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Shigella is a Gram-negative bacterium that causes bacillary dysentery worldwide. It invades the intestinal epithelium to elicit intense inflammation and tissue damage, yet the underlying mechanisms of its host selectivity and low infectious inoculum remain perplexing. Here, we report that Shigella co-opts human α-defensin 5 (HD5), a host defense peptide important for intestinal homeostasis and innate immunity, to enhance its adhesion to and invasion of mucosal tissues. HD5 promoted Shigella infection in vitro in a structure-dependent manner. Shigella, commonly devoid of an effective host-adhesion apparatus, preferentially targeted HD5 to augment its ability to colonize the intestinal epithelium through interactions with multiple bacterial membrane proteins. HD5 exacerbated infectivity and Shigella-induced pathology in a culture of human colorectal tissues and three animal models. Our findings illuminate how Shigella exploits innate immunity by turning HD5 into a virulence factor for infection, unveiling a mechanism of action for this highly proficient human pathogen.
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Aderência Bacteriana/fisiologia , Disenteria Bacilar/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Shigella/patogenicidade , alfa-Defensinas , Animais , HumanosRESUMO
Exocytosis and endocytosis are tightly coupled. In addition to initiating exocytosis, Ca2+ plays critical roles in exocytosisendocytosis coupling in neurons and nonneuronal cells. Both positive and negative roles of Ca2+ in endocytosis have been reported; however, Ca2+ inhibition in endocytosis remains debatable with unknown mechanisms. Here, we show that synaptotagmin-1 (Syt1), the primary Ca2+ sensor initiating exocytosis, plays bidirectional and opposite roles in exocytosisendocytosis coupling by promoting slow, small-sized clathrin-mediated endocytosis but inhibiting fast, large-sized bulk endocytosis. Ca2+-binding ability is required for Syt1 to regulate both types of endocytic pathways, the disruption of which leads to inefficient vesicle recycling under mild stimulation and excessive membrane retrieval following intense stimulation. Ca2+-dependent membrane tubulation may explain the opposite endocytic roles of Syt1 and provides a general membrane-remodeling working model for endocytosis determination. Thus, Syt1 is a primary bidirectional Ca2+ sensor facilitating clathrin-mediated endocytosis but clamping bulk endocytosis, probably by manipulating membrane curvature to ensure both efficient and precise coupling of endocytosis to exocytosis.
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Endocitose , Transmissão Sináptica , Sinaptotagmina I , Cálcio/metabolismo , Endocitose/fisiologia , Exocitose/fisiologia , Neurônios/metabolismo , Sinaptotagmina I/metabolismoRESUMO
Mitochondrial Membrane Chromatography (MMC) is a bioaffinity chromatography technique developed to study the interaction between target proteins embedded in the mitochondrial membrane and their ligand compounds. However, the MMC stationary phases (MMSP) prepared by chemical immobilization are prone to nonspecific binding in candidate agent screening inevitably. To address these challenges, Twin Strep-Tag/Strep Tactin was employed to establish a specific affinity system in the present study. We prepared a carnitine palmitoyltransferase 1A (CPT1A) MMSP by specifically linking Strep-tactin-modified silica gel with the Twin Strep-Tag on the CPT1A-oriented mitochondrial membrane. This Twin Strep-Tag/Strep Tactin modified CPT1A/MMC method exhibited remarkably better retention behavior, longer stationary phase lifespan, and higher screening specificity compared with previous MMC systems with glutaraldehyde immobilization. We adopted the CPT1A-specific MMC system in screening CPT1A ligands from traditional Chinese medicines, and successfully identified novel candidate ligands: ononin, isoliquiritigenin, and aloe-emodin, from Glycyrrhiza uralensis Fisch and Senna tora (L.) Roxb extracts. Biological assessments illustrated that the compounds screened promote CPT1A enzyme activity without affecting CPT1A protein expression, as well as effectively reduce the lipid droplets and triglyceride levels in the high fat induction HepG2 cells. The results suggest that we have developed an MMC system, which is promising for studying the bioaffinity of mitochondrial membrane proteins to candidate compounds. This system provides a platform for a key step in mitochondrial medicine discovery, especially for bioactive molecule screening from complex herbal extracts.
Assuntos
Carnitina O-Palmitoiltransferase , Metabolismo dos Lipídeos , Membranas Mitocondriais , Humanos , Carnitina O-Palmitoiltransferase/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Cromatografia de Afinidade , LigantesRESUMO
Acrolein is a ubiquitous gaseous air pollutant and endogenous toxicant, which poses strong risk for oxidative stress-related diseases such as cardiovascular disease. Adenosine has been identified as potential therapeutic agent for age-related cardiovascular disease, while the molecular mechanisms underlying its cardioprotection remain elusive. In the present study, we investigated the myocardial protective effects and the mechanism of adenosine on acrolein-induced toxicity in H9c2 cells and primary neonatal rat cardiomyocytes. We found that acrolein caused apoptosis of cardiomyocytes resulting from oxidative damage, autophagy defect, and mitochondrial dysfunction, as evidenced by loss of mitochondrial membrane potential, impairment of mitochondrial biogenesis, dynamics, and oxidative phosphorylation, decrease of mitochondrial deoxyribonucleic acid (mtDNA) copy number and adenosine 5'-triphosphate (ATP) production. Adenosine pretreatment protected against acrolein-induced cardiotoxicity by maintaining mitochondrial homeostasis, activating the phase II detoxifying enzyme system, promoting autophagic flux, and alleviating mitochondrial-dependent apoptosis. We further demonstrated that the up-regulation of forkhead box protein O1 (FoxO1) mediated by extracellular regulated protein kinases (ERK) activation contributes to the cardioprotection of adenosine. These results expand the application of adenosine in cardioprotection to preventing myocardial damages induced by environmental pollutant acrolein exposure, and uncover the adenosine-ERK-FoxO1 axis as the underlying mechanism mediating the protection of mitochondrial homeostasis, Nrf2-mediated antioxidant defense and autophagic flux, shedding light on the better understanding about the pathological mechanism of cardiovascular disease caused by environmental pollutants and applications of adenosine in cardioprotection.
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Epidemiologic studies have demonstrated a direct correlation between fine particulate matter (FPM) exposure and the high risk of respiratory diseases. FPM can penetrate deep into the lung and deposit in the alveoli with breath, where it directly interacts with alveolar epithelial cell (APC). However, we know little about the effects nor mechanisms of FPM on APC. Here, using human APC A549 cells, we found that FPM resulted in blockade of autophagic flux, redox imbalance and oxidative stress, mitochondrial fragmentation, increased mitophagy and impaired mitochondrial respiration. Further we showed that activation of JNK signaling (c-Jun N-terminal kinase) and excessive ROS (reactive oxygen species) release contribute to these adverse effects, with the former being upstream of the latter. More importantly, we found that scavenging ROS or inhibiting JNK activation could restore those effects as well as ameliorate FPM-induced inhibition of cell proliferation, and epithelial-mesenchymal transformation (EMT) in A549 cells. Taken together, our findings indicate that FPM leads to toxicity in alveolar type II cells via JNK activation, and JNK-targeting or antioxidant strategies might be beneficial for prevention or treatment of FPM-related pulmonary diseases.
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Alzheimer's disease (AD) has become a major public health problem that affects the elderly population. Therapeutic compounds with curative effects are not available due to the complex pathogenesis of AD. Daphnetin, a natural coumarin derivative and inhibitor of various kinases, has anti-inflammatory and antioxidant activities. In this study, we found that daphnetin improved spatial learning and memory in an amyloid precursor protein (APP)/presenilin 1 (PS1) double-transgenic mouse model of AD. Daphnetin markedly decreased the levels of amyloid-ß peptide 1-40 (Aß40) and 1-42 (Aß42) in the cerebral cortex, downregulated the expressions of enzymes involved in APP processing, e.g., beta-site APP-cleaving enzyme (BACE), nicastrin and presenilin enhancer protein 2 (PEN2). We further found the reduced serum levels of inflammatory factors, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and chemokine (C-C motif) ligand 3 (CCL3), while daphnetin increased total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) levels in the serum. Interestingly, daphnetin markedly decreased the expression of glial fibrillary acidic protein (GFAP) and the upstream regulatory molecule- phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in APP/PS1 mice, and mainly inhibited the phosphorylation of STAT3 at Ser727 to decrease GFAP expression evidenced in a LPS-activated glial cell model. These results suggest that daphnetin ameliorates cognitive deficits and that Aß deposition in APP/PS1 mice is mainly correlated with astrocyte activation and APP processing.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Idoso , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Antioxidantes/uso terapêutico , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-1/uso terapêutico , Fator de Transcrição STAT3/metabolismo , UmbeliferonasRESUMO
BACKGROUND: Background: Acute lung injury (ALI) is one kind of frequently occurred emergency in Intensive Care Unite with a high mortality. The underlying causes are uncontrolled inflammatory reactions and intractable hypoxemia, which are difficult to control and improve. In the past 10 years, gas medical studies have found that both hydrogen molecules and oxygen molecules have protective effects on acute lung injury by improving inflammatory reactions and hypoxia, respectively. Oxygen is an oxidant and hydrogen is an antioxidant. In this study, we investigated the combined effect of above two-gas molecular on lipopolysaccharide (LPS) -induced acute lung injury. METHODS: To clarify whether the combination of hydrogen and oxygen could increase or cancel out the protective effect, an ALI mice model induced by intraperitoneal injection of LPS was established, and the degree of lung tissue and mitochondria damage was evaluated based on the pathological sections, inflammatory factors, wet-dry ratio, bronchoalveolar lavage fluid (BALF). Immunohistochemistry, electron microscopy, western blotting and other detection methods also used to evaluate the therapeutic effect on acute lung injury model. RESULTS: We observed that the combined protective effect of hydrogen and oxygen was superior to their respective protective effects, and the specific molecular mechanisms of the two therapies might be different. CONCLUSION: Hydrogen plays a more important role in the inflammatory and anti-apoptosis mechanisms, while oxygen improves hypoxia of the body, and thus, its molecular mechanism may be closely associated to the hypoxia pathways.
Assuntos
Lesão Pulmonar Aguda , Hidrogênio , Oxigênio , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/prevenção & controle , Animais , Hidrogênio/farmacologia , Hipóxia/metabolismo , Inflamação/metabolismo , Lipopolissacarídeos , Pulmão , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Oxigênio/farmacologia , Transdução de SinaisRESUMO
Sleep deprivation has profound influence on several aspects of health and disease. Mitochondria dysfunction has been implicated to play an essential role in the neuronal cellular damage induced by sleep deprivation, but little is known about how neuronal mitochondrial ultrastructure is affected under sleep deprivation. In this report, we utilized electron cryo-tomography to reconstruct the three-dimensional (3-D) mitochondrial structure and extracted morphometric parameters to quantitatively characterize its reorganizations. Isolated mitochondria from the hippocampus and cerebral cortex of adult male Sprague-Dawley rats after 72 h of paradoxical sleep deprivation (PSD) were reconstructed and analyzed. Statistical analysis of six morphometric parameters specific to the mitochondrial inner membrane topology revealed identical pattern of changes in both the hippocampus and cerebral cortex but with higher significance levels in the hippocampus. The structural differences were indistinguishable by conventional phenotypic methods based on two-dimensional electron microscopy images or 3-D electron tomography reconstructions. Furthermore, to correlate structure alterations with mitochondrial functions, high-resolution respirometry was employed to investigate the effects of PSD on mitochondrial respiration, which showed that PSD significantly suppressed the mitochondrial respiratory capacity of the hippocampus, whereas the isolated mitochondria from the cerebral cortex were less affected. These results demonstrate the capability of the morphometric parameters for quantifying complex structural reorganizations and suggest a correlation between PSD and inner membrane architecture/respiratory functions of the brain mitochondria with variable effects in different brain regions.
Assuntos
Córtex Cerebral/ultraestrutura , Hipocampo/ultraestrutura , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/ultraestrutura , Privação do Sono/fisiopatologia , Sono REM/fisiologia , Animais , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , Tomografia com Microscopia Eletrônica , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Processamento de Imagem Assistida por Computador/métodos , Masculino , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Especificidade de Órgãos , Consumo de Oxigênio/fisiologia , Ratos , Ratos Sprague-Dawley , Privação do Sono/metabolismoRESUMO
Mounting evidence from epidemiological and clinical studies has revealed marked correlations between the air pollutant fine particulate matter (FPM) and respiratory diseases. FPM reaches distal airways and deposits in alveolar regions where it can act directly on alveolar macrophages. However, the detailed effect of FPM on the physiological function of alveolar macrophages and the underlying mechanisms remain unclear. In this study, we showed that exposing THP-1-derived macrophages to FPM led to autophagy dysfunction. FPM activated the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway, which promoted the expression of autophagy-related 2A (ATG2A) and reactive oxygen species generation. The overexpression of ATG2A enhanced the synthesis of autophagic membranes, and the excessive production of reactive oxygen species caused autophagy flux inhibition through disrupting the lysosomal activity. More importantly, FPM impaired the phagocytic ability of macrophages on Escherichia coli and apoptotic neutrophils. Finally, we showed that restoring autophagy rescued the impairment of phagocytic ability induced by FPM. In summary, these results reveal the molecular mechanism of autophagy dysfunction caused by FPM and provide a novel approach to resolve the impaired function of macrophages in respiratory diseases induced by FPM.
Assuntos
Autofagia/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Material Particulado/farmacologia , Fagocitose/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Escherichia coli/metabolismo , Células HEK293 , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células THP-1RESUMO
Pneumonia is a chronic disorder of the respiratory system associated with worsening quality of life and a significant economic burden. Pinitol, a plant cyclic polyol, has been documented for immune-inflammatory potential. The aim of present investigation was to evaluate the potential and possible mechanism of action of pinitol against lipopolysaccharide (LPS)-induced pneumonia in the experimental animal model. Pneumonia was induced in Sprague-Dawley rats by intratracheal administration of LPS (2 mg/kg). Animals were treated with either vehicle or dexamethasone or pinitol (5 or 10 or 20 mg/kg). Potential of pinitol against LPS-induced pulmonary insult was assessed based on behavioral, biochemical, molecular, and ultrastructural studies. Intratracheal instillation of LPS induced significant (P < .05) inflammatory infiltration in bronchoalveolar lavage fluid (BALF) and lung tissue reflected by elevated pleural effusion volume, lung edema, BALF polymorphonuclear leukocytes count and lung myeloperoxidase levels, which was attenuated by pinitol (10 and 20 mg/kg) administration. Pinitol also markedly (P < .05) inhibited LPS-induced alterations in electrocardiographic, hemodynamic changes, right ventricular, and lung function tests. The LPS-induced downregulated nuclear factor erythroid 2-related factor 2 (Nrf-2) and heme oxygenase-1 (HO-1), whereas upregulated transforming growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3), and inducible nitric oxide synthase (iNOs) lung messenger RNA expressions were significantly (P < .05) inhibited by pinitol. Western blot analysis suggested pinitol markedly (P < .05) decreased nuclear factor-κB (NF-κB), inhibitor of nuclear factor κB (IkBα), toll-like receptor 4 (TLR-4), and cyclooxygenase-II (COX-II) protein expressions in the lung. These findings were further supported by histological and ultrastructural analyses of lung tissue that show pinitol significantly (P < .05) ameliorates LPS-induced aberrations in lung tissue. In conclusion, pinitol attenuated LPS-induced pneumonia via inhibition of TLR-4 to downregulate the NF-κB/IκBα signaling cascade and thus ameliorated the production of proinflammatory cytokines (TNF-α, ILs, NLRP3, and TGF-ß), inflammatory mediators (COX-II and iNOs) and elevated oxidative stress (Nrf-2 and HO-1).
Assuntos
Inositol/análogos & derivados , Lipopolissacarídeos/toxicidade , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Pneumonia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Inositol/farmacologia , Masculino , Pneumonia/induzido quimicamente , Pneumonia/tratamento farmacológico , Pneumonia/metabolismo , Pneumonia/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Growing evidence has suggested that autophagy-related protein 7 (ATG7) plays an important role in insulin signaling, but the mechanism of ATG7 in hepatic insulin sensitivity is not fully understood. The purpose of the present study is to clarify the underlying molecular mechanisms of ATG7 in obesity development. Serum and liver samples from mice fed a high fat diet (HFD) were evaluated for metabolic profile data and ATG expressions during obesity development. We found that compared with other ATGs, ATG7 expression increased earlier with lower hepatic insulin sensitivity in the 4-wk HFD-fed mice. For in vitro analyses, silencing ATG7 significantly up-regulated insulin-stimulated phosphorylation of protein kinase B (Akt) and down-regulated phosphatase and tension homolog deleted on chromosome ten (PTEN) in HepG2 cells. Replenishing PTEN to ATG7-silenced hepatocytes restored the phosphorylated Akt level. Furthermore, ATG7 silencing led to higher c-JUN expression, which transcriptionally reduced PTEN expression. These results reveal a novel mechanism by which ATG7 regulates Akt phosphorylation via the c-JUN/PTEN pathway at the early stage of HFD-induced metabolic disorder.-Zhao, D., Zhang, S., Wang, X., Gao, D., Liu, J., Cao, K., Chen, L., Liu, R., Liu, J., Long, J. ATG7 regulates hepatic Akt phosphorylation through the c-JUN/PTEN pathway in high fat diet-induced metabolic disorder.
Assuntos
Proteína 7 Relacionada à Autofagia/metabolismo , Dieta Hiperlipídica/efeitos adversos , Fígado/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proteína 7 Relacionada à Autofagia/genética , Gorduras na Dieta/efeitos adversos , Regulação para Baixo , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Células Hep G2 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Doenças Metabólicas/induzido quimicamente , Doenças Metabólicas/metabolismo , Camundongos , Células PC-3 , PTEN Fosfo-Hidrolase/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
BACKGROUND: Growing evidence suggests that leptin is critical for glycemic control. Impaired leptin signaling may also contribute to low adiponectin expression in obese individuals. We assessed the association of leptin and adiponectin with incident type 2 diabetes (T2D), their interactions with sex and obesity status, and mediation by insulin resistance. METHODS: We included study participants from the Jackson Heart Study, a prospective cohort of adult African Americans in Jackson, Mississippi, that were free of T2D at the baseline Exam 1. Incident T2D was defined as new cases at Exam 2 or Exam 3. We created separate Cox regression models (hazard ratios per log-transformed ng/mL of leptin and adiponectin) with and without insulin resistance, HOMA-IR. Mediation by insulin resistance was analyzed. Several interactions were assessed, including by sex, HbA1c, and obesity. RESULTS: Among our 3363 participants (mean age 53 years, 63% women), 584 developed incident T2D. Leptin was directly associated with incident T2D when modeled without HOMA-IR (HR = 1.29, 95% CI = 1.05-1.58). This direct association between leptin and T2D was significant among men (HR = 1.33, 95% CI = 1.05-1.69), but nonsignificant among women (HR = 1.24, 95% CI = 0.94-1.64); statistical interaction with sex was nonsignificant (p = 0.65). The associations in all participants and in men were nullified by HOMA-IR (HR = 0.99, 95% CI = 0.80-1.22; HR = 1.00, 95% CI = 0.78-1.28, respectively), indicating mediation through insulin resistance (proportion mediated: 1.04), and were not observed in abdominally obese participants. Adiponectin was inversely associated with T2D even after adjustment for HOMA-IR in women (HR = 0.68, 95% CI = 0.55-0.84), but not in men (HR = 0.80, 95% CI = 0.62-1.04). The inverse association was present only among abdominally obese participants, and persisted after adjustment for HOMA-IR. CONCLUSIONS: Among African Americans in the Jackson Heart Study the association of leptin with incident type 2 diabetes was mediated by insulin resistance. This association was present only among abdominally non-obese participants. Differences by sex appeared: men showed a significant association mediated by insulin resistance. Among abdominally obese participants, adiponectin was inversely associated with incident T2D even after adjustment for HOMA-IR. Our results should inform future clinical trials that aim to reduce the burden of type 2 diabetes through the modification of serum levels of leptin and adiponectin.
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Adiponectina/sangue , Biomarcadores/sangue , Negro ou Afro-Americano/estatística & dados numéricos , Diabetes Mellitus Tipo 2/epidemiologia , Resistência à Insulina , Leptina/sangue , Obesidade/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Glicemia/análise , Índice de Massa Corporal , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/patologia , Feminino , Seguimentos , Humanos , Incidência , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Fatores de Risco , Estados Unidos/epidemiologia , Adulto JovemRESUMO
SKP1-cullin-1-F-box-protein (SCF) E3 ubiquitin ligase complex is responsible for the degradation of proteins in a strictly regulated manner, through which it exerts pivotal roles in regulating various key cellular processes including cell cycle and division, apoptosis, and differentiation. The substrate specificity of the SCF complex largely depends on the distinct F-box proteins, which function in either tumor promotion or suppression or in a context-dependent manner. Among the 69 F-box proteins identified in human genome, FBW7, SKP2, and ß-TRCP have been extensively investigated among various types of cancer in respective of their roles in cancer development, progression, and metastasis. Moreover, several specific inhibitors have been developed to target those E3 ligases, and their efficiency in tumors has been determined. In this review, we provide a summary of the roles of SCF E3 ligases in cancer development, as well as the potential application of miRNA or specific inhibitors for cancer therapy.
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Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas F-Box/metabolismo , Humanos , Neoplasias/patologiaRESUMO
DNA methylation is a well-defined epigenetic modification that regulates gene transcription. However, the role of DNA methylation in the cardiac hypertrophy seen in hypertension is unclear. This study was performed to investigate genome-wide DNA methylation profiles in spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKY), and the cardioprotective effect of choline. Eight-week-old male SHRs received intraperitoneal injections of choline (8â¯mg/kg/day) for 8â¯weeks. SHRs showed aberrant methylation distribution on chromosomes and genome regions, with decreased methylation levels at CHG and CHH sites. A total of 91,559 differentially methylated regions (DMRs) were detected between SHRs and WKY rats, of which 28,197 were demethylated and 63,362 were methylated. Choline treatment partly restored the DMRs in SHRs, which were related to 131 genes. Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis of DMRs suggested that choline partly reversed the dysfunctions of biological processes, cellular components and molecular functions in SHRs. Moreover, the inhibition of 2-oxoglutarate accumulation by choline, thereby inhibiting excessive activation of ten-eleven translocation methylcytosine dioxygenase enzymes, may correlate with the beneficial effects of choline on methylation levels, cardiac hypertrophy and cardiac function of SHRs, as indicated by decreased heart rate and blood pressure, and increased ejection fraction and fractional shortening. This study provides the first genome-wide DNA methylation profile of the hypertrophic myocardium of SHRs and suggests a novel role for this epigenetic modification in hypertension. Choline treatment may represent a promising approach for modification of DNA methylation and optimization of the epigenetic profile for antihypertensive therapy.
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Cardiomegalia/tratamento farmacológico , Colina/farmacologia , Metilação de DNA/genética , Hipertensão/tratamento farmacológico , Animais , Pressão Sanguínea/efeitos dos fármacos , Cardiomegalia/genética , Cardiomegalia/patologia , Metilação de DNA/efeitos dos fármacos , Modelos Animais de Doenças , Epigênese Genética/efeitos dos fármacos , Genoma/efeitos dos fármacos , Humanos , Hipertensão/genética , Hipertensão/patologia , Ácidos Cetoglutáricos/metabolismo , Ratos , Ratos Endogâmicos SHR , Transdução de Sinais/efeitos dos fármacos , Sequenciamento Completo do GenomaRESUMO
Obesity is associated with skeletal muscle loss and impaired myogenesis. Increased infiltration of proinflammatory macrophages in skeletal muscle is noted in obesity and is associated with muscle insulin resistance. However, whether the infiltrated macrophages can contribute to obesity-induced muscle loss is unclear. In this study, we investigate macrophage and muscle differentiation markers in the quadriceps (QC), gastrocnemius, tibia anterior, and soleus muscles from obese mice that were fed a high-fat diet for 16 weeks. Then, we examined the effect and mediator of macrophage-secreted factors on myoblast differentiation in vitro. We found markedly increased levels of proinflammatory macrophage markers (F4/80 and CD11c) in the QC muscle compared with the other three muscle groups. Consistent with the increased levels of proinflammatory macrophage infiltration, the QC muscle also showed a significant reduction in the expression of muscle differentiation makers MYOD1 and myosin heavy chain. In in vitro studies, treatment of C2C12 myoblasts with Raw 264.7 macrophage-conditioned medium (CM) significantly promoted cell proliferation and inhibited myoblast differentiation. Neutralization of tumor necrosis factor α (TNF-α) in Raw 264.7 macrophage CM reversed the reduction of myoblast differentiation. Finally, we found that both macrophage CM and TNF-α induced sustained activation of p38 mitogen-activated protein kinase (MAPK) in C2C12 myoblasts. Together, our findings suggest that the increased infiltration of proinflammatory macrophages could contribute toward obesity-induced muscle loss by secreting inflammatory cytokine TNF-α via the p38 MAPK signaling pathway.
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Diferenciação Celular/fisiologia , Desenvolvimento Muscular/fisiologia , Obesidade/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Meios de Cultivo Condicionados/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Sprouting angiogenesis is a major form of neovascularization of tissues suffering from hypoxia and other related stress. Endothelial cells (ECs) undergo proliferation, differentiation, programmed death, and migration during angiogenic sprouting, but the underlying molecular mechanisms regulating ECs in angiogenesis have been incompletely elucidated. Here we report that the transmembrane protein 215 (TMEM215) is involved in angiogenesis by regulating EC survival. The murine TMEM215 gene, which possesses two transcriptional starting sites as determined by 5'-rapid amplification of complementary DNA (cDNA) ends (RACE), encodes a two-pass TMEM. The TMEM215 transcripts were detected in ECs in addition to other tissues by quantitative reverse transcription-polymerase chain reaction. Immunofluorescence showed that TMEM215 was expressed in the vasculature in retina, liver, and tumor, and colocalized with EC markers. We show that knockdown of TMEM215 in ECs induced strong cell death of ECs in vitro without affecting cell proliferation and migration, suggesting that TMEM215 was required for EC survival. Downregulation of TMEM215 expression compromised lumen formation and sprouting capacities of ECs in vitro. Moreover, intravitreous injection of TMEM215 small interfering RNA resulted in delayed and abnormal development of retinal vasculature with poor perfusion. These results identified TMEM215 as a novel molecule involved in angiogenesis by regulating the survival of ECs.
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Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteínas de Membrana/metabolismo , Neovascularização Fisiológica , Sequência de Aminoácidos , Animais , Sequência de Bases , Sobrevivência Celular , Células Endoteliais/ultraestrutura , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Retina/metabolismoRESUMO
Variations in mitochondrial DNA (mtDNA) have been fundamental for understanding human evolution and are causative for a plethora of inherited mitochondrial diseases, but the mutation signatures of germline mtDNA and their value in understanding mitochondrial pathogenicity remain unknown. Here, we carried out a systematic analysis of mutation patterns in germline mtDNA based on 97,566 mtDNA variants from 45,494 full-length sequences and revealed a highly non-stochastic and replication-coupled mutation signature characterized by nucleotide-specific mutation pressure (G > T>A > C) and position-specific selection pressure, suggesting the existence of an intensive mutation-selection interplay in germline mtDNA. We provide evidence that this mutation-selection interplay has strongly shaped the mtDNA sequence during evolution, which not only manifests as an oriented alteration of amino acid compositions of mitochondrial encoded proteins, but also explains the long-lasting mystery of CpG depletion in mitochondrial genome. Finally, we demonstrated that these insights may be integrated to better understand the pathogenicity of disease-implicated mitochondrial variants.
Assuntos
DNA Mitocondrial/genética , Evolução Molecular , Genoma Mitocondrial/genética , Mutação em Linhagem Germinativa , Doenças Mitocondriais/genética , Sequência de Bases , Mapeamento Cromossômico/métodos , Análise Mutacional de DNA/métodos , Humanos , Doenças Mitocondriais/diagnóstico , Proteínas Mitocondriais/genéticaRESUMO
Impairment of retinal pigment epithelial (RPE) cells is considered a key contributor to the development of age-related macular degeneration. Apoptosis-related protein 3 (APR3) was recently discovered after treatment with all- trans retinoic acid, a pivotal molecule in RPE cells. However, the function of APR3 remains poorly understood. In the present study, we found that APR3 could interact with nuclear factor (erythroid-derived 2)-like 2, which is a regulator of phase II enzymes, and that knockdown of APR3 promoted nuclear factor (erythroid-derived 2)-like 2 nuclear translocation and activated expression of phase II enzymes, which was accompanied by improved redox status and mitochondrial activity. Overexpression of APR3 revealed its mitochondrial localization and induced a robust production of reactive oxygen species that was accompanied by impaired mitochondrial oxygen consumption, complex activity, and lower ATP content, resulting in significant changes in mitochondrial structure, which may contribute to cell apoptosis. High doses of all- trans retinoic acid treatment were found to significantly induce APR3 expression, increase reactive oxygen species levels, and decrease ATP content, which were abolished by knockdown of APR3. These results indicate that APR3 plays a vital role in regulating redox status and mitochondrial activity and thus suggest APR3 might be a potential novel target for study of treatment of age-related macular degeneration.-Li, Y., Zou, X., Gao, J., Cao, K., Feng, Z., Liu, J. APR3 modulates oxidative stress and mitochondrial function in ARPE-19 cells.
RESUMO
AIMS: Metformin could have benefits on cardiovascular disease and kidney disease progression but is often withheld from individuals with diabetes and chronic kidney disease (CKD) because of a concern that it may increase the risk of lactic acidosis. MATERIALS AND METHODS: All-cause mortality, cardiovascular death, cardiovascular events (death, hospitalization for heart failure, myocardial infarction, stroke or myocardial ischemia), end stage renal disease (ESRD) and the kidney disease composite (ESRD or death) were compared in metformin users and non-users with diabetes and CKD enrolled in the Trial to Reduce Cardiovascular Events with Aranesp (darbepoeitin-alfa) Therapy (TREAT) (NCT00093015). Outcomes were compared after propensity matching of users and non-users and in multivariable proportional hazards models. RESULTS: There were 591 individuals who used metformin at baseline and 3447 non-users. Among propensity-matched users, the crude incidence rate for mortality, cardiovascular mortality, cardiovascular events and the combined endpoint was lower in metformin users than in non-users, but ESRD was marginally higher (4.0% vs 3.6%). Metformin use was independently associated with a reduced risk of all-cause mortality (HR, 0.49; 95% CI, 0.36-0.69), cardiovascular death (HR, 0.49; 95% CI, 0.32-0.74), the cardiovascular composite (HR, 0.67, 95% CI, 0.51-0.88) and the kidney disease composite (HR, 0.77; 95% CI, 0.61-0.98). Associations with ESRD (HR, 1.01; 95% CI, 0.65-1.55) were not significant. Results were qualitatively similar in adjusted analyses of the full population. Two cases of lactic acidosis were observed. CONCLUSIONS: Metformin may be safer for use in CKD than previously considered and may lower the risk of death and cardiovascular events in individuals with stage 3 CKD.
Assuntos
Darbepoetina alfa/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Angiopatias Diabéticas/epidemiologia , Angiopatias Diabéticas/prevenção & controle , Metformina/uso terapêutico , Insuficiência Renal Crônica/tratamento farmacológico , Idoso , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/prevenção & controle , Causas de Morte , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/epidemiologia , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/epidemiologia , Feminino , Insuficiência Cardíaca/epidemiologia , Insuficiência Cardíaca/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mortalidade , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/epidemiologia , Medição de Risco , Fatores de Risco , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/etiologiaRESUMO
Accurate data imputation requires ethnicity-matched reference panels. However, recent admixtures have created mosaic human genomes, different chromosomal segments have different ethnic backgrounds, so it is impossible for a single-ethnicity reference panel to be the matched for data imputation. In this study, we explored a novel strategy for imputation. We created individualized mosaic reference panel for each person according to his/her ethnic backgrounds at each genomic locus. We examined on datasets with 70% missing values on haplotypes and 50% missing values on genotypes. Results showed that the imputation with mosaic references steadily yielded high imputation accuracy and outperforms the other strategies. With the mosaic reference panels, the imputation accuracy was 98.8±0.1% (CEU), 98.7±0.1% (YRI), 98.5±0.1% (CHB), 98.6±0.1% (ASW), 97.3±0.1% (MKK) and 98.2±0.1% (MXL). Mosaic reference panel will be one option for future missing value imputation in big data era.