RESUMO
BACKGROUND: Infection-induced preterm birth is a major cause of neonatal mortality and morbidity and leads to preterm premature rupture of placental chorioamniotic membranes. The loss of amniotic epithelial cells and tensile strength preceding membrane rupture is poorly understood. We hypothesized that intrauterine bacterial infection induces changes in microRNA (miRNA) expression, leading to amniotic epithelial cell loss and membrane weakening. METHODS: Ten pregnant pigtail macaques received choriodecidual inoculation of either group B Streptococcus (GBS) or saline (nâ =â 5/group). Placental chorioamniotic membranes were studied using RNA microarray and immunohistochemistry. Chorioamniotic membranes from women with preterm premature rupture of membranes (pPROM) and normal term pregnancies were studied using transmission electron microscopy. RESULTS: In our model, an experimental GBS infection was associated with changes in the miRNA profile in the chorioamniotic membranes consistent with epithelial to mesenchymal transition (EMT) with loss of epithelial (E-cadherin) and gain of mesenchymal (vimentin) markers. Similarly, loss of desmosomes (intercellular junctions) was seen in placental tissues from women with pPROM. CONCLUSIONS: We describe EMT as a novel mechanism for infection-associated chorioamniotic membrane weakening, which may be a common pathway for many etiologies of pPROM. Therapy based on anti-miRNA targeting of EMT may prevent pPROM due to perinatal infection.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Ruptura Prematura de Membranas Fetais/metabolismo , MicroRNAs/metabolismo , Infecções Estreptocócicas/metabolismo , Âmnio/patologia , Animais , Corioamnionite/microbiologia , Modelos Animais de Doenças , Feminino , Ruptura Prematura de Membranas Fetais/etiologia , Ruptura Prematura de Membranas Fetais/microbiologia , Ruptura Prematura de Membranas Fetais/patologia , Humanos , Imuno-Histoquímica , Macaca nemestrina , MicroRNAs/genética , Gravidez , Nascimento Prematuro , Infecções Estreptocócicas/complicações , Streptococcus agalactiaeRESUMO
Inhibition of mammalian target of rapamycin complex I (mTORC1) by rapamycin improves cardiac function in both aging and heart failure. While the protective mechanisms are not fully understood in mammals, they are presumably mediated through metabolic regulation and suppression of protein translation by reduced phosphorylation of 4EBP1, a target of mTORC1. Using transverse aortic constriction (TAC) and Gαq overexpression-induced heart failure models, we examined the effect of cardiac-specific heterozygous deletion (het) of Raptor, a component of mTORC1, and cardiac-specific transgenic overexpression of wild type or phosphorylation site mutant 4EBP1. In wild-type mice with TAC-induced heart failure, quantitative shotgun proteomics revealed decreased abundance of proteins of mitochondrial metabolism and increased abundance of proteins in oxidative stress response, ubiquitin, and other pathways. The Raptor het ameliorated both TAC- and Gαq overexpression-induced heart failure and the associated proteomic remodeling, especially those pathways involved in mitochondrial function, citric acid cycle, and ubiquitination. In contrast, transgenic overexpression of either wild type or mutant 4EBP1 aggravated TAC and Gαq, consistent with reduced adaptive hypertrophy by suppression of protein translation, in parallel with adverse remodeling of left ventricular proteomes. Partial mTORC1 inhibition by Raptor heterozygous deletion ameliorates heart failure and is associated with better preservation of the mitochondrial proteome; however, this effect does not appear to be mediated through suppression of protein translation by increased 4EBP1. Increased activity of 4EBP1 reduced adaptive hypertrophy and aggravated heart failure, suggesting that protein translation is essential for adaptive hypertrophy in pressure overload.
Assuntos
Regulação da Expressão Gênica , Insuficiência Cardíaca/tratamento farmacológico , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Sirolimo/farmacologia , Animais , Western Blotting , DNA/genética , Modelos Animais de Doenças , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Imunossupressores/farmacologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Transgênicos , Proteoma , Transdução de SinaisRESUMO
Accumulation of protein aggregates with age was first described in aged human tissue over 150 years ago and has since been described in virtually every human tissue. Ubiquitin modifications are a canonical marker of insoluble protein aggregates; however, the composition of most age-related inclusions remains relatively unknown. To examine the landscape of age-related protein aggregation in vivo, we performed an antibody-based pulldown of ubiquitinated proteins coupled with metabolic labeling and mass spectrometry on young and old mice on calorie restriction (CR), rapamycin (RP)-supplemented, and control diets. We show increased abundance of many ubiquitinated proteins in old mice and greater retention of preexisting (unlabeled) ubiquitinated proteins relative to their unmodified counterparts-fitting the expected profile of age-increased accumulation of long-lived aggregating proteins. Both CR and RP profoundly affected ubiquitinome composition, half-live, and the insolubility of proteins, consistent with their ability to mobilize these age-associated accumulations. Finally, confocal microscopy confirmed the aggregation of two of the top predicted aggregating proteins, keratins 8/18 and catalase, as well as their attenuation by CR and RP. Stable-isotope labeling is a powerful tool to gain novel insights into proteostasis mechanisms, including protein aggregation, and could be used to identify novel therapeutic targets in aging and protein aggregation diseases.
Assuntos
Envelhecimento/metabolismo , Restrição Calórica , Marcação por Isótopo , Agregados Proteicos/efeitos dos fármacos , Sirolimo/farmacologia , Ubiquitina/metabolismo , Animais , Feminino , Meia-Vida , Leucina/farmacologia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Biossíntese de Proteínas/efeitos dos fármacos , Proteoma/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
The FDA approved drug rapamycin increases lifespan in rodents and delays age-related dysfunction in rodents and humans. Nevertheless, important questions remain regarding the optimal dose, duration, and mechanisms of action in the context of healthy aging. Here we show that 3 months of rapamycin treatment is sufficient to increase life expectancy by up to 60% and improve measures of healthspan in middle-aged mice. This transient treatment is also associated with a remodeling of the microbiome, including dramatically increased prevalence of segmented filamentous bacteria in the small intestine. We also define a dose in female mice that does not extend lifespan, but is associated with a striking shift in cancer prevalence toward aggressive hematopoietic cancers and away from non-hematopoietic malignancies. These data suggest that a short-term rapamycin treatment late in life has persistent effects that can robustly delay aging, influence cancer prevalence, and modulate the microbiome.
Assuntos
Antibacterianos/administração & dosagem , Antibióticos Antineoplásicos/administração & dosagem , Microbioma Gastrointestinal/efeitos dos fármacos , Longevidade/efeitos dos fármacos , Neoplasias/prevenção & controle , Sirolimo/administração & dosagem , Animais , CamundongosRESUMO
Reactive oxygen species (ROS) are highly reactive oxygen-containing molecules associated with aging and a broad spectrum of pathologies. We have previously shown that transgenic expression of the antioxidant enzyme catalase targeted to the mitochondria (mCAT) in mice reduces ROS, attenuates age-related disease, and increases lifespan. However, it has been increasingly recognized that ROS also has beneficial roles in signaling, hormesis, stress response, and immunity. We therefore hypothesized that mCAT might be beneficial only when ROS approaches pathological levels in older age and might not be advantageous at a younger age when basal ROS is low. We analyzed abundance and turnover of the global proteome in hearts and livers of young (4 month) and old (20 month) mCAT and wild-type (WT) mice. In old hearts and livers of WT mice, protein half-lives were reduced compared to young, while in mCAT mice the reverse was observed; the longest half-lives were seen in old mCAT mice and the shortest in young mCAT. Protein abundance of old mCAT hearts recapitulated a more youthful proteomic expression profile (P-value < 0.01). However, young mCAT mice partially phenocopied the older wild-type proteome (P-value < 0.01). Age strongly interacts with mCAT, consistent with antagonistic pleiotropy in the reverse of the typical direction. These findings underscore the contrasting roles of ROS in young vs. old mice and indicate the need for better understanding of the interaction between dose and age in assessing the efficacy of therapeutic interventions in aging, including mitochondrial antioxidants.
Assuntos
Envelhecimento/metabolismo , Catalase/metabolismo , Pleiotropia Genética , Mitocôndrias/metabolismo , Proteoma/metabolismo , Animais , Biomarcadores/metabolismo , Meia-Vida , Fígado/metabolismo , Redes e Vias Metabólicas , Camundongos Endogâmicos C57BL , Miocárdio/metabolismoRESUMO
The Werner syndrome (WS) is a prototypic adult Mendelian progeroid syndrome in which signs of premature aging are associated with genomic instability and an elevated risk of cancer. The WRN RECQ helicase protein binds and unwinds G-quadruplex (G4) DNA substrates in vitro, and we identified significant enrichment in G4 sequence motifs at the transcription start site and 5' ends of first introns (false discovery rate < 0.001) of genes down-regulated in WS patient fibroblasts. This finding provides strong evidence that WRN binds G4 DNA structures at many chromosomal sites to modulate gene expression. WRN appears to bind a distinct subpopulation of G4 motifs in human cells, when compared with the related Bloom syndrome RECQ helicase protein. Functional annotation of the genes and miRNAs altered in WS provided new insight into WS disease pathogenesis. WS patient fibroblasts displayed altered expression of multiple, mechanistically distinct, senescence-associated gene expression programs, with altered expression of disease-associated miRNAs, and dysregulation of canonical pathways that regulate cell signaling, genome stability and tumorigenesis. WS fibroblasts also displayed a highly statistically significant and distinct gene expression signature, with coordinate overexpression of nearly all of the cytoplasmic tRNA synthetases and associated ARS-interacting multifunctional protein genes. The 'non-canonical' functions of many of these upregulated tRNA charging proteins may together promote WS disease pathogenesis. Our results identify the human WRN RECQ protein as a G4 helicase that modulates gene expression in G4-dependent fashion at many chromosomal sites and provide several new and unexpected mechanistic insights into WS disease pathogenesis.
Assuntos
Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , Neoplasias/genética , RecQ Helicases/genética , Síndrome de Werner/genética , Carcinogênese/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos , Quadruplex G , Regulação da Expressão Gênica , Genoma Humano , Humanos , MicroRNAs , Neoplasias/patologia , Motivos de Nucleotídeos , RecQ Helicases/metabolismoRESUMO
Rapamycin, an inhibitor of mTOR signaling, has been shown to reverse diastolic dysfunction in old mice in 10 weeks, highlighting its therapeutic potential for a poorly treatable condition. However, the mechanisms and temporal regulation of its cardiac benefits remain unclear. We show that improved diastolic function in old mice begins at 2-4 weeks, progressing over the course of 10-week treatment. While TORC1-mediated S6 phosphorylation and TORC2 mediated AKT and PKCα phosphorylation are inhibited throughout the course of treatment, rapamycin inhibits ULK phosphorylation and induces autophagy during just the first week of treatment, returning to baseline at two weeks and after. Concordantly, markers of mitochondrial biogenesis increase over the first two weeks of treatment and return to control levels thereafter. This transient induction of autophagy and mitochondrial biogenesis suggests that damaged mitochondria are replaced by newly synthesized ones to rejuvenate mitochondrial homeostasis. This remodeling is shown to rapidly reverse the age-related reduction in fatty acid oxidation to restore a more youthful substrate utilization and energetic profile in old isolated perfused hearts, and modulates the myocardial metabolomein vivo. This study demonstrates the differential and dynamic mechanisms following rapamycin treatment and highlights the importance of understanding the temporal regulation of rapamycin effects.
Assuntos
Envelhecimento/fisiologia , Metabolismo Energético/efeitos dos fármacos , Coração/efeitos dos fármacos , Mitocôndrias/metabolismo , Sirolimo/farmacologia , Animais , Autofagia/efeitos dos fármacos , Feminino , Immunoblotting , Imunossupressores/farmacologia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Miocárdio/metabolismo , Ressonância Magnética Nuclear Biomolecular , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The use of sentinel species for population and ecosystem health assessments has been advocated as part of a One Health perspective. The Arctic is experiencing rapid change, including climate and environmental shifts, as well as increased resource development, which will alter exposure of biota to environmental agents of disease. Arctic canid species have wide geographic ranges and feeding ecologies and are often exposed to high concentrations of both terrestrial and marine-based contaminants. The domestic dog (Canis lupus familiaris) has been used in biomedical research for a number of years and has been advocated as a sentinel for human health due to its proximity to humans and, in some instances, similar diet. Exploiting the potential of molecular tools for describing the toxicogenomics of Arctic canids is critical for their development as biomedical models as well as environmental sentinels. Here, we present three approaches analyzing toxicogenomics of Arctic contaminants in both domestic and free-ranging canids (Arctic fox, Vulpes lagopus). We describe a number of confounding variables that must be addressed when conducting toxicogenomics studies in canid and other mammalian models. The ability for canids to act as models for Arctic molecular toxicology research is unique and significant for advancing our understanding and expanding the tool box for assessing the changing landscape of environmental agents of disease in the Arctic.
Assuntos
Ecotoxicologia/métodos , Exposição Ambiental/análise , Raposas , Perfilação da Expressão Gênica/métodos , Animais , Regiões Árticas , Cães/genética , Ecossistema , Monitoramento Ambiental/métodos , Poluentes Ambientais/toxicidade , Peixes , Raposas/genética , Mercúrio/toxicidade , Bifenilos Policlorados/toxicidadeRESUMO
Changes in mitochondrial function with age vary between different muscle types, and mechanisms underlying this variation remain poorly defined. We examined whether the rate of mitochondrial protein turnover contributes to this variation. Using heavy label proteomics, we measured mitochondrial protein turnover and abundance in slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) from young and aged mice. We found that mitochondrial proteins were longer lived in EDL than SOL at both ages. Proteomic analyses revealed that age-induced changes in protein abundance differed between EDL and SOL with the largest change being increased mitochondrial respiratory protein content in EDL. To determine how altered mitochondrial proteomics affect function, we measured respiratory capacity in permeabilized SOL and EDL. The increased mitochondrial protein content in aged EDL resulted in reduced complex I respiratory efficiency in addition to increased complex I-derived H2 O2 production. In contrast, SOL maintained mitochondrial quality, but demonstrated reduced respiratory capacity with age. Thus, the decline in mitochondrial quality with age in EDL was associated with slower protein turnover throughout life that may contribute to the greater decline in mitochondrial dysfunction in this muscle. Furthermore, mitochondrial-targeted catalase protected respiratory function with age suggesting a causal role of oxidative stress. Our data clearly indicate divergent effects of age between different skeletal muscles on mitochondrial protein homeostasis and function with the greatest differences related to complex I. These results show the importance of tissue-specific changes in the interaction between dysregulation of respiratory protein expression, oxidative stress, and mitochondrial function with age.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Homeostase/fisiologia , Mitocôndrias/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Envelhecimento , Animais , Feminino , Camundongos Endogâmicos C57BL , ProteômicaRESUMO
Quantum dots (QDs) are engineered semiconductor nanoparticles with unique physicochemical properties that make them potentially useful in clinical, research and industrial settings. However, a growing body of evidence indicates that like other engineered nanomaterials, QDs have the potential to be respiratory hazards, especially in the context of the manufacture of QDs and products containing them, as well as exposures to consumers using these products. The overall goal of this study was to investigate the role of mouse strain in determining susceptibility to QD-induced pulmonary inflammation and toxicity. Male mice from 8 genetically diverse inbred strains (the Collaborative Cross founder strains) were exposed to CdSe-ZnS core-shell QDs stabilized with an amphiphilic polymer. QD treatment resulted in significant increases in the percentage of neutrophils and levels of cytokines present in bronchoalveolar lavage fluid (BALF) obtained from NOD/ShiLtJ and NZO/HlLtJ mice relative to their saline (Sal) treated controls. Cadmium measurements in lung tissue indicated strain-dependent differences in disposition of QDs in the lung. Total glutathione levels in lung tissue were significantly correlated with percent neutrophils in BALF as well as with lung tissue Cd levels. Our findings indicate that QD-induced acute lung inflammation is mouse strain dependent, that it is heritable, and that the choice of mouse strain is an important consideration in planning QD toxicity studies. These data also suggest that formal genetic analyses using additional strains or recombinant inbred strains from these mice could be useful for discovering potential QD-induced inflammation susceptibility loci.
Assuntos
Compostos de Cádmio/toxicidade , Pulmão/efeitos dos fármacos , Pneumonia/induzido quimicamente , Pontos Quânticos/toxicidade , Compostos de Selênio/toxicidade , Sulfetos/toxicidade , Compostos de Zinco/toxicidade , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Análise por Conglomerados , Citocinas/metabolismo , Predisposição Genética para Doença , Glutationa/metabolismo , Hereditariedade , Pulmão/imunologia , Pulmão/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fenótipo , Pneumonia/genética , Pneumonia/imunologia , Pneumonia/metabolismo , Fatores de Risco , Especificidade da Espécie , Fatores de TempoRESUMO
Many genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C. elegans and M. musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C. elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is nonadditive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging.
Assuntos
Envelhecimento/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Longevidade/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Caenorhabditis elegans/genética , Restrição Calórica , Dano ao DNA/genética , Deleção de Genes , Regulação da Expressão Gênica/genética , Genoma , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genéticaRESUMO
Most Pacific salmonids undergo smoltification and transition from freshwater to saltwater, making various adjustments in metabolism, catabolism, osmotic, and ion regulation. The molecular mechanisms underlying this transition are largely unknown. In the present study, we acclimated coho salmon (Oncorhynchus kisutch) to four different salinities and assessed gene expression through microarray analysis of gills, liver, and olfactory rosettes. Gills are involved in osmotic regulation, liver plays a role in energetics, and olfactory rosettes are involved in behavior. Between all salinity treatments, liver had the highest number of differentially expressed genes at 1616, gills had 1074, and olfactory rosettes had 924, using a 1.5-fold cutoff and a false discovery rate of 0.5. Higher responsiveness of liver to metabolic changes after salinity acclimation to provide energy for other osmoregulatory tissues such as the gills may explain the differences in number of differentially expressed genes. Differentially expressed genes were tissue- and salinity-dependent. There were no known genes differentially expressed that were common to all salinity treatments and all tissues. Gene ontology term analysis revealed biological processes, molecular functions, and cellular components that were significantly affected by salinity, a majority of which were tissue-dependent. For liver, oxygen binding and transport terms were highlighted. For gills, muscle, and cytoskeleton-related terms predominated and for olfactory rosettes, immune response-related genes were accentuated. Interaction networks were examined in combination with GO terms and determined similarities between tissues for potential osmosensors, signal transduction cascades, and transcription factors.
Assuntos
Regulação da Expressão Gênica/fisiologia , Brânquias/metabolismo , Fígado/metabolismo , Oncorhynchus kisutch/fisiologia , Aclimatação/genética , Aclimatação/fisiologia , Animais , Regulação da Expressão Gênica/genética , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Oncorhynchus kisutch/genética , Oncorhynchus kisutch/metabolismo , Osmorregulação/genética , Osmorregulação/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Salinidade , Olfato/genética , Olfato/fisiologiaRESUMO
The mechanisms underlying fetal lung injury remain poorly defined. MicroRNAs (miRNAs) are small noncoding, endogenous RNAs that regulate gene expression and have been implicated in the pathogenesis of lung disease. Using a nonhuman primate model of choriodecidual infection, we sought to determine if differentially expressed miRNAs were associated with acute fetal lung injury. After inoculating 10 chronically catheterized pregnant monkeys (Macaca nemestrina) with either group B streptococcus (GBS) at 1 × 10(6) CFU (n = 5) or saline (n = 5) in the choriodecidual space, we extracted fetal lung mRNA and miRNA and profiled the changes in expression by microarray analysis. We identified 9 differentially expressed miRNAs in GBS-exposed fetal lungs, but of these, only miR-155-5p was validated by quantitative reverse transcription-PCR (P = 0.02). Significantly elevated miR-155-5p expression was also observed when immortalized human fetal airway epithelial (FeAE) cells were exposed to proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]). Overexpression of miR-155-5p in FeAE cells in turn increased the production of IL-6 and CXCL10/gamma interferon-induced protein 10, which are implicated in leukocyte recruitment but also in protection from lung injury. Interestingly, while miR-155-5p decreased fibroblast growth factor 9 (FGF9) expression in a luciferase reporter assay, FGF9 levels were actually increased in GBS-exposed fetal lungs in vivo. FGF9 overexpression is associated with abnormal lung development. Thus, upregulation of miR-155-5p may serve as a compensatory mechanism to lessen the increase in FGF9 and prevent aberrant lung development. Understanding the complicated networks regulating lung development in the setting of infection is a key step in identifying how to prevent fetal lung injury leading to bronchopulmonary dysplasia.
Assuntos
Doenças Fetais/genética , Doenças Fetais/microbiologia , Pulmão/metabolismo , Infecções Estreptocócicas/embriologia , Infecções Estreptocócicas/genética , Streptococcus/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Doenças Fetais/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Pulmão/crescimento & desenvolvimento , Pulmão/microbiologia , Macaca nemestrina , Masculino , Gravidez , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Silymarin, a characterized extract of the seeds of milk thistle (Silybum marianum), suppresses cellular inflammation. To define how this occurs, transcriptional profiling, metabolomics, and signaling studies were performed in human liver and T cell lines. Cellular stress and metabolic pathways were modulated within 4 h of silymarin treatment: activation of Activating Transcription Factor 4 (ATF-4) and adenosine monophosphate protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR) signaling, the latter being associated with induction of DNA-damage-inducible transcript 4 (DDIT4). Metabolomics analyses revealed silymarin suppression of glycolytic, tricarboxylic acid (TCA) cycle, and amino acid metabolism. Anti-inflammatory effects arose with prolonged (i.e., 24 h) silymarin exposure, with suppression of multiple pro-inflammatory mRNAs and signaling pathways including nuclear factor kappa B (NF-κB) and forkhead box O (FOXO). Studies with murine knock out cells revealed that silymarin inhibition of both mTOR and NF-κB was partially AMPK dependent, whereas silymarin inhibition of mTOR required DDIT4. Other natural products induced similar stress responses, which correlated with their ability to suppress inflammation. Thus, natural products activate stress and repair responses that culminate in an anti-inflammatory cellular phenotype. Natural products like silymarin may be useful as tools to define how metabolic, stress, and repair pathways regulate cellular inflammation.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Silybum marianum/química , Silimarina/farmacologia , Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Antioxidantes/farmacologia , Ciclo do Ácido Cítrico/efeitos dos fármacos , Fatores de Transcrição Forkhead/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Células Jurkat , Fígado/metabolismo , Camundongos , Estrutura Molecular , NF-kappa B/antagonistas & inibidores , NF-kappa B/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II , Transdução de Sinais/efeitos dos fármacos , Silimarina/química , Linfócitos T/metabolismoRESUMO
The goal of the present study was to compare hepatic toxicogenomic signatures across in vitro and in vivo mouse models following exposure to acetaminophen (APAP) or its relatively nontoxic regioisomer 3'-hydroxyacetanilide (AMAP). Two different Affymetrix microarray platforms and one Agilent Oligonucleotide microarray were utilized. APAP and AMAP treatments resulted in significant and large changes in gene expression that were quite disparate, and likely related to their different toxicologic profiles. Ten transcripts, all of which have been implicated in p53 signaling, were identified as differentially regulated at all time-points following APAP and AMAP treatments across multiple microarray platforms. Protein-level quantification of p53 activity aligned with results from the transcriptomic analysis, thus supporting the implicated mechanism of APAP-induced toxicity. Therefore, the results of this study provide good evidence that APAP-induced p53 phosphorylation and an altered p53-driven transcriptional response are fundamental steps in APAP-induced toxicity.
RESUMO
Morphine is used to sedate critically ill infants to treat painful or stressful conditions associated with intensive care. Whether neonatal morphine exposure affects microRNA (miR) expression and thereby alters mRNA regulation is unknown. We tested the hypothesis that repeated morphine treatment in stress-exposed neonatal mice alters hippocampal mRNA and miR expression. C57BL/6 male mice were treated from postnatal day (P) 5 to P9 with morphine sulfate at 2 or 5 mg/kg ip twice daily and then exposed to stress consisting of hypoxia (100% N2 1 min and 100% O2 5 min) followed by 2h maternal separation. Control mice were untreated and dam-reared. mRNA and miR expression profiling was performed on hippocampal tissues at P9. Overall, 2 and 5 mg/kg morphine treatment altered expression of a total of 150 transcripts (>1.5 fold change, P<0.05) from which 100 unique mRNAs were recognized (21 genes were up- and 79 genes were down-regulated), and 5 mg/kg morphine affected 63 mRNAs exclusively. The most upregulated mRNAs were fidgetin, arginine vasopressin, and resistin-like alpha, and the most down-regulated were defensin beta 11, aquaporin 1, calmodulin-like 4, chloride intracellular channel 6, and claudin 2. Gene Set Enrichment Analysis revealed that morphine treatment affected pathways related to cell cycle, membrane function, signaling, metabolism, cell death, transcriptional regulation, and immune response. Morphine decreased expression of miR-204-5p, miR-455-3p, miR-448-5p, and miR-574-3p. Nine morphine-responsive mRNAs that are involved in neurodevelopment, neurotransmission, and inflammation are predicted targets of the aforementioned differentially expressed miRs. These data establish that morphine produces dose-dependent changes in both hippocampal mRNA and miR expression in stressed neonatal mice. If permanent, morphine-mediated neuroepigenetic effects may affect long-term hippocampal function, and this provides a mechanism for the neonatal morphine-related impairment of adult learning.
Assuntos
Analgésicos Opioides/administração & dosagem , Hipocampo/efeitos dos fármacos , MicroRNAs/genética , Morfina/administração & dosagem , RNA Mensageiro/genética , Analgésicos Opioides/farmacologia , Animais , Animais Recém-Nascidos , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/metabolismo , Camundongos , Morfina/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Psicológico/genética , Estresse Psicológico/patologiaRESUMO
Single suture craniosynostosis (SSC) is the premature fusion of one calvarial suture and occurs in 1-1700-2500 live births. Congenital fusion of either the sagittal, metopic, or coronal sutures represents 95% of all cases of SSC. Sagittal and metopic synostosis have a male preponderance (3:1) while premature fusion of the coronal suture has a female preponderance (2:1). Although environmental and genetic factors contribute to SSC, the etiology of the majority of SSC cases remains unclear. In this study, 227 primary calvarial osteoblast cell lines from patients with coronal, metopic, or sagittal synostosis and unaffected controls were established and assayed for ALP activity and BrdU incorporation (n = 226) as respective measures of early stage osteoblast differentiation and proliferation. Primary osteoblast cell lines from individuals with sagittal synostosis demonstrated higher levels of ALP activity and reduced proliferation when compared to control lines. In order to address the sex differences in SSC types, the data was further stratified by sex. Osteoblasts from males and females with sagittal synostosis as well as males with metopic synostosis demonstrated higher levels of ALP activity when compared to sex matched controls, and males with sagittal or metopic synostosis demonstrated reduced levels of proliferation. In order to elucidate genes and pathways involved in these observed phenotypes, correlation analyses comparing ALP activity and proliferation to global gene expression was performed. Transcripts related to osteoblast differentiation were identified both differentially up and downregulated, correlated with ALP activity when compared to controls, and demonstrated a striking sex specific gene expression pattern. These data support that the dysregulation of osteoblast differentiation plays a role in the development of SSC and that genetic factors contribute to the observed sex related differences.
Assuntos
Diferenciação Celular , Craniossinostoses/genética , Perfilação da Expressão Gênica , Osteoblastos/patologia , Fatores Sexuais , Proliferação de Células , Células Cultivadas , Craniossinostoses/patologia , Feminino , Humanos , Masculino , Osteoblastos/metabolismoRESUMO
Calorie restriction (CR) and rapamycin (RP) extend lifespan and improve health across model organisms. Both treatments inhibit mammalian target of rapamycin (mTOR) signaling, a conserved longevity pathway and a key regulator of protein homeostasis, yet their effects on proteome homeostasis are relatively unknown. To comprehensively study the effects of aging, CR, and RP on protein homeostasis, we performed the first simultaneous measurement of mRNA translation, protein turnover, and abundance in livers of young (3 month) and old (25 month) mice subjected to 10-week RP or 40% CR. Protein abundance and turnover were measured in vivo using (2) H3 -leucine heavy isotope labeling followed by LC-MS/MS, and translation was assessed by polysome profiling. We observed 35-60% increased protein half-lives after CR and 15% increased half-lives after RP compared to age-matched controls. Surprisingly, the effects of RP and CR on protein turnover and abundance differed greatly between canonical pathways, with opposite effects in mitochondrial (mt) dysfunction and eIF2 signaling pathways. CR most closely recapitulated the young phenotype in the top pathways. Polysome profiles indicated that CR reduced polysome loading while RP increased polysome loading in young and old mice, suggesting distinct mechanisms of reduced protein synthesis. CR and RP both attenuated protein oxidative damage. Our findings collectively suggest that CR and RP extend lifespan in part through the reduction of protein synthetic burden and damage and a concomitant increase in protein quality. However, these results challenge the notion that RP is a faithful CR mimetic and highlight mechanistic differences between the two interventions.
Assuntos
Envelhecimento/genética , Restrição Calórica , Fígado/efeitos dos fármacos , Proteoma/genética , Sirolimo/farmacologia , Envelhecimento/metabolismo , Animais , Deutério , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Regulação da Expressão Gênica , Meia-Vida , Homeostase , Marcação por Isótopo , Leucina/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Estabilidade Proteica , Proteólise , Proteoma/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Espectrometria de Massas em TandemRESUMO
AIM: We sought to discover endogenous urinary biomarkers of human CYP2D6 activity. PATIENTS & METHODS: Healthy pediatric subjects (n = 189) were phenotyped using dextromethorphan and randomized for candidate biomarker selection and validation. Global urinary metabolomics was performed using liquid chromatography quadrupole time-of-flight mass spectrometry. Candidate biomarkers were tested in adults receiving fluoxetine, a CYP2D6 inhibitor. RESULTS: A biomarker, M1 (m/z 444.3102) was correlated with CYP2D6 activity in both the pediatric training and validation sets. Poor metabolizers had undetectable levels of M1, whereas it was present in subjects with other phenotypes. In adult subjects, a 9.56-fold decrease in M1 abundance was observed during CYP2D6 inhibition. CONCLUSION: Identification and validation of M1 may provide a noninvasive means of CYP2D6 phenotyping.
Assuntos
Biomarcadores/urina , Citocromo P-450 CYP2D6/genética , Fluoxetina/administração & dosagem , Metabolômica , Adolescente , Adulto , Criança , Inibidores do Citocromo P-450 CYP2D6/administração & dosagem , Dextrometorfano/urina , Dextrorfano/urina , Feminino , Voluntários Saudáveis , Humanos , MasculinoRESUMO
Domoic acid is an algal-derived seafood toxin that functions as a glutamate agonist and exerts excitotoxicity via overstimulation of glutamate receptors (AMPA, NMDA) in the central nervous system (CNS). At high (symptomatic) doses, domoic acid is well-known to cause seizures, brain lesions and memory loss; however, a significant knowledge gap exists regarding the health impacts of repeated low-level (asymptomatic) exposure. Here, we investigated the impacts of low-level repetitive domoic acid exposure on gene transcription and mitochondrial function in the vertebrate CNS using a zebrafish model in order to: (1) identify transcriptional biomarkers of exposure; and (2) examine potential pathophysiology that may occur in the absence of overt excitotoxic symptoms. We found that transcription of genes related to neurological function and development were significantly altered, and that asymptomatic exposure impaired mitochondrial function. Interestingly, the transcriptome response was highly variable across the exposure duration (36 weeks), with little to no overlap of specific genes across the six exposure time points (2, 6, 12, 18, 24, and 36 weeks). Moreover, there were no apparent similarities at any time point with the gene transcriptome profile exhibited by the glud1 mouse model of chronic moderate excess glutamate release. These results suggest that although the fundamental mechanisms of toxicity may be similar, gene transcriptome responses to domoic acid exposure do not extrapolate well between different exposure durations. However, the observed impairment of mitochondrial function based on respiration rates and mitochondrial protein content suggests that repetitive low-level exposure does have fundamental cellular level impacts that could contribute to chronic health consequences.