RESUMEN
Toxicity mechanisms of metal oxide nanoparticles towards bacteria and underlying roles of membrane composition are still debated. Herein, the response of lipopolysaccharide-truncated Escherichia coli K12 mutants to TiO2 nanoparticles (TiO2NPs, exposure in dark) is addressed at the molecular, single cell, and population levels by transcriptomics, fluorescence assays, cell nanomechanics and electrohydrodynamics. We show that outer core-free lipopolysaccharides featuring intact inner core increase cell sensitivity to TiO2NPs. TiO2NPs operate as membrane strippers, which induce osmotic stress, inactivate cell osmoregulation and initiate lipid peroxidation, which ultimately leads to genesis of membrane vesicles. In itself, truncation of lipopolysaccharide inner core triggers membrane permeabilization/depolarization, lipid peroxidation and hypervesiculation. In turn, it favors the regulation of TiO2NP-mediated changes in cell Turgor stress and leads to efficient vesicle-facilitated release of damaged membrane components. Remarkably, vesicles further act as electrostatic baits for TiO2NPs, thereby mitigating TiO2NPs toxicity. Altogether, we highlight antagonistic lipopolysaccharide-dependent bacterial responses to nanoparticles and we show that the destabilized membrane can generate unexpected resistance phenotype.
Asunto(s)
Vesículas Citoplasmáticas/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Nanopartículas del Metal/toxicidad , Presión Osmótica/efectos de los fármacos , Titanio/toxicidad , Vesículas Citoplasmáticas/metabolismo , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Microscopía de Fuerza Atómica/métodos , MutaciónRESUMEN
The vector-borne flaviviruses (VBFVs) are well known for causing great misery and death in humans worldwide. The VBFVs include those transmitted by mosquitos, such as Zika virus (ZIKV), dengue virus; and those transmitted by ticks including the tick-borne flavivirus serocomplex and Powassan virus (POWV). Two of our recent reports showed that intracranial POWV infection in the reservoir host, Peromyscus leucopus, was restricted and caused no overt clinical disease. Several modes of analyses suggested activation of the LXR pathway. Activation of the LXR pathway leads to increased efflux of cholesterol from cells and consequent disturbances in membrane biogenesis. Because VBFV replication is dependent on membrane biogenesis, we evaluated the effect of an LXR agonist (LXR623) on POWV and ZIKV infection and observed that the compound impaired permissive replication of both viruses in a human neuroblastoma SK-N-SH cell line. The LXR agonist resulted in failure of the viruses to induce ER expansion and elaborate vesicle formation, suggesting that the efflux of cholesterol was part of the antiviral mechanism. We also observed that the LXR agonist contributed to the mechanism of virus suppression by increased expression of mRNAs encoding for the antiviral cytokines CXCL10, RANTES and IFN1ß. In sharp contrast, a LXR antagonist (GSK2033) had no significant effect on VBFV replication. We conclude that LXR623 impairs flavivirus replication by stimulating cellular antiviral factors.
Asunto(s)
Virus de la Encefalitis Transmitidos por Garrapatas/efectos de los fármacos , Indazoles/farmacología , Receptores X del Hígado/agonistas , Virus Zika/efectos de los fármacos , Antivirales/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Efecto Citopatogénico Viral/efectos de los fármacos , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , Virus de la Encefalitis Transmitidos por Garrapatas/fisiología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Humanos , Receptores X del Hígado/metabolismo , Replicación Viral/efectos de los fármacos , Virus Zika/fisiologíaRESUMEN
The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro; in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways.
Asunto(s)
Antivirales/farmacología , G-Cuádruplex/efectos de los fármacos , Herpesvirus Humano 1/efectos de los fármacos , Porfirinas/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/química , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , ADN Viral/química , ADN Viral/efectos de los fármacos , Herpesvirus Humano 1/fisiología , Ligandos , Estructura Molecular , Porfirinas/química , Células Vero , Virión/efectos de los fármacos , Virión/metabolismoAsunto(s)
Movimiento Celular/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , Fibroblastos/metabolismo , Fosfohidrolasa PTEN/metabolismo , ARN Mensajero/metabolismo , Animales , Línea Celular , Vesículas Citoplasmáticas/efectos de los fármacos , Ratones , Transporte de Proteínas/efectos de los fármacos , Ribonucleasas/farmacologíaRESUMEN
Drosophila nephrocytes share functional, structural and molecular similarities with human podocytes. It is known that podocytes express the rabphilin 3A (RPH3A)-RAB3A complex, and its expression is altered in mouse and human proteinuric disease. Furthermore, we previously identified a polymorphism that suggested a role for RPH3A protein in the development of urinary albumin excretion. As endocytosis and vesicle trafficking are fundamental pathways for nephrocytes, the objective of this study was to assess the role of the RPH3A orthologue in Drosophila, Rabphilin (Rph), in the structure and function of nephrocytes. We confirmed that Rph is required for the correct function of the endocytic pathway in pericardial Drosophila nephrocytes. Knockdown of Rph reduced the expression of the cubilin and stick and stones genes, which encode proteins that are involved in protein uptake and filtration. We also found that reduced Rph expression resulted in a disappearance of the labyrinthine channel structure and a reduction in the number of endosomes, which ultimately leads to changes in the number and volume of nephrocytes. Finally, we demonstrated that the administration of retinoic acid to IR-Rph nephrocytes rescued some altered aspects, such as filtration and molecular uptake, as well as the maintenance of cell fate. According to our data, Rph is crucial for nephrocyte filtration and reabsorption, and it is required for the maintenance of the ultrastructure, integrity and differentiation of the nephrocyte.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Podocitos/metabolismo , Animales , Linaje de la Célula/efectos de los fármacos , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , Drosophila melanogaster/efectos de los fármacos , Endocitosis/efectos de los fármacos , Femenino , Humanos , Larva/citología , Larva/efectos de los fármacos , Podocitos/citología , Podocitos/efectos de los fármacos , Podocitos/ultraestructura , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , Nitrato de Plata/toxicidad , Análisis de Supervivencia , Tretinoina/metabolismoRESUMEN
The demonstrated expression of endocannabinoid receptors in myofascial tissue suggested the role of fascia as a source and modulator of pain. Fibroblasts can modulate the production of the various components of the extracellular matrix, according to type of stimuli: physical, mechanical, hormonal, and pharmacological. In this work, fascial fibroblasts were isolated from small samples of human fascia lata of the thigh, collected from three volunteer patients (two men, one woman) during orthopedic surgery. This text demonstrates for the first time that the agonist of cannabinoid receptor 2, HU-308, can lead to in vitro production of hyaluronan-rich vesicles only 3-4 h after treatment, being rapidly released into the extracellular environment. We demonstrated that these vesicles are rich in hyaluronan after Alcian blue and Toluidine blue stainings, immunocytochemistry, and transmission electron microscopy. In addition, incubation with the antagonist AM630 blocked vesicles production by cells, confirming that release of hyaluronan is a cannabinoid-mediated effect. These results may show how fascial cells respond to the endocannabinoid system by regulating and remodeling the formation of the extracellular matrix. This is a first step in our understanding of how therapeutic applications of cannabinoids to treat pain may also have a peripheral effect, altering the biosynthesis of the extracellular matrix in fasciae and, consequently, remodeling the tissue and its properties.
Asunto(s)
Endocannabinoides/farmacología , Fascia/efectos de los fármacos , Fascia/metabolismo , Anciano , Biomarcadores , Cannabinoides/farmacología , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Humanos , Ácido Hialurónico/metabolismo , Inmunohistoquímica , MasculinoRESUMEN
Alveolar rhabdomyosarcoma (aRMS) is a highly malicious childhood malignancy characterized by specific chromosomal translocations mostly encoding the oncogenic transcription factor PAX3-FOXO1 and therefore also referred to as fusion-positive RMS (FP-RMS). Previously, we have identified fenretinide (retinoic acid p-hydroxyanilide) to affect PAX3-FOXO1 expression levels as well as FP-RMS cell viability. Here, we characterize the mode of action of fenretinide in more detail. First, we demonstrate that fenretinide-induced generation of reactive oxygen species (ROS) depends on complex II of the mitochondrial respiratory chain, since ROS scavenging as well as complexing of iron completely abolished cell death. Second, we co-treated cells with a range of pharmacological inhibitors of specific cell death pathways including z-vad (apoptosis), necrostatin-1 (necroptosis), 3-methyladenine (3-MA) (autophagy), and ferrostatin-1 (ferroptosis) together with fenretinide. Surprisingly, none of these inhibitors was able to prevent cell death. Also genetic depletion of key players in the apoptotic and necroptotic pathway (BAK, BAX, and RIPK1) confirmed the pharmacological data. Interestingly however, electron microscopy of fenretinide-treated cells revealed an excessive accumulation of cytoplasmic vacuoles, which were distinct from autophagosomes. Further flow cytometry and fluorescence microscopy experiments suggested a hyperstimulation of macropinocytosis, leading to an accumulation of enlarged early and late endosomes. Surprisingly, pharmacological inhibition as well as genetic depletion of large dynamin GTPases completely abolished fenretinide-induced vesicle formation and subsequent cell death, suggesting a new form of dynamin-dependent programmed cell death. Taken together, our data identify a new form of cell death mediated through the production of ROS by fenretinide treatment, highlighting the value of this compound for treatment of sarcoma patients including FP-RMS.
Asunto(s)
Dinaminas/metabolismo , Fenretinida/farmacología , Sarcoma/metabolismo , Sarcoma/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Niño , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , Dinaminas/genética , Complejo II de Transporte de Electrones/metabolismo , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Endosomas/ultraestructura , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sarcoma/genética , Sarcoma/ultraestructuraRESUMEN
AIMS: Nanoparticles (NPs)-based drugs have been recently introduced to improve the efficacy of current therapeutic strategies for the treatment of cancer; however, the molecular mechanisms by which a NP interacts with cellular systems still need to be delineated. Here, we utilize the autophagic potential of TiO2 NPs for improving chemotherapeutic effects of 5-fluorouracil (5-FU) in human AGS gastric cells. MATERIALS AND METHODS: Cell growth and viability were determined by trypan blue exclusion test and MTT assay, respectively. Vesicular organelles formation was evaluated by acridine orange staining of cells. Cell cycle and apoptosis were monitored by flow cytometry. Reactive oxygen species (ROS) level were measured by DCHF-DA staining. Autophagy was examined by q-PCR and western blotting. Molecular docking was used for studying NP interaction with autophagic proteins. KEY FINDINGS: TiO2 NPs increase ROS production, impair lysosomal function and subsequently block autophagy flux in AGS cells. In addition, the autophagy blockade induced by non-toxic concentrations of TiO2 NPs (1 µg/ml) can promote cytotoxic and apoptotic effects of 5-FU in AGS cells. SIGNIFICANCE: These results confirm the beneficial effects of TiO2 NPs in combination with chemotherapy in in vitro model of gastric cancer, which may pave the way to develop a possible solution to circumvent chemoresistance in cancer.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Fluorouracilo/farmacología , Regulación Neoplásica de la Expresión Génica , Nanopartículas/química , Titanio/farmacología , Antimetabolitos Antineoplásicos/química , Apoptosis/efectos de los fármacos , Apoptosis/genética , Autofagia/genética , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Beclina-1/genética , Beclina-1/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , Sinergismo Farmacológico , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fluorouracilo/química , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Simulación del Acoplamiento Molecular , Nanopartículas/ultraestructura , Conformación Proteica , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/agonistas , Especies Reactivas de Oxígeno/metabolismo , Proteína Sequestosoma-1/antagonistas & inhibidores , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Titanio/química , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Dysregulation of vesicle trafficking in muscle is one of the factors responsible for the pathogenesis of insulin resistance (IR). Ferulic acid (FER) and resveratrol (RSV) are known to have hypoglycemic property. In this study, differentiated L6 myotubes were induced with palmitate as a model of IR. Chemical ablation of muscle vesicles was used to investigate how FER and RSV influence glucose utilization. Results showed that both FER and RSV elicit glucose uptake and promote glycogen synthesis in insulin-resistant muscle cells. Mechanistic studies further showed that FER markedly enhances the transferrin receptor-containing endosomal compartment activities via phosphoinositide 3-kinase (PI3K)/atypical protein kinase C-dependent pathway, while RSV facilitates glucose transporter storage vesicles (GSV) trafficking via an exercise-like effect of conventional protein kinase C/5'-adenosine monophosphate-activated protein kinase (AMPK) modulation. Therefore, these two phenolic compounds promoted glucose transport through two separate routes, and they had an additive effect on the increase of glucose uptake in insulin-resistant muscle cells. These findings provide a basis for the understanding of the antidiabetic potential of RSV and FER combination.
Asunto(s)
Ácidos Cumáricos/farmacología , Vesículas Citoplasmáticas/efectos de los fármacos , Glucosa/metabolismo , Resistencia a la Insulina , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Resveratrol/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Vesículas Citoplasmáticas/metabolismo , Sinergismo Farmacológico , Metabolismo Energético/efectos de los fármacos , Células Hep G2 , Humanos , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , RatasRESUMEN
Cerebral choline metabolism is crucial for normal brain function, and its homoeostasis depends on carrier-mediated transport. Here, we report on four individuals from three families with neurodegenerative disease and homozygous frameshift mutations (Asp517Metfs*19, Ser126Metfs*8, and Lys90Metfs*18) in the SLC44A1 gene encoding choline transporter-like protein 1. Clinical features included progressive ataxia, tremor, cognitive decline, dysphagia, optic atrophy, dysarthria, as well as urinary and bowel incontinence. Brain MRI demonstrated cerebellar atrophy and leukoencephalopathy. Moreover, low signal intensity in globus pallidus with hyperintensive streaking and low signal intensity in substantia nigra were seen in two individuals. The Asp517Metfs*19 and Ser126Metfs*8 fibroblasts were structurally and functionally indistinguishable. The most prominent ultrastructural changes of the mutant fibroblasts were reduced presence of free ribosomes, the appearance of elongated endoplasmic reticulum and strikingly increased number of mitochondria and small vesicles. When chronically treated with choline, those characteristics disappeared and mutant ultrastructure resembled healthy control cells. Functional analysis revealed diminished choline transport yet the membrane phosphatidylcholine content remained unchanged. As part of the mechanism to preserve choline and phosphatidylcholine, choline transporter deficiency was implicated in impaired membrane homeostasis of other phospholipids. Choline treatments could restore the membrane lipids, repair cellular organelles and protect mutant cells from acute iron overload. In conclusion, we describe a novel childhood-onset neurometabolic disease caused by choline transporter deficiency with autosomal recessive inheritance.
Asunto(s)
Antígenos CD/genética , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Proteínas de Transporte de Catión Orgánico/genética , Adolescente , Ataxia/genética , Ataxia/fisiopatología , Atrofia , Cerebelo/diagnóstico por imagen , Cerebelo/patología , Colina/farmacología , Disfunción Cognitiva/genética , Disfunción Cognitiva/fisiopatología , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/ultraestructura , Trastornos de Deglución/genética , Trastornos de Deglución/fisiopatología , Disartria/genética , Disartria/fisiopatología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/ultraestructura , Incontinencia Fecal/genética , Incontinencia Fecal/fisiopatología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/ultraestructura , Mutación del Sistema de Lectura , Globo Pálido/diagnóstico por imagen , Trastornos Heredodegenerativos del Sistema Nervioso/diagnóstico por imagen , Trastornos Heredodegenerativos del Sistema Nervioso/patología , Trastornos Heredodegenerativos del Sistema Nervioso/fisiopatología , Homocigoto , Humanos , Leucoencefalopatías/diagnóstico por imagen , Leucoencefalopatías/genética , Leucoencefalopatías/fisiopatología , Imagen por Resonancia Magnética , Masculino , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Nootrópicos/farmacología , Atrofia Óptica/genética , Atrofia Óptica/fisiopatología , Linaje , Ribosomas/efectos de los fármacos , Ribosomas/ultraestructura , Sustancia Negra/diagnóstico por imagen , Síndrome , Temblor/genética , Temblor/fisiopatología , Incontinencia Urinaria/genética , Incontinencia Urinaria/fisiopatologíaRESUMEN
MicroRNAs plays important role in the development of myocardial infarction (MI). The aim of this study was to analyze whether miR-429 has effect on the process of autophagy in myocardial anoxia/reoxygenation (AR) or ischemia/reperfusion (IR) injury and explore the underlying mechanism. The results showed that miR-429 was significantly decreased in MI mouse hearts and AR treated cardiomyocytes. Dual luciferase activity assay proved that MO25 was the direct target of miR-429. MO25 was dramatically decreased in AR treated cardiomyocytes. Overexpression of miR-429 dramatically decreased the expression of MO25, whereas inhibition of miR-429 noticeably increased the expression of MO25. In addition, overexpression of miR-429 reduced GFP-LC3B labelled cells, decreased the number of vesicle and autophagosome in each cardiomyocyte, and induced cell apoptosis in AR treated cardiomyocytes. In contrast, inhibition of miR-429 had the opposite effect. The further in vivo study showed that when mouse in IR group were injected with antagomiR-429, the weight of left ventricular was increased and infarct size was significantly decreased. Finally, both the in vitro and in vivo study showed that the expression of MO25, LKB1, pAMPKa, ATG13, p62 and LC3BI/II was noticeably increased by antagomiR-429. In conclusion, our results suggested that antagonism of miR-429 ameliorates anoxia/reoxygenation injury in cardiomyocytes by enhancing MO25/LKB1/AMPK mediated autophagy.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia/efectos de los fármacos , Proteínas de Unión al Calcio/metabolismo , Hipoxia/metabolismo , MicroARNs/antagonistas & inhibidores , Miocitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Antagomirs/farmacología , Antagomirs/uso terapéutico , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagosomas/efectos de los fármacos , Recuento de Células , Vesículas Citoplasmáticas/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Ratones , MicroARNs/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Daño por Reperfusión Miocárdica , Miocardio/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismoRESUMEN
Previous work with isolated outer membrane vesicles of lobster branchiostegite epithelial cells has shown that 45Ca2+ uptake by these structures is significantly (p < 0.02) reduced by an incremental decrease in saline pH (increased proton concentration) and that this decrease is due to competitive inhibition between carrier-mediated transport of 45Ca2+ and hydrogen ions. The present paper extends these previous findings and describes the combined effects of pH and cationic heavy metals on branchiostegite uptake of 45Ca2+. Partially purified membrane vesicles of branchiostegite cells were produced by a homogenization/centrifugation method and were loaded with mannitol at pH 7.0. The time course of 1 mM 45Ca2+ uptake in a mannitol medium at pH 8.5 containing 100 µM verapamil (Ca2+ channel blocker) was hyperbolic and approached equilibrium at 30 min. This uptake was either significantly reduced (p < 0.05) by the addition of 5 µM Zn2+ or essentially abolished with the addition of 5 µM Cu2+. Increasing zinc concentrations (5-500 µM) reduced 1 mM 45Ca2+ uptake at pH 8.5 or 7.5 in a hyperbolic fashion with the remaining non-inhibited uptake due to apparent non-specific binding. Uptake of 1 mM 45Ca2+ at pH 8.5, 7.5, 7.5 + Zn2+, and 7.5 + Zn2+ + Cu2+ + Cd2+ in the presence of 100 µM verapamil displayed a stepwise reduction of 45Ca2+ uptake with the addition of each treatment until only non-specific isotope binding occurred with all cation inhibitors. 45Ca2+ influxes (15 s uptakes; 0.25-5.0 mM calcium + 100 µM verapamil) in the presence and absence of 10 µM Zn2+ were both hyperbolic functions of calcium concentration. The curve with Zn2+ displayed a transport Km twice that of the control (p < 0.05), while inhibitor and control curve Jmax values were not significantly different (p > 0.05), suggesting competitive inhibition between 45Ca2+ and Zn2+ influxes. Analysis of the relative inhibitory effects of increased proton or heavy metal interaction with 45Ca2+ uptake suggests that divalent metals may reduce the calcium transport about twice as much as a drop in pH, but together, they appear to abolish carrier-mediated transport.
Asunto(s)
Radioisótopos de Calcio/farmacocinética , Vesículas Citoplasmáticas/efectos de los fármacos , Metales Pesados/farmacología , Nephropidae/metabolismo , Protones , Agua de Mar/química , Contaminantes Químicos del Agua/farmacología , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Células Epiteliales/metabolismoRESUMEN
Dengue fever is one of the most important mosquito-borne viral infections in large parts of tropical and subtropical countries and is a significant public health concern and socioeconomic burden. There is an urgent need to develop antivirals that can effectively reduce dengue virus (DENV) replication and decrease viral load. Niclosamide, an antiparasitic drug approved for human use, has been recently identified as an effective antiviral agent against a number of pH-dependent viruses, including flaviviruses. Here, we reveal that neutralization of low-pH intracellular compartments by niclosamide affects multiple steps of the DENV infectious cycle. Specifically, niclosamide-induced endosomal neutralization not only prevents viral RNA replication but also affects the maturation of DENV particles, rendering them non-infectious. We found that niclosamide-induced endosomal neutralization prevented E glycoprotein conformational changes on the virion surface of flaviviruses, resulting in the release of non-infectious immature virus particles with uncleaved pr peptide from host cells. Collectively, our findings support the potential application of niclosamide as an antiviral agent against flavivirus infection and highlight a previously uncharacterized mechanism of action of the drug.
Asunto(s)
Vesículas Citoplasmáticas/efectos de los fármacos , Virus del Dengue/efectos de los fármacos , Endosomas/efectos de los fármacos , Espacio Intracelular/efectos de los fármacos , Niclosamida/farmacología , Animales , Antivirales/farmacología , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Vesículas Citoplasmáticas/química , Vesículas Citoplasmáticas/virología , Virus del Dengue/genética , Virus del Dengue/crecimiento & desarrollo , Endosomas/química , Endosomas/virología , Humanos , Concentración de Iones de Hidrógeno , Espacio Intracelular/química , Espacio Intracelular/virología , Estadios del Ciclo de Vida/efectos de los fármacos , Células Vero , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Virión/efectos de los fármacos , Virión/genética , Virión/crecimiento & desarrollo , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
Presently, curcumin derivatives had been paid more attention in view of their high bioavailability or water solubility, which herein possibly replaced the curcumin for their functional applications in future. Here, one novel chemically synthesized curcumin derivative, ZYX01, was used to identify anti-proliferation activity of human non-small lung cancer cells A549 and its anti-proliferative mechanism. Our study showed that ZYX01 could induce autophagic death of A549 cells by morphological observation, MTT assay, acridine orange staining and MDC assay, which possess a dose-and time-dependent manner. ZYX01-treated A549 cells possessed an increase in LC3-II/LC3-I ratio, upregulation of beclin-1 and downregulation of p62 expression. We further confirmed the cellular AMPK/ULK1/Beclin-1 signaling pathway in A549 cells after ZYX01 treatment. The anti-migration effect of ZYX01 in A549 cells was also explored by wound healing assay and transwell experiment. Current results had confirmed that ZYX01 induced A549 cells autophagy through AMPK/ULK1/Beclin-1 pathway and shed light on the future study on the anti-cancer molecular mechanism.
Asunto(s)
Adenilato Quinasa/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia/efectos de los fármacos , Beclina-1/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Curcumina/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Pulmonares/patología , Transducción de Señal , Células A549 , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Curcumina/química , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , Humanos , Neoplasias Pulmonares/enzimología , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
Acamprosate, also known as N-acetyl homotaurine, is an N-methyl-d-aspartate receptor antagonist that is used for treating alcohol dependence. Although the exact mechanism of acamprosate has not been clearly established, it appears to work by promoting a balance between the excitatory and inhibitory neurotransmitters, glutamate, and gamma-aminobutyric acid, respectively. Several studies have demonstrated that acamprosate provides neuroprotection against ischemia-induced brain injury. However, no studies have been performed evaluating the effect of acamprosate on traumatic brain injury (TBI). In the present study, we sought to evaluate the therapeutic potential of acamprosate to protect against neuronal death following TBI. Rats were given oral acamprosate (200 mg/kg/d for 2weeks) and then subjected to a controlled cortical impact injury localized over the parietal cortex. Histologic analysis was performed at 3hours, 24hours, and 7days after TBI. We found that acamprosate treatment reduced the concentration of vesicular glutamate and zinc in the hippocampus. Consequently, this reduced vesicular glutamate and zinc level resulted in a reduction of reactive oxygen species production after TBI. When evaluated 24hours after TBI, acamprosate administration reduced the number of degenerating neurons, zinc accumulation, blood-brain barrier disruption, neutrophil infiltration, and dendritic loss. Acamprosate also reduced glial activation and neuronal loss at 7days after TBI. In addition, acamprosate rescued TBI-induced neurologic and cognitive dysfunction. The present study demonstrates that acamprosate attenuates TBI-induced brain damage by depletion of vesicular glutamate and zinc levels. Therefore, this study suggests that acamprosate may have high therapeutic potential for prevention of TBI-induced neuronal death.
Asunto(s)
Acamprosato/uso terapéutico , Alcoholismo/tratamiento farmacológico , Lesiones Traumáticas del Encéfalo/patología , Lesiones Traumáticas del Encéfalo/prevención & control , Vesículas Citoplasmáticas/metabolismo , Neuronas/patología , Zinc/metabolismo , Acamprosato/farmacología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Lesiones Traumáticas del Encéfalo/fisiopatología , Muerte Celular/efectos de los fármacos , Cognición/efectos de los fármacos , Vesículas Citoplasmáticas/efectos de los fármacos , Dendritas/efectos de los fármacos , Dendritas/metabolismo , Dendritas/patología , Hipocampo/metabolismo , Masculino , Modelos Biológicos , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuroprotección/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Infiltración Neutrófila/efectos de los fármacos , Ratas Sprague-Dawley , Superóxidos/metabolismoRESUMEN
Astroglia, the primary homeostatic cells of the central nervous system, play an important role in neuroinflammation. They act as facultative immunocompetent antigen-presenting cells (APCs), expressing major histocompatibility complex (MHC) class II antigens upon activation with interferon (IFN)-γ and possibly other proinflammatory cytokines that are upregulated in disease states, including multiple sclerosis (MS). We characterized the anti-inflammatory effects of fingolimod (FTY720), an established drug for MS, and its phosphorylated metabolite (FTY720-P) in IFN-γ-activated cultured rat astrocytes. The expression of MHC class II compartments, ß2 adrenergic receptor (ADR-ß2), and nuclear factor kappa-light-chain enhancer of activated B cells subunit p65 (NF-κB p65) was quantified in immunofluorescence images acquired by laser scanning confocal microscopy. In addition, MHC class II-enriched endocytotic vesicles were labeled by fluorescent dextran and their mobility analyzed in astrocytes subjected to different treatments. FTY720 and FTY720-P treatment significantly reduced the number of IFN-γ-induced MHC class II compartments and substantially increased ADR-ß2 expression, which is otherwise small or absent in astrocytes in MS. These effects could be partially attributed to the observed decrease in NF-κB p65 expression, because the NF-κB signaling cascade is activated in inflammatory processes. We also found attenuated trafficking and secretion from dextran-labeled endo-/lysosomes that may hinder efficient delivery of MHC class II molecules to the plasma membrane. Our data suggest that FTY720 and FTY720-P at submicromolar concentrations mediate anti-inflammatory effects on astrocytes by suppressing their action as APCs, which may further downregulate the inflammatory process in the brain, constituting the therapeutic effect of fingolimod in MS.
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Astrocitos/patología , Clorhidrato de Fingolimod/uso terapéutico , Inflamación/tratamiento farmacológico , Inflamación/patología , Interferón gamma/farmacología , Adenosina Trifosfato/farmacología , Animales , Astrocitos/efectos de los fármacos , Recuento de Células , Células Cultivadas , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , Dextranos/metabolismo , Femenino , Clorhidrato de Fingolimod/farmacología , Antígenos de Histocompatibilidad Clase II/metabolismo , Ratas Wistar , Receptores Adrenérgicos beta 2/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
Proteins and lipids of the plasma membrane underlie constant remodeling via a combination of the secretory- and the endocytic pathway. In the yeast endocytic pathway, cargo is sorted for recycling to the plasma membrane or degradation in vacuoles. Previously we have shown a role for the GARP complex in sphingolipid sorting and homeostasis (Fröhlich et al. 2015). However, the majority of cargo sorted in a GARP dependent process remain largely unknown. Here we use auxin induced degradation of GARP combined with mass spectrometry based vacuolar proteomics and lipidomics to show that recycling of two specific groups of proteins, the amino-phospholipid flippases and cell wall synthesis proteins depends on a functional GARP complex. Our results suggest that mis-sorting of flippases and remodeling of the lipid composition are the first occurring defects in GARP mutants. Our assay can be adapted to systematically map cargo of the entire endocytic pathway.
Asunto(s)
Endosomas/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Ácidos Indolacéticos/farmacología , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Vacuolas/efectos de los fármacos , Proteínas de Transporte Vesicular/genética , Transporte Biológico/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , Endocitosis/efectos de los fármacos , Endocitosis/genética , Endosomas/metabolismo , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Lipidómica/métodos , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteolisis , Proteómica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esfingolípidos/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/deficienciaRESUMEN
Bacterial membrane vesicle research has so far focused mainly on Gram-negative bacteria. Only recently have Gram-positive bacteria been demonstrated to produce and release extracellular membrane vesicles (MVs) that contribute to bacterial virulence. Although treatment of bacteria with antibiotics is a well-established trigger of bacterial MV formation, the underlying mechanisms are poorly understood. In this study, we show that antibiotics can induce MVs through different routes in the important human pathogen Staphylococcus aureus DNA-damaging agents and antibiotics inducing the SOS response triggered vesicle formation in lysogenic strains of S. aureus but not in their phage-devoid counterparts. The ß-lactam antibiotics flucloxacillin and ceftaroline increased vesicle formation in a prophage-independent manner by weakening the peptidoglycan layer. We present evidence that the amount of DNA associated with MVs formed by phage lysis is greater than that for MVs formed by ß-lactam antibiotic-induced blebbing. The purified MVs derived from S. aureus protected the bacteria from challenge with daptomycin, a membrane-targeting antibiotic, both in vitro and ex vivo in whole blood. In addition, the MVs protected S. aureus from killing in whole blood, indicating that antibiotic-induced MVs function as a decoy and thereby contribute to the survival of the bacterium.
Asunto(s)
Antibacterianos/farmacología , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/virología , Lisogenia/fisiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/virología , Bacteriófagos/fisiología , Cefalosporinas/farmacología , ADN Bacteriano/efectos de los fármacos , ADN Bacteriano/genética , Daptomicina/farmacología , Floxacilina/farmacología , Humanos , Lisogenia/genética , Peptidoglicano/efectos de los fármacos , CeftarolinaRESUMEN
Once infected by HIV-1, microglia abundantly produce accessory protein Nef that enhances virus production and infectivity, but little is known about its intracellular compartmentalization, trafficking mode(s), and release from microglia. Here, we transfected immortalized human microglia with a plasmid encoding Nef tagged with green fluorescent protein (Nef.GFP) to biochemically and microscopically identify Nef.GFP-associated cellular compartments and examine their mobility and Nef release from cultured cells. Immunoblotting revealed that Nef.GFP confined to subcellular fractions with a buoyant density similar to organelles positive for lysosomal-associated membrane protein 1 (LAMP1) but structurally segregated from dextran-laden and LysoTracker-laden endo-/lysosomes in live cells. As revealed by confocal microscopy, Nef.GFP-positive vesicle-like structures were smaller than dextran-laden vesicles and displayed slow and non-directional mobility, in contrast to the faster and directional mobility of dextran-laden vesicles. Ionomycin-evoked elevation in intracellular free Ca2+ concentration ([Ca2+]i) negligibly affected mobility of Nef.GFP structures but strongly and irrecoverably attenuated mobility of dextran-laden vesicles. A slow time-dependent decrease in the number of Nef.GFP-positive structures was observed in non-stimulated controls (5 ± 1 structures/min), but not in ionomycin-stimulated cells (0 ± 2 structures/min; P < 0.05), indicating that elevated [Ca2+]i inhibits the release of Nef.GFP structures. The latter significantly co-localized with membrane sites immunopositive for the tetraspanins CD9 (36 ± 4%) and CD81 (22 ± 1%). This is the first report to demonstrate that microglial CD9- and CD81-positive plasma membrane-derived compartments are associated with biogenesis and Nef release.
Asunto(s)
Calcio/metabolismo , Vesículas Citoplasmáticas/metabolismo , Citosol/metabolismo , Microglía/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Recuento de Células , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Vesículas Citoplasmáticas/efectos de los fármacos , Citosol/efectos de los fármacos , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ionomicina/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Microglía/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Tetraspanina 28/metabolismo , Tetraspanina 29/metabolismoRESUMEN
Single cell amperometry and intracellular vesicle impact electrochemical cytometry were used to examine whether lidocaine can regulate neurotransmitter release or storage for PC12 cells to explain the biphasic effects whereby it can protect neurons and improve cognitive outcome at low concentration, but can cause neurotoxicity at high concentration. We show that lidocaine affects the behavior of PC12 cell exocytosis in a concentration dependent way, which exactly corresponds to its biphasic effects. At a relatively high concentration, it shows a much narrower pore size and a longer-duration fusion pore with less monoamine released than control cells. However, at a relatively low concentration, the fusion pore is open even longer than at high concentration, and with more monoamine released than control cells. Furthermore, intracellular vesicle impact electrochemical cytometry was used to confirm that lidocaine did not change the catecholamine content of the vesicles. These data provide a mechanism for the observed biphasic effects of the drug and suggest that lidocaine influences exocytosis through multiple mechanisms.