RESUMO
Structural variations in genomes are commonly studied by (micro)array-based comparative genomic hybridization. The data analysis methods to infer copy number variation in model organisms (human, mouse) are established. In principle, the procedures are based on signal ratios between test and reference samples and the order of the probe targets in the genome. These procedures are less applicable to experiments with non-model organisms, which frequently comprise non-sequenced genomes with an unknown order of probe targets. We therefore present an additional analysis approach, which does not depend on the structural information of a reference genome, and quantifies the presence or absence of a probe target in an unknown genome. The principle is that intensity values of target probes are compared with the intensities of negative-control probes and positive-control probes from a control hybridization, to determine if a probe target is absent or present. In a test, analyzing the genome content of a known bacterial strain: Staphylococcus aureus MRSA252, this approach proved to be successful, demonstrated by receiver operating characteristic area under the curve values larger than 0.9995. We show its usability in various applications, such as comparing genome content and validating next-generation sequencing reads from eukaryotic non-model organisms.
Assuntos
Hibridização Genômica Comparativa/métodos , Variação Estrutural do Genoma , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Interpretação Estatística de Dados , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Modelos Genéticos , Sondas de Oligonucleotídeos , Staphylococcus aureus/genéticaRESUMO
OBJECTIVE: The objective of this study was to investigate the dose-dependent therapeutic effect of the orally administrated antioxidant drugs [4-hydroxy alpha-phenyl-tert-butylnitrone (4-OHPBN) and N-acetyl-L-cysteine (NAC)] on acute noise-induced hearing loss because oral administration is the most commonly used method of drug administration due to its convenience, safety, and economical efficiency. METHODS: Thirty chinchilla were exposed to a 105 dB octave band noise centered at 4 kHz for 6 h and randomly assigned to a control group (saline only) and three experimental groups [4-OHPBN (10 mg/kg) plus NAC (20 mg/kg), 4-OHPBN (20 mg/kg) plus NAC (50 mg/kg), and 4-OHPBN (50 mg/kg) plus NAC (100 mg/kg)]. The drugs were orally administrated beginning 4 h after noise exposure and then administered twice daily for the next 2 days. Permanent auditory brainstem response threshold shifts, distortion product otoacoustic emission threshold shifts, and the percentage of missing outer hair cell were determined. RESULTS: The oral administration significantly reduced permanent hearing threshold shift, distortion product otoacoustic emission threshold shift, and the percentage of missing outer hair cell in a dose-dependent manner. DISCUSSION: This result demonstrates that orally administered drugs can treat acute noise-induced hearing loss in a dose-dependent manner. This suggests that oral administration was effective in treating acute noise-induced hearing loss as in intraperitoneal administration.
Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Perda Auditiva Provocada por Ruído/tratamento farmacológico , Iminas/farmacologia , Fenóis/farmacologia , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Chinchila , Relação Dose-Resposta a Droga , Feminino , Perda Auditiva Provocada por Ruído/etiologia , Perda Auditiva Provocada por Ruído/patologia , Distribuição AleatóriaRESUMO
Starvation elicits a complex adaptive response in an organism. No information on transcriptional regulation of metabolic adaptations is available. We, therefore, studied the gene expression profiles of brain, small intestine, kidney, liver, and skeletal muscle in mice that were subjected to 0-72 h of fasting. Functional-category enrichment, text mining, and network analyses were employed to scrutinize the overall adaptation, aiming to identify responsive pathways, processes, and networks, and their regulation. The observed transcriptomics response did not follow the accepted "carbohydrate-lipid-protein" succession of expenditure of energy substrates. Instead, these processes were activated simultaneously in different organs during the entire period. The most prominent changes occurred in lipid and steroid metabolism, especially in the liver and kidney. They were accompanied by suppression of the immune response and cell turnover, particularly in the small intestine, and by increased proteolysis in the muscle. The brain was extremely well protected from the sequels of starvation. 60% of the identified overconnected transcription factors were organ-specific, 6% were common for 4 organs, with nuclear receptors as protagonists, accounting for almost 40% of all transcriptional regulators during fasting. The common transcription factors were PPARα, HNF4α, GCRα, AR (androgen receptor), SREBP1 and -2, FOXOs, EGR1, c-JUN, c-MYC, SP1, YY1, and ETS1. Our data strongly suggest that the control of metabolism in four metabolically active organs is exerted by transcription factors that are activated by nutrient signals and serves, at least partly, to prevent irreversible brain damage.
Assuntos
Jejum/metabolismo , Regulação da Expressão Gênica , Metabolismo dos Lipídeos , Inanição/metabolismo , Esteroides/metabolismo , Transcrição Gênica , Animais , Perfilação da Expressão Gênica , Masculino , Camundongos , Especificidade de Órgãos , Fatores de Transcrição/biossínteseRESUMO
The chemical legislation of the EU, Registration, Evaluation, and Authorization of Chemicals (REACH), stipulates that about 30 000 chemical substances are to be assessed on their possible risks. Toxicological evaluation of these compounds will at least partly be based on animal testing. In particular, the assessment of reproductive toxicity is a very complicated, time-consuming and animal-demanding process. Introducing microarray-based technologies can potentially refine in vivo toxicity testing. If compounds of a distinct chemical class induce reproducible gene-expression responses with a recognizable overlap, these gene-expression signatures may indicate intrinsic features of certain compounds, including specific toxicity. In the present study, we have set out the first steps towards this approach for the reproductive toxicity of phthalates. Male rats were treated with a single dose of either reprotoxic or non-reprotoxic phthalates, and were analyzed 24 h afterwards. Subsequently, histopathological and gene-expression profiling analyses were performed. Despite ambiguous histopathological observations, we were able to identify genes with differential expression profiles between the reprotoxic phthalates and the non-reprotoxic counterparts. This shows that differences in gene-expression profiles, indicative of the type of exposure, may be detected earlier, or at lower doses, than classical pathological endpoints. These findings are promising for 'early warning' biomarker analyses and for using toxicogenomics in a category approach. Ultimately, this could lead to a more cost-effective approach for prioritizing the toxicity testing of large numbers of chemicals in a short period of time in hazard assessment of chemicals, which is one of the objectives of the REACH chemical legislation.
Assuntos
Antagonistas de Hormônios/toxicidade , Ácidos Ftálicos/toxicidade , Reprodução/efeitos dos fármacos , Testículo/efeitos dos fármacos , Toxicogenética/métodos , Transcriptoma/efeitos dos fármacos , Administração Oral , Alternativas aos Testes com Animais , Animais , Expressão Gênica , Perfilação da Expressão Gênica , Antagonistas de Hormônios/classificação , Masculino , Ácidos Ftálicos/classificação , Análise Serial de Proteínas , Ratos , Ratos Endogâmicos , Reprodução/genética , Transcriptoma/genéticaRESUMO
BACKGROUND: The Gram negative anaerobic bacterium Porphyromonas gingivalis has long been recognized as a causative agent of periodontitis. Periodontitis is a chronic infectious disease of the tooth supporting tissues eventually leading to tooth-loss. Capsular polysaccharide (CPS) of P. gingivalis has been shown to be an important virulence determinant. Seven capsular serotypes have been described. Here, we used micro-array based comparative genomic hybridization analysis (CGH) to analyze a representative of each of the capsular serotypes and a non-encapsulated strain against the highly virulent and sequenced W83 strain. We defined absent calls using Arabidopsis thaliana negative control probes, with the aim to distinguish between aberrations due to mutations and gene gain/loss. RESULTS: Our analyses allowed us to call aberrant genes, absent genes and divergent regions in each of the test strains. A conserved core P. gingivalis genome was described, which consists of 80% of the analyzed genes from the sequenced W83 strain. The percentage of aberrant genes between the test strains and control strain W83 was 8.2% to 13.7%. Among the aberrant genes many CPS biosynthesis genes were found. Most other virulence related genes could be found in the conserved core genome. Comparing highly virulent strains with less virulent strains indicates that hmuS, a putative CobN/Mg chelatase involved in heme uptake, may be a more relevant virulence determinant than previously expected. Furthermore, the description of the 39 W83-specific genes could give more insight in why this strain is more virulent than others. CONCLUSION: Analyses of the genetic content of the P. gingivalis capsular serotypes allowed the description of a P. gingivalis core genome. The high resolution data from three types of analysis of triplicate hybridization experiments may explain the higher divergence between P. gingivalis strains than previously recognized.
Assuntos
Genoma Bacteriano , Porphyromonas gingivalis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Hibridização Genômica Comparativa , DNA Bacteriano/genética , Genes Bacterianos , Anotação de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/patogenicidade , Virulência/genéticaRESUMO
Cortical tubers in patients with tuberous sclerosis complex are associated with disabling neurological manifestations, including intractable epilepsy. While these malformations are believed to result from the effects of TSC1 or TSC2 gene mutations, the molecular mechanisms leading to tuber formation, as well as the onset of seizures, remain largely unknown. We used the Affymetrix Gene Chip platform to provide the first genome-wide investigation of gene expression in surgically resected tubers, compared with histological normal perituberal tissue from the same patients or autopsy control tissue. We identified 2501 differentially expressed genes in cortical tubers compared with autopsy controls. Expression of genes associated with cell adhesion, for example, VCAM1, integrins and CD44, or with the inflammatory response, including complement factors, serpinA3, CCL2 and several cytokines, was increased in cortical tubers, whereas genes related to synaptic transmission, for example, the glial glutamate transporter GLT-1, and voltage-gated channel activity, exhibited lower expression. Gene expression in perituberal cortex was distinct from autopsy control cortex suggesting that even in the absence of tissue pathology the transcriptome is altered in TSC. Changes in gene expression yield insights into new candidate genes that may contribute to tuber formation or seizure onset, representing new targets for potential therapeutic development.
Assuntos
Adesão Celular/genética , Córtex Cerebral/metabolismo , Inflamação/genética , Esclerose Tuberosa/genética , Adolescente , Adulto , Idoso , Biomarcadores/metabolismo , Córtex Cerebral/patologia , Criança , Pré-Escolar , Feminino , Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Imuno-Histoquímica , Lactente , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologiaRESUMO
Current detection methods (computed tomography, ultrasound, and MRI) for hepatocarcinogenesis in humans rely on visual confirmation of neoplastic formations. A more effective early detection method is needed. Using in vivo magnetic resonance spectroscopy (MRS), we show that alterations in the integral ratios of the bis-allyl to vinyl hydrogen protons in unsaturated lipid fatty acyl groups correlate with the development of neoplastic formations in vivo in a TGFalpha/c-myc mouse hepatocellular carcinoma (HCC) model. HPLC analysis of the TGFalpha/c-myc mice liver tissue revealed a significant increase in the amount of oleic acid, along with alterations in linoleic and gamma-linolenic acids, as compared with control CD1 mice. Electrospray ionization tandem mass spectrometry analysis indicated a significant increase in the abundance of specific glycerol phosphatidylcholine (GPCho) lipids containing palmitic and oleic acids between control CD1 and TGFalpha/c-myc mice liver tissue extracts. Western blot analysis of the mice liver tissue indicates alterations in the desaturase enzyme stearoyl CoA desaturase (SCD)1, responsible for palmitic and oleic acid formation. Microarray analysis detected alterations in several genes involved with fatty acid metabolism, particularly SCD2, in transgenic mouse liver tissue. In correlation with the HPLC, mass spectrometry, Western blot, and microarray analyses, we are able to confirm the ability of in vivo MRS to detect precancerous lesions in the mouse liver before visual neoplastic formations were detectable by MRI.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Genes myc , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Fator de Crescimento Transformador alfa/genética , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Ácidos Graxos Insaturados/química , Neoplasias Hepáticas Experimentais/patologia , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.
Assuntos
Intoxicação por Tetracloreto de Carbono/metabolismo , Tetracloreto de Carbono/toxicidade , DNA/metabolismo , Desoxiguanosina/análogos & derivados , Metabolismo dos Lipídeos , Estresse Oxidativo , 8-Hidroxi-2'-Desoxiguanosina , Animais , Ensaio Cometa , Dano ao DNA , Desoxiguanosina/farmacologia , Radicais Livres , Cromatografia Gasosa-Espectrometria de Massas , Peróxido de Hidrogênio/metabolismo , Imunoensaio , Immunoblotting , Fígado/metabolismo , Masculino , Malondialdeído/farmacologia , Metionina/metabolismo , Oxigênio/metabolismo , Ratos , Ratos Endogâmicos F344 , Espectrofotometria , Substâncias Reativas com Ácido Tiobarbitúrico , Fatores de Tempo , Tirosina/química , Tirosina/metabolismoRESUMO
Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Primers do DNA/genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Interferência de RNA , RNA de Plantas/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Primers do DNA/química , DNA de Plantas/genética , Bases de Dados Genéticas , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/genéticaRESUMO
The transient system deficit hypothesis (TSDH) of specific reading disability [Percept. Psychophys. 40 (1986) 440] remains contentious. As part of a study examining multiple measures of transient and sustained system function, heterochromatic flicker matching (HFM) and brightness matching (HBM) were assessed in 30 poor readers (9.11+/-0.68 years) and 30 age, grade and sex matched controls (9.24+/-0.73 years). HBM and HFM are known to reflect the processing of brightness and luminance information and have been related to the function of magnocellular and parvocellular visual sub-systems. Flicker and brightness matches were determined for blue, green, yellow and red stimuli on Macintosh colour displays using 2AFC and double interleaved random staircases. A ratio of the luminances for brightness and flicker matches represented performance. A significant difference between controls and poor readers in performance for red and blue stimuli was found indicating different visual function in poor readers. While not providing direct support for the transient system deficit hypothesis, this effect implies a mismatch between those achromatic systems that subserve HFM and those more complex mechanisms involved in HBM. The most important aspect of this finding is that poor readers and normal controls could be differentiated on the basis of a paradigm known to be contingent upon magnocellular and parvocellular functioning.
Assuntos
Sensibilidades de Contraste/fisiologia , Dislexia/fisiopatologia , Percepção Visual/fisiologia , Análise de Variância , Criança , Percepção de Cores/fisiologia , Feminino , Fusão Flicker , Humanos , Masculino , Acuidade Visual/fisiologiaRESUMO
Following induction of acute neuroinflammation by intracisternal injection of endotoxin (lipopolysaccharide) in rats, quantitative autoradiography was used to assess the regional level of microglial activation and glutamate (NMDA) receptor binding. The possible protective action of the antioxidant phenyl-tert-butyl nitrone in this model was tested by administering the drug in the drinking water for 6 days starting 24 hafter endotoxin injection. Animals were killed 7 days post-injection and consecutive cryostat brain sections labeled with [3H]PK11195 as a marker of activated microglia and [125I]iodoMK801 as a marker of the open-channel, activated state of NMDA receptors. Lipopolysaccharide increased [3H]PK11195 binding in the brain, with the largest increases (two- to threefold) in temporal and entorhinal cortex, hippocampus, and substantia innominata. A significant (> 50%) decrease in [125I]iodoMK801 binding was found in the same brain regions. Phenyl-tert-butyl nitrone treatment resulted in a partial inhibition (approx. 25% decrease) of the lipopolysaccharide-induced increase in [3H]PK11195 binding but completely reversed the lipopolysaccharide-induced decrease in [125I]iodoMK80 binding in the entorhinal cortex, hippocampus, and substantia innominata. Loss of NMDA receptor function in cortical and hippocampal regions may contribute to the cognitive deficits observed in diseases with a neuroinflammatory component, such as meningitis or Alzheimer's disease.
Assuntos
Antioxidantes/uso terapêutico , Encéfalo/metabolismo , Encefalite/metabolismo , Óxidos de Nitrogênio/uso terapêutico , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Antioxidantes/administração & dosagem , Autorradiografia , Ligação Competitiva/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Óxidos N-Cíclicos , Modelos Animais de Doenças , Maleato de Dizocilpina/análogos & derivados , Maleato de Dizocilpina/farmacocinética , Vias de Administração de Medicamentos , Encefalite/induzido quimicamente , Encefalite/tratamento farmacológico , Encefalite/patologia , Antagonistas de Aminoácidos Excitatórios/farmacocinética , Isoquinolinas/farmacocinética , Lipopolissacarídeos , Masculino , Microglia/patologia , Óxidos de Nitrogênio/administração & dosagem , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Distribuição Tecidual/efeitos dos fármacosRESUMO
INTRODUCTION: Proinflammatory cytokines may play a pivotal role in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). In vitro, the formation of nitric oxide (NO) catalyzed by inducible NO synthase (iNOS) has been shown to be involved in the cytotoxic effects of cytokines on pancreatic beta cells. Cytokines have also been shown to cause the expression of inducible cyclooxygenase (COX-2) in isolated islets. AIMS: To describe a novel in vivo model that allows investigation of the effects of direct cytokine administration to the pancreas. METHODOLOGY AND RESULTS: By using this method, we demonstrate that administration of interleukin-1beta and interferon-gamma to rat pancreas results in the generation of NO in the treated pancreata as detected by NO trapping and electron paramagnetic resonance spectroscopy. Beta cells were identified as the source of the formed NO. Reverse transcription and polymerase chain reaction analyses showed that administration of cytokines to the pancreas leads to the expression of iNOS and COX-2 mRNA in the pancreas tissue as well as the islets isolated from such tissues. The compound phenyl N-tert-butylnitrone, which protects mice against streptozotocin-induced IDDM, inhibits NO formation and downregulates both iNOS and COX-2 mRNA levels.
Assuntos
Interferon gama/administração & dosagem , Interleucina-1/administração & dosagem , Isoenzimas/genética , Óxido Nítrico Sintase/genética , Óxido Nítrico/biossíntese , Pâncreas/metabolismo , Prostaglandina-Endoperóxido Sintases/genética , Aloxano/farmacologia , Animais , Óxidos N-Cíclicos , Ciclo-Oxigenase 2 , Expressão Gênica , Ilhotas Pancreáticas/metabolismo , Masculino , Óxido Nítrico Sintase Tipo II , Óxidos de Nitrogênio/farmacologia , RNA Mensageiro/análise , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult beta-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the beta-globin promoter at -153 to -148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress beta-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the beta-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant beta-globin promoter for the -150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the -150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human beta-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases.
Assuntos
Ferritinas/fisiologia , Globinas/genética , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Linhagem Celular , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ferritinas/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like , Proteínas Nucleares/metabolismo , Ligação Proteica , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Phosphorylation of the serine/threonine kinase Akt has previously been shown to be increased by treatment of cells with H2O2; the target of H2O2 has not been clearly identified. Here we show that treatment of rat primary astrocytes with H2O2 resulted in increased Akt phosphorylation that was blocked by wortmannin. The thiol-reducing agent N-acetylcysteine had only a slight inhibitory effect. Treatment with rotenone or antimycin A also resulted in increased wortmannin-sensitive Akt phosphorylation, probably by increasing intracellular H2O2 generation by blocking mitochondrial electron transport. Addition of phosphatidylinositol 3,4-bisphosphate to cells also resulted in an increase in Akt phosphorylation. This increase was additive to that induced by H2O2 and was also blocked by wortmannin. These results suggest that activation of Akt by H2O2 occurs upstream of phosphatidylinositol 3-kinase (PI 3-K) activity in astrocytes. The data indicate that major oxidative effects do not occur at the level of the PI 3-K-antagonizing phosphatase PTEN.
Assuntos
Astrócitos/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras de Tumor , Acetilcisteína/farmacologia , Androstadienos/farmacologia , Animais , Antimicina A/farmacologia , Astrócitos/citologia , Astrócitos/enzimologia , Astrócitos/metabolismo , Western Blotting , Células Cultivadas , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/metabolismo , PTEN Fosfo-Hidrolase , Fosfatidilinositol 4,5-Difosfato/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Rotenona/farmacologia , Desacopladores/farmacologia , WortmaninaRESUMO
Clues as to why long-lived species live so much longer than short-lived species may reside in the amount of reactive oxygen species (ROS) produced and their effect on damaging cell components (especially proteins) and alterations of crucial cellular processes. Rigorous evaluation of these concepts required critical comparisons of oxidative damage markers and/or parameters with assess difference in ROS flux and the critical age-modifying processes they influence. The limited experimental comparative results available implicate that ROS production per unit weight of total oxygen consumed is much less in the longer-lived species than in shorter-lived species. Mitochondria are the major site of ROS production. They are also the functional nexus for intracellular signaling thus modulating stress and growth factor mediated cellular survival, proliferation and apoptotic processes. Mitochondrial DNA mutations, perhaps caused by ROS, increase with age. Mutant mitochondria possess comparative replicative advantage, which leads to age-specific intracellular swarms. General inflammatory stress tends to increase with age. Disruption in coordinated cell-to-cell signaling triggered by alterations in intracellular signaling may be the basis of the age-related increases in tissue inflammation, which may explain some of the differences between long-lived species and short-lived species.
Assuntos
Envelhecimento/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Envelhecimento/fisiologia , Animais , Biomarcadores , Radicais Livres/metabolismo , Humanos , Longevidade , Mitocôndrias/metabolismo , Proteínas/metabolismo , PesquisaRESUMO
Caloric restriction (CR) retards diseases and aging in laboratory rodents and is now being tested in nonhuman primates. One way to apply these findings to human health is to identify and test agents that may mimic critical actions of CR. Panel 2 focused on two outcomes of CR, reduction of oxidative stress and improved glucoregulation, for which candidate metabolic mimics exist. It was recommended that studies on oxidative stress should emphasize mitochondrial function and to test the efficacy of nitrone and other antioxidants in mimicking CR's effects. Studies should also focus on the long-term effects of compounds known to lower circulating glucose and insulin concentrations or to increase insulin sensitivity. Also, four other developing areas were identified: intermediary metabolism, response to infection, stress responses, and source of dietary fat. These areas are important because either they hold promise for the discovery of new mimetics or they need to be explored prior to initiation of CR trials in humans. Other recommendations were that transgenic approaches and adult-onset CR should be emphasized in future studies.
Assuntos
Glicemia/metabolismo , Ingestão de Energia , Estresse Oxidativo/fisiologia , Animais , Animais Geneticamente Modificados , Humanos , Insulina/fisiologia , Mitocôndrias/fisiologiaRESUMO
Sleep deprivation is reported to have both beneficial and harmful effects upon host defenses. In the work reported herein, we address the effects of sleep deprivation on the mucosal anti-influenza defenses of both immune and nonimmune BALB/c mice. Sleep deprivation does not depress existing mucosal antiviral defenses in the respiratory tracts of BALB/c mice; in fact, it may actually be beneficial. Nasal mucosal immunity is not adversely affected in immune mice by sleep deprivation. In nonimmune mice, sleep deprivation slows or prevents the progress of nasal influenza viral infection down the trachea into the lungs. By 72 hours post-infection, 12 of 12 control mice shed virus into bronchioalveolar lavages (BAL) while only 2 of 12 sleep deprived mice shed virus (p<0.001). BAL levels of IL-1beta and interferon alpha were increased in sleep deprived animals, suggesting that sleep deprivation may exert its beneficial effects on the respiratory tract by upregulating the production of antiviral cytokines.
Assuntos
Orthomyxoviridae/imunologia , Infecções Respiratórias , Privação do Sono/etiologia , Animais , Anticorpos Antivirais/imunologia , Lavagem Broncoalveolar , Feminino , Interferons/análise , Interferons/imunologia , Interferons/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Nasal/química , Mucosa Nasal/imunologia , Infecções Respiratórias/complicações , Infecções Respiratórias/imunologia , Infecções Respiratórias/virologia , Regulação para Cima/fisiologiaRESUMO
Oxidative stress has traditionally been viewed as a stochastic process of cell damage resulting from aerobic metabolism, and antioxidants have been viewed simply as free radical scavengers. Only recently has it been recognized that reactive oxygen species (ROS) are widely used as second messengers to propagate proinflammatory or growth-stimulatory signals. With this knowledge has come the corollary realization that oxidative stress and chronic inflammation are related, perhaps inseparable phenomena. New pharmacological strategies aimed at supplementing antioxidant defense systems while antagonizing redox-sensitive signal transduction may allow improved clinical management of chronic inflammatory or degenerative conditions, including Alzheimer's disease. Introduction of antioxidant therapies into mainstream medicine is possible and promising, but will require significant advances in basic cell biology, pharmacology, and clinical bioanalysis.
Assuntos
Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Antioxidantes/farmacologia , Sequestradores de Radicais Livres/farmacologia , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/terapia , Oxirredução , Estresse Oxidativo , Sistemas do Segundo MensageiroRESUMO
Cytokines and nitric oxide (NO) have been implicated in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). We have shown that the spin-trapping agent phenyl N-tert-butylnitrone (PBN) protects against streptozotocin (STZ)-induced IDDM in mice. In order to gain more insights into the mechanism(s) of the protective action of PBN against IDDM, we have investigated the effect of this compound on the cytokine-induced NO generation (measured as nitrite) in rat insulinoma RIN-5F cells. Our results demonstrate that PBN cotreatment prevents the generation of nitrite by RIN-5F cells induced by treatment with tumor necrosis factor-alpha, interleukin 1beta, and interferon-gamma in a dose-dependent fashion. The generation of NO as a result of cytokine treatment and the inhibitory effect of PBN were further confirmed by electron paramagnetic resonance spectroscopy. Aminoguanidine, a selective inhibitor of inducible nitric oxide synthase (iNOS), abolished the cytokine-induced nitrite generation whereas N-nitro-l-arginine, an inhibitor more selective for other NOS isoforms, was significantly less effective. Western and Northern analyses demonstrated that PBN inhibits the cytokine-mediated expression of iNOS at the transcriptional level. Cytokine-induced nitrite formation was also inhibited by the two antioxidant agents alpha-lipoic acid and N-acetylcysteine. These results indicate that PBN protects against IDDM at least in part by prevention of cytokine-induced NO generation by pancreatic beta-cells.
Assuntos
Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/biossíntese , Óxidos de Nitrogênio/farmacologia , Acetilcisteína/farmacologia , Animais , Northern Blotting , Sobrevivência Celular/efeitos dos fármacos , Óxidos N-Cíclicos , Diabetes Mellitus Tipo 1/prevenção & controle , Indução Enzimática , Sequestradores de Radicais Livres/farmacologia , Immunoblotting , Insulinoma , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Nitritos/metabolismo , Óxidos de Nitrogênio/uso terapêutico , Substâncias Protetoras/farmacologia , Substâncias Protetoras/uso terapêutico , RNA Mensageiro/biossíntese , Ratos , Ácido Tióctico/farmacologia , Células Tumorais CultivadasRESUMO
The mechanistic basis of the neuroprotective activity of the nitrone-based free radical trap PBN (alpha-phenyl-N-tert-butyl nitrone) has been investigated extensively. Key observations exclude its simple mass action spin trapping of free radicals activity as the key mechanism of action. These include: A) the fact that it protects in experimental stroke even if administered several hours after the event and B) the fact that its chronic low-level administration to old experimental animals reverses their age-enhanced susceptibility to stroke even several days after the last dosage. PBN was found to inhibit gene induction in several models including stroke and an LPS-mediated septic shock model. Stoke causes inducible nitric oxide synthase (iNOS) to be expressed. High levels of nitric oxide and peroxynitrite (formed from nitric oxide), produced by iNOS, is particularly neurotoxic. PBN inhibits iNOS induction. Therefore, it seems that prevention of the formation of neurotoxic products is a rational mechanism of action of PBN in the stroke model. There is strong rationale to consider that there is an enhanced propensity for a "smoldering" neuro-inflammatory state in the old brain. Reversal of this state by PBN may explain its action in preventing age-enhanced stroke susceptibility in old experimental animals. Significant new findings underscore the importance of neuro-inflammatory processes in neuronal death or dysfunction in Alzheimer's disease. Neuro-inflammatory processes implicate enhanced signal transduction processes. Strong evidence for this is the enhanced p38 kinase activation in neurons near plaques and tangles of the Alzheimer's brain in contrast to normal aged-matched control brain which did not show p38 activation. In rat primary astrocytes p38 activation by the pro-inflammatory cytokine IL-1 beta, as well as by H2O2, was significantly suppressed by PBN. Mechanistically it was shown that PBN suppresses the amount of reactive oxygen species (ROS) produced in mitochondrial respiration. Much evidence indicates that ROS are signaling molecules and that they also are involved to maintaining brain phosphatases in an inactive state. We argue that finding a specific high affinity site mechanism for the neuroprotective action of PBN is unlikely based on the complexity of the system reflecting ROS generation and signal transduction processes that have apparently evolved to maintain adaptive responses. The promising pharmacological activity of molecules like PBN is not diminished by this however, for only excessive amounts of ROS is considered detrimental. The action of PBN in suppressing signal transduction processes, most likely by suppressing ROS production in mitochondrial respiration, effectively controls excessive oxidative damage and prevents induction of genes that form neurotoxic products.