RESUMEN
The gastrointestinal (GI) system is highly susceptible to irradiation. Currently, there is no Food and Drug Administration (FDA)-approved medical countermeasures for GI radiation injury. The vitamin E analog gamma-tocotrienol (GT3) is a promising radioprotector in mice and nonhuman primates (NHP). We evaluated GT3-mediated GI recovery in total-body irradiated (TBI) NHPs. Sixteen rhesus macaques were divided into two groups; eight received vehicle and eight GT3 24 h prior to 12 Gy TBI. Proximal jejunum was assessed for structural injuries and crypt survival on day 4 and 7. Apoptotic cell death and crypt cell proliferation were assessed with TUNEL and Ki-67 immunostaining. Irradiation induced significant shortening of the villi and reduced mucosal surface area. GT3 induced an increase in crypt depth at day 7, suggesting that more stem cells survived and proliferated after irradiation. GT3 did not influence crypt survival after irradiation. GT3 treatment caused a significant decline in TUNEL-positive cells at both day 4 (p < 0.03) and 7 (p < 0.0003). Importantly, GT3 induced a significant increase in Ki-67-positive cells at day 7 (p < 0.05). These data suggest that GT3 has radioprotective function in intestinal epithelial and crypt cells. GT3 should be further explored as a prophylactic medical countermeasure for radiation-induced GI injury.
Asunto(s)
Síndrome de Radiación Aguda , Cromanos , Protectores contra Radiación , Vitamina E , Síndrome de Radiación Aguda/tratamiento farmacológico , Síndrome de Radiación Aguda/prevención & control , Animales , Cromanos/uso terapéutico , Modelos Animales de Enfermedad , Intestinos/patología , Intestinos/efectos de la radiación , Antígeno Ki-67 , Macaca mulatta , Protectores contra Radiación/farmacología , Protectores contra Radiación/uso terapéutico , Vitamina E/análogos & derivados , Vitamina E/uso terapéuticoRESUMEN
DNA fragmentation produced by apoptotic DNases (endonucleases) leads to irreversible cell death. Although apoptotic DNases are simultaneously induced following toxic/oxidative cell injury and/or failed DNA repair, the study of DNases in apoptosis has generally been reductionist in approach, focusing on individual DNases rather than their possible cooperativity. Coordinated induction of DNases would require a mechanism of communication; however, mutual DNase induction or activation of DNases by enzymatic or non-enzymatic mechanisms is not currently recognized. The evidence presented in this review suggests apoptotic DNases operate in a network in which members induce each other through the DNA breaks they produce. With DNA breaks being a common communicator among DNases, it would be logical to propose that DNA breaks from other sources such as oxidative DNA damage or actions of DNA repair endonucleases and DNA topoisomerases may also serve as triggers for a cooperative DNase feedback loop leading to elevated DNA fragmentation and subsequent cell death. Therefore, mutual induction of apoptotic DNases has serious implications for studies focused on activation or inhibition of specific DNases as a strategy for therapeutic intervention aimed at modulation of cell death.
Asunto(s)
Apoptosis/genética , Reparación del ADN/genética , ADN-Topoisomerasas/genética , Estrés Oxidativo/genética , Fragmentación del ADN , Desoxirribonucleasas/genética , HumanosRESUMEN
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay is a long-established assay used to detect cell death-associated DNA fragmentation (3'-OH DNA termini) by endonucleases. Because these enzymes are particularly active in the kidney, TUNEL is widely used to identify and quantify DNA fragmentation and cell death in cultured kidney cells and animal and human kidneys resulting from toxic or hypoxic injury. The early characterization of TUNEL as an apoptotic assay has led to numerous misinterpretations of the mechanisms of kidney cell injury. Nevertheless, TUNEL is becoming increasingly popular for kidney injury assessment because it can be used universally in cultured and tissue cells and for all mechanisms of cell death. Furthermore, it is sensitive, accurate, quantitative, easily linked to particular cells or tissue compartments, and can be combined with immunohistochemistry to allow reliable identification of cell types or likely mechanisms of cell death. Traditionally, TUNEL analysis has been limited to the presence or absence of a TUNEL signal. However, additional information on the mechanism of cell death can be obtained from the analysis of TUNEL patterns.
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Apoptosis/genética , Fragmentación del ADN , Etiquetado Corte-Fin in Situ/métodos , Enfermedades Renales/diagnóstico , Animales , Células Cultivadas , Desoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Humanos , Riñón/citología , Riñón/enzimología , Riñón/lesiones , Riñón/patología , Enfermedades Renales/enzimología , Enfermedades Renales/fisiopatologíaRESUMEN
Endonuclease-mediated DNA fragmentation is both an immediate cause and a result of apoptosis and of all other types of irreversible cell death after injury. It is produced by nine enzymes including DNase I, DNase 2, their homologs, caspase-activated DNase (CAD) and endonuclease G (EndoG). The endonucleases act simultaneously during cell death; however, regulatory links between these enzymes have not been established. We hypothesized that DNase I, the most abundant of endonucleases, may regulate other endonucleases. To test this hypothesis, rat kidney tubular epithelial NRK-52E cells were transfected with the DNase I gene or its inactive mutant in a pECFP expression vector, while control cells were transfected with the empty vector. mRNA expression of all nine endonucleases was studied using real-time RT-PCR; DNA strand breaks in endonuclease genes were determined by PCR and protein expression of the enzymes was measured by Western blotting and quantitative immunocytochemistry. Our data showed that DNase I, but not its inactive mutant, induces all other endonucleases at varying time periods after transfection, causes DNA breaks in endonuclease genes, and elevates protein expression of several endonucleases. This is the first evidence that endonucleases seem to be induced by the DNA-degrading activity of DNase I.
Asunto(s)
Roturas del ADN , Fragmentación del ADN , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Células Epiteliales/enzimología , Túbulos Renales/enzimología , Animales , Línea Celular , ADN/genética , Desoxirribonucleasa I/genética , RatasRESUMEN
The recent identification of profilin1 mutations in 25 familial ALS cases has linked altered function of this cytoskeleton-regulating protein to the pathogenesis of motor neuron disease. To investigate the pathological role of mutant profilin1 in motor neuron disease, we generated transgenic lines of mice expressing human profilin1 with a mutation at position 118 (hPFN1G118V). One of the mouse lines expressing high levels of mutant human PFN1 protein in the brain and spinal cord exhibited many key clinical and pathological features consistent with human ALS disease. These include loss of lower (ventral horn) and upper motor neurons (corticospinal motor neurons in layer V), mutant profilin1 aggregation, abnormally ubiquitinated proteins, reduced choline acetyltransferase (ChAT) enzyme expression, fragmented mitochondria, glial cell activation, muscle atrophy, weight loss, and reduced survival. Our investigations of actin dynamics and axonal integrity suggest that mutant PFN1 protein is associated with an abnormally low filamentous/globular (F/G)-actin ratio that may be the underlying cause of severe damage to ventral root axons resulting in a Wallerian-like degeneration. These observations indicate that our novel profilin1 mutant mouse line may provide a new ALS model with the opportunity to gain unique perspectives into mechanisms of neurodegeneration that contribute to ALS pathogenesis.
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Esclerosis Amiotrófica Lateral/metabolismo , Encéfalo/metabolismo , Mutación Missense , Profilinas/biosíntesis , Médula Espinal/metabolismo , Sustitución de Aminoácidos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Transgénicos , Profilinas/genética , Médula Espinal/patologíaRESUMEN
Microbial resistance to drugs is an unresolved global concern, which is present in every country. Developing new antibiotics is one of the guidelines of the Centers for Disease Control and Preventions (CDC) to combat bacterial resistance to drugs. Based on our lead molecules, we report the synthesis and antimicrobial studies of 27 new pyrazole derivatives. These new coumarin-pyrazole-hydrazone hybrids are readily synthesized from commercially available starting materials and reagents using benign reaction conditions. All the synthesized molecules were tested against 14 Gram-positive and Gram-negative bacterial strains. Several of these molecules have been found to be potent growth inhibitors of several strains of these tested bacteria with minimum inhibitory concentrations as low as 1.56 µg/mL. Furthermore, active molecules are non-toxic in in vitro and in vivo toxicity studies.
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Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Hidrazonas/síntesis química , Hidrazonas/farmacología , Staphylococcus aureus/efectos de los fármacos , Acinetobacter baumannii/metabolismo , Animales , Antibacterianos/química , Técnicas de Química Sintética , Humanos , Hidrazonas/química , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Staphylococcus aureus/metabolismoRESUMEN
Graphene, a crystalline allotrope or carbon, presents numerous useful properties; however, its toxicity is yet to be determined. One of the most dramatic and irreversible toxic abilities of carbon nanomaterials is the induction of DNA fragmentation produced by endogenous cellular endonucleases. This study demonstrated that pristine graphene exposed to cultured kidney tubular epithelial cells is capable of inducing DNA fragmentation measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which is usually associated with cell death. TUNEL (cell death) and endonuclease activity measured using a near infrared fluorescence probe was significantly higher in cells containing graphene aggregates detected by Raman spectroscopy. The elevation of TUNEL coincided with the increased abundance of heme oxygenase 1 (HO-1), heat shock protein 90 (HSP90), active caspase-3 and endonucleases (deoxyribonuclease I [DNase I] and endonuclease G [EndoG]), as measured by quantitative immunocytochemistry. Specific inhibitors for HO-1, HSP90, caspase-3, DNase I and EndoG almost completely blocked the DNA fragmentation induced by graphene exposure. Therefore, graphene induces cell death through oxidative injury, caspase-mediated and caspase-independent pathways; and endonucleases DNase I and EndoG are important for graphene toxicity. Inhibition of these pathways may ameliorate cell injury produced by graphene. Copyright © 2017 John Wiley & Sons, Ltd.
Asunto(s)
Daño del ADN , Desoxirribonucleasa I/metabolismo , Endodesoxirribonucleasas/metabolismo , Células Epiteliales/efectos de los fármacos , Grafito/toxicidad , Túbulos Renales/efectos de los fármacos , Nanopartículas/toxicidad , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular , Desoxirribonucleasa I/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Endodesoxirribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Células Epiteliales/enzimología , Células Epiteliales/patología , Proteínas HSP90 de Choque Térmico/metabolismo , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Hemo Oxigenasa (Desciclizante)/metabolismo , Túbulos Renales/enzimología , Túbulos Renales/patología , Estrés Oxidativo/efectos de los fármacos , Ratas , Medición de Riesgo , Factores de TiempoRESUMEN
AMP kinase (AMPK) plays an important role in the regulation of energy metabolism in cardiac cells. Furthermore, activation of AMPK protects the heart from myocardial infarction and heart failure. The present study examines whether or not AMPK affects the peroxisome proliferator-activated receptor-α (PPARα)/mitochondria pathway in response to acute oxidative stress in cultured cardiomyocytes. Cultured H9c2 rat embryonic cardioblasts were exposed to H2O2-induced acute oxidative stress in the presence or absence of metformin, compound C (AMPK inhibitor), GW6471 (PPARα inhibitor), or A-769662 (AMPK activator). Results showed that AMPK activation by metformin reverted oxidative stress-induced inactivation of AMPK and prevented oxidative stress-induced cell death. In addition, metformin attenuated reactive oxygen species generation and depolarization of the inner mitochondrial membrane. The antioxidative effects of metformin were associated with the prevention of mitochondrial DNA damage in cardiomyocytes. Coimmunoprecipitation studies revealed that metformin abolished oxidative stress-induced physical interactions between PPARα and cyclophilin D (CypD), and the abolishment of these interactions was associated with inhibition of permeability transition pore formation. The beneficial effects of metformin were not due to acetylation or phosphorylation of PPARα in response to oxidative stress. In conclusion, this study demonstrates that the protective effects of metformin-induced AMPK activation against oxidative stress converge on mitochondria and are mediated, at least in part, through the dissociation of PPARα-CypD interactions, independent of phosphorylation and acetylation of PPARα and CypD.
Asunto(s)
Adenilato Quinasa/metabolismo , Antioxidantes/farmacología , Ciclofilinas/metabolismo , Activadores de Enzimas/farmacología , Metformina/farmacología , Miocitos Cardíacos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , PPAR alfa/metabolismo , Adenilato Quinasa/antagonistas & inhibidores , Animales , Compuestos de Bifenilo , Muerte Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Activación Enzimática , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/enzimología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Oxidantes/farmacología , PPAR alfa/antagonistas & inhibidores , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Pironas/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Tiofenos/farmacologíaRESUMEN
A need exists for developing new therapies to improve cardiovascular outcomes in end-stage kidney disease. Three new areas that address novel pathophysiological mechanisms and/or therapeutic approaches toward cardiovascular events in chronic kidney disease patients include the use of an anti-inflammatory agent, the role of catalytic iron, and protein carbamylation. In preliminary studies, hydroxychloroquine, which has multiple anti-inflammatory properties, preserved vascular compliance for the aorta and major vessels, as well as reduced the extent of severity of atherosclerosis in ApoE-/- mice. The ability of iron to rapidly and reversibly cycle between 2 oxidation states makes iron potentially hazardous by enabling it to participate in the generation of powerful oxidant species. We have shown that high catalytic iron in the general population is associated with a 4-fold increase in prevalent cardiovascular disease (CVD), even after accounting for traditional risk factors. In addition, the highest levels of catalytic iron are present in dialysis patients and, more specifically, patients with prevalent CVD have several-fold higher catalytic iron levels compared with controls without CVD. These data suggest the utility of iron chelators for preventing and treating CVD in patients with chronic kidney disease and should be further investigated. Carbamylation of proteins results from nonenzymatic chemical modification by isocyanic acid derived from urea and an alternative route, the myeloperoxidase-catalyzed oxidation of thiocyanate. We have shown carbamylated low-density lipoprotein to have all the major biological effects relevant to atherosclerosis including endothelial cell injury, increased expression of cell adhesion molecules, and vascular smooth muscle cell proliferation. In 2 separate clinical studies, plasma levels of carbamylated protein independently predicted an increased risk of CVD and death.
Asunto(s)
Aterosclerosis/complicaciones , Aterosclerosis/fisiopatología , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/fisiopatología , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Factores de RiesgoRESUMEN
About one-fourth of patients with pancreatic ductal adenocarcinoma (PDAC) are categorized as borderline resectable (BR) or locally advanced (LA). Chemotherapy and radiation therapy have not yielded the anticipated outcomes in curing patients with BR/LA PDAC. The surgical resection of these tumors presents challenges owing to the unpredictability of the resection margin, involvement of vasculature with the tumor, the likelihood of occult metastasis, a higher ratio of positive lymph nodes, and the relatively larger size of tumor nodules. Oncolytic virotherapy has shown promising activity in preclinical PDAC models. Unfortunately, the desmoplastic stroma within the PDAC tumor microenvironment establishes a barrier, hindering the infiltration of oncolytic viruses and various therapeutic drugs-such as antibodies, adoptive cell therapy agents, and chemotherapeutic agents-in reaching the tumor site. Recently, a growing emphasis has been placed on targeting major acellular components of tumor stroma, such as hyaluronic acid and collagen, to enhance drug penetration. Oncolytic viruses can be engineered to express proteolytic enzymes that cleave hyaluronic acid and collagen into smaller polypeptides, thereby softening the desmoplastic stroma, ultimately leading to increased viral distribution along with increased oncolysis and subsequent tumor size regression. This approach may offer new possibilities to improve the resectability of patients diagnosed with BR and LA PDAC.
RESUMEN
Cold storage of kidneys before transplantation is problematic because of the limited survival time of the allografts. In this study, zinc-N-acetylcysteine (ZnNAC) was shown to be a potent endonuclease inhibitor and antioxidant, and it was tested as a potential additive to a cold storage solution for kidney preservation. Exposure of normal rat kidney NRK-52E cells to ZnNAC resulted in zinc delivery to the cells as determined by TFL-Zn fluorophore and partial protection of the cells against injury by cold storage in University of Wisconsin solution (UWS) as measured by propidium iodide assay. Ex vivo, rat kidneys demonstrated time- and temperature-dependent DNA fragmentation as assessed by TUNEL assay, indicating irreversible cell death. DNA fragmentation was faster in the medulla than in the cortex, and tubules were affected more than glomeruli. Perfusion of rat kidneys with cold ZnNAC solution in UWS significantly inhibited cell death both in the cortex and medulla at concentrations of 0.3-30 mM compared with UWS alone, with a maximum effect at 1-10 mM ZnNAC. Cold storage of the kidney significantly increased quantities of cleaved caspase-3 and endonuclease G (EndoG) in the tissue, which were abolished by 10 mM ZnNAC, indicating its ability to suppress both caspase-dependent and -independent cell death. Therefore, supplementation of UWS with ZnNAC can decrease DNA fragmentation and protect kidney allografts from cell death due to cold storage.
Asunto(s)
Acetilcisteína/farmacología , Riñón/efectos de los fármacos , Preservación de Órganos , Acetilcisteína/química , Animales , Antioxidantes/análisis , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Fragmentación del ADN/efectos de los fármacos , Endonucleasas/antagonistas & inhibidores , Células Epiteliales/enzimología , Etiquetado Corte-Fin in Situ , Riñón/enzimología , Masculino , Soluciones Preservantes de Órganos/química , Ratas , Ratas Sprague-Dawley , Refrigeración , Acetato de Zinc/químicaRESUMEN
The high cardiovascular morbidity and mortality associated with chronic kidney disease (CKD) cannot be explained entirely by traditional risk factors. Urea spontaneously dissociates to form cyanate, which modifies proteins in a process referred to as carbamylation. Carbamylated low-density lipoprotein (cLDL) has been shown to have all of the major biological effects relevant to atherosclerosis, including endothelial cell injury, increased expression of cell adhesion molecules, and vascular smooth muscle cell proliferation. Recent studies indicate that cLDL leads to endonuclease G activation, which participates in cellular injury. In addition, cLDL has been shown to enhance generation of oxidants. Limited human data have demonstrated high levels of cLDL in hemodialysis patients, with the highest levels in patients who have atherosclerosis. In 2 separate clinical studies, plasma levels of carbamylated protein independently predicted an increased risk of coronary artery disease, future myocardial infarction, stroke, and death. Future prospective studies to examine the association and/or predictive value of cLDL and studies to establish cause-effect relationship in patients with CKD are needed.
Asunto(s)
Enfermedades Cardiovasculares/etiología , Fallo Renal Crónico/complicaciones , Lipoproteínas LDL/sangre , Animales , Aterosclerosis/etiología , Enfermedades Cardiovasculares/sangre , Humanos , Fallo Renal Crónico/sangre , Fallo Renal Crónico/terapia , Lipoproteínas LDL/fisiología , Diálisis Renal , Factores de RiesgoRESUMEN
Exposure to high doses of radiation, accidental or therapeutic, often results in gastrointestinal (GI) injury. To date, there are no therapies available to mitigate GI injury after radiation exposure. Gamma-tocotrienol (GT3) is a promising radioprotector under investigation in nonhuman primates (NHP). We have shown that GT3 has radioprotective function in intestinal epithelial and crypt cells in NHPs exposed to 12 Gy total-body irradiation (TBI). Here, we determined GT3 potential in accelerating the GI recovery in partial-body irradiated (PBI) NHPs using X-rays, sparing 5% bone marrow. Sixteen rhesus macaques were treated with either vehicle or GT3 24 h prior to 12 Gy PBI. Structural injuries and crypt survival were examined in proximal jejunum on days 4 and 7. Plasma citrulline was assessed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Crypt cell proliferation and apoptotic cell death were evaluated using Ki-67 and TUNEL staining. PBI significantly decreased mucosal surface area and reduced villous height. Interestingly, GT3 increased crypt survival and enhanced stem cell proliferation at day 4; however, the effects seemed to be minimized by day 7. GT3 did not ameliorate a radiation-induced decrease in citrulline levels. These data suggest that X-rays induce severe intestinal injury post-PBI and that GT3 has minimal radioprotective effect in this novel model.
RESUMEN
Carbon-based nanomaterials (CBNs) are often used for potential agricultural applications. Since CBNs applied to plants can easily enter plant organs and reach the human diet, the consequences of the introduction of CBNs into the food chain need to be investigated. We created a platform for a comprehensive investigation of the possible health risks of multiwalled carbon nanotubes (CNTs) accumulated in the organs of exposed tomato plants. Quantification and visualization of CNTs absorbed by plant organs were determined by microwave-induced heating (MIH) and radio frequency (RF) heating methods. Feeding mice with CNT-contaminated tomatoes showed an absence of toxicity for all assessed animal organs. The amount of CNTs accumulated inside the organs of mice fed with CNT-containing fruits was assessed by an RF heating technique and was found to be negligible. Our work provides the experimental evidence that the amount of CNTs accumulated in plant organs as a result of nanofertilization is not sufficient to induce toxicity in mice.
Asunto(s)
Nanotubos de Carbono , Solanum lycopersicum , Humanos , Ratones , Animales , Nanotubos de Carbono/toxicidad , Plantas , Agricultura , Medición de RiesgoRESUMEN
Mice with the type I interferon (IFN) receptor gene knocked out (IFNAR KO mice) or deficient for alpha/beta IFN (IFN-α/ß) signaling clear chlamydial infection earlier than control mice and develop less oviduct pathology. Initiation of host IFN-ß transcription during an in vitro chlamydial infection requires interferon regulatory transcription factor 3 (IRF3). The goal of the present study was to characterize the influence of IRF3 on chlamydial genital infection and its relationship to IFN-ß expression in the mouse model. IRF3 KO mice were able to resolve infection as well as control mice, overcoming increased chlamydial colonization and tissue burden early during infection. As previously observed for IFNAR KO mice, IRF3 KO mice generated a potent antigen-specific T cell response. However, in contrast to IFNAR KO mice, IRF3 KO mice exhibited unusually severe dilatation and pathology in the uterine horns but normal oviduct pathology after infection. Although IFN-ß expression in vivo was dependent on the presence of IRF3 early in infection (before day 4), the IFN-independent function of IRF3 was likely driving this phenotype. Specifically, early during infection, the number of apoptotic cells and the number of inflammatory cells were significantly less in uterine horns from IRF3 KO mice than in those from control mice, despite an increased chlamydial burden. To delineate the effects of IFN-ß versus IRF3, neutralizing IFN-ß antibody was administered to wild-type (WT) mice during chlamydial infection. IFN-ß depletion in WT mice mimicked that in IFNΑR KO mice but not that in IRF3 KO mice with respect to both chlamydial clearance and reduced oviduct pathology. These data suggest that IRF3 has a role in protection from uterine horn pathology that is independent of its function in IFN-ß expression.
Asunto(s)
Infecciones por Chlamydia/inmunología , Chlamydia muridarum/inmunología , Enfermedades de los Genitales Femeninos/inmunología , Factor 3 Regulador del Interferón/inmunología , Útero/patología , Animales , Infecciones por Chlamydia/microbiología , Chlamydia muridarum/patogenicidad , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Enfermedades de los Genitales Femeninos/microbiología , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/genética , Interferón beta/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Linfocitos T/inmunología , Útero/microbiologíaRESUMEN
Male Sprague-Dawley rats were chronically fed a high-unsaturated-fat diet for 130 days by using total enteral nutrition (TEN), or the same diet in which ethanol (EtOH) isocalorically replaced carbohydrate calories. Additional groups were supplemented with the antioxidant N-acetylcysteine (NAC) at 1.7 g·kg(-1)·day(-1). Relative to an ad libitum chow-fed group, the high-fat-fed controls had three- to fourfold greater expression of fatty acid transporter CD36 mRNA and developed mild steatosis but little other hepatic pathology. NAC treatment resulted in increased somatic growth relative to controls (4.0 ± 0.1 vs. 3.1 ± 0.1 g/day) and increased hepatic steatosis score (3.5 ± 0.6 vs. 2.7 ± 1.2), associated with suppression of the triglyceride hydrolyzing protein adiponutrin, but produced no elevation in serum alanine aminotransferase (ALT). Chronic EtOH treatment increased expression of fatty acid transport protein FATP-2 mRNA twofold, resulting in marked hepatic steatosis, oxidative stress, and a twofold elevation in serum ALT. However, no changes in tumor necrosis factor-α or transforming growth factor-ß expression were observed. Fibrosis, as measured by Masson's trichrome and picrosirius red staining, and a twofold increase in expression of type I and type III collagen mRNA, was only observed after EtOH treatment. Long-term EtOH treatment increased hepatocyte proliferation but did not modify the hepatic mRNAs for hedgehog pathway ligands or target genes or genes regulating epithelial-to-mesenchymal transition. Although the effects of NAC on EtOH-induced fibrosis could not be fully evaluated, NAC had additive effects on hepatocyte proliferation and prevented EtOH-induced oxidative stress and necrosis, despite a failure to reverse hepatic steatosis.
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Etanol/toxicidad , Hígado Graso/etiología , Hepatopatías Alcohólicas/metabolismo , Hígado/efectos de los fármacos , Acetilcisteína/farmacología , Acetilcisteína/uso terapéutico , Animales , Proliferación Celular/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Grasas de la Dieta/administración & dosificación , Nutrición Enteral , Etanol/farmacología , Hígado Graso/patología , Proteínas Hedgehog/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/patología , Hepatopatías Alcohólicas/patología , Regeneración Hepática/efectos de los fármacos , Masculino , Estrés Oxidativo/efectos de los fármacos , RatasRESUMEN
End-stage kidney disease is a terminal stage of chronic kidney disease, which is associated with a high incidence of cardiovascular disease. Cardiovascular disease frequently results from endothelial injury caused by carbamylated LDL (cLDL), the product of LDL modification by urea-derived cyanate. Our previous data suggested that cLDL induces mitogen-activated protein kinase-dependent mitotic DNA fragmentation and cell death. However, the mechanism of this pathway is unknown. The current study demonstrated that cLDL-induced endothelial mitotic cell death is independent of caspase-3. The expression of endonuclease G (EndoG), the nuclease implicated in caspase-independent DNA fragmentation, was significantly increased in response to cLDL exposure to the cells. The inhibition of EndoG by RNAi protected cLDL-induced DNA fragmentation, whereas the overexpression of EndoG induced more DNA fragmentation in endothelial cells. Ex vivo experiments with primary endothelial cells isolated from wild-type (WT) and EndoG knockout (KO) mice demonstrated that EndoG KO cells are partially protected against cLDL toxicity compared with WT cells. To determine cLDL toxicity in vivo, we administered cLDL or native LDL (nLDL) intravenously to the WT and EndoG KO mice and then measured floating endothelial cells in blood using flow cytometry. The results showed an increased number of floating endothelial cells after cLDL versus nLDL injection in WT mice but not in EndoG KO mice. Finally, the inhibitors of MEK-ERK1/2 and JNK-c-jun pathways decreased cLDL-induced EndoG overexpression and DNA fragmentation. In summary, our data suggest that cLDL-induced endothelial toxicity is caspase independent and results from EndoG-dependent DNA fragmentation.
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Apoptosis/efectos de los fármacos , Vasos Coronarios/patología , Endodesoxirribonucleasas/fisiología , Endotelio Vascular/patología , Lipoproteínas LDL/farmacología , Animales , Apoptosis/fisiología , Células Cultivadas , Vasos Coronarios/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Endodesoxirribonucleasas/genética , Endotelio Vascular/efectos de los fármacos , Humanos , MAP Quinasa Quinasa 4/antagonistas & inhibidores , MAP Quinasa Quinasa 4/fisiología , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Modelos Animales , Transducción de Señal/fisiologíaRESUMEN
Carbamylated LDL (cLDL) is a potential atherogenic factor in chronic kidney disease (CKD). However, whether elevated plasma cLDL associates with atherosclerosis in vivo is unknown. Here, we induced CKD surgically in apolipoprotein E-deficient (ApoE(-/-)) mice fed a high-fat diet to promote the development of atherosclerosis. These mice had two- to threefold higher plasma levels of both oxidized LDL (oxLDL) and cLDL compared with control mice. Oral administration of urea increased cLDL approximately eightfold in ApoE(-/-) mice subjected to unilateral nephrectomy and a high-fat diet, but oxLDL did not rise. Regardless of the model, the uremic mice with high plasma cLDL had more severe atherosclerosis as measured by intravital ultrasound echography and en face aortic staining of lipid deposits. Furthermore, cLDL accumulated in the aortic wall and colocalized with ICAM-1 and macrophage infiltration. In summary, these data demonstrate that elevated plasma cLDL may represent an independent risk factor for uremia-induced atherosclerosis.
Asunto(s)
Aterosclerosis/sangre , Aterosclerosis/epidemiología , Enfermedades Renales/complicaciones , Lipoproteínas LDL/sangre , Uremia/complicaciones , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/inducido químicamente , Enfermedad Crónica , Grasas de la Dieta/efectos adversos , Modelos Animales de Enfermedad , Molécula 1 de Adhesión Intercelular/sangre , Enfermedades Renales/sangre , Enfermedades Renales/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nefrectomía/efectos adversos , Factores de Riesgo , Urea/efectos adversos , Uremia/sangre , Uremia/inducido químicamenteRESUMEN
The discovery of chimeric anti-melanoma agents is reported. These molecules are potent growth suppressors of melanoma cells in vitro with growth inhibition of 50% (GI50) values as low as 1.32 µM. Compounds were more toxic to melanoma cells in vitro than commonly used anti-melanoma agent dacarbazine as measured by TUNEL assay. They induced both caspase-independent apoptosis evident by colocalization of TUNEL with endonuclease G (EndoG) and caspase-mediated apoptosis measured by colocalization of TUNEL with caspase-activated DNase (CAD). In addition, compounds 3 and 5 strongly induced oxidative injury to melanoma cells as measured by TUNEL colocalization with heme oxygenase-1 (HO1). Dacarbazine induced only caspase-independent apoptosis, which may explain why it is less cytotoxic to melanoma cells than compounds 3, 4 and 5.
RESUMEN
Endonuclease G (ENDOG), a mitochondrial nuclease, is known to participate in many cellular processes, including apoptosis and paternal mitochondrial elimination, while its role in autophagy remains unclear. Here, we report that ENDOG released from mitochondria promotes autophagy during starvation, which we find to be evolutionally conserved across species by performing experiments in human cell lines, mice, Drosophila and C. elegans. Under starvation, Glycogen synthase kinase 3 beta-mediated phosphorylation of ENDOG at Thr-128 and Ser-288 enhances its interaction with 14-3-3γ, which leads to the release of Tuberin (TSC2) and Phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34) from 14-3-3γ, followed by mTOR pathway suppression and autophagy initiation. Alternatively, ENDOG activates DNA damage response and triggers autophagy through its endonuclease activity. Our results demonstrate that ENDOG is a crucial regulator of autophagy, manifested by phosphorylation-mediated interaction with 14-3-3γ, and its endonuclease activity-mediated DNA damage response.