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‣ Cellular responses in the Malpighian tubules of Scaptotrigona postica (Latreille, 1807) exposed to low doses of fipronil and boric acid

Ferreira, Rafael Alexandre Costa; Silva Zacarin, Elaine Cristina Mathias; Malaspina, Osmar; Bueno, Odair Correa; Tomotake, Maria Eliza Miyoko; Pereira, Andrigo Monroe
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 57-65
Português
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Studies of sub-lethal effects of pesticide residues on stingless bees are scarce and morphological analysis of organs would add information to toxicological analysis in order to clarify the continuous exposure of Scaptotrigona postica to insecticides. The aim of this study was to evaluate the morphology and histochemistry of the Malpighian tubules (excretory organ) of S. postica exposed to fipronil or boric acid to detect cellular responses that indicate toxicity or adaptative mechanisms to stress induced by exposure of worker bees to low doses of these chemical compounds. Newly emerged bees were submitted to toxicological bioassays and morphological analyses by optical microscopy and Transmission Electron Microscopy, as well as histochemical methods, were performed to detect proteins and glycoconjugates. Additionally, immunohistochemical detection of DNA fragmentation and HSP70 (70-kDa Heat shock protein) were performed to detect cell death and stress response, respectively. Statistical analysis, for the bioassays conducted with ingestion of contaminated diet with boric acid at 0.75% (w/w) or with fipronil at 0.1μg/kg of food, showed that the survival of bees that ingested the contaminated diets were significantly different to the survival rate presented by the control group (P<0.0001). Although some characteristics indicative of initiation of cell death were observed...

‣ Curcumin Stimulates Proliferation of Embryonic Neural Progenitor Cells and Neurogenesis in the Adult Hippocampus*S⃞

Kim, So Jung; Son, Tae Gen; Park, Hee Ra; Park, Mikyung; Kim, Min-Sun; Kim, Hyung Sik; Chung, Hae Young; Mattson, Mark P.; Lee, Jaewon
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 23/05/2008 Português
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Curcumin is a natural phenolic component of yellow curry spice, which is used in some cultures for the treatment of diseases associated with oxidative stress and inflammation. Curcumin has been reported to be capable of preventing the death of neurons in animal models of neurodegenerative disorders, but its possible effects on developmental and adult neuroplasticity are unknown. In the present study, we investigated the effects of curcumin on mouse multi-potent neural progenitor cells (NPC) and adult hippocampal neurogenesis. Curcumin exerted biphasic effects on cultured NPC; low concentrations stimulated cell proliferation, whereas high concentrations were cytotoxic. Curcumin activated extracellular signal-regulated kinases (ERKs) and p38 kinases, cellular signal transduction pathways known to be involved in the regulation of neuronal plasticity and stress responses. Inhibitors of ERKs and p38 kinases effectively blocked the mitogenic effect of curcumin in NPC. Administration of curcumin to adult mice resulted in a significant increase in the number of newly generated cells in the dentate gyrus of hippocampus, indicating that curcumin enhances adult hippocampal neurogenesis. Our findings suggest that curcumin can stimulate developmental and adult hippocampal neurogenesis...

‣ Pharmacologic Profiling of Phosphoinositide 3-Kinase Inhibitors as Mitigators of Ionizing Radiation–Induced Cell Death

Lazo, John S.; Sharlow, Elizabeth R.; Epperly, Michael W.; Lira, Ana; Leimgruber, Stephanie; Skoda, Erin M.; Wipf, Peter; Greenberger, Joel S.
Fonte: The American Society for Pharmacology and Experimental Therapeutics Publicador: The American Society for Pharmacology and Experimental Therapeutics
Tipo: Artigo de Revista Científica
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Ionizing radiation (IR) induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade. Pluripotent cells are the most important population affected by IR because they are required for cellular replenishment. Despite the clear danger to large population centers, we still lack safe and effective therapies to abrogate the life-threatening effects of any accidental or intentional IR exposure. Therefore, we computationally analyzed the chemical structural similarity of previously published small molecules that, when given after IR, mitigate cell death and found a chemical cluster that was populated with PI3K inhibitors. Subsequently, we evaluated structurally diverse PI3K inhibitors. It is remarkable that 9 of 14 PI3K inhibitors mitigated γIR-induced death in pluripotent NCCIT cells as measured by caspase 3/7 activation. A single intraperitoneal dose of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], administered to mice at 4 or 24 hours, or PX-867 [(4S,4aR,5R,6aS,9aR,Z)-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-2,7,10-trioxo-1-(pyrrolidin-1-ylmethylene)-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroindeno[4,5-H]isochromen-5-yl acetate (CID24798773)]...

‣ In vivo siRNA delivery of Keap1 modulates death and survival signaling pathways and attenuates concanavalin-A-induced acute liver injury in mice

González-Rodríguez, Águeda; Reibert, Bjorn; Amann, Thomas; Constien, Rainier; Rondinone, Cristina M.; Valverde, Ángela M.
Fonte: The Company of Biologists Limited Publicador: The Company of Biologists Limited
Tipo: Artigo de Revista Científica
Português
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723.6405%
Oxidative stress contributes to the progression of acute liver failure (ALF). Transcription factor nuclear factor-erythroid 2-related factor (Nrf2) serves as an endogenous regulator by which cells combat oxidative stress. We have investigated liver damage and the balance between death and survival signaling pathways in concanavalin A (ConA)-mediated ALF using in vivo siRNA delivery targeting Keap1 in hepatocytes. For that goal, mice were injected with Keap1- or luciferase-siRNA-containing liposomes via the tail vein. After 48 hours, ALF was induced by ConA. Liver histology, pro-inflammatory mediators, antioxidant responses, cellular death, and stress and survival signaling were assessed. Keap1 mRNA and protein levels significantly decreased in livers of Keap1-siRNA-injected mice. In these animals, histological liver damage was less evident than in control mice when challenged with ConA. Likewise, markers of cellular death (FasL and caspases 8, 3 and 1) decreased at 4 and 8 hours post-injection. Nuclear Nrf2 and its target, hemoxygenase 1 (HO1), were elevated in Keap1-siRNA-injected mice compared with control animals, resulting in reduced oxidative stress in the liver. Similarly, mRNA levels of pro-inflammatory cytokines were reduced in livers from Keap1-siRNA-injected mice. At the molecular level...

‣ Clinical implications of cellular stress responses

Poljšak, Borut; Milisav, Irina
Fonte: Association of Basic Medical Sciences of Federation of Bosnia and Herzegovina Publicador: Association of Basic Medical Sciences of Federation of Bosnia and Herzegovina
Tipo: Artigo de Revista Científica
Publicado em /05/2012 Português
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Cellular stress response is a reaction to changes or fluctuations of extracellular conditions that damage the structure and function of macromolecules. Different stressors trigger different cellular responses, namely induce cell repair mechanisms, induce cell responses that result in temporary adaptation to some stressors, induce autophagy or trigger cell death. Inability to repair the damage or exposure to prolonged stress may contribute to aging. Persistent cell stress often enhances susceptibility to cancer and aging associated diseases. Cells and tissues are increasingly being used for transplantations and other novel therapeutic methods in which the quality and well being of cells is of paramount importance for the treatment to succeed. Therefore, discovering the mechanisms of cellular stress responses and the ability to detect and ameliorate them is important in prevention of development of disorders developed by persistent stress and for the success of transplantation and other cell related methods of regenerative medicine.

‣ Cell Encapsulation in Sub-mm Sized Gel Modules Using Replica Molding

McGuigan, Alison P.; Butte, Manish; Whitesides, George; Bruzewicz, Derek A.; Glavan, Ana
Fonte: Public Library of Science Publicador: Public Library of Science
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For many types of cells, behavior in two-dimensional (2D) culture differs from that in three-dimensional (3D) culture. Among biologists, 2D culture on treated plastic surfaces is currently the most popular method for cell culture. In 3D, no analogous standard method—one that is similarly convenient, flexible, and reproducible—exists. This paper describes a soft-lithographic method to encapsulate cells in 3D gel objects (modules) in a variety of simple shapes (cylinders, crosses, rectangular prisms) with lateral dimensions between 40 and 1000 ?m, cell densities of 105 – 108 cells/cm3, and total volumes between 1×10?7 and 8×10?4 cm3. By varying (i) the initial density of cells at seeding, and (ii) the dimensions of the modules, the number of cells per module ranged from 1 to 2500 cells. Modules were formed from a range of standard biopolymers, including collagen, Matrigel™, and agarose, without the complex equipment often used in encapsulation. The small dimensions of the modules allowed rapid transport of nutrients by diffusion to cells at any location in the module, and therefore allowed generation of modules with cell densities near to those of dense tissues (108 – 109 cells/cm3). This modular method is based on soft lithography and requires little special equipment; the method is therefore accessible...

‣ The SIRT1 Deacetylase Suppresses Intestinal Tumorigenesis and Colon Cancer Growth

Firestein, Ron; Blander, Gil; Michan, Shaday; Oberdoerffer, Philipp; Luikenhuis, Sandra; de Cabo, Rafael; Guarente, Leonard P.; Ogino, Shuji; Campbell, Jennifer; Bhimavarapu, Anupama; Fuchs, Charles Stewart; Hahn, William C.; Sinclair, David Andrew
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
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780.8714%
Numerous longevity genes have been discovered in model organisms and altering their function results in prolonged lifespan. In mammals, some have speculated that any health benefits derived from manipulating these same pathways might be offset by increased cancer risk on account of their propensity to boost cell survival. The Sir2/SIRT1 family of NAD+-dependent deacetylases is proposed to underlie the health benefits of calorie restriction (CR), a diet that broadly suppresses cancer in mammals. Here we show that CR induces a two-fold increase SIRT1 expression in the intestine of rodents and that ectopic induction of SIRT1 in a β-catenin-driven mouse model of colon cancer significantly reduces tumor formation, proliferation, and animal morbidity in the absence of CR. We show that SIRT1 deacetylates β-catenin and suppresses its ability to activate transcription and drive cell proliferation. Moreover, SIRT1 promotes cytoplasmic localization of the otherwise nuclear-localized oncogenic form of β-catenin. Consistent with this, a significant inverse correlation was found between the presence of nuclear SIRT1 and the oncogenic form of β−catenin in 81 human colon tumor specimens analyzed. Taken together, these observations show that SIRT1 suppresses intestinal tumor formation in vivo and raise the prospect that therapies targeting SIRT1 may be of clinical use in β−catenin-driven malignancies.

‣ Rule-Based Cell Systems Model of Aging using Feedback Loop Motifs Mediated by Stress Responses

Kriete, Andres; Booker, Glenn; Bosl, William
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
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Investigating the complex systems dynamics of the aging process requires integration of a broad range of cellular processes describing damage and functional decline co-existing with adaptive and protective regulatory mechanisms. We evolve an integrated generic cell network to represent the connectivity of key cellular mechanisms structured into positive and negative feedback loop motifs centrally important for aging. The conceptual network is casted into a fuzzy-logic, hybrid-intelligent framework based on interaction rules assembled from a priori knowledge. Based upon a classical homeostatic representation of cellular energy metabolism, we first demonstrate how positive-feedback loops accelerate damage and decline consistent with a vicious cycle. This model is iteratively extended towards an adaptive response model by incorporating protective negative-feedback loop circuits. Time-lapse simulations of the adaptive response model uncover how transcriptional and translational changes, mediated by stress sensors NF-κB and mTOR, counteract accumulating damage and dysfunction by modulating mitochondrial respiration, metabolic fluxes, biosynthesis, and autophagy, crucial for cellular survival. The model allows consideration of lifespan optimization scenarios with respect to fitness criteria using a sensitivity analysis. Our work establishes a novel extendable and scalable computational approach capable to connect tractable molecular mechanisms with cellular network dynamics underlying the emerging aging phenotype.

‣ The Acid Test of Fluoride: How pH Modulates Toxicity

Tsuchiya, Masahiro; Skobe, Ziedonis; Sharma, Ramaswamy Narayanaswamy; Tannous, Bakhos A.; Bartlett, John D.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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Background: It is not known why the ameloblasts responsible for dental enamel formation are uniquely sensitive to fluoride ((F^−)). Herein, we present a novel theory with supporting data to show that the low pH environment of maturating stage ameloblasts enhances their sensitivity to a given dose of (F^−). Enamel formation is initiated in a neutral pH environment (secretory stage); however, the pH can fall to below 6.0 as most of the mineral precipitates (maturation stage). Low pH can facilitate entry of (F^−) into cells. Here, we asked if (F^−) was more toxic at low pH, as measured by increased cell stress and decreased cell function. Methodology/Principal Findings: Treatment of ameloblast-derived LS8 cells with (F^−) at low pH reduced the threshold dose of (F^−) required to phosphorylate stress-related proteins, PERK, eIF2α, JNK and c-jun. To assess protein secretion, LS8 cells were stably transduced with a secreted reporter, Gaussia luciferase, and secretion was quantified as a function of (F^−) dose and pH. Luciferase secretion significantly decreased within 2 hr of (F^−) treatment at low pH versus neutral pH, indicating increased functional toxicity. Rats given 100 ppm (F^−) in their drinking water exhibited increased stress-mediated phosphorylation of eIF2α in maturation stage ameloblasts (pH<6.0) as compared to secretory stage ameloblasts (pH∼7.2). Intriguingly...

‣ RNAi Screening Implicates a SKN-1–Dependent Transcriptional Response in Stress Resistance and Longevity Deriving from Translation Inhibition

Robida-Stubbs, Stacey; Tullet, Jennifer M. A.; Rual, Jean-Francois; Vidal, Marc; Blackwell, Thomas Keith; Wang, Jinling
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
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605.1965%
Caenorhabditis elegans SKN-1 (ortholog of mammalian Nrf1/2/3) is critical for oxidative stress resistance and promotes longevity under reduced insulin/IGF-1–like signaling (IIS), dietary restriction (DR), and normal conditions. SKN-1 inducibly activates genes involved in detoxification, protein homeostasis, and other functions in response to stress. Here we used genome-scale RNA interference (RNAi) screening to identify mechanisms that prevent inappropriate SKN-1 target gene expression under non-stressed conditions. We identified 41 genes for which knockdown leads to activation of a SKN-1 target gene (gcs-1) through skn-1-dependent or other mechanisms. These genes correspond to multiple cellular processes, including mRNA translation. Inhibition of translation is known to increase longevity and stress resistance and may be important for DR–induced lifespan extension. One model postulates that these effects derive from reduced energy needs, but various observations suggest that specific longevity pathways are involved. Here we show that translation initiation factor RNAi robustly induces SKN-1 target gene transcription and confers skn-1-dependent oxidative stress resistance. The accompanying increases in longevity are mediated largely through the activities of SKN-1 and the transcription factor DAF-16 (FOXO)...

‣ Induction of Erythroid Differentiation in Human Erythroleukemia Cells by Depletion of Malic Enzyme 2

Everett, Peter; Clish, Clary B.; Polymenis, Michael; Ren, Jian-Guo; Seth, Pankaj; Sukhatme, Vikas Pandurang
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
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680.825%
Malic enzyme 2 (ME2) is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and CO2 and uses NAD as a cofactor. Higher expression of this enzyme correlates with the degree of cell de-differentiation. We found that ME2 is expressed in K562 erythroleukemia cells, in which a number of agents have been found to induce differentiation either along the erythroid or the myeloid lineage. We found that knockdown of ME2 led to diminished proliferation of tumor cells and increased apoptosis in vitro. These findings were accompanied by differentiation of K562 cells along the erythroid lineage, as confirmed by staining for glycophorin A and hemoglobin production. ME2 knockdown also totally abolished growth of K562 cells in nude mice. Increased ROS levels, likely reflecting increased mitochondrial production, and a decreased (NADPH/NADP^+) ratio were noted but use of a free radical scavenger to decrease inhibition of ROS levels did not reverse the differentiation or apoptotic phenotype, suggesting that ROS production is not causally involved in the resultant phenotype. As might be expected, depletion of ME2 induced an increase in the (NAD^+/NADH) ratio and ATP levels fell significantly. Inhibition of the malate-aspartate shuttle was insufficient to induce K562 differentiation. We also examined several intracellular signaling pathways and expression of transcription factors and intermediate filament proteins whose expression is known to be modulated during erythroid differentiation in K562 cells. We found that silencing of ME2 leads to phospho-ERK1/2 inhibition...

‣ Polychlorinated Biphenyls (PCBs) Enhance Metastatic Properties of Breast Cancer Cells by Activating Rho-Associated Kinase (ROCK)

Du, Yuguo; Li, Shitao; Liu, Sijin
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
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680.6178%
Background: Polychlorinated biphenyls (PCBs) are a family of structurally related chlorinated aromatic hydrocarbons. Numerous studies have documented a wide spectrum of biological effects of PCBs on human health, such as immunotoxicity, neurotoxocity, estrogenic or antiestrogenic activity, and carcinogensis. The role of PCBs as etiologic agents for breast cancer has been intensively explored in a variety of in vivo, animal and epidemiologic studies. A number of investigations indicated that higher levels of PCBs in mammary tissues or sera correlated to breast cancer risk, and PCBs might be implicated in advancing breast cancer progression. Methodology/Principal Findings: In the current study, we for the first time report that PCBs greatly promote the ROCK activity and therefore increase cell motility for both non-metastatic and metastatic human breast cancer cells in vitro. In the in vivo study, PCBs significantly advance disease progression, leading to enhanced capability of metastatic breast cancer cells to metastasize to bone, lung and liver. Additionally, PCBs robustly induce the production of intracellular reactive oxygen species (ROS) in breast cancer cells; ROS mechanistically elevate ROCK activity. Conclusions/Significance: PCBs enhance the metastatic propensity of breast cancer cells by activating the ROCK signaling...

‣ Oxidative Stress Impairs the Heat Stress Response and Delays Unfolded Protein Recovery

Adachi, Masaaki; Liu, Yaohua; Fujii, Kyoko; Nakai, Akira; Imai, Kohzoh; Shinomura, Yasuhisa; Calderwood, Stuart K.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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Background: Environmental changes, air pollution and ozone depletion are increasing oxidative stress, and global warming threatens health by heat stress. We now face a high risk of simultaneous exposure to heat and oxidative stress. However, there have been few studies investigating their combined adverse effects on cell viability. Principal Findings: Pretreatment of hydrogen peroxide ((H_2O_2)) specifically and highly sensitized cells to heat stress, and enhanced loss of mitochondrial membrane potential. (H_2O_2) exposure impaired the HSP40/HSP70 induction as heat shock response (HSR) and the unfolded protein recovery, and enhanced (eIF2alpha) phosphorylation and/or XBP1 splicing, land marks of ER stress. These (H_2O_2)-mediated effects mimicked enhanced heat sensitivity in HSF1 knockdown or knockout cells. Importantly, thermal preconditioning blocked (H_2O_2)–mediated inhibitory effects on refolding activity and rescued HSF1 +/+ MEFs, but neither blocked the effects nor rescued HSF1 -/- MEFs. These data strongly suggest that inhibition of HSR and refolding activity is crucial for (H_2O_2)–mediated enhanced heat sensitivity. Conclusions: (H_2O_2) blocks HSR and refolding activity under heat stress, thereby leading to insufficient quality control and enhancing ER stress. These uncontrolled stress responses may enhance cell death. Our data thus highlight oxidative stress as a crucial factor affecting heat tolerance.

‣ Insulin Signaling Regulates Mitochondrial Function in Pancreatic β-Cells

Liu, Siming; Okada, Terumasa; Assmann, Anke; Soto, Jamie; Bugger, Heiko; Shirihai, Orian S.; Abel, E. Dale; Liew, Chong-Wee; Kulkarni, Rohit Narayan
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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680.7695%
Insulin/IGF-I signaling regulates the metabolism of most mammalian tissues including pancreatic islets. To dissect the mechanisms linking insulin signaling with mitochondrial function, we first identified a mitochondria-tethering complex in β-cells that included glucokinase (GK), and the pro-apoptotic protein, BADS. Mitochondria isolated from β-cells derived from β-cell specific insulin receptor knockout (βIRKO) mice exhibited reduced BADS, GK and protein kinase A in the complex, and attenuated function. Similar alterations were evident in islets from patients with type 2 diabetes. Decreased mitochondrial GK activity in βIRKOs could be explained, in part, by reduced expression and altered phosphorylation of BADS. The elevated phosphorylation of p70S6K and JNK1 was likely due to compensatory increase in IGF-1 receptor expression. Re-expression of insulin receptors in βIRKO cells partially restored the stoichiometry of the complex and mitochondrial function. These data indicate that insulin signaling regulates mitochondrial function and have implications for β-cell dysfunction in type 2 diabetes.

‣ Mitogen-Activated Protein Kinases Regulate Susceptibility to Ventilator-Induced Lung Injury

Dolinay, Tamás; Kaminski, Naftali; Ifedigbo, Emeka; Kaynar, A. Murat; Szilasi, Mária; Watkins, Simon C.; Hoetzel, Alexander; Wu, Wei; Ryter, Stefan W.; Choi, Augustine M.K.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
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680.825%
Background: Mechanical ventilation causes ventilator-induced lung injury in animals and humans. Mitogen-activated protein kinases have been implicated in ventilator-induced lung injury though their functional significance remains incomplete. We characterize the role of p38 mitogen-activated protein kinase/mitogen activated protein kinase kinase-3 and c-Jun-NH2-terminal kinase-1 in ventilator-induced lung injury and investigate novel independent mechanisms contributing to lung injury during mechanical ventilation. Methodology and Principle Findings: C57/BL6 wild-type mice and mice genetically deleted for mitogen-activated protein kinase kinase-3 (mkk-3−/−) or c-Jun-NH2-terminal kinase-1 (jnk1−/−) were ventilated, and lung injury parameters were assessed. We demonstrate that mkk3−/− or jnk1−/− mice displayed significantly reduced inflammatory lung injury and apoptosis relative to wild-type mice. Since jnk1−/− mice were highly resistant to ventilator-induced lung injury, we performed comprehensive gene expression profiling of ventilated wild-type or jnk1−/− mice to identify novel candidate genes which may play critical roles in the pathogenesis of ventilator-induced lung injury. Microarray analysis revealed many novel genes differentially expressed by ventilation including matrix metalloproteinase-8 (MMP8) and GADD45α. Functional characterization of MMP8 revealed that mmp8−/− mice were sensitized to ventilator-induced lung injury with increased lung vascular permeability. Conclusions: We demonstrate that mitogen-activated protein kinase pathways mediate inflammatory lung injury during ventilator-induced lung injury. C-Jun-NH2-terminal kinase was also involved in alveolo-capillary leakage and edema formation...

‣ Egr-1 Regulates Autophagy in Cigarette Smoke-induced Chronic Obstructive Pulmonary Disease

Chen, Zhi-Hua; Kim, Hong Pyo; Sciurba, Frank C.; Feghali-Bostwick, Carol; Stolz, Donna B.; Dhir, Rajiv; Landreneau, Rodney J.; Schuchert, Mathew J.; Yousem, Samuel A.; Pilewski, Joseph M.; Lee, Janet S.; Zhang, Yingze; Lee, Seon-Jin; Nakahira, Kiichi; Ryt
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
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Background: Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by abnormal cellular responses to cigarette smoke, resulting in tissue destruction and airflow limitation. Autophagy is a degradative process involving lysosomal turnover of cellular components, though its role in human diseases remains unclear. Methodology and Principal Findings: Increased autophagy was observed in lung tissue from COPD patients, as indicated by electron microscopic analysis, as well as by increased activation of autophagic proteins (microtubule-associated protein-1 light chain-3B, LC3B, Atg4, Atg5/12, Atg7). Cigarette smoke extract (CSE) is an established model for studying the effects of cigarette smoke exposure in vitro. In human pulmonary epithelial cells, exposure to CSE or histone deacetylase (HDAC) inhibitor rapidly induced autophagy. CSE decreased HDAC activity, resulting in increased binding of early growth response-1 (Egr-1) and E2F factors to the autophagy gene LC3B promoter, and increased LC3B expression. Knockdown of E2F-4 or Egr-1 inhibited CSE-induced LC3B expression. Knockdown of Egr-1 also inhibited the expression of Atg4B, a critical factor for LC3B conversion. Inhibition of autophagy by LC3B-knockdown protected epithelial cells from CSE-induced apoptosis. Egr-1(^{−/−}) mice...

‣ In vivo siRNA delivery of Keap1 modulates death and survival signaling pathways and attenuates concanavalin-A-induced acute liver injury in mice

González-Rodríguez, Agueda; Reibert, Bjorn; Amann, Thomas; Constien, Rainier; Rondinone, Cristina M.; Valverde, Angela M.
Fonte: The Company of Biologists Publicador: The Company of Biologists
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
723.6405%
Oxidative stress contributes to the progression of acute liver failure (ALF). Transcription factor nuclear factor-erythroid 2-related factor (Nrf2) serves as an endogenous regulator by which cells combat oxidative stress. We have investigated liver damage and the balance between death and survival signaling pathways in concanavalin A (ConA)-mediated ALF using in vivo siRNA delivery targeting Keap1 in hepatocytes. For that goal, mice were injected with Keap1- or luciferase-siRNA-containing liposomes via the tail vein. After 48 hours, ALF was induced by ConA. Liver histology, proinflammatory mediators, antioxidant responses, cellular death, and stress and survival signaling were assessed. Keap1 mRNA and protein levels significantly decreased in livers of Keap1-siRNAinjected mice. In these animals, histological liver damage was less evident than in control mice when challenged with ConA. Likewise, markers of cellular death (FasL and caspases 8, 3 and 1) decreased at 4 and 8 hours post-injection. Nuclear Nrf2 and its target, hemoxygenase 1 (HO1), were elevated in Keap1-siRNA-injected mice compared with control animals, resulting in reduced oxidative stress in the liver. Similarly, mRNA levels of pro-inflammatory cytokines were reduced in livers from Keap1-siRNA-injected mice. At the molecular level...

‣ An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor

Tsui, Melody; Carpenter, Anne E.; Xie, Tiao; Orth, James Daniel; Rudnicki, Stewart; Kim, Suejong; Shamu, Caroline Elizabeth; Mitchison, Timothy J.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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794.6634%
Kinesin-5 (also known as Eg5, KSP and Kif11) is required for assembly of a bipolar mitotic spindle. Small molecule inhibitors of Kinesin-5, developed as potential anti-cancer drugs, arrest cell in mitosis and promote apoptosis of cancer cells. We performed a genome-wide siRNA screen for enhancers and suppressors of a Kinesin-5 inhibitor in human cells to elucidate cellular responses, and thus identify factors that might predict drug sensitivity in cancers. Because the drug's actions play out over several days, we developed an intermittent imaging screen. Live HeLa cells expressing GFP-tagged histone H2B were imaged at 0, 24 and 48 hours after drug addition, and images were analyzed using open-source software that incorporates machine learning. This screen effectively identified siRNAs that caused increased mitotic arrest at low drug concentrations (enhancers), and vice versa (suppressors), and we report siRNAs that caused both effects. We then classified the effect of siRNAs for 15 genes where 3 or 4 out of 4 siRNA oligos tested were suppressors as assessed by time lapse imaging, and by testing for suppression of mitotic arrest in taxol and nocodazole. This identified 4 phenotypic classes of drug suppressors, which included known and novel genes. Our methodology should be applicable to other screens...

‣ Characterization of Age-Dependent and Progressive Cortical Neuronal Degeneration in Presenilin Conditional Mutant Mice

Wines-Samuelson, Mary; Schulte, Eva C.; Smith, Miriam J.; Aoki, Chiye; Liu, Xinran; Cookson, Mark R.; Kelleher, Raymond J.; Shen, Jie
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
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903.785%
Presenilins are the major causative genes of familial Alzheimer's disease (AD). Our previous study has demonstrated essential roles of presenilins in memory and neuronal survival. Here, we explore further how loss of presenilins results in age-related, progressive neurodegeneration in the adult cerebral cortex, where the pathogenesis of AD occurs. To circumvent the requirement of presenilins for embryonic development, we used presenilin conditional double knockout (Psen cDKO) mice, in which presenilin inactivation is restricted temporally and spatially to excitatory neurons of the postnatal forebrain beginning at 4 weeks of age. Increases in the number of degenerating (Fluoro-Jade B+, 7.6-fold) and apoptotic (TUNEL+, 7.4-fold) neurons, which represent ∼0.1% of all cortical neurons, were first detected at 2 months of age when there is still no significant loss of cortical neurons and volume in Psen cDKO mice. By 4 months of age, significant loss of cortical neurons (∼9%) and gliosis was found in Psen cDKO mice. The apoptotic cell death is associated with caspase activation, as shown by increased numbers of cells immunoreactive for active caspases 9 and 3 in the Psen cDKO cortex. The vulnerability of cortical neurons to loss of presenilins is region-specific with cortical neurons in the lateral cortex most susceptible. Compared to the neocortex...

‣ Catastrophic NAD+ Depletion in Activated T Lymphocytes Through Nampt Inhibition Reduces Demyelination and Disability in EAE

Bruzzone, Santina; Fruscione, Floriana; Morando, Sara; Ferrando, Tiziana; Poggi, Alessandro; Garuti, Anna; D'Urso, Agustina; Selmo, Martina; Benvenuto, Federica; Cea, Michele; Zoppoli, Gabriele; Moran, Eva; Soncini, Debora; Ballestrero, Alberto; Sordat, B
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
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Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD+ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD+ depletion upon FK866-mediated Nampt inhibition. As a consequence, impaired proliferation, reduced IFN-γ and TNF-α production, and finally autophagic cell demise result. We demonstrate that upregulation of the NAD+-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD+ depletion. In addition, we relate defective IFN-γ and TNF-α production in response to FK866 to impaired Sirt6 activity. Finally...