors had greater inhibitory effects on the HCV RNA and core protein levels measured in the medium than inside the cells. This inhibitory effect was observed in the presence of a low level of a known RNA replication inhibitor (CsA or interferon-α). Treating the cells with a combination of radicicol and cyclosporin A for 24 h resulted in significant synergy (CI < 1) that affected the release of both the viral RNA and the core protein. CONCLUSION: In addition to having an inhibitory effect on RNA replication, HSP90 inhibitors may interfere with an HCV replication step that occurs after the synthesis of viral RNA, such as assembly and release. PMID:26925202

  2. Long noncoding RNA H19 interacts with polypyrimidine tract-binding protein 1 to reprogram hepatic lipid homeostasis.

    PubMed

    Liu, Chune; Yang, Zhihong; Wu, Jianguo; Zhang, Li; Lee, Sangmin; Shin, Dong-Ju; Tran, Melanie; Wang, Li

    2018-05-01

    H19 is an imprinted long noncoding RNA abundantly expressed in embryonic liver and repressed after birth. We show that H19 serves as a lipid sensor by synergizing with the RNA-binding polypyrimidine tract-binding protein 1 (PTBP1) to modulate hepatic metabolic homeostasis. H19 RNA interacts with PTBP1 to facilitate its association with sterol regulatory element-binding protein 1c mRNA and protein, leading to increased stability and nuclear transcriptional activity. H19 and PTBP1 are up-regulated by fatty acids in hepatocytes and in diet-induced fatty liver, which further augments lipid accumulation. Ectopic expression of H19 induces steatosis and pushes the liver into a "pseudo-fed" state in response to fasting by promoting sterol regulatory element-binding protein 1c protein cleavage and nuclear translocation. Deletion of H19 or knockdown of PTBP1 abolishes high-fat and high-sucrose diet-induced steatosis. Our study unveils an H19/PTBP1/sterol regulatory element-binding protein 1 feedforward amplifying signaling pathway to exacerbate the development of fatty liver. (Hepatology 2018;67:1768-1783). © 2017 by the American Association for the Study of Liver Diseases.

  3. A homologous series of regioselectively tetradeprotonated group 8 metallocenes: new inverse crown ring compounds synthesized via a mixed sodium-magnesium tris(diisopropylamide) synergic base.

    PubMed

    Andrikopoulos, Prokopis C; Armstrong, David R; Clegg, William; Gilfillan, Carly J; Hevia, Eva; Kennedy, Alan R; Mulvey, Robert E; O'Hara, Charles T; Parkinson, John A; Tooke, Duncan M

    2004-09-22

    Subjecting ferrocene, ruthenocene, or osmocene to the synergic amide base sodium-magnesium tris(diisopropylamido) affords a unique homologous series of metallocene derivatives of general formula [(M(C(5)H(3))(2))Na(4)Mg(4)(i-Pr(2)N)(8)] (where M = Fe (1), Ru (2), or Os (3)). X-ray crystallographic studies of 1-3 reveal a common molecular "inverse crown" structure comprising a 16-membered [(NaNMgN)(4)](4+) "host" ring and a metallocenetetraide [M(C(5)H(3))(2)](4-) "guest" core, the cleaved protons of which are lost selectively from the 1, 1', 3, and 3'-positions. Variable-temperature NMR spectroscopic studies indicate that 1, 2, and 3 each exist as two distinct interconverting conformers in arene solution, the rates of exchange of which have been calculated using coalescence and EXSY NMR measurements.

  4. Expression of inducible heat shock proteins Hsp27 and Hsp70 in the visual pathway of rats subjected to various models of retinal ganglion cell injury.

    PubMed

    Chidlow, Glyn; Wood, John P M; Casson, Robert J

    2014-01-01

    Inducible heat shock proteins (Hsps) are upregulated in the central nervous system in response to a wide variety of injuries. Surprisingly, however, no coherent picture has emerged regarding the magnitude, duration and cellular distribution of inducible Hsps in the visual system following injury to retinal ganglion cells (RGCs). The current study sought, therefore, to achieve the following two objectives. The first aim of this study was to systematically characterise the patterns of Hsp27 and -70 expression in the retina and optic nerve in four discrete models of retinal ganglion cell (RGC) degeneration: axonal injury (ON crush), somato-dendritic injury (NMDA-induced excitotoxicity), chronic hypoperfusion (bilateral occlusion of the carotid arteris) and experimental glaucoma. The second aim was to document Hsp27 and -70 expression in the optic tract, the subcortical retinorecipient areas of the brain, and the visual cortex during Wallerian degeneration of RGC axons. Hsp27 was robustly upregulated in the retina in each injury paradigm, with the chronic models, 2VO and experimental glaucoma, displaying a more persistent Hsp27 transcriptional response than the acute models. Hsp27 expression was always associated with astrocytes and with a subset of RGCs in each of the models excluding NMDA. Hsp27 was present within astrocytes of the optic nerve/optic tract in control rats. During Wallerian degeneration, Hsp27 was upregulated in the optic nerve/optic tract and expressed de novo by astrocytes in the lateral geniculate nucleus and the stratum opticum of the superior colliculus. Conversely, the results of our study indicate Hsp70 was minimally induced in any of the models of injury, either in the retina, or in the optic nerve/optic tract, or in the subcortical, retinorecipient areas of the brain. The findings of the present study augment our understanding of the involvement of Hsp27 and Hsp70 in the response of the visual system to RGC degeneration.

  5. Expression of Inducible Heat Shock Proteins Hsp27 and Hsp70 in the Visual Pathway of Rats Subjected to Various Models of Retinal Ganglion Cell Injury

    PubMed Central

    Chidlow, Glyn; Wood, John P. M.; Casson, Robert J.

    2014-01-01

    Inducible heat shock proteins (Hsps) are upregulated in the central nervous system in response to a wide variety of injuries. Surprisingly, however, no coherent picture has emerged regarding the magnitude, duration and cellular distribution of inducible Hsps in the visual system following injury to retinal ganglion cells (RGCs). The current study sought, therefore, to achieve the following two objectives. The first aim of this study was to systematically characterise the patterns of Hsp27 and −70 expression in the retina and optic nerve in four discrete models of retinal ganglion cell (RGC) degeneration: axonal injury (ON crush), somato-dendritic injury (NMDA-induced excitotoxicity), chronic hypoperfusion (bilateral occlusion of the carotid arteris) and experimental glaucoma. The second aim was to document Hsp27 and −70 expression in the optic tract, the subcortical retinorecipient areas of the brain, and the visual cortex during Wallerian degeneration of RGC axons. Hsp27 was robustly upregulated in the retina in each injury paradigm, with the chronic models, 2VO and experimental glaucoma, displaying a more persistent Hsp27 transcriptional response than the acute models. Hsp27 expression was always associated with astrocytes and with a subset of RGCs in each of the models excluding NMDA. Hsp27 was present within astrocytes of the optic nerve/optic tract in control rats. During Wallerian degeneration, Hsp27 was upregulated in the optic nerve/optic tract and expressed de novo by astrocytes in the lateral geniculate nucleus and the stratum opticum of the superior colliculus. Conversely, the results of our study indicate Hsp70 was minimally induced in any of the models of injury, either in the retina, or in the optic nerve/optic tract, or in the subcortical, retinorecipient areas of the brain. The findings of the present study augment our understanding of the involvement of Hsp27 and Hsp70 in the response of the visual system to RGC degeneration. PMID:25535743

  6. 27 CFR 27.121 - Containers.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Containers. 27.121 Section... Spirits In Bulk § 27.121 Containers. Imported distilled spirits may be bottled in either domestic or imported containers conforming to the provisions of subpart N of this part. (72 Stat. 1374; 26 U.S.C. 5301...

  7. SKP2 siRNA inhibits the degradation of P27kip1 and down-regulates the expression of MRP in HL-60/A cells.

    PubMed

    Xiao, Jie; Yin, Songmei; Li, Yiqing; Xie, Shuangfeng; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan; Feng, Jianhong

    2009-08-01

    S-phase kinase-associated protein 2 (SKP2) gene is a tumor suppressor gene, and is involved in the ubiquitin-mediated degradation of P27kip1. SKP2 and P27kip1 affect the proceeding and prognosis of leukemia through regulating the proliferation, apoptosis and differentiation of leukemia cells. In this study, we explored the mechanism of reversing of HL-60/A drug resistance through SKP2 down-regulation. HL-60/A cells were nucleofected by Amaxa Nucleofector System with SKP2 siRNA. The gene and protein expression levels of Skp2, P27kip1, and multi-drug resistance associated protein (MRP) were determined by reverse transcription-polymerase chain reaction and western blot analysis, respectively. The cell cycle was analyzed by flow cytometry. The 50% inhibitory concentration value was calculated using cytotoxic analysis according to the death rate of these two kinds of cells under different concentrations of chemotherapeutics to compare the sensitivity of the cells. HL-60/A cells showed multi-drug resistance phenotype characteristic by cross-resistance to adriamycin, daunorubicin, and arabinosylcytosine, due to the expression of MRP. We found that the expression of SKP2 was higher in HL-60/A cells than in HL-60 cells, but the expression of P27kip1 was lower. The expression of SKP2 in HL-60/A cells nucleofected by SKP2 siRNA was down-regulated whereas the protein level of P27kip1 was up-regulated. Compared with the MRP expression level in the control group (nucleofected by control siRNA), the mRNA and protein expression levels of MRP in HL-60/A cells nucleofected by SKP2 siRNA were lower, and the latter cells were more sensitive to adriamycin, daunorubicin, and arabinosylcytosine. Down-regulating the SKP2 expression and arresting cells in the G0/G1 phase improve drug sensitivity of leukemia cells with down-regulated MRP expression.

  8. Characterization of hsp27 kinases activated by elevated aortic pressure in heart

    PubMed Central

    Boivin, Benoit; Khairallah, Maya; Cartier, Raymond; Allen, Bruce G.

    2013-01-01

    Chronic hemodynamic overload results in left ventricular hypertrophy, fibroblast proliferation, and interstitial fibrosis. The small heat shock protein hsp27 has been shown to be cardioprotective and this requires a phosphorylatable form of this protein. To further understand the regulation of hsp27 in heart in response to stress, we investigated the ability of elevated aortic pressure to activate hsp27-kinase activities. Isolated hearts were subjected to retrograde perfusion and then snap-frozen. Hsp27-kinase activity was measured in vitro as hsp27 phosphorylation. Immune complex assays revealed that MK2 activity was low in non-perfused hearts and increased following crystalline perfusion at 60 or 120 mmHg. Hsp27-kinase activities were further studied following ion-exchange chromatography. Anion exchange chromatography on Mono Q revealed 2 peaks (‘b’ and ‘c’) of hsp27-kinase activity. A third peak ‘a’ was detected upon chromatography of the Mono Q flow-through fractions on the cation exchange resin, Mono S. The hsp27-kinase activity underlying peaks ‘a’ and ‘c’ increased as perfusion pressure was increased from 40 to 120 mmHg. In contrast, peak ‘b’ increased over pressures 60–100 mmHg but was decreased at 120 mmHg. Peaks ‘a’, ‘b’, and ‘c’ contained MK2 immunoreactivity, whereas MK3 and MK5 immunoreactivity was detected in peak ‘a’. p38 MAPK and phospho-p38 MAPK were also detected in peaks ‘b’ and ‘c’ but absent from peak ‘a’. Hsp27-kinase activity in peaks ‘b’ and ‘c’ (120 mmHg) eluted from a Superose 12 gel filtration column with an apparent molecular mass of 50-kDa. Hence, peaks ‘b’ and ‘c’ were not a result of MK2 forming complexes. In-gel hsp27-kinase assays revealed a single 49-kDa renaturable hsp27-kinase activity in peaks ‘b’ and ‘c’ at 60 mmHg, whereas several hsp27-kinases (p43, p49, p54, p66) were detected in peaks ‘b’ and ‘c’ from hearts perfused at 120 mmHg. Thus

  9. Differential Protection of Cry1Fa Toxin against Spodoptera frugiperda Larval Gut Proteases by Cadherin Orthologs Correlates with Increased Synergism

    PubMed Central

    Rahman, Khalidur; Abdullah, Mohd Amir F.; Ambati, Suresh; Taylor, Milton D.

    2012-01-01

    The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin. PMID:22081566

  10. Differential protection of Cry1Fa toxin against Spodoptera frugiperda larval gut proteases by cadherin orthologs correlates with increased synergism.

    PubMed

    Rahman, Khalidur; Abdullah, Mohd Amir F; Ambati, Suresh; Taylor, Milton D; Adang, Michael J

    2012-01-01

    The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin.

  11. Metformin synergizes 5-fluorouracil, epirubicin, and cyclophosphamide (FEC) combination therapy through impairing intracellular ATP production and DNA repair in breast cancer stem cells.

    PubMed

    Soo, Jaslyn Sian-Siu; Ng, Char-Hong; Tan, Si Hoey; Malik, Rozita Abdul; Teh, Yew-Ching; Tan, Boon-Shing; Ho, Gwo-Fuang; See, Mee-Hoong; Taib, Nur Aishah Mohd; Yip, Cheng-Har; Chung, Felicia Fei-Lei; Hii, Ling-Wei; Teo, Soo-Hwang; Leong, Chee-Onn

    2015-10-01

    Metformin, an AMPK activator, has been reported to improve pathological response to chemotherapy in diabetic breast cancer patients. To date, its mechanism of action in cancer, especially in cancer stem cells (CSCs) have not been fully elucidated. In this study, we demonstrated that metformin, but not other AMPK activators (e.g. AICAR and A-769662), synergizes 5-fluouracil, epirubicin, and cyclophosphamide (FEC) combination chemotherapy in non-stem breast cancer cells and breast cancer stem cells. We show that this occurs through an AMPK-dependent mechanism in parental breast cancer cell lines. In contrast, the synergistic effects of metformin and FEC occurred in an AMPK-independent mechanism in breast CSCs. Further analyses revealed that metformin accelerated glucose consumption and lactate production more severely in the breast CSCs but the production of intracellular ATP was severely hampered, leading to a severe energy crisis and impairs the ability of CSCs to repair FEC-induced DNA damage. Indeed, addition of extracellular ATP completely abrogated the synergistic effects of metformin on FEC sensitivity in breast CSCs. In conclusion, our results suggest that metformin synergizes FEC sensitivity through distinct mechanism in parental breast cancer cell lines and CSCs, thus providing further evidence for the clinical relevance of metformin for the treatment of cancers.

  12. p27kip1 expression distinguishes papillary hyperplasia in Graves' disease from papillary thyroid carcinoma.

    PubMed

    Erickson, L A; Yousef, O M; Jin, L; Lohse, C M; Pankratz, V S; Lloyd, R V

    2000-09-01

    In most cases, the histopathologic and cytologic distinction between Graves' disease and papillary thyroid carcinoma is relatively easy, but on occasion Graves' disease may simulate a thyroid papillary carcinoma. For example, papillary fronds with fibrovascular cores may be present in both Graves' disease and papillary carcinoma. p27kip1 (p27) is a cyclin-dependent kinase inhibitory protein that has been shown to be an independent prognostic factor in a variety of human tumors. Our previous studies of p27 expression in hyperplastic and neoplastic endocrine lesions showed that the level of p27 was quite different in these two conditions. To determine if this distinction could also be made between Graves' disease and papillary carcinoma, we analyzed expression of p27 and other cell cycle proteins in a series of cases of Graves' disease with papillary hyperplasia and a series of papillary thyroid carcinomas. Formalin-fixed paraffin-embedded tissues from 61 randomly selected patients with thyroid disease, including 29 cases of Graves' disease with papillary architectural features and 32 cases of papillary carcinoma, were analyzed for expression of p27, Ki-67, and DNA topoisomerase II alpha (topo II alpha) by immunostaining. The distribution of immunoreactivity was analyzed by quantifying the percentage of positive nuclei that was expressed as the labeling index (LI) plus or minus the standard error of the mean. The papillary hyperplasia of Graves' disease had a p27 LI of 68.2 +/- 3.1 (range, 24 to 88), whereas papillary carcinomas had a LI of 25.6 +/- 2.5 (range, 12 to 70) (P < .0001). No significant differences in Ki-67 or topo II alpha expression were identified between papillary hyperplasia in Graves' disease and papillary carcinoma. These results indicate that p27 protein expression is significantly higher in papillary hyperplasia of Graves' disease compared to papillary carcinoma, which may be diagnostically useful in difficult cases.

  13. Immunohistochemical expression of heat shock protein27 in the mouse dental pulp after immediate teeth separation

    PubMed Central

    2011-01-01

    Aim After immediate teeth separation, expression of HSP27 in the mouse dental pulp was examined. Immunohistochemistry was performed to examine the incidence of HSP27 expression. Materials and methods A total of 36 8-week-old ddY mice were used as experimental subjects and a wedge was inserted in between maxillary right molars. The wedge was removed 30 min or 3 h after insertion. Animals were immediately sacrificed after the removal of wedge or until 1 week later and serial sections from paraffin-embedded tissues were prepared. Immunohistochemistry was carried out to examine the expression of HSP27. The untreated side served as the control. Results In the control group, the endothelial cells and some pulp fibroblasts weakly expressed HSP27 suggesting that the expression is due to mechanical stress brought about by physiological masticatory force and pressure from the tongue. In both 30 min and 3 h experimental groups, HSP27 expression was highest at 24 h after wedge removal and the expression remained the same or started to decrease thereafter. The expression decreased at the same level as that of the control group 1 week after wedge removal. Conclusion HSP27 may serve as an indicator of stimulus strong enough to show its expression. PMID:22027643

  14. Relaxin receptor antagonist AT-001 synergizes with docetaxel in androgen-independent prostate xenografts.

    PubMed

    Neschadim, Anton; Pritzker, Laura B; Pritzker, Kenneth P H; Branch, Donald R; Summerlee, Alastair J S; Trachtenberg, John; Silvertown, Joshua D

    2014-06-01

    Androgen hormones and the androgen receptor (AR) pathway are the main targets of anti-hormonal therapies for prostate cancer. However, resistance inevitably develops to treatments aimed at the AR pathway resulting in androgen-independent or hormone-refractory prostate cancer (HRPC). Therefore, there is a significant unmet need for new, non-androgen anti-hormonal strategies for the management of prostate cancer. We demonstrate that a relaxin hormone receptor antagonist, AT-001, an analog of human H2 relaxin, represents a first-in-class anti-hormonal candidate treatment designed to significantly curtail the growth of androgen-independent human prostate tumor xenografts. Chemically synthesized AT-001, administered subcutaneously, suppressed PC3 xenograft growth by up to 60%. AT-001 also synergized with docetaxel, standard first-line chemotherapy for HRPC, to suppress tumor growth by more than 98% in PC3 xenografts via a mechanism involving the downregulation of hypoxia-inducible factor 1 alpha and the hypoxia-induced response. Our data support developing AT-001 for clinical use as an anti-relaxin hormonal therapy for advanced prostate cancer.

  15. MIR-27a regulates the TGF-β signaling pathway by targeting SMAD2 and SMAD4 in lung cancer.

    PubMed

    Chae, Dong-Kyu; Ban, Eunmi; Yoo, Young Sook; Kim, Eunice EunKyeong; Baik, Ja-Hyun; Song, Eun Joo

    2017-08-01

    The transforming growth factor-β (TGF-β) signaling pathway is associated with carcinogenesis and various biological processes. SMAD2 and SMAD4, which are putative tumor suppressors, have an important role in TGF-β signaling. The aberrant expression of these genes is implicated in some cancers. However, the mechanisms of SMAD2 and SMAD4 dysregulation are poorly understood. In this study, we observed that miR-27a was upregulated in lung cancer cell lines and patients. In addition, SMAD2 and SMAD4 genes were identified as targets of miR-27a by several target prediction databases and experimental validation. Functional studies revealed that miR-27a overexpression decreased SMAD2 and SMAD4 mRNA and protein levels. Furthermore, miR-27a contributed to cell proliferation and invasion by inhibiting TGF-β-induced cell cycle arrest. These results suggest that miR-27a may function as an oncogene by regulating SMAD2 and SMAD4 in lung cancer. Thus, miR-27a may be a potential target for cancer therapy. © 2017 Wiley Periodicals, Inc.

  16. The transcription factor Lc-Maf participates in Col27a1 regulation during chondrocyte maturation

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

    Mayo, Jaime L.; Holden, Devin N.; Barrow, Jeffery R.

    2009-08-01

    The transcription factor Lc-Maf, which is a splice variant of c-Maf, is expressed in cartilage undergoing endochondral ossification and participates in the regulation of type II collagen through a cartilage-specific Col2a1 enhancer element. Type XXVII and type XI collagens are also expressed in cartilage during endochondral ossification, and so enhancer/reporter assays were used to determine whether Lc-Maf could regulate cartilage-specific enhancers from the Col27a1 and Col11a2 genes. The Col27a1 enhancer was upregulated over 4-fold by Lc-Maf, while the Col11a2 enhancer was downregulated slightly. To confirm the results of these reporter assays, rat chondrosarcoma (RCS) cells were transiently transfected with anmore » Lc-Maf expression plasmid, and quantitative RT-PCR was performed to measure the expression of endogenous Col27a1 and Col11a2 genes. Endogenous Col27a1 was upregulated 6-fold by Lc-Maf overexpression, while endogenous Col11a2 was unchanged. Finally, in situ hybridization and immunohistochemistry were performed in the radius and ulna of embryonic day 17 mouse forelimbs undergoing endochondral ossification. Results demonstrated that Lc-Maf and Col27a1 mRNAs are coexpressed in proliferating and prehypertrophic regions, as would be predicted if Lc-Maf regulates Col27a1 expression. Type XXVII collagen protein was also most abundant in prehypertrophic and proliferating chondrocytes. Others have shown that mice that are null for Lc-Maf and c-Maf have expanded hypertrophic regions with reduced ossification and delayed vascularization. Separate studies have indicated that Col27a1 may serve as a scaffold for ossification and vascularization. The work presented here suggests that Lc-Maf may affect the process of endochondral ossification by participating in the regulation of Col27a1 expression.« less

  17. Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier–dependent pathway

    PubMed Central

    Ahner, Annette; Gong, Xiaoyan; Schmidt, Bela Z.; Peters, Kathryn W.; Rabeh, Wael M.; Thibodeau, Patrick H.; Lukacs, Gergely L.; Frizzell, Raymond A.

    2013-01-01

    Small heat shock proteins (sHsps) bind destabilized proteins during cell stress and disease, but their physiological functions are less clear. We evaluated the impact of Hsp27, an sHsp expressed in airway epithelial cells, on the common protein misfolding mutant that is responsible for most cystic fibrosis. F508del cystic fibrosis transmembrane conductance regulator (CFTR), a well-studied protein that is subject to cytosolic quality control, selectively associated with Hsp27, whose overexpression preferentially targeted mutant CFTR to proteasomal degradation. Hsp27 interacted physically with Ubc9, the small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, implying that F508del SUMOylation leads to its sHsp-mediated degradation. Enhancing or disabling the SUMO pathway increased or blocked Hsp27’s ability to degrade mutant CFTR. Hsp27 promoted selective SUMOylation of F508del NBD1 in vitro and of full-length F508del CFTR in vivo, which preferred endogenous SUMO-2/3 paralogues that form poly-chains. The SUMO-targeted ubiquitin ligase (STUbL) RNF4 recognizes poly-SUMO chains to facilitate nuclear protein degradation. RNF4 overexpression elicited F508del degradation, whereas Hsp27 knockdown blocked RNF4’s impact on mutant CFTR. Similarly, the ability of Hsp27 to degrade F508del CFTR was lost during overexpression of dominant-negative RNF4. These findings link sHsp-mediated F508del CFTR degradation to its SUMOylation and to STUbL-mediated targeting to the ubiquitin–proteasome system and thereby implicate this pathway in the disposal of an integral membrane protein. PMID:23155000

  18. HL-A27 and anterior uveitis.

    PubMed

    Woodrow, J C; Mapstone, R; Anderson, J; Usher, N

    1975-09-01

    HL-A types were determined in 90 successive patients with non-granulomatous uveitis. Fifty-one were HL-A27 positive (55.7%) compared to 8.2% of controls. Of 16 patients with ankylosing spondylitis, 13 were HL-A27 positive, as were two patients with a history of Reiter's syndrome. Twenty-eight patients were HL-A27 positive but had no evidence of rheumatic disease. The findings are discussed in relation to the possible pathogenesis of uveitis.

  19. A unique PDZ domain and arrestin-like fold interaction reveals mechanistic details of endocytic recycling by SNX27-retromer.

    PubMed

    Gallon, Matthew; Clairfeuille, Thomas; Steinberg, Florian; Mas, Caroline; Ghai, Rajesh; Sessions, Richard B; Teasdale, Rohan D; Collins, Brett M; Cullen, Peter J

    2014-09-02

    The sorting nexin 27 (SNX27)-retromer complex is a major regulator of endosome-to-plasma membrane recycling of transmembrane cargos that contain a PSD95, Dlg1, zo-1 (PDZ)-binding motif. Here we describe the core interaction in SNX27-retromer assembly and its functional relevance for cargo sorting. Crystal structures and NMR experiments reveal that an exposed β-hairpin in the SNX27 PDZ domain engages a groove in the arrestin-like structure of the vacuolar protein sorting 26A (VPS26A) retromer subunit. The structure establishes how the SNX27 PDZ domain simultaneously binds PDZ-binding motifs and retromer-associated VPS26. Importantly, VPS26A binding increases the affinity of the SNX27 PDZ domain for PDZ- binding motifs by an order of magnitude, revealing cooperativity in cargo selection. With disruption of SNX27 and retromer function linked to synaptic dysfunction and neurodegenerative disease, our work provides the first step, to our knowledge, in the molecular description of this important sorting complex, and more broadly describes a unique interaction between a PDZ domain and an arrestin-like fold.

  20. 27 CFR 27.77 - Standard effective tax rate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Standard effective tax rate. 27.77 Section 27.77 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE... Requirements Wine and Flavors Content of Distilled Spirits § 27.77 Standard effective tax rate. (a) In lieu of...

  1. Switch on the engine: how the eukaryotic replicative helicase MCM2-7 becomes activated.

    PubMed

    Tognetti, Silvia; Riera, Alberto; Speck, Christian

    2015-03-01

    A crucial step during eukaryotic initiation of DNA replication is the correct loading and activation of the replicative DNA helicase, which ensures that each replication origin fires only once. Unregulated DNA helicase loading and activation, as it occurs in cancer, can cause severe DNA damage and genomic instability. The essential mini-chromosome maintenance proteins 2-7 (MCM2-7) represent the core of the eukaryotic replicative helicase that is loaded at DNA replication origins during G1-phase of the cell cycle. The MCM2-7 helicase activity, however, is only triggered during S-phase once the holo-helicase Cdc45-MCM2-7-GINS (CMG) has been formed. A large number of factors and several kinases interact and contribute to CMG formation and helicase activation, though the exact mechanisms remain unclear. Crucially, upon DNA damage, this reaction is temporarily halted to ensure genome integrity. Here, we review the current understanding of helicase activation; we focus on protein interactions during CMG formation, discuss structural changes during helicase activation, and outline similarities and differences of the prokaryotic and eukaryotic helicase activation process.

  2. Ac-SDKP suppresses epithelial-mesenchymal transition in A549 cells via HSP27 signaling.

    PubMed

    Deng, Haijing; Yang, Fang; Xu, Hong; Sun, Yue; Xue, Xinxin; Du, Shipu; Wang, Xiaojun; Li, Shifeng; Liu, Yan; Wang, Ruimin

    2014-08-01

    The synthetic tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) has been shown to be a modulator of molecular aspects of the fibrosis pathway. This study reveals that Ac-SDKP exerts an anti-fibrotic effect on human type II alveolar epithelial cells (A549), which are a source of myofibroblasts once exposed to TGF-β1, by decreasing the expression of heat shock protein 27 (HSP27). We used A549 cells in vitro to detect morphological evidence of epithelial-mesenchymal transition (EMT) by phase-contrast microscopy. Immunocytochemical and western blot analysis determined the distributions of cytokeratin 8 (CK8), α-smooth muscle actin (α-SMA), and SNAI1. Confocal laser scanning microscopy revealed a colocalization of HSP27 and SNAI1 on TGF-β1-induced A549 cells. These results also demonstrated that A549 cells became spindle-like when exposed to TGF-β1. Coincident with these morphological changes, expression levels of CK8 and E-cad decreased, while those of vimentin and α-SMA increased. This process was accompanied by increases in levels of HSP27, SNAI1, and type I and type III collagen. In vitro transfection experiments demonstrated that the inhibition of HSP27 in cultured A549 cells could decrease the expression of SNAI1 and α-SMA while increasing the expression of E-cad. A noticeable reduction in collagen types I and III was also evident. Our results found that Ac-SDKP inhibited the transition of cultured A549 cells to myofibroblasts and attenuated collagen synthesis through modulating the expression of HSP27. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Detecting Selection on Protein Stability through Statistical Mechanical Models of Folding and Evolution

    PubMed Central

    Bastolla, Ugo

    2014-01-01

    The properties of biomolecules depend both on physics and on the evolutionary process that formed them. These two points of view produce a powerful synergism. Physics sets the stage and the constraints that molecular evolution has to obey, and evolutionary theory helps in rationalizing the physical properties of biomolecules, including protein folding thermodynamics. To complete the parallelism, protein thermodynamics is founded on the statistical mechanics in the space of protein structures, and molecular evolution can be viewed as statistical mechanics in the space of protein sequences. In this review, we will integrate both points of view, applying them to detecting selection on the stability of the folded state of proteins. We will start discussing positive design, which strengthens the stability of the folded against the unfolded state of proteins. Positive design justifies why statistical potentials for protein folding can be obtained from the frequencies of structural motifs. Stability against unfolding is easier to achieve for longer proteins. On the contrary, negative design, which consists in destabilizing frequently formed misfolded conformations, is more difficult to achieve for longer proteins. The folding rate can be enhanced by strengthening short-range native interactions, but this requirement contrasts with negative design, and evolution has to trade-off between them. Finally, selection can accelerate functional movements by favoring low frequency normal modes of the dynamics of the native state that strongly correlate with the functional conformation change. PMID:24970217

  4. Next-generation proteasome inhibitor oprozomib synergizes with modulators of the unfolded protein response to suppress hepatocellular carcinoma.

    PubMed

    Vandewynckel, Yves-Paul; Coucke, Céline; Laukens, Debby; Devisscher, Lindsey; Paridaens, Annelies; Bogaerts, Eliene; Vandierendonck, Astrid; Raevens, Sarah; Verhelst, Xavier; Van Steenkiste, Christophe; Libbrecht, Louis; Geerts, Anja; Van Vlierberghe, Hans

    2016-06-07

    Hepatocellular carcinoma (HCC) responds poorly to conventional systemic therapies. The first-in-class proteasome inhibitor bortezomib has been approved in clinical use for hematologic malignancies and has shown modest activity in solid tumors, including HCC. However, a considerable proportion of patients fail to respond and experience important adverse events. Recently, the next-generation orally bioavailable irreversible proteasome inhibitor oprozomib was developed. Here, we assessed the efficacy of oprozomib and its effects on the unfolded protein response (UPR), a signaling cascade activated through the ATF6, PERK and IRE1 pathways by accumulation of unfolded proteins in the endoplasmic reticulum, in HCC. The effects of oprozomib and the role of the UPR were evaluated in HCC cell lines and in diethylnitrosamine-induced and xenograft mouse models for HCC. Oprozomib dose-dependently reduced the viability and proliferation of human HCC cells. Unexpectedly, oprozomib-treated cells displayed diminished cytoprotective ATF6-mediated signal transduction as well as unaltered PERK and IRE1 signaling. However, oprozomib increased pro-apoptotic UPR-mediated protein levels by prolonging their half-life, implying that the proteasome acts as a negative UPR regulator. Supplementary boosting of UPR activity synergistically improved the sensitivity to oprozomib via the PERK pathway. Oral oprozomib displayed significant antitumor effects in the orthotopic and xenograft models for HCC, and importantly, combining oprozomib with different UPR activators enhanced the antitumor efficacy by stimulating UPR-induced apoptosis without cumulative toxicity. In conclusion, next-generation proteasome inhibition by oprozomib results in dysregulated UPR activation in HCC. This finding can be exploited to enhance the antitumor efficacy by combining oprozomib with clinically applicable UPR activators.

  5. Fully utilizing high power diode lasers by synergizing diode laser light sources and beam shaping micro-optics

    NASA Astrophysics Data System (ADS)

    Fan, Yingmin; Wang, Jingwei; Cai, Lei; Mitra, Thomas; Hauschild, Dirk; Zah, Chung-En; Liu, Xingsheng

    2018-02-01

    High power diode lasers (HPDLs) offer the highest wall-plug efficiency, highest specific power (power-to-weight ratio), arguably the lowest cost and highest reliability among all laser types. However, the poor beam quality of commercially HPDLs is the main bottleneck limiting their direct applications requiring high brightness at least in one dimension. In order to expand the applications of HPDLs, beam shaping and optical design are essential. In this work, we report the recent progresses on maximizing applications of HPDLs by synergizing diode laser light source and beam shaping micro-optics. Successful examples of matching of diode laser light sources and beam shaping micro-optics driving new applications are presented.

  6. Phenobarbital Induction and Chemical Synergism Demonstrate the Role of UDP-Glucuronosyltransferases in Detoxification of Naphthalophos by Haemonchus contortus Larvae

    PubMed Central

    Ruffell, Angela P.; Ingham, Aaron B.

    2014-01-01

    We used an enzyme induction approach to study the role of detoxification enzymes in the interaction of the anthelmintic compound naphthalophos with Haemonchus contortus larvae. Larvae were treated with the barbiturate phenobarbital, which is known to induce the activity of a number of detoxification enzymes in mammals and insects, including cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UDPGTs), and glutathione (GSH) S-transferases (GSTs). Cotreatment of larvae with phenobarbital and naphthalophos resulted in a significant increase in the naphthalophos 50% inhibitory concentration (IC50) compared to treatment of larvae with the anthelmintic alone (up to a 28-fold increase). The phenobarbital-induced drug tolerance was reversed by cotreatment with the UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, probenecid, and sulfinpyrazone. Isobologram analysis of the interaction of 5-nitrouracil with naphthalophos in phenobarbital-treated larvae clearly showed the presence of strong synergism. The UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, and probenecid also showed synergistic effects with non-phenobarbital-treated worms (synergism ratio up to 3.2-fold). This study indicates that H. contortus larvae possess one or more UDPGT enzymes able to detoxify naphthalophos. In highlighting the protective role of this enzyme group, this study reveals the potential for UDPGT enzymes to act as a resistance mechanism that may develop under drug selection pressure in field isolates of this species. In addition, the data indicate the potential for a chemotherapeutic approach utilizing inhibitors of UDPGT enzymes as synergists to increase the activity of naphthalophos against parasitic worms and to combat detoxification-mediated drug resistance if it arises in the field. PMID:25288079

  7. Phenobarbital induction and chemical synergism demonstrate the role of UDP-glucuronosyltransferases in detoxification of naphthalophos by Haemonchus contortus larvae.

    PubMed

    Kotze, Andrew C; Ruffell, Angela P; Ingham, Aaron B

    2014-12-01

    We used an enzyme induction approach to study the role of detoxification enzymes in the interaction of the anthelmintic compound naphthalophos with Haemonchus contortus larvae. Larvae were treated with the barbiturate phenobarbital, which is known to induce the activity of a number of detoxification enzymes in mammals and insects, including cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UDPGTs), and glutathione (GSH) S-transferases (GSTs). Cotreatment of larvae with phenobarbital and naphthalophos resulted in a significant increase in the naphthalophos 50% inhibitory concentration (IC50) compared to treatment of larvae with the anthelmintic alone (up to a 28-fold increase). The phenobarbital-induced drug tolerance was reversed by cotreatment with the UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, probenecid, and sulfinpyrazone. Isobologram analysis of the interaction of 5-nitrouracil with naphthalophos in phenobarbital-treated larvae clearly showed the presence of strong synergism. The UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, and probenecid also showed synergistic effects with non-phenobarbital-treated worms (synergism ratio up to 3.2-fold). This study indicates that H. contortus larvae possess one or more UDPGT enzymes able to detoxify naphthalophos. In highlighting the protective role of this enzyme group, this study reveals the potential for UDPGT enzymes to act as a resistance mechanism that may develop under drug selection pressure in field isolates of this species. In addition, the data indicate the potential for a chemotherapeutic approach utilizing inhibitors of UDPGT enzymes as synergists to increase the activity of naphthalophos against parasitic worms and to combat detoxification-mediated drug resistance if it arises in the field. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  8. p27{sup Kip1} inhibits tissue factor expression

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

    Breitenstein, Alexander, E-mail: alexander.breitenstein@usz.ch; Cardiovascular Research, Physiology Institute, University of Zurich; Center for Integrative Human Physiology

    2013-10-04

    Highlights: •p27{sup Kip1}regulates the expression of tissue factor at the transcriptional level. •This inhibitory effect of p27{sup Kip1} is independently of its cell regulatory action. •The current study provides new insights into a pleiotrophic function of p27{sup Kip1}. -- Abstract: Background: The cyclin-dependent kinase inhibitor (CDKI) p27{sup Kip1} regulates cell proliferation and thus inhibits atherosclerosis and vascular remodeling. Expression of tissue factor (TF), the key initator of the coagulation cascade, is associated with atherosclerosis. Yet, it has not been studied whether p27{sup Kip1} influences the expression of TF. Methods and results: p27{sup Kip1} overexpression in human aortic endothelial cells wasmore » achieved by adenoviral transfection. Cells were rendered quiescent for 24 h in 0.5% fetal-calf serum. After stimulation with TNF-α (5 ng/ml), TF protein expression and activity was significantly reduced (n = 4; P < 0.001) in cells transfected with p27{sup Kip1}. In line with this, p27{sup Kip1} overexpression reduced cytokine-induced TF mRNA expression (n = 4; P < 0.01) and TF promotor activity (n = 4; P < 0.05). In contrast, activation of the MAP kinases p38, ERK and JNK was not affected by p27{sup Kip1} overexpression. Conclusion: This in vitro study suggests that p27{sup Kip1} inhibits TF expression at the transcriptional level. These data indicate an interaction between p27{sup Kip1} and TF in important pathological alterations such as atherosclerosis and vascular remodeling.« less

  9. [SYNERGISM OF PRECONCEPTIVE RADIATION EXPOSURE AND PARENTS' ONCO-PATHOLOGY IN THE RISE OF CARCINOGENIC RISK IN THE OFFSPRINGS OF PROFESSIONAL EMPLOYEES].

    PubMed

    Telnov, V I; Kabirova, N R; Okatenko, P V

    2015-01-01

    The problem of carcinogenic risk in offsprings of individuals exposed to radiation is challenging and insufficiently studied. In that there are no evaluations of the interaction between radiation and non-radiation factors. The aim of the study was the analysis of interaction of preconceptive radiation exposure and parents' onco-pathology in cancer mortality in offsprings (F1) of workers (fathers) of the Mayak Production Association exposed to a wide range of doses of radiation over a year prior conception. The number of offspring is 8191 individuals (4180 men and 4011 women). The analysis was performed with the use of fourfold table and eightfold tables. The interaction offactors was estimated on the base of the additive and multiplicative models. The studied factors were independent. The odds ratio (OR) of cancer mortality in the offspring with parents' oncopathology (1.43) was insignificant. OR of cancer mortality in preconceptive radiation exposure in a dose over 110 mGy and without parents' onco-pathology was 2.61 (1.52-4.49), and in their combination--3.86 (1.93-7.71). Index of synergism of preconceptive radiation exposure and parents' onco-patholog in the rise of carcinogenic risk in the offspring was 1.34 and the character of their interaction was multiplicative. Thus, for the first time there was established the interaction between radiation and non-radiation factors in the synergism sort in the increase of carcinogenic risk in the offspring of people exposed to radiation.

  10. A minimalist model protein with multiple folding funnels

    PubMed Central

    Locker, C. Rebecca; Hernandez, Rigoberto

    2001-01-01

    Kinetic and structural studies of wild-type proteins such as prions and amyloidogenic proteins provide suggestive evidence that proteins may adopt multiple long-lived states in addition to the native state. All of these states differ structurally because they lie far apart in configuration space, but their stability is not necessarily caused by cooperative (nucleation) effects. In this study, a minimalist model protein is designed to exhibit multiple long-lived states to explore the dynamics of the corresponding wild-type proteins. The minimalist protein is modeled as a 27-monomer sequence confined to a cubic lattice with three different monomer types. An order parameter—the winding index—is introduced to characterize the extent of folding. The winding index has several advantages over other commonly used order parameters like the number of native contacts. It can distinguish between enantiomers, its calculation requires less computational time than the number of native contacts, and reduced-dimensional landscapes can be developed when the native state structure is not known a priori. The results for the designed model protein prove by existence that the rugged energy landscape picture of protein folding can be generalized to include protein “misfolding” into long-lived states. PMID:11470921

  11. Systematic chemical-genetic and chemical-chemical interaction datasets for prediction of compound synergism

    PubMed Central

    Wildenhain, Jan; Spitzer, Michaela; Dolma, Sonam; Jarvik, Nick; White, Rachel; Roy, Marcia; Griffiths, Emma; Bellows, David S.; Wright, Gerard D.; Tyers, Mike

    2016-01-01

    The network structure of biological systems suggests that effective therapeutic intervention may require combinations of agents that act synergistically. However, a dearth of systematic chemical combination datasets have limited the development of predictive algorithms for chemical synergism. Here, we report two large datasets of linked chemical-genetic and chemical-chemical interactions in the budding yeast Saccharomyces cerevisiae. We screened 5,518 unique compounds against 242 diverse yeast gene deletion strains to generate an extended chemical-genetic matrix (CGM) of 492,126 chemical-gene interaction measurements. This CGM dataset contained 1,434 genotype-specific inhibitors, termed cryptagens. We selected 128 structurally diverse cryptagens and tested all pairwise combinations to generate a benchmark dataset of 8,128 pairwise chemical-chemical interaction tests for synergy prediction, termed the cryptagen matrix (CM). An accompanying database resource called ChemGRID was developed to enable analysis, visualisation and downloads of all data. The CGM and CM datasets will facilitate the benchmarking of computational approaches for synergy prediction, as well as chemical structure-activity relationship models for anti-fungal drug discovery. PMID:27874849

  12. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

    PubMed Central

    Hostetler, Jessica B.; Sharma, Sumana; Bartholdson, S. Josefin; Wright, Gavin J.; Fairhurst, Rick M.; Rayner, Julian C.

    2015-01-01

    Background A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion. Methodology/Principal Findings We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further

  13. The interaction between RACK1 and WEE1 regulates the growth of gastric cancer cell line HGC27

    PubMed Central

    Liu, Chao; Ren, Lili; Wang, Yizhao; Liu, Yimeng; Xiao, Jianying

    2017-01-01

    Receptor of activated C Kinase 1 (RACK1) is an essential scaffold and anchoring protein, which serves an important role in multiple tumorigenesis signaling pathways. The present study aimed to investigate the expression of RACK1 in gastric cancer (GC), and its association with the occurrence and development of GC. In addition, the effect and mechanism of RACK1 overexpression on the growth, and proliferation of GC cells was examined. Firstly, the protein expression of RACK1 was detected in 70 cases of GC tissues and 30 cases of noncancerous tissues using immunohistochemical staining, and the association between clinical and pathological features of GC was analyzed. Secondly, the mRNA and protein expression of RACK1 was determined in the poorly-differentiated human gastric cancer cell line HGC27 and gastric epithelial cell line GES-1. The growth of HGC27 cells following the upregulation of RACK1 was detected using MTT method. Subsequently, the interaction and co-location between RACK1, and WEE1 homolog (S. pombe) (WEE1) in HGC27 cells was confirmed using co-immunoprecipitation and indirect immunofluorescence. The expression level of RACK1 in GC was significantly lower compared with that in pericarcinous tissues (P<0.05). The protein level of RACK1 expression correlated with tumor node metastasis stage, tumor differentiation and lymph node metastasis. The mRNA and protein levels of RACK1 in HGC27 cells were significantly reduced, and overexpressed RACK1 downregulated WEE1 protein expression, thus inhibiting the growth of HGC27 cells. Co-immunoprecipitation and immunofluorescence confirmed that RACK1, and WEE1 interacted and co-located in the cytoplasm of HGC27 cells. Therefore, the abnormal expression of RACK1 in GC tissues was identified to be involved in the occurrence and development of GC. Overexpression of RACK1 was able to inhibit the growth of HGC27 cells. The current study suggests that low expression of RACK1 is an important indicator of poor prognosis of GC

  14. 27 CFR 9.27 - Lime Kiln Valley.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Lime Kiln Valley. 9.27... OF THE TREASURY LIQUORS AMERICAN VITICULTURAL AREAS Approved American Viticultural Areas § 9.27 Lime Kiln Valley. (a) Name. The name of the viticultural area described in this section is “Lime Kiln Valley...

  15. 27 CFR 21.52 - Formula No. 27.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.52 Formula No. 27. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Formula No. 27. 21.52...

  16. 27 CFR 21.52 - Formula No. 27.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.52 Formula No. 27. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Formula No. 27. 21.52...

  17. 27 CFR 21.52 - Formula No. 27.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.52 Formula No. 27. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Formula No. 27. 21.52...

  18. 27 CFR 21.52 - Formula No. 27.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... OF THE TREASURY LIQUORS FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.52 Formula No. 27. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Formula No. 27. 21.52...

  19. 27 CFR 21.52 - Formula No. 27.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... OF THE TREASURY ALCOHOL FORMULAS FOR DENATURED ALCOHOL AND RUM Specially Denatured Spirits Formulas and Authorized Uses § 21.52 Formula No. 27. (a) Formula. To every 100 gallons of alcohol add: One... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Formula No. 27. 21.52...

  20. Identification of peptides in human Hsp20 and Hsp27 that possess molecular chaperone and anti-apoptotic activities.

    PubMed

    Nahomi, Rooban B; DiMauro, Michael A; Wang, Benlian; Nagaraj, Ram H

    2015-01-01

    Previous studies have identified peptides in the 'crystallin-domain' of the small heat-shock protein (sHSP) α-crystallin with chaperone and anti-apoptotic activities. We found that peptides in heat-shock protein Hsp20 (G71HFSVLLDVKHFSPEEIAVK91) and Hsp27 (D93RWRVSLDVNHFAPDELTVK113) with sequence homology to α-crystallin also have robust chaperone and anti-apoptotic activities. Both peptides inhibited hyperthermic and chemically induced aggregation of client proteins. The scrambled peptides of Hsp20 and Hsp27 showed no such effects. The chaperone activities of the peptides were better than those from αA- and αB-crystallin. HeLa cells took up the FITC-conjugated Hsp20 peptide and, when the cells were thermally stressed, the peptide was translocated from the cytoplasm to the nucleus. The two peptides inhibited apoptosis in HeLa cells by blocking cytochrome c release from the mitochondria and caspase-3 activation. We found that scrambling the last four amino acids in the two peptides (KAIV in Hsp20 and KTLV in Hsp27) made them unable to enter cells and ineffective against stress-induced apoptosis. Intraperitoneal injection of the peptides prevented sodium-selenite-induced cataract formation in rats by inhibiting protein aggregation and oxidative stress. Our study has identified peptides from Hsp20 and Hsp27 that may have therapeutic benefit in diseases where protein aggregation and apoptosis are contributing factors.

  1. Structural and Functional Features of a Developmentally Regulated Lipopolysaccharide-Binding Protein

    PubMed Central

    Krasity, Benjamin C.; Troll, Joshua V.; Lehnert, Erik M.; Hackett, Kathleen T.; Dillard, Joseph P.; Apicella, Michael A.; Goldman, William E.

    2015-01-01

    ABSTRACT Mammalian lipopolysaccharide (LPS) binding proteins (LBPs) occur mainly in extracellular fluids and promote LPS delivery to specific host cell receptors. The function of LBPs has been studied principally in the context of host defense; the possible role of LBPs in nonpathogenic host-microbe interactions has not been well characterized. Using the Euprymna scolopes-Vibrio fischeri model, we analyzed the structure and function of an LBP family protein, E. scolopes LBP1 (EsLBP1), and provide evidence for its role in triggering a symbiont-induced host developmental program. Previous studies showed that, during initial host colonization, the LPS of V. fischeri synergizes with peptidoglycan (PGN) monomer to induce morphogenesis of epithelial tissues of the host animal. Computationally modeled EsLBP1 shares some but not all structural features of mammalian LBPs that are thought important for LPS binding. Similar to human LBP, recombinant EsLBP1 expressed in insect cells bound V. fischeri LPS and Neisseria meningitidis lipooligosaccharide (LOS) with nanomolar or greater affinity but bound Francisella tularensis LPS only weakly and did not bind PGN monomer. Unlike human LBP, EsLBP1 did not bind N. meningitidis LOS:CD14 complexes. The eslbp1 transcript was upregulated ~22-fold by V. fischeri at 24 h postinoculation. Surprisingly, this upregulation was not induced by exposure to LPS but, rather, to the PGN monomer alone. Hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) and immunocytochemistry (ICC) localized eslbp1 transcript and protein in crypt epithelia, where V. fischeri induces morphogenesis. The data presented here provide a window into the evolution of LBPs and the scope of their roles in animal symbioses. PMID:26463160

  2. Inhibitory effect of SJSZ glycoprotein (38 kDa) on expression of heat shock protein 27 and 70 in chromium (VI)-treated hepatocytes.

    PubMed

    Lee, Jin; Lim, Kye-Taek

    2012-01-01

    Chromium (VI) is as an extremely toxic chemical substance, and is also an internationally recognized human carcinogen. The principal objective of this study was to determine whether or not Styrax japonica Siebold et al. Zuccarini (SJSZ) glycoprotein prevents hepatocarcinogenesis in chromium-treated BNL CL.2 cells and ICR mice. Firstly, it was evaluated that SJSZ glycoprotein has strong antioxidant character and scavenges radicals. In an effort to assess the chemopreventive effects of SJSZ glycoprotein on hepatocarcinogenesis, ICR mice were intraperitoneally injected with chromium (10 mg/kg, BW) for 8 weeks. After sacrifice, we evaluated indicators of liver tissue damage [the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT), and thiobarbituric acid-reactive substances (TBARS)], antioxidative enzymes [activities of superoxide dismutase (SOD), catalase (CAT) and gluthathione peroxidase (GPx)], and initiating hepatocarcinogenic indicator [heat shock protein (HSP) 27 and 70] and protein kinase C (PKC), p38 MAPK and PCNA via biochemical methods and immunoblot analysis. The results obtained from this study demonstrated that the SJSZ glycoprotein (50 μg/ml) inhibited the production of intracellular ROS in BNL CL.2 cells. In addition, the SJSZ glycoprotein (10 mg/kg, BW) attenuated the levels of LDH, ALT, and TBARS, whereas it increased antioxidative enzymes in mouse serum. SJSZ glycoprotein attenuated the activity of HSP27, HSP70, PKC, MAPKs, and PCNA in BNL CL.2 cells and liver tissue. Taken together, our results indicate that SJSZ glycoprotein might be have a potent preventive effect against hepatocarcinogenesis induced by oxidative stress.

  3. MKBP, a Novel Member of the Small Heat Shock Protein Family, Binds and Activates the Myotonic Dystrophy Protein Kinase

    PubMed Central

    Suzuki, Atsushi; Sugiyama, Yuki; Hayashi, Yukiko; Nyu-i, Nobuo; Yoshida, Michihiko; Nonaka, Ikuya; Ishiura, Sho-ichi; Arahata, Kiichi; Ohno, Shigeo

    1998-01-01

    Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as αB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by αB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM. PMID:9490724

  4. Characterization of a Proteasome and TAP-independent Presentation of Intracellular Epitopes by HLA-B27 Molecules*

    PubMed Central

    Magnacca, Adriana; Persiconi, Irene; Nurzia, Elisa; Caristi, Silvana; Meloni, Francesca; Barnaba, Vincenzo; Paladini, Fabiana; Raimondo, Domenico; Fiorillo, Maria Teresa; Sorrentino, Rosa

    2012-01-01

    Nascent HLA-class I molecules are stabilized by proteasome-derived peptides in the ER and the new complexes proceed to the cell surface through the post-ER vesicles. It has been shown, however, that less stable complexes can exchange peptides in the Trans Golgi Network (TGN). HLA-B27 are the most studied HLA-class I molecules due to their association with Ankylosing Spondylitis (AS). Chimeric proteins driven by TAT of HIV have been exploited by us to deliver viral epitopes, whose cross-presentation by the HLA-B27 molecules was proteasome and TAP-independent and not restricted to Antigen-Presenting Cells (APC). Here, using these chimeric proteins as epitope suppliers, we compared with each other and with the HLA-A2 molecules, the two HLA-B*2705 and B*2709 alleles differing at residue 116 (D116H) and differentially associated with AS. We found that the antigen presentation by the two HLA-B27 molecules was proteasome-, TAP-, and APC-independent whereas the presentation by the HLA-A2 molecules required proteasome, TAP and professional APC. Assuming that such difference could be due to the unpaired, highly reactive Cys-67 distinguishing the HLA-B27 molecules, C67S mutants in HLA-B*2705 and B*2709 and V67C mutant in HLA-A*0201 were also analyzed. The results showed that this mutation did not influence the HLA-A2-restricted antigen presentation while it drastically affected the HLA-B27-restricted presentation with, however, remarkable differences between B*2705 and B*2709. The data, together with the occurrence on the cell surface of unfolded molecules in the case of C67S-B*2705 mutant but not in that of C67S-B*2709 mutant, indicates that Cys-67 has a more critical role in stabilizing the B*2705 rather than the B*2709 complexes. PMID:22807446

  5. 27 CFR 18.27 - Additional requirements.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Additional requirements. 18.27 Section 18.27 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Qualification Application...

  6. 27 CFR 18.27 - Additional requirements.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Additional requirements. 18.27 Section 18.27 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Qualification Application...

  7. 27 CFR 18.27 - Additional requirements.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Additional requirements. 18.27 Section 18.27 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Qualification Application...

  8. 27 CFR 18.27 - Additional requirements.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Additional requirements. 18.27 Section 18.27 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Qualification Application...

  9. 27 CFR 18.27 - Additional requirements.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Additional requirements. 18.27 Section 18.27 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Qualification Application...

  10. The use of a P. falciparum specific coiled-coil domain to construct a self-assembling protein nanoparticle vaccine to prevent malaria.

    PubMed

    Karch, Christopher P; Doll, Tais A P F; Paulillo, Sara M; Nebie, Issa; Lanar, David E; Corradin, Giampietro; Burkhard, Peter

    2017-09-06

    The parasitic disease malaria remains a major global public health concern and no truly effective vaccine exists. One approach to the development of a malaria vaccine is to target the asexual blood stage that results in clinical symptoms. Most attempts have failed. New antigens such as P27A and P27 have emerged as potential new vaccine candidates. Multiple studies have demonstrated that antigens are more immunogenic and are better correlated with protection when presented on particulate delivery systems. One such particulate delivery system is the self-assembling protein nanoparticle (SAPN) that relies on coiled-coil domains of proteins to form stable nanoparticles. In the past we have used de novo designed amino acid domains to drive the formation of the coiled-coil scaffolds which present the antigenic epitopes on the particle surface. Here we use naturally occurring domains found in the tex1 protein to form the coiled-coil scaffolding of the nanoparticle. Thus, by engineering P27A and a new extended form of the coiled-coil domain P27 onto the N and C terminus of the SAPN protein monomer we have developed a particulate delivery system that effectively displays both antigens on a single particle that uses malaria tex1 sequences to form the nanoparticle scaffold. These particles are immunogenic in a murine model and induce immune responses similar to the ones observed in seropositive individuals in malaria endemic regions. We demonstrate that our P27/P27A-SAPNs induce an immune response akin to the one in seropositive individuals in Burkina Faso. Since P27 is highly conserved among different Plasmodium species, these novel SAPNs may even provide cross-protection between Plasmodium falciparum and Plasmodium vivax the two major human malaria pathogens. As the SAPNs are also easy to manufacture and store they can be delivered to the population in need without complication thus providing a low cost malaria vaccine.

  11. Synthesis, antifungal activity of caffeic acid derivative esters, and their synergism with fluconazole and nystatin against Candida spp.

    PubMed

    Sardi, Janaína de Cássia Orlandi; Gullo, Fernanda Patrícia; Freires, Irlan Almeida; Pitangui, Nayla de Souza; Segalla, Maicon Petrônio; Fusco-Almeida, Ana Marisa; Rosalen, Pedro Luiz; Regasini, Luís Octávio; Mendes-Giannini, Maria José Soares

    2016-12-01

    We tested the antifungal potential of caffeic acid and 8 of its derivative esters against Candidaalbicans ATCC 90028 and 9 clinical isolatesand carried out a synergism assay with fluconazole and nystatin. Propyl caffeate (C3) showed the best antifungal activity against the tested strains. When in combination, C3 markedly reduced the MIC of fluconazole and nystatin with synergistic effect up to 64-fold. Finally, C3 showed a high IC 50 value and selective indexagainst oral keratinocytes, demonstrating low toxicity against this cell type and selectivity for yeast cells. Further research should confirm its antifungal potential for development of combined therapy to treat C. albicans infections. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Dietary Protein and Calcium Interact to Influence Calcium Retention: A Controlled Feeding Study

    USDA-ARS?s Scientific Manuscript database

    Objective: To test the effect of dietary protein on Ca (Ca) retention at low and high Ca intakes. Methods: In a randomized, controlled feeding study with a 2x2 factorial crossover design, healthy post-menopausal women (n=27), consumed either ~675 or ~1510 mg Ca/d, with both low and high protein (pro...

  13. Nucks1 synergizes with Trp53 to promote radiation lymphomagenesis in mice

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

    Yue, Yangbo; Leung, Stanley G.; Liu, Yueyong

    NUCKS1 is a 27 kD vertebrate-specific protein, with a role in the DNA damage response. Here, we show that after 4 Gy total-body X-irradiation, Trp53+/- Nucks1+/- mice more rapidly developed tumors, particularly thymic lymphoma (TL), than Trp53+/- mice. TLs in both cohorts showed loss of heterozygosity (LOH) of the Trp53+ allele in essentially all cases. In contrast, LOH of the Nucks1+ allele was rare. Nucks1 expression correlated well with Nucks1 gene dosage in normal thymi, but was increased in the majority of TLs from Trp53+/- Nucks1+/- mice, suggesting that elevated Nucks1 message may be associated with progression towards malignancy inmore » vivo. Trp53+/- Nucks1+/- mice frequently succumbed to CD4- CD8- TLs harboring translocations involving Igh but not Tcra/d, indicating TLs in Trp53+/- Nucks1+/- mice mostly originated prior to the double positive stage and at earlier lineage than TLs in Trp53+/- mice. Monoclonal rearrangements at Tcrb were more prevalent in TLs from Trp53+/- Nucks1+/- mice, as was infiltration of primary TL cells to distant organs (liver, kidney and spleen). We propose that, in the context of Trp53 deficiency, wild type levels of Nucks1 are required to suppress radiation-induced TL, likely through the role of the NUCKS1 protein in the DNA damage response.« less

  14. Nucks1 synergizes with Trp53 to promote radiation lymphomagenesis in mice

    DOE PAGES

    Yue, Yangbo; Leung, Stanley G.; Liu, Yueyong; ...

    2016-08-16

    NUCKS1 is a 27 kD vertebrate-specific protein, with a role in the DNA damage response. Here, we show that after 4 Gy total-body X-irradiation, Trp53+/- Nucks1+/- mice more rapidly developed tumors, particularly thymic lymphoma (TL), than Trp53+/- mice. TLs in both cohorts showed loss of heterozygosity (LOH) of the Trp53+ allele in essentially all cases. In contrast, LOH of the Nucks1+ allele was rare. Nucks1 expression correlated well with Nucks1 gene dosage in normal thymi, but was increased in the majority of TLs from Trp53+/- Nucks1+/- mice, suggesting that elevated Nucks1 message may be associated with progression towards malignancy inmore » vivo. Trp53+/- Nucks1+/- mice frequently succumbed to CD4- CD8- TLs harboring translocations involving Igh but not Tcra/d, indicating TLs in Trp53+/- Nucks1+/- mice mostly originated prior to the double positive stage and at earlier lineage than TLs in Trp53+/- mice. Monoclonal rearrangements at Tcrb were more prevalent in TLs from Trp53+/- Nucks1+/- mice, as was infiltration of primary TL cells to distant organs (liver, kidney and spleen). We propose that, in the context of Trp53 deficiency, wild type levels of Nucks1 are required to suppress radiation-induced TL, likely through the role of the NUCKS1 protein in the DNA damage response.« less

  15. Nucks1 synergizes with Trp53 to promote radiation lymphomagenesis in mice

    PubMed Central

    Yue, Yangbo; Leung, Stanley G.; Liu, Yueyong; Huang, Yurong; Grundt, Kirsten; Østvold, Anne-Carine; Jen, Kuang-Yu; Schild, David; Mao, Jian-Hua; Wiese, Claudia

    2016-01-01

    NUCKS1 is a 27 kD vertebrate-specific protein, with a role in the DNA damage response. Here, we show that after 4 Gy total-body X-irradiation, Trp53+/− Nucks1+/− mice more rapidly developed tumors, particularly thymic lymphoma (TL), than Trp53+/− mice. TLs in both cohorts showed loss of heterozygosity (LOH) of the Trp53+ allele in essentially all cases. In contrast, LOH of the Nucks1+ allele was rare. Nucks1 expression correlated well with Nucks1 gene dosage in normal thymi, but was increased in the majority of TLs from Trp53+/− Nucks1+/− mice, suggesting that elevated Nucks1 message may be associated with progression towards malignancy in vivo. Trp53+/− Nucks1+/− mice frequently succumbed to CD4- CD8- TLs harboring translocations involving Igh but not Tcra/d, indicating TLs in Trp53+/− Nucks1+/− mice mostly originated prior to the double positive stage and at earlier lineage than TLs in Trp53+/- mice. Monoclonal rearrangements at Tcrb were more prevalent in TLs from Trp53+/− Nucks1+/− mice, as was infiltration of primary TL cells to distant organs (liver, kidney and spleen). We propose that, in the context of Trp53 deficiency, wild type levels of Nucks1 are required to suppress radiation-induced TL, likely through the role of the NUCKS1 protein in the DNA damage response. PMID:27542204

  16. Characterization of a common wheat (Triticum aestivum L.) TaSnRK2.7 gene involved in abiotic stress responses

    PubMed Central

    Zhang, Hongying; Mao, Xinguo; Jing, Ruilian; Chang, Xiaoping; Xie, Huimin

    2011-01-01

    Sucrose non-fermenting-1-related protein kinase 2 (SnRK2) plays a key role in the plant stress signalling transduction pathway via phosphorylation. Here, a SnRK2 member of common wheat, TaSnRK2.7, was cloned and characterized. Southern blot analysis suggested that the common wheat genome contains three copies of TaSnRK2.7. Subcellular localization showed the presence of TaSnRK2.7 in the cell membrane, cytoplasm, and nucleus. Expression patterns revealed that TaSnRK2.7 is expressed strongly in roots, and responds to polyethylene glycol, NaCl, and cold stress, but not to abscisic acid (ABA) application, suggesting that TaSnRK2.7 might participate in non-ABA-dependent signal transduction pathways. TaSnRK2.7 was transferred to Arabidopsis under the control of the CaMV-35S promoter. Function analysis showed that TaSnRK2.7 is involved in carbohydrate metabolism, decreasing osmotic potential, enhancing photosystem II activity, and promoting root growth. Its overexpression results in enhanced tolerance to multi-abiotic stress. Therefore, TaSnRK2.7 is a multifunctional regulatory factor in plants, and has the potential to be utilized in transgenic breeding to improve abiotic stress tolerance in crop plants. PMID:21030389

  17. HLA-B27 Misfolding and Ankylosing Spondylitis

    PubMed Central

    Colbert, Robert A.; Tran, Tri M.; Layh-Schmitt, Gerlinde

    2013-01-01

    Understanding how HLA-B27 contributes to the pathogenesis of spondyloarthritis continues to be an important goal. Current efforts are aimed largely on three areas of investigation; peptide presentation to CD8 T cells, abnormal forms of the HLA-B27 heavy chain and their recognition by leukocyte immunoglobulin-like receptors on immune effector cells, and HLA-B27 heavy chain misfolding and intrinsic biological effects on affected cells. In this chapter we review our current understanding of the causes and consequences of HLA-B27 misfolding, which can be defined biochemically as a propensity to oligomerize and form complexes in the endoplasmic reticulum (ER) with the chaperone BiP (HSPA5/GRP78). HLA-B27 misfolding is linked to an unusual combination of polymorphisms that identify this allele, and cause the heavy chain to fold and load peptides inefficiently. Misfolding can result in ER-associated degradation (ERAD) of heavy chains, which is mediated in part by the E3 ubiquitin ligase HRD1 (SYVN1), and the ubiquitin conjugating enzyme UBE2JL. Upregulation of HLA-B27 and accumulation of misfolded heavy chains can activate ER stress signaling pathways that orchestrate the unfolded protein response. In transgenic rats where HLA-B27 is overexpressed, UPR activation is prominent. However, it is specific for heavy chain misfolding, since overexpression of HLA-B7, an allele that does not misfold, fails to generate ER stress. UPR activation has been linked to cytokine dysregulation, promoting lL-23, IFNβ, and lL-1α production, and may activate the IL-23/IL-17 axis in these rats. IL-1α and IFNβ are pro- and anti-osteoclastogenic cytokines, respectively, that modulate osteoclast development in HLA-B27-expressing transgenic rat monocytes. Translational studies of patient derived cells expressing HLA-B27 at physiologic levels have provided evidence that ER stress and UPR activation can occur in peripheral blood, but this has not been reported to date in isolated macrophages

  18. HLA-B27 misfolding and ankylosing spondylitis.

    PubMed

    Colbert, Robert A; Tran, Tri M; Layh-Schmitt, Gerlinde

    2014-01-01

    Understanding how HLA-B27 contributes to the pathogenesis of spondyloarthritis continues to be an important goal. Current efforts are aimed largely on three areas of investigation; peptide presentation to CD8T cells, abnormal forms of the HLA-B27 heavy chain and their recognition by leukocyte immunoglobulin-like receptors on immune effector cells, and HLA-B27 heavy chain misfolding and intrinsic biological effects on affected cells. In this chapter we review our current understanding of the causes and consequences of HLA-B27 misfolding, which can be defined biochemically as a propensity to oligomerize and form complexes in the endoplasmic reticulum (ER) with the chaperone BiP (HSPA5/GRP78). HLA-B27 misfolding is linked to an unusual combination of polymorphisms that identify this allele, and cause the heavy chain to fold and load peptides inefficiently. Misfolding can result in ER-associated degradation (ERAD) of heavy chains, which is mediated in part by the E3 ubiquitin ligase HRD1 (SYVN1), and the ubiquitin conjugating enzyme UBE2JL. Upregulation of HLA-B27 and accumulation of misfolded heavy chains can activate ER stress signaling pathways that orchestrate the unfolded protein response. In transgenic rats where HLA-B27 is overexpressed, UPR activation is prominent. However, it is specific for heavy chain misfolding, since overexpression of HLA-B7, an allele that does not misfold, fails to generate ER stress. UPR activation has been linked to cytokine dysregulation, promoting lL-23, IFNβ, and lL-1α production, and may activate the IL-23/IL-17 axis in these rats. IL-1α and IFNβ are pro- and anti-osteoclastogenic cytokines, respectively, that modulate osteoclast development in HLA-B27-expressing transgenic rat monocytes. Translational studies of patient derived cells expressing HLA-B27 at physiologic levels have provided evidence that ER stress and UPR activation can occur in peripheral blood, but this has not been reported to date in isolated macrophages

  19. Discovery of prenylated flavonoids with dual activity against influenza virus and Streptococcus pneumoniae.

    PubMed

    Grienke, Ulrike; Richter, Martina; Walther, Elisabeth; Hoffmann, Anja; Kirchmair, Johannes; Makarov, Vadim; Nietzsche, Sandor; Schmidtke, Michaela; Rollinger, Judith M

    2016-06-03

    Influenza virus neuraminidase (NA) is the primary target for influenza therapeutics. Severe complications are often related to secondary pneumonia caused by Streptococcus pneumoniae (pneumococci), which also express NAs. Recently, a NA-mediated lethal synergism between influenza A viruses and pneumococci was described. Therefore, dual inhibitors of both viral and bacterial NAs are expected to be advantageous for the treatment of influenza. We investigated the traditional Chinese herbal drug sāng bái pí (mulberry root bark) as source for anti-infectives. Two prenylated flavonoid derivatives, sanggenon G (4) and sanggenol A (5) inhibited influenza A viral and pneumococcal NAs and, in contrast to the approved NA inhibitor oseltamivir, also planktonic growth and biofilm formation of pneumococci. Evaluation of 27 congeners of 5 revealed a correlation between the degree of prenylation and bioactivity. Abyssinone-V 4'-methyl ether (27) inhibited pneumococcal NA with IC50 = 2.18 μM, pneumococcal growth with MIC = 5.63 μM, and biofilm formation with MBIC = 4.21 μM, without harming lung epithelial cells. Compounds 5 and 27 also disrupt the synergism between influenza A virus and pneumococcal NA in vitro, hence functioning as dual-acting anti-infectives. The results warrant further studies on whether the observed disruption of this synergism is transferable to in vivo systems.

  20. Discovery of prenylated flavonoids with dual activity against influenza virus and Streptococcus pneumoniae

    PubMed Central

    Grienke, Ulrike; Richter, Martina; Walther, Elisabeth; Hoffmann, Anja; Kirchmair, Johannes; Makarov, Vadim; Nietzsche, Sandor; Schmidtke, Michaela; Rollinger, Judith M.

    2016-01-01

    Influenza virus neuraminidase (NA) is the primary target for influenza therapeutics. Severe complications are often related to secondary pneumonia caused by Streptococcus pneumoniae (pneumococci), which also express NAs. Recently, a NA-mediated lethal synergism between influenza A viruses and pneumococci was described. Therefore, dual inhibitors of both viral and bacterial NAs are expected to be advantageous for the treatment of influenza. We investigated the traditional Chinese herbal drug sāng bái pí (mulberry root bark) as source for anti-infectives. Two prenylated flavonoid derivatives, sanggenon G (4) and sanggenol A (5) inhibited influenza A viral and pneumococcal NAs and, in contrast to the approved NA inhibitor oseltamivir, also planktonic growth and biofilm formation of pneumococci. Evaluation of 27 congeners of 5 revealed a correlation between the degree of prenylation and bioactivity. Abyssinone-V 4′-methyl ether (27) inhibited pneumococcal NA with IC50 = 2.18 μM, pneumococcal growth with MIC = 5.63 μM, and biofilm formation with MBIC = 4.21 μM, without harming lung epithelial cells. Compounds 5 and 27 also disrupt the synergism between influenza A virus and pneumococcal NA in vitro, hence functioning as dual-acting anti-infectives. The results warrant further studies on whether the observed disruption of this synergism is transferable to in vivo systems. PMID:27257160