RESUMO
Human NLRP1 (NACHT, LRR, and PYD domain-containing protein 1) is an innate immune sensor predominantly expressed in the skin and airway epithelium. Here, we report that human NLRP1 senses the ultraviolet B (UVB)- and toxin-induced ribotoxic stress response (RSR). Biochemically, RSR leads to the direct hyperphosphorylation of a human-specific disordered linker region of NLRP1 (NLRP1DR) by MAP3K20/ZAKα kinase and its downstream effector, p38. Mutating a single ZAKα phosphorylation site in NLRP1DR abrogates UVB- and ribotoxin-driven pyroptosis in human keratinocytes. Moreover, fusing NLRP1DR to CARD8, which is insensitive to RSR by itself, creates a minimal inflammasome sensor for UVB and ribotoxins. These results provide insight into UVB sensing by human skin keratinocytes, identify several ribotoxins as NLRP1 agonists, and establish inflammasome-driven pyroptosis as an integral component of the RSR.
Assuntos
Inflamassomos , MAP Quinase Quinase Quinases , Proteínas NLR , Piroptose , Ribossomos , Estresse Fisiológico , Anisomicina/toxicidade , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Humanos , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Inflamassomos/efeitos da radiação , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , MAP Quinase Quinase Quinases/metabolismo , Mutação , Proteínas NLR/genética , Proteínas NLR/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Piroptose/efeitos dos fármacos , Piroptose/efeitos da radiação , Ribossomos/efeitos dos fármacos , Ribossomos/efeitos da radiação , Raios UltravioletaRESUMO
Ototoxicity of drugs is an important inducement for hearing loss. Anisomycin is a candidate drug for parasite, cancer, immunosuppression, and mental disease. However, the ototoxicity of anisomycin has not been examined. In this study, the ototoxicity of anisomycin was evaluated using zebrafish lateral line. We found the zebrafish treated with anisomycin during lateral line development could inhibit hair cell formation in a time- and dose-dependent manner. After neuromasts are mature with differentiated hair cells by 5 day post-fertilization, anisomycin could induce hair cell loss effectively through chronic exposure rather than acute exposure. TUNEL assay and qPCR of apoptosis related genes tp53, casp8, casp3a, and casp3b indicated that cell apoptotic was induced by chronic anisomycin exposure. Furthermore, knocking down tp53 with antisense morpholino could attenuate the hair cell loss induced by anisomycin. In addition, we found that anisomycin chronic exposure also inhibited the proliferation of supporting cell. Together, these results indicate that chronic anisomycin exposure could induce hair cell death and block supporting cell proliferation, which causes hair cell loss in zebrafish neuromast. This study provides primary ototoxicity evaluation for anisomycin.
Assuntos
Anisomicina/toxicidade , Morte Celular/efeitos dos fármacos , Células Ciliadas Auditivas/efeitos dos fármacos , Sistema da Linha Lateral/efeitos dos fármacos , Peixe-Zebra/crescimento & desenvolvimento , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacosRESUMO
MultiFlow® DNA Damage-p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/ß-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose-response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.
Assuntos
Ativação Metabólica/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Animais , Anisomicina/toxicidade , Brefeldina A/toxicidade , Linhagem Celular , Cicloeximida/toxicidade , Ensaios de Triagem em Larga Escala/métodos , Histonas/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Ratos , Proteína Supressora de Tumor p53/genéticaRESUMO
The involvement of protein synthesis in the mechanisms of conditioned food aversion memory impairment and recovery in grape snails was studied. It was found that protein synthesis inhibitor (cycloheximide) injections before a reminder by the conditioned stimulus (CS) caused amnesia development. Three days after amnesia induction, injections of cycloheximide or another protein synthesis inhibitor, anisomycin, combined with a reminder by four CSs resulted in memory retrieval, which was saved for 24 h. Cycloheximide injections and the administration of one CS as a reminder to an amnestic animals caused the memory expression only in response to this CS, while it was absent the next day. The isolated administration of a reminder or inhibitor injections without a reminder was not effective. It is suggested that amnesia is an active process and that one of its mechanisms may be a protein-dependent amnesia reactivation caused by a reminder. The administration of protein synthesis inhibitors led to impairment of amnesia reactivation and to recovery of the state formed before amnesia induction and thus to the recovery of conditioned food aversion memory.
Assuntos
Anisomicina/toxicidade , Cicloeximida/toxicidade , Transtornos da Memória/induzido quimicamente , Inibidores da Síntese de Proteínas/toxicidade , Recuperação de Função Fisiológica/efeitos dos fármacos , Animais , Condicionamento Clássico/efeitos dos fármacos , Modelos Animais de Doenças , Esquema de Medicação , Estimulação Elétrica/efeitos adversos , Alimentos , Transdução de Sinais/efeitos dos fármacos , Caramujos , Estatísticas não Paramétricas , Fatores de TempoRESUMO
Short syntheses of oxa-preussin, racemic preussin and (-)-preussin are reported. Starting from a racemic 3-nonyl-substituted methoxyallene derivative, its lithiation and addition to phenylethanal provided the corresponding allenyl alcohol that was converted into two diastereomeric dihydrofuran derivatives by silver nitrate-catalyzed 5-endo-trig cyclization. The acid hydrolysis of the enol ether moiety gave heterocyclic ketones and subsequent highly stereoselective reductions with l-selectride furnished 2-benzyl-5-nonylfuran-3-ol derivatives in good overall yield. The major all-cis-diastereomer has the skeleton and relative configuration of preussin and is hence called oxa-preussin. An analogous sequence with the same allene, but an N-sulfonyl imine as the electrophile, finally led to racemic preussin. The stereoselectivities of the individual steps are discussed in detail. With an enantiopure 2-benzyl-5-nonylpyrrolidin-3-one intermediate the preparation of (-)-preussin with an enantiomeric ratio of >95 : 5 could be accomplished in a few steps. The sign of the optical rotation of this product finally proved the absolute configurations of its precursors and demonstrated that our chiral auxiliary-based route led to the antipode of the natural product. The cytotoxicity of several of the prepared heterocycles against MCF-7 tumor cells was investigated and five compounds, including racemic and enantiopure (-)-preussin, were identified as highly cytotoxic with IC50 values in the range of 3-6 µM.
Assuntos
Alcadienos/química , Anisomicina/análogos & derivados , Álcoois , Anisomicina/síntese química , Anisomicina/toxicidade , Catálise , Citotoxinas/síntese química , Humanos , Hidrólise , Concentração Inibidora 50 , Cetonas , Células MCF-7 , EstereoisomerismoRESUMO
Motor rehabilitative training after stroke can improve motor function and promote topographical reorganization of remaining motor cortical movement representations, but this reorganization follows behavioral improvements. A more detailed understanding of the neural bases of rehabilitation efficacy is needed to inform therapeutic efforts to improve it. Using a rat model of upper extremity impairments after ischemic stroke, we examined effects of motor rehabilitative training at the ultrastructural level in peri-infarct motor cortex. Extensive training in a skilled reaching task promoted improved performance and recovery of more normal movements. This was linked with greater axodendritic synapse density and ultrastructural characteristics of enhanced synaptic efficacy that were coordinated with changes in perisynaptic astrocytic processes in the border region between head and forelimb areas of peri-infarct motor cortex. Disrupting synapses and motor maps by infusions of anisomycin (ANI) into anatomically reorganized motor, but not posterior parietal, cortex eliminated behavioral gains from rehabilitative training. In contrast, ANI infusion in the equivalent cortical region of intact animals had no effect on reaching skills. These results suggest that rehabilitative training efficacy for improving manual skills is mediated by synaptic plasticity in a region of motor cortex that, before lesions, is not essential for manual skills, but becomes so as a result of the training. These findings support that experience-driven synaptic structural reorganization underlies functional vicariation in residual motor cortex after motor cortical infarcts.SIGNIFICANCE STATEMENT Stroke is a leading cause of long-term disability. Motor rehabilitation, the main treatment for physical disability, is of variable efficacy. A better understanding of neural mechanisms underlying effective motor rehabilitation would inform strategies for improving it. Here, we reveal synaptic underpinnings of effective motor rehabilitation. Rehabilitative training improved manual skill in the paretic forelimb and induced the formation of special synapse subtypes in coordination with structural changes in astrocytes, a glial cell that influences neural communication. These changes were found in a region that is nonessential for manual skill in intact animals, but came to mediate this skill due to training after stroke. Therefore, motor rehabilitation efficacy depends on synaptic changes that enable remaining brain regions to assume new functions.
Assuntos
Astrócitos/patologia , Infarto Cerebral/patologia , Córtex Motor/patologia , Plasticidade Neuronal , Prática Psicológica , Sinapses/patologia , Animais , Anisomicina/toxicidade , Mapeamento Encefálico , Infarto Cerebral/psicologia , Modelos Animais de Doenças , Membro Anterior/inervação , Membro Anterior/fisiopatologia , Masculino , Destreza Motora/efeitos dos fármacos , Inibidores da Síntese de Proteínas/toxicidade , Ratos , Ratos Long-Evans , Acidente Vascular Cerebral/patologia , Reabilitação do Acidente Vascular CerebralRESUMO
To identify an individual as familiar, rodents form a specific type of memory named social recognition memory. The olfactory bulb (OB) is an important structure for social recognition memory, while the hippocampus recruitment is still controversial. The present study was designed to elucidate the OB and the dorsal hippocampus contribution to the consolidation of social memory. For that purpose, we tested the effect of anisomycin (ANI), which one of the effects is the inhibition of protein synthesis, on the consolidation of social recognition memory. Swiss adult mice with cannulae implanted into the CA1 region of the dorsal hippocampus or into the OB were exposed to a juvenile during 5 min (training session; TR), and once again 1.5 h or 24 h later to test social short-term memory (S-STM) or social long-term memory (S-LTM), respectively. To study S-LTM consolidation, mice received intra-OB or intra-CA1 infusion of saline or ANI immediately, 3, 6 or 18 h after TR. ANI impaired S-LTM consolidation in the OB, when administered immediately or 6h after TR. In the dorsal hippocampus, ANI was amnesic only if administered 3 h after TR. Furthermore, the infusion of ANI in either OB or CA1, immediately after training, did not affect S-STM. Moreover, ANI administered into the OB did not alter the animal's performance in the buried food-finding task. Altogether, our results suggest the consolidation of S-LTM requires both OB and hippocampus participation, although in different time points. This study may help shedding light on the specific roles of the OB and dorsal hippocampus in social recognition memory.
Assuntos
Anisomicina/toxicidade , Hipocampo/efeitos dos fármacos , Transtornos da Memória/induzido quimicamente , Inibidores da Síntese de Ácido Nucleico/toxicidade , Bulbo Olfatório/efeitos dos fármacos , Reconhecimento Psicológico/efeitos dos fármacos , Comportamento Social , Fatores Etários , Animais , Condicionamento Psicológico/efeitos dos fármacos , Modelos Animais de Doenças , Medo/psicologia , Comportamento Alimentar/efeitos dos fármacos , Masculino , Camundongos , Tempo de Reação/efeitos dos fármacos , Estatísticas não Paramétricas , Fatores de TempoRESUMO
Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is a critical upstream mediator of the ribotoxic stress response (RSR) to the trichothecene deoxynivalenol (DON) and other translational inhibitors. Here, we employed HeLa cell lysates to: (1) characterize PKR's interactions with the ribosome and ribosomal RNA (rRNA); (2) demonstrate cell-free activation of ribosomal-associated PKR and (3) integrate these findings in a unified model for RSR. Robust PKR-dependent RSR was initially confirmed in intact cells. PKR basally associated with 40S, 60S, 80S and polysome fractions at molar ratios of 7, 2, 23 and 3, respectively. Treatment of ATP-containing HeLa lysates with DON or the ribotoxins anisomycin and ricin concentration-dependently elicited phosphorylation of PKR and its substrate eIF2α. These phosphorylations could be blocked by PKR inhibitors. rRNA immunoprecipitation (RNA-IP) of HeLa lysates with PKR-specific antibody and sequencing revealed that in the presence of DON or not, the kinase associated with numerous discrete sites on both the 18S and 28S rRNA molecules, a number of which contained double-stranded hairpins. These findings are consistent with a sentinel model whereby multiple PKR molecules basally associate with the ribosome positioning them to respond to ribotoxin-induced alterations in rRNA structure by dimerizing, autoactivating and, ultimately, evoking RSR.
Assuntos
Anisomicina/toxicidade , Ricina/toxicidade , Tricotecenos/toxicidade , eIF-2 Quinase/metabolismo , Clonagem Molecular , Células HeLa , Humanos , Fosforilação , Inibidores da Síntese de Proteínas , RNA Ribossômico 18S/química , RNA Ribossômico 28S/química , Ribossomos/metabolismo , Análise de Sequência de RNA , eIF-2 Quinase/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Previously, we studied the effects of deoxynivalenol (DON) and tributyltin oxide (TBTO) on whole genome mRNA expression profiles of human T lymphocyte Jurkat cells. These studies indicated that DON induces ribotoxic stress and both DON and TBTO induced ER stress which resulted into T-cell activation and apoptosis. The first goal of the present study was to provide final proof for these mode of actions by comparing the effects of 6 h exposure to DON and TBTO on mRNA expression to those of positive controls of ribotoxic stress (anisomycin), ER stress (thapsigargin) and T cell activation (ionomycin). Genes affected by anisomycin and the majority of genes affected by thapsigargin were affected in the same direction by DON and TBTO, respectively, confirming the expected modes of action. Pathway analysis further sustained that DON induces ribotoxic stress and both DON and TBTO induce unfolded protein response (UPR), ER stress, T cell activation and apoptosis. The second goal was to assess whether DON and/or TBTO affect other pathways above those detected before. TBTO induced groups of genes that are involved in DNA packaging and heat shock response that were not affected by thapsigargin. DON did not affect other genes than anisomycin indicating the effect of DON to be restricted to ribotoxic stress. This study also demonstrates that comparative gene expression analysis is a very promising tool for the identification of modes of action of immunotoxic compounds.
Assuntos
Anisomicina/toxicidade , Carcinógenos/toxicidade , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Perfilação da Expressão Gênica , Inibidores da Síntese de Ácido Nucleico/toxicidade , Tapsigargina/toxicidade , Compostos de Trialquitina/toxicidade , Tricotecenos/toxicidade , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Mapeamento Cromossômico , Interpretação Estatística de Dados , Proteínas de Choque Térmico/metabolismo , Humanos , Ionomicina/farmacologia , Células Jurkat , Análise em Microsséries , Proteínas Mitocondriais/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , RNA Neoplásico/biossíntese , RNA Neoplásico/isolamento & purificação , Linfócitos T/efeitos dos fármacosRESUMO
The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥25ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥10ng/ml) and ribosome-inactivating protein ricin (≥300ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activated kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-µ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspases 8, 9 and 3 concurrently with apoptosis further suggested that rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors of cathepsins L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism.
Assuntos
Clivagem do RNA/efeitos dos fármacos , RNA Ribossômico/efeitos dos fármacos , Tricotecenos/toxicidade , Animais , Anisomicina/toxicidade , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 8/efeitos dos fármacos , Catepsina L/farmacologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidores da Síntese de Ácido Nucleico/toxicidade , Proteínas Proto-Oncogênicas c-hck/metabolismo , RNA Ribossômico/isolamento & purificação , Ricina/toxicidade , Tricotecenos/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Our current understanding of brain mechanisms involved in learning and memory has been derived largely from studies using experimentally naïve animals. However, it is becoming increasingly clear that not all identified mechanisms may generalize to subsequent learning. For example, N-methyl-D-aspartate glutamate (NMDA) receptors in the dorsal hippocampus are required for contextual fear conditioning in naïve animals but not in animals previously trained in a similar task. Here we investigated how animals learn contextual fear conditioning for a second time-a response which is not due to habituation or generalization. We found that dorsal hippocampus infusions of voltage-dependent calcium channel blockers or the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) agonist impaired the first, not the second contextual learning. Only manipulations of the entire hippocampus led to an impairment in second learning. Specifically, inactivation of either the dorsal or ventral hippocampus caused the remaining portion of the hippocampus to acquire and consolidate the second learning. Thus, dorsal hippocampus seems necessary for initial contextual fear conditioning, but either the dorsal or ventral hippocampus is sufficient for subsequent conditioning in a different context. Together, these findings suggest that prior training experiences can change how the hippocampus processes subsequent similar learning.
Assuntos
Condicionamento Clássico/fisiologia , Medo/fisiologia , Hipocampo/fisiologia , Retenção Psicológica/fisiologia , 2-Amino-5-fosfonovalerato/análogos & derivados , 2-Amino-5-fosfonovalerato/farmacologia , 2-Amino-5-fosfonovalerato/toxicidade , Amnésia/induzido quimicamente , Amnésia/fisiopatologia , Animais , Anisomicina/farmacologia , Anisomicina/toxicidade , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/toxicidade , Condicionamento Clássico/efeitos dos fármacos , Eletrochoque , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/toxicidade , Reação de Congelamento Cataléptica/efeitos dos fármacos , Reação de Congelamento Cataléptica/fisiologia , Agonistas GABAérgicos/farmacologia , Agonistas GABAérgicos/toxicidade , Hipocampo/efeitos dos fármacos , Hipocampo/ultraestrutura , Masculino , Modelos Neurológicos , Modelos Psicológicos , Muscimol/farmacologia , Muscimol/toxicidade , Inibidores da Síntese de Proteínas/farmacologia , Inibidores da Síntese de Proteínas/toxicidade , Ratos Sprague-Dawley , Retenção Psicológica/efeitos dos fármacos , Verapamil/farmacologia , Verapamil/toxicidadeRESUMO
Anisomycin is a pyrrolidine antibiotic isolated from Streptomyces griseolus. Recent studies have shown that Anisomycin as a novel immunosuppressive agent is superior to Cyclosporine A (J. Immunother. 31, 858-870, 2008). In order to make toxicological evaluation of Anisomycin, acute and four-week continuously intravenous toxicity studies were performed in mice. IC(50) value tested on peripheral lymphocytes was 25.44 ng/ml. The calculated LD(50) for Anisomycin was 119.64 mg/kg. The mice were intravenously injected through mouse tail vein with a total dose of 5, 15, 30 and 60 mg/kg/mice of Anisomycin every other day for 4 weeks. Just in the high-dose mice, death of three mice happened and body weight of the mice was significantly decreased. Statistically significant changes in organ index included increases in ratios of the spleen, liver, lung and brain to the body weight, and decrease in ratio of the thymus to the body weight. Changes in clinical biochemistry parameters included increases in the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, and decreases in the glucose (GLU) activity. The distinct inflammation appeared in the lung, liver and kidney, and the number and size of megakaryocytes in the spleen were significantly increased. Anisomycin did not induce formation of the peripheral blood micronucleus, but increased the number of micronucleated polychromatic erythrocytes in bone marrow and sperm aberrations. However, the above aberrant changes occurred only in the mice treated with the high-dose Anisomycin. These results indicate that although Anisomycin has no significant side effects at effectively therapeutic doses, its over-dosage may lead to toxicity, particularly pulmo-, nephro- and hepato-toxicity.
Assuntos
Anisomicina/toxicidade , Antibacterianos/toxicidade , Animais , Anisomicina/administração & dosagem , Antibacterianos/administração & dosagem , Peso Corporal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Concentração Inibidora 50 , Injeções Intravenosas , Rim/efeitos dos fármacos , Dose Letal Mediana , Fígado/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Aleatória , Espermatozoides/efeitos dos fármacos , Testes de Toxicidade AgudaRESUMO
CCAAT/enhancer-binding protein homologous protein (CHOP) is a crucial stress-responsive factor in various mucosal injuries, including cellular translational stress conditions. In this study, chemical ribosome-inactivating stresses were assessed for their effects on stress-inducible CHOP expression and its association with epithelial inflammatory cytokine production. Several representative ribotoxic agents (deoxynivalenol, anisomycin, and 15-acetyldeoxynivalenol) enhanced CHOP expression and its nuclear translocation in human intestinal epithelial cells. Moreover, CHOP was a strong positive regulator of IL-8 production, but CHOP-mediated IL-8 production was inversely associated with expression of the mucosal regulatory factor peroxisome proliferator-activated receptor γ (PPARγ). Based on our recent report that PPARγ is a negative regulator of mRNA stability of IL-8, PPARγ was linked to a notable mRNA stabilizing protein, HuR, since ribotoxin-induced IL-8 mRNA is stabilized by HuR protein. Expression of exogenous PPARγ suppressed ribotoxin-triggered cytoplasmic translocation of HuR. In contrast, PPARγ-regulating CHOP was a positive modulator of HuR protein export from nuclei. Taken together, the results indicate that ribotoxin-induced CHOP protein is positively associated with production of proinflammatory cytokine IL-8, but it downregulates PPARγ action, subsequently allowing the cytosolic translocation of HuR protein and stabilization of IL-8 mRNA in gut epithelial cells. CHOP and PPARγ may represent critical mechanistic links between ribotoxic stress and proinflammatory cytokine production, and they may have a broader functional significance with regard to gastrointestinal stresses by toxic mucosal insults.
Assuntos
Regulação da Expressão Gênica/imunologia , Mucosa Intestinal/imunologia , PPAR gama/metabolismo , Fator de Transcrição CHOP/biossíntese , Anisomicina/toxicidade , Antígenos de Superfície/metabolismo , Western Blotting , Linhagem Celular , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Ensaio de Imunoadsorção Enzimática , Expressão Gênica/efeitos dos fármacos , Humanos , Imunidade nas Mucosas/genética , Imunidade nas Mucosas/imunologia , Interleucina-8/biossíntese , Interleucina-8/imunologia , Mucosa Intestinal/metabolismo , Microscopia Confocal , PPAR gama/genética , PPAR gama/imunologia , Inibidores da Síntese de Proteínas/toxicidade , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/imunologia , Transfecção , Tricotecenos/toxicidadeRESUMO
To this day, it remains unresolved whether experimental amnesia reflects failed memory storage or the inability to retrieve otherwise intact memory. Methodological as well as conceptual reasons prevented deciding between these two alternatives: The absence of recovery from amnesia is typically taken as supporting storage impairment interpretations; however, this absence of recovery does not positively demonstrate nonexistence of memory, allowing for alternative interpretations of amnesia as impairment of memory retrieval. To address this shortcoming, we present a novel approach to study the nature of amnesia that makes positive, i.e., falsifiable, predictions for the absence of memory. Applying this paradigm, we demonstrate here that infusing anisomycin into the dorsal hippocampus induces amnesia by impairing memory storage, not retrieval.
Assuntos
Amnésia/fisiopatologia , Hipocampo/fisiopatologia , Memória/fisiologia , Amnésia/induzido quimicamente , Animais , Anisomicina/administração & dosagem , Anisomicina/toxicidade , Condicionamento Operante/efeitos dos fármacos , Condicionamento Operante/fisiologia , Extinção Psicológica/efeitos dos fármacos , Extinção Psicológica/fisiologia , Hipocampo/efeitos dos fármacos , Injeções Intraventriculares , Masculino , Inibidores da Síntese de Proteínas/administração & dosagem , Inibidores da Síntese de Proteínas/toxicidade , Ratos , Ratos Sprague-DawleyRESUMO
Ribotoxic stress responses lead to the expression of genes important for cellular homeostasis by modulating cell survival, proliferation and differentiation. ATF3 was investigated for its modulation of the epithelial cellular integrity in response to mucosal ribotoxic stress. ATF3 expression was up-regulated by chemical agents causing ribotoxic stress such as deoxynivalenol and anisomycin in different types of intestinal epithelial cells. Moreover, reduction of ATF3 gene expression promoted ribotoxic stress-triggered programmed cell death, implicating a protective role of ATF3 in epithelial cell survival. Mechanistically, stabilization of ATF3 messenger RNA and protein played a critical role in maintaining enhanced levels of ATF3 production in response to the ribotoxic chemical agent. For ATF3 mRNA stability, p38 specific inhibitor SB203580 was the most efficient agent for suppression, suggesting the involvement of the p38 MAP kinase in ATF3 mRNA stabilization. In addition, the p38 MAP kinase as well as its downstream mediator glycogen synthase kinase 3beta (GSK3beta) was involved in ATF3 protein stabilization caused by chemical ribotoxic stress in human epithelial cells. As another separate signaling cascade, double-stranded RNA (dsRNA)-activated protein kinase (PKR) was demonstrated to translationally modulate ATF3 expression and contribute to the epithelial cell survival. PKR interference caused cells to be more susceptible to cell death caused by the chemical ribotoxic stress. The results of this study showed that enhanced ATF3 production was associated with cellular defenses by maintaining the epithelial survival after ribotoxic mucosal insults.
Assuntos
Fator 3 Ativador da Transcrição/biossíntese , Dano ao DNA/fisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Ribossomos/metabolismo , Fator 3 Ativador da Transcrição/genética , Anisomicina/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Dano ao DNA/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Ribossomos/efeitos dos fármacos , Tricotecenos/toxicidade , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Initiation of adaptive immunity requires activation of dendritic cells (DC) by "danger" signals. This study examines the functional consequences of activating a cellular stress response in human DC. Anisomycin, a potent inducer of this "stress" response, selectively activates p38 kinase in DC at low concentrations, and both p38 kinases and JNKs at higher concentrations. Activation of p38, was accompanied by an increase in the potency of dendritic cells to activate T cells. In contrast to LPS, anisomycin had no effect on the expression of several DC activation markers. Anisomycin synergised with LPS in driving release of IL-12 and TNF-alpha. Anisomycin also enhanced the formation of clusters between DC and T cells. Enhanced cytokine release and clustering were both inhibited by the selective p38 alpha and p38 beta inhibitor SB203580. This study demonstrates that the cellular stress response, mediated via p38 kinases, plays an important role in the regulation of several aspects of DC function.
Assuntos
Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Anisomicina/toxicidade , Apresentação de Antígeno , Células Dendríticas/efeitos dos fármacos , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Humanos , Técnicas In Vitro , Interleucina-12/biossíntese , Interleucina-6/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno , Lipopolissacarídeos/toxicidade , Modelos Imunológicos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The retina of newborn rats consists of the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL) containing amacrine cells and the neuroblastic layer (NBL). In retinal explants, the GCL enters cell death after sectioning of the optic nerve, whereas there is almost no cell death in the NBL. When protein synthesis is inhibited with anisomycin, cell death is blocked in the GCL and induced in the NBL. We tested the roles of nitric oxide (NO) on cell death in the retina in vitro. Either L-arginine, the substrate for NO synthase or the NO donor S:-nitroso-acetylpenicillamine (SNAP) blocked cell death induced by anisomycin in the NBL, but had no effect in the GCL. Sepiapterin, a precursor of the nitric oxide synthase (NOS)-cofactor tetrahydrobiopterin also had a protective effect against anisomycin. The use of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble form of guanylyl cyclase, showed that anti-apoptotic effect of SNAP is partially mediated by cGMP generated by activation of guanylyl cyclase. NADPH-diaphorase histochemistry stained cells only in the GCL and INL. Thus, the degenerative effect of anisomycin is observed within the NBL, whereas the localization of NOS is restricted to the GCL and INL. The protective effect of both the NO substrate and cofactor upon cell death induced by anisomycin in the NBL, indicates that NO produced by amacrine and ganglion cells is a paracrine modulator of cell death within the retinal tissue.
Assuntos
Biopterinas/análogos & derivados , Fármacos Neuroprotetores/metabolismo , Óxido Nítrico/metabolismo , Comunicação Parácrina/fisiologia , Pterinas , Retina/metabolismo , Animais , Animais Recém-Nascidos , Anisomicina/toxicidade , Arginina/metabolismo , Arginina/farmacologia , Biopterinas/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Técnicas In Vitro , NADPH Desidrogenase/metabolismo , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico/farmacologia , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Pteridinas/farmacologia , Ratos , Ratos Endogâmicos , Retina/citologia , Retina/efeitos dos fármacos , Retina/crescimento & desenvolvimento , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , S-Nitroso-N-AcetilpenicilaminaRESUMO
Eukaryotic initiation factor (eIF) 4E binds to the 5'-cap structure of eukaryotic mRNA and has a central role in the control of cell proliferation. We have shown previously that the stimulation of cultured Xenopus kidney cells with serum resulted in the activation of protein synthesis, enhanced phosphorylation of eIF4E and increased binding of the adapter protein, eIF4G, and poly(A)-binding protein (PABP) to eIF4E to form the functional initiation factor complex, eIF4F/PABP. We now show that cellular stresses such as arsenite, anisomycin and heat shock also result in increased phosphorylation of eIF4E, eIF4F complex formation and the association of PABP with eIF4G, in conditions under which the rate of protein synthesis is severely inhibited. In contrast with reported effects on mammalian cells, the stress-induced increase in eIF4F complex formation occurs in the absence of changes in the association of eIF4E with its binding proteins 4E-BP1 or 4E-BP2. The stress-induced changes in eIF4E phosphorylation were totally abrogated by the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580, and were partly inhibited by the phosphoinositide 3-kinase inhibitor LY294002 and the mammalian target of rapamycin (mTOR) inhibitor rapamycin. However, eIF4E phosphorylation was unaffected by extracellular signal-regulated protein kinase (MAP kinase) inhibitor PD98059. These results indicate that cellular stresses activate multiple signalling pathways that converge at the level of eIF4F complex formation to influence the interactions between eIF4E, eIF4G and PABP.
Assuntos
Rim/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas Quinases , Proteínas de Ligação a RNA/metabolismo , Animais , Anisomicina/toxicidade , Arsenitos/toxicidade , Células Cultivadas , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 4E em Eucariotos , Fator de Iniciação 4F em Eucariotos , Temperatura Alta , Imidazóis/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Proteínas de Ligação a Poli(A) , Piridinas/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Xenopus laevis , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The effects of anisomycin (ANM) on newborn chicks have been studied with respect to brain protein synthesis, growth, EEG, toxicity, and several passive avoidance learning tasks. It was found that intracerebral ANM (80 nmol) gave a maximum inhibition of brain protein synthesis of 30%, while a combination of subcutaneous (10 mumol; 53 mg/kg) plus intracerebral (80 nmol; 21 mug) ANM inhibited by 91% in the first 2 hr and by 75% in the subsequent 2 hr period. Cycloheximide (CXM) also in combined injections at the same doses as ANM, inhibited by 97% in the 4 hr that followed injection. However, all the CXM-injected chicks were dead by 18 hr, while the lethality of ANM did not differ from that of saline. ANM also did not affect EEG measured at 1, 3, 5, or 24 hr following the subcutaneous plus intracerebral injections, nor did ANM affect body or brain growth curves or brain protein accretion. In the learning experiments, animals were initially trained to peck at water-coated metal spheres (type A learning) or at water imbibed birdseed (types B and C learning) in less than 1 sec, and were exposed to the same lures treated with the aversant methylanthranilate (MeA) one day later on one occasion (types A and B learning) or exposed twice (type C learning) and tested for learning retention one day later. Learning criterion was set as failure to peck at the lure during the first 20 sec of presentation. If ANM was injected 1 hr prior to MeA exposure, large and highly significant memory deficits were found during the retention test, as compared with saline injected controls. No effect of ANM was seen, however, if it was injected one day after learning, indicating that it did not interfere with retrieval mechanisms. ANM also decreased the external manifestations of fear or displeasure that chicks express during retention testing. Such manifestations have a high correlation with pecking suppression (r = 0.88, P less than 0.001).