RESUMEN
Propionic acid (PA) predominantly accumulates in tissues and biological fluids of patients affected by propionic acidemia that may manifest chronic renal failure along development. High urinary excretion of maleic acid (MA) has also been described. Considering that the underlying mechanisms of renal dysfunction in this disorder are poorly known, the present work investigated the effects of PA and MA (1-5 mM) on mitochondrial functions and cellular viability in rat kidney and cultured human embryonic kidney (HEK-293) cells. Mitochondrial membrane potential (∆ψm), NAD(P)H content, swelling and ATP production were measured in rat kidney mitochondrial preparations supported by glutamate or glutamate plus malate, in the presence or absence of Ca2+. MTT reduction and propidium iodide (PI) incorporation were also determined in intact renal cells pre-incubated with MA or PA for 24 h. MA decreased Δψm and NAD(P)H content and induced swelling in Ca2+-loaded mitochondria either respiring with glutamate or glutamate plus malate. Noteworthy, these alterations were fully prevented by cyclosporin A plus ADP, suggesting the involvement of mitochondrial permeability transition (mPT). MA also markedly inhibited ATP synthesis in kidney mitochondria using the same substrates, implying a strong bioenergetics impairment. In contrast, PA only caused milder changes in these parameters. Finally, MA decreased MTT reduction and increased PI incorporation in intact HEK-293 cells, indicating a possible association between mitochondrial dysfunction and cell death in an intact cell system. It is therefore presumed that the MA-induced disruption of mitochondrial functions involving mPT pore opening may be involved in the chronic renal failure occurring in propionic acidemia.
Asunto(s)
Fallo Renal Crónico , Acidemia Propiónica , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Ciclosporina/metabolismo , Ciclosporina/farmacología , Ácido Glutámico/farmacología , Células HEK293 , Humanos , Riñón , Fallo Renal Crónico/metabolismo , Malatos/metabolismo , Malatos/farmacología , Maleatos , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , NAD/metabolismo , Permeabilidad , Propidio/metabolismo , Propidio/farmacología , Acidemia Propiónica/metabolismo , Ratas , Ratas WistarRESUMEN
Lectins are a group of widely distributed and structurally heterogeneous proteins of nonimmune origin. These proteins have the ability to interact with glycans present on cell surfaces and elicit diverse biological activities. Machaerium acutifolium lectin (MaL) is an N-acetyl-D-glucosamine-binding lectin that exhibits antinociceptive activity via transient receptor potential cation channel subfamily V member 1 (TRPV1). Lectins that have the ability to recognize and interact with N-acetyl-D-glucosamine residues are potential candidates for studies of fungicidal activity. In this work, we show that MaL has antifungal activity against Candida species, and we describe its mode of action towards Candida parapsilosis. MaL inhibited the growth of C. albicans and C. parapsilosis. However, MaL was more potent against C. parapsilosis. The candidacidal mode of action of MaL on C. parapsilosis involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+-ATPase), induction of oxidative stress, and DNA damage. MaL also exhibited antibiofilm activity and noncytotoxicity to Vero cells. These results indicate that MaL is a promising candidate for the future development of a new, natural, and safe drug for the treatment of infections caused by C. parapsilosis.
Asunto(s)
Antifúngicos/farmacología , Candida parapsilosis/metabolismo , Estructuras de la Membrana Celular/química , Fabaceae/química , Lectinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Antifúngicos/administración & dosificación , Antifúngicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Biopelículas/efectos de los fármacos , Candida albicans/efectos de los fármacos , Candida albicans/metabolismo , Candida parapsilosis/citología , Candida parapsilosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Estructuras de la Membrana Celular/metabolismo , Chlorocebus aethiops , Medios de Cultivo/análisis , Medios de Cultivo/química , Daño del ADN , Lectinas/administración & dosificación , Lectinas/aislamiento & purificación , Microscopía Electrónica de Rastreo , Propidio/metabolismo , Semillas/química , Células VeroRESUMEN
Azospirillum brasilense is a plant growth promoting bacteria used as an inoculant in diverse crops. Accurate analytical methods are required to enumerate viable cells in inoculant formulations or in planta. We developed a quantitative polymerase chain reaction (qPCR) assay associated to propidium monoazide (PMA) to evaluate the cell viability of A. brasilense in inoculant and in maize roots. A. brasilense was grown in culture medium and was exposed to 50 â. Maize roots were grown in vitro and harvested 7 days after inoculation. Quantification was performed by qPCR, PMA-qPCR, and plate counting. Standard curves efficiency values ranged from 85 to 99%. The limit of detection was 104 CFU per gram of fresh root. Enumeration obtained in maize roots by qPCR where higher than enumeration by PMA-qPCR and by plate counting. PMA-qPCR assay was efficient in quantifying inoculant viable cells and provides reliable results in a quickly and accurately way compared to culture-dependent methods.
Asunto(s)
Azidas/metabolismo , Azospirillum brasilense/fisiología , Microbiología Industrial/métodos , Viabilidad Microbiana , Raíces de Plantas/microbiología , Propidio/análogos & derivados , Reacción en Cadena en Tiempo Real de la Polimerasa , Propidio/metabolismo , Zea mays/microbiologíaRESUMEN
BACKGROUND: The increasing prevalence of antibiotic resistant bacteria has raised an urgent need for substitute remedies. Antimicrobial peptides (AMPs) are considered promising candidates to address infections by multidrug-resistant bacteria through new mechanisms of action that require a careful evaluation of their performance. OBJECTIVE: Identification of effective AMPs against Neisseria meningitidis, which represents a pathogen of great public health importance worldwide that is intrinsically resistant to some AMPs, such as polymyxin B. METHODS: A cationic 11-residue peptide (KLKLLLLLKLK), referred to as poly-Leu, was synthesized and its antimeningococcal activity was compared to cecropin A and poly-P (KLKPPPPPKLK) through a variety of assays. Flow cytometry was used to measure propidium iodide uptake by N. meningitidis serotype B as an indicator of the effectiveness of each peptide when added to cultures at different concentrations. RESULTS: The addition of the poly-Leu peptide led to a 90.3% uptake of the dye with an EC50 value of 7.9 µg mL-1. In contrast, uptake was <10% in cells grown in the absence of peptides or with an identical concentration of cecropin and poly-Pro peptides. Electron micrographs indicated that the integrity of the cellular wall and internal membrane was impacted in relation to peptide concentrations, which was confirmed by the detection of released alkaline phosphatase from the periplasmic space due to disruption of the external membrane. CONCLUSION: Poly-Leu peptide demonstrated definitive antimicrobial activity against N. meningitidis.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Neisseria meningitidis/efectos de los fármacos , Antibacterianos/síntesis química , Péptidos Catiónicos Antimicrobianos/síntesis química , Farmacorresistencia Bacteriana Múltiple , Humanos , Pruebas de Sensibilidad Microbiana , Propidio/metabolismoRESUMEN
Brazilian Northeast is the world's largest producer of Agave sisalana Perrine for the supply of the sisal fiber. About 95% of plant biomass, which comprise the mucilage and sisal juice, is considered a waste residual is discarded in the soil. However, the sisal juice is rich in steroidal saponins, which exhibits different pharmacological properties. Despite this, natural products are not necessarily safe. Based on this, this study analyzed the antioxidant, cytotoxic and mutagenic potential of three extracts derived from acid hydrolysis (AHAS), dried precipitate (DPAS) and hexanic of A. sisalana (HAS). These analyses were performed by in vitro and in vivo methods, using Vero cells, human lymphocytes and mice. Results showed that AHAS 50 and 100 can be considered a useful antineoplastic candidate due to their antioxidant and cytotoxic activity, with no genotoxic/clastogenic potential in Vero cells and mice. Although the comet assay in human lymphocytes has showed that the AHAS 25, AHAS 50 and AHAS 100 can lead to DNA breaks, these extracts did not promote DNA damages in mice bone marrow. Considering the different mutagenic responses obtained with the different methods employed, this study suggest that the metabolizing pathways can produce by-products harmful to health. For this reason, it is mandatory to analyze the mutagenic potential by both in vitro and in vivo techniques, using cells derived from different species and origins.
Asunto(s)
Agave/química , Antioxidantes/farmacología , Eritrocitos/metabolismo , Linfocitos/metabolismo , Mutagénesis , Extractos Vegetales/farmacología , Animales , Anexina A5/metabolismo , Muerte Celular/efectos de los fármacos , Chlorocebus aethiops , Cromatografía Liquida , Ensayo Cometa , Roturas del ADN de Doble Cadena/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Fluoresceínas/metabolismo , Histonas/metabolismo , Humanos , Linfocitos/efectos de los fármacos , Espectrometría de Masas , Ratones , Hojas de la Planta/química , Propidio/metabolismo , Saponinas/análisis , Células VeroRESUMEN
Available treatments against human fungal pathogens present high levels of resistance, motivating the development of new antifungal therapies. In this context, the present work aimed to analyze direct electric current (DC) antifungal action, using an in vitro apparatus equipped with platinum electrodes. Candida albicans yeast cells were submitted to three distinct conditions of DC treatment (anodic flow-AF; electroionic flow-EIF; and cathodic flow-CF), as well as different charges, ranging from 0.03 to 2.40 C. Our results indicated C. albicans presented distinct sensibility depending on the DC intensity and polarity applied. Both the colony-forming unit assay and the cytometry flow with propidium iodide indicated a drastic reduction on cellular viability after AF treatment with 0.15 C, while CF- and EIF-treated cells stayed alive when DC doses were increased up to 2.40 C. Additionally, transmission electron microscopy revealed important ultrastructural alterations in AF-treated yeasts, including cell structure disorganization, ruptures in plasmatic membrane, and cytoplasmic rarefaction. This work emphasizes the importance of physical parameters (polarity and doses) in cellular damage, and brings new evidence for using electrotherapy to treat C. albicans pathology process. Bioelectromagnetics. 38:95-108, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Candida albicans/citología , Electricidad , Candida albicans/metabolismo , Candida albicans/fisiología , Candida albicans/ultraestructura , Adhesión Celular , Línea Celular , Electrodos , Células Epiteliales/citología , Humanos , Masculino , Viabilidad Microbiana , Persona de Mediana Edad , Platino (Metal)/química , Propidio/metabolismoRESUMEN
Elevated plasma homocysteine (Hcy) levels have been detected in patients with various neurodegenerative conditions. Studies of brain tissue have revealed that hyperhomocysteinemia may impair energy metabolism, resulting in neuronal damage. In addition, new evidence has indicated that vitamin D plays crucial roles in brain development, brain metabolism and neuroprotection. The aim of this study was to investigate the neuroprotective effects of 1,25-dihydroxivitamin D3 (calcitriol) in cerebral cortex slices that were incubated with a mild concentration of Hcy. Cerebral cortex slices from adult rats were first pre-treated for 30 min with one of three different concentrations of calcitriol (50 nM, 100 nM and 250 nM), followed by Hcy for 1h to promote cellular dysfunction. Hcy caused changes in bioenergetics parameters (e.g., respiratory chain enzymes) and mitochondrial functions by inducing changes in mitochondrial mass and swelling. Here, we used flow cytometry to analyze neurons that were double-labelled with Propidium Iodide (PI) and found that Hcy induced an increase in NeuN(+)/PI cells but did not affect GFAP(+)/Pi cells. Hcy also induced oxidative stress by increasing reactive oxygen species generation, lipid peroxidation and protein damage and reducing the activity of antioxidant enzymes (e.g., SOD, CAT and GPx). Calcitriol (50 nM) prevented these alterations by increasing the level of the vitamin D receptor. Our findings suggest that using calcitriol may be a therapeutic strategy for treating the cerebral complications caused by Hcy.
Asunto(s)
Calcitriol/farmacología , Corteza Cerebral/efectos de los fármacos , Homocisteína/farmacología , Fármacos Neuroprotectores/farmacología , Animales , Antioxidantes/metabolismo , Relación Dosis-Respuesta a Droga , Complejo II de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Citometría de Flujo , Proteína Ácida Fibrilar de la Glía/metabolismo , Técnicas In Vitro , Masculino , Fosfopiruvato Hidratasa/metabolismo , Propidio/metabolismo , Ratas , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
BACKGROUND: The bone marrow is a hematopoietic tissue that, in the presence of cytokines and growth factors, generates all of the circulating blood cells. These cells are important for protecting the organism against pathogens and for establishing an effective immune response. Previous studies have shown immunomodulatory effects of different products isolated from plant extracts. This study aimed to evaluate the immunomodulatory properties of aqueous Physalis angulata (AEPa) extract on the differentiation of bone marrow cells. RESULTS: Increased cellular area, higher spreading ability and several cytoplasmatic projections were observed in the treated cells, using optical microscopy, suggesting cell differentiation. Furthermore, AEPa did not promote the proliferation of lymphocytes and polymorphonuclear leukocytes, however promotes increased the number of macrophages in the culture. The ultrastructural analysis by Transmission Electron Microscopy of treated cells showed spreading ability, high number of cytoplasmatic projections and increase of autophagic vacuoles. Moreover, a high level of LC3b expression by treated cells was detected by flow cytometry, suggesting an autophagic process. Cell surface expression of F4/80 and CD11b also indicated that AEPa may stimulate differentiation of bone marrow cells mainly into macrophages. In addition, AEPa did not differentiate cells into dendritic cells, as assessed by CD11c analysis. Furthermore, no cytotoxic effects were observed in the cells treated with AEPa. CONCLUSION: Results demonstrate that AEPa promotes the differentiation of bone marrow cells, particularly into macrophages and may hold promise as an immunomodulating agent.
Asunto(s)
Células de la Médula Ósea/citología , Diferenciación Celular , Macrófagos/citología , Physalis/química , Animales , Anexina A5/metabolismo , Autofagia/efectos de los fármacos , Biomarcadores/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/ultraestructura , Antígeno CD11b/metabolismo , Adhesión Celular/efectos de los fármacos , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones Endogámicos BALB C , Extractos Vegetales/farmacología , Propidio/metabolismoRESUMEN
PURPOSE: Retinal degeneration caused by a defect in the phototransduction cascade leads to the apoptosis of photoreceptor cells, although the precise molecular mechanism is still unknown. In addition, constant low light exposure produces photoreceptor cell death through the activation of downstream phototransduction. The authors investigated the time course and molecular mechanisms of death and the rhodopsin phosphorylation occurring during retinal degeneration after exposure to continuous low-intensity light. METHODS: Wistar rats were exposed to constant cool white 200 lx intensity LED light (LL) for one to ten days and compared with animals kept in the dark (DD) or controls exposed to a regular 12:12 h (LD) cycle. One eye from each rat was used for histological and quantitative outer nuclear layer (ONL) analysis and the other for biochemical assays. RESULTS: The histological analysis showed a significant reduction in the ONL of LL-exposed rats after seven days compared with LD- or DD-exposed rats. Retinal analysis by flow cytometer and the TUNEL assay revealed an increase in cell death in the ONL, the in vitro enzymatic activity assay and western blot analysis showing no caspase-3 activation. The rhodopsin analysis demonstrated more phosphorylation in serine 334 residues (Ser(334)) in LL-exposed than in LD- or DD-exposed rats. However, for all times studied, rhodopsin was completely dephosphorylated after four days of DD treatment. CONCLUSIONS: Constant light exposure for seven days produces ONL reduction by photoreceptor cell death through a capase-3-independent mechanism. Increases in rhodopsin-phospho-Ser(334) levels were observed, supporting the notion that changes in the regulation of the phototransduction cascade occur during retinal degeneration.
Asunto(s)
Luz , Mamíferos/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Degeneración Retiniana/patología , Animales , Anexina A5/metabolismo , Caspasa 3/metabolismo , Muerte Celular/efectos de la radiación , Modelos Animales de Enfermedad , Etiquetado Corte-Fin in Situ , Fosforilación/efectos de la radiación , Fosfoserina/metabolismo , Células Fotorreceptoras de Vertebrados/enzimología , Propidio/metabolismo , Ratas , Ratas Wistar , Degeneración Retiniana/enzimología , Rodopsina/metabolismoRESUMEN
The N-methyl-D-aspartate receptor (NMDAR) is involved in synaptic plasticity, learning, memory, and neurological diseases like epilepsy and it is the major mediator of excitotoxicity. Functional NMDARs in the mature brain are heteromeric complexes composed of different subunits: GluN1 and GluN2. There are four different GluN2 subunits (A-D) and each of them critically determines the pharmacological and electrophysiological properties of NMDARs. GluN1 is ubiquitously expressed in the central nervous system while the highest GluN2A expression is in the hippocampus. Adenosine, an endogenous anticonvulsant, is a neuromodulator with a critical role in the regulation of neuronal activity, mediating its effect on specific receptors, among which adenosine A1 receptor is highly expressed in the hippocampus. In the present work hippocampal GluN2A expression after the convulsant drug 3-mercaptopropionic acid (MP) induced seizures and the effect of cyclopentyladenosine (CPA) given alone or prior to MP (CPA + MP) in an acute or repetitive experimental model was studied. CPA administered to rats for one or 4 days increases seizure threshold induced by MP. After one administration of MP, no significant difference in GluN2A expression was observed in CPA and CPA + MP by Western blot, although immunohistochemistry revealed an increase in CA2/3 area. However, repetitive MP administration during 4 days showed a significant increase of GluN2A expression, and the repetitive administration of CPA 30 min prior to MP caused a significant decrease of GluN2A expression with respect to MP treatment, returning to control levels. These results show that GluN2A subunit is involved in repetitive MP-induced seizures, while CPA administration displays a protective effect against it.
Asunto(s)
Adenosina/análogos & derivados , Hipocampo/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsiones/tratamiento farmacológico , Convulsiones/metabolismo , Ácido 3-Mercaptopropiónico , Adenosina/administración & dosificación , Adenosina/farmacología , Adenosina/uso terapéutico , Animales , Hipocampo/efectos de los fármacos , Hipocampo/patología , Immunoblotting , Inmunohistoquímica , Masculino , Fármacos Neuroprotectores/farmacología , Propidio/metabolismo , Ratas , Ratas WistarRESUMEN
Methylglyoxal is a dicarbonyl compound that is physiologically produced by enzymatic and non-enzymatic reactions. It can lead to cytotoxicity, which is mainly related to Advanced Glycation End Products (AGEs) formation. Methylglyoxal and AGEs are involved in the pathogenesis of Neurodegenerative Diseases (ND) and, in these situations, can cause the impairment of energetic metabolism. Astroglial cells play critical roles in brain metabolism and the appropriate functioning of astrocytes is essential for the survival and function of neurons. However, there are only a few studies evaluating the effect of methylglyoxal on astroglial cells. The aim of this study was to evaluate the effect of methylglyoxal exposure, over short (1 and 3 h) and long term (24 h) periods, on glucose, glycine and lactate metabolism in C6 glioma cells, as well as investigate the glyoxalase system and AGEs formation. Glucose uptake and glucose oxidation to CO(2) increased in 1 h and the conversion of glucose to lipids increased at 3 h. In addition, glycine oxidation to CO(2) and conversion of glycine to lipids increased at 1 h, whereas the incorporation of glycine in proteins decreased at 1 and 3 h. Methylglyoxal decreased glyoxalase I and II activities and increased AGEs content within 24 h. Lactate oxidation and lactate levels were not modified by methylglyoxal exposure. These data provide evidence that methylglyoxal may impair glucose metabolism and can affect glyoxalase activity. In periods of increased methylglyoxal exposure, such alterations could be exacerbated, leading to further increases in intracellular methylglyoxal and AGEs, and therefore triggering and/or worsening ND.
Asunto(s)
Glioma/metabolismo , Glucosa/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Piruvaldehído/farmacología , Línea Celular Tumoral , Colorantes , Metabolismo Energético/efectos de los fármacos , Glicina/metabolismo , Humanos , Ácido Láctico/metabolismo , Lactoilglutatión Liasa/metabolismo , Rojo Neutro , Oxidación-Reducción , Propidio/metabolismo , Sales de Tetrazolio , Tiazoles , Tioléster Hidrolasas/metabolismoRESUMEN
The main symptoms of leaf senescence are the degradation of chlorophyll and proteins (which may be accompanied by ammonium accumulation), and an increase of electrolyte leakage (EL), which has been traditionally attributed to disruption of cell membranes. The aim of this study was to determine if ammonium efflux contributes to the increase EL in senescing barley leaves. During senescence of detached leaves the increase of EL correlated with ammonium leakage (r(2) = 0.82) and ammonium content in tissues (r(2) = 0.73), but not with K(1+) leakage (r(2) = 0.23). Although lower amounts of ammonium accumulated in senescing attached leaves, again changes in EL paralleled ammonium accumulation. EL increased early during senescence even though ion leakage was selective (leaves leaked proportionally more ammonium than K(1+)), and membranes appeared intact as judged from staining with the cell impermeant stain propidium iodide. Detached leaves maintained their capacity to regreen after 3 days of senescence-acceleration in darkness, i.e., membrane integrity was not severely compromised. During the early stages of senescence, EL increases due to ammonium accumulation (possibly resulting from protein degradation) even if there is no massive disruption of cell membranes. Therefore, increased EL in senescing leaves is not an unequivocal symptom of cell membrane damage.
Asunto(s)
Membrana Celular/metabolismo , Electrólitos/metabolismo , Hordeum/metabolismo , Hojas de la Planta/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Muerte Celular , Membrana Celular/fisiología , Clorofila/análisis , Clorofila/metabolismo , Oscuridad , Hordeum/fisiología , Microscopía Fluorescente , Permeabilidad , Hojas de la Planta/fisiología , Proteínas de Plantas/análisis , Proteínas de Plantas/metabolismo , Potasio/análisis , Potasio/metabolismo , Propidio/metabolismo , Compuestos de Amonio Cuaternario/análisis , Factores de TiempoRESUMEN
Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the copolymer concentration and hydrophobicity was found. No inhibitory effect against these ABC pumps was observed with the hydrophilic T1107. These findings further evidence the potential usefulness of these Trojan horses as both drug nanocarriers and ABC inhibitors in hepatic MDR tumors and infections that involve the activity of these efflux transporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Polímeros/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Anexina A5/metabolismo , Apoptosis/efectos de los fármacos , Carbocianinas/metabolismo , Línea Celular Tumoral , Etilenodiaminas/química , Humanos , Peso Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Polietilenglicoles/química , Polímeros/química , Propidio/metabolismo , Glicoles de Propileno/química , Rodamina 123/metabolismo , Vinblastina/metabolismoRESUMEN
Kinetics of permeation through connexin 43-EGFP hemichannels (Cx43-EGFP HCs) were evaluated in divalent cation-free solutions, which enhance HC open probability and thus, allow measurements during initial velocity. Three cations that become fluorescent upon binding to intracellular nucleic acids [ethidium (Etd), propidium (Prd) and 4',6-diamidino-2-phenylindole (DAPI)] and Cx43-EGFP or Cx43 wild type HeLa cell transfectants (Cx43-EGFP- and Cx43-WT-HeLa cells, respectively) were used. Levels of Cx43-EGFP at the cell periphery and rate of dye uptake were directly related. The rate of uptake of each dye reached saturation consistent with a facilitated transport mechanism. Before saturation, the relation between rate of uptake and concentration of each dye was sigmoidal with Hill coefficients >1, indicating positive cooperativity of transport at low concentrations. The maximal rate of Etd uptake was not affected by the presence of DAPI and vice versa, but under each condition the apparent affinity constant of the main permeant molecule increased significantly consistent with competitive inhibition or competition for binding sites within the channel. Moreover, Cx43-EGFP and Cx43-WT HCs had similar permeability properties, indicating that EGFP bound to the C-terminal of Cx43 does not significantly alter the permeability of Cx43 HCs to positively charged molecules. Thus, competitive inhibition of permeation through hemichannels might contribute to cellular retention of essential molecules and/or uptake inhibition of toxic compounds.
Asunto(s)
Permeabilidad de la Membrana Celular , Conexina 43/metabolismo , Animales , Cationes/metabolismo , Conexina 43/genética , Etidio/metabolismo , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Indoles/metabolismo , Ratones , Propidio/metabolismoRESUMEN
Here, we observed the uptake of membrane-impermeant molecules by cercariae as they penetrate the skin and are transformed into schistosomula. We propose that membrane-impermeant molecules, Lucifer Yellow, Propidium iodide and Hoechst 33258 enter the parasite through both thenephridiopore and the surface membrane and then diffuse throughout the body of the parasite. We present a hypothesis that the internal cells of the body of the schistosomulum represent a new host-parasite interface, at which skin-derived growth factors may stimulate receptors on internal membranes during transformation of the cercariae into the schistosomulum.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Interacciones Huésped-Parásitos/fisiología , Schistosoma mansoni/crecimiento & desarrollo , Piel/parasitología , Animales , Isoquinolinas/metabolismo , Ratones , Microscopía Fluorescente , Propidio/metabolismoRESUMEN
Alzheimer's disease (AD) is a progressive neurodegenerative disorder marked by accumulation of extracellular deposits of amyloid-beta (Abeta) peptide in brain regions that are important for memory and cognition. The buildup of Abeta aggregates in the AD is followed by the formation of intracellular neurofibrillary tangles and activation of neuroinflammatory reactions. The present study investigated whether melatonin possesses a neuroprotective effect against Abeta-induced toxicity. For this purpose, organotypic hippocampal slices were cultured and exposed to 25 microm of Abeta(25-35) in the absence or in the presence of melatonin (25, 50, or 100 microm). In addition, the authors have investigated the involvement of GSK-3beta, tau protein, astroglial, and microglial activation, and cytokine levels in the melatonin protection against Abeta-induced neurotoxicity. Melatonin prevented the cell damage in hippocampus induced by the exposure to Abeta(25-35). In addition, melatonin significantly reduced the activation of GSK-3beta, the phosphorylation of tau protein, the glial activation and the Abeta-induced increase of TNF-alpha and IL-6 levels. On the basis of these findings, we speculate that melatonin may provide an effective therapeutic strategy for AD, by attenuating Abeta-induced phosphorylation of tau protein, and preventing GSK-3beta activation and neuroinflammation.
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Péptidos beta-Amiloides/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Melatonina/farmacología , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/toxicidad , Análisis de Varianza , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Muerte Celular/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Histocitoquímica , Interleucina-6/metabolismo , Masculino , Proteínas del Tejido Nervioso/metabolismo , Propidio/metabolismo , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Guanine derivatives modulate the glutamatergic system through displacement of binding of glutamate to its receptors acting as antagonist of glutamate receptors in moderate to high micromolar concentrations. Guanosine-5'-monophosphate (GMP) is shown to be neuroprotective against glutamate- or oxygen/glucose deprivation-induced neurotoxicity and also against NMDA-induced apoptosis in hippocampal slices. However, in this study we are showing that high extracellular GMP concentrations (5mM) reduced cell viability in hippocampal brain slices. The toxic effect of GMP was not blocked by dipyridamole, a nucleoside transport inhibitor, nor mimicked by guanosine, suggesting an extracellular mode of action to GMP which does not involve its hydrolysis to guanosine. GMP-dependent cell damage was not blocked by P1 purinergic receptor antagonists, neither altered by adenosine A(1) or A(2A) receptor agonists. The blockage of the ionotropic glutamate receptors AMPA or NMDA, but not KA or metabotropic glutamate receptors, reversed the toxicity induced by GMP. GMP (5mM) induced a decrease in glutamate uptake into hippocampal slices, which was reversed by dl-TBOA. Therefore, GMP-induced hippocampal cell damage involves activation of ionotropic glutamate receptors and inhibition of glutamate transporters activity.
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Agonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/metabolismo , Guanosina Monofosfato/toxicidad , Hipocampo/citología , Receptores de Glutamato/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colorantes , Dipiridamol/farmacología , Hipocampo/efectos de los fármacos , Indicadores y Reactivos , Masculino , Técnicas de Cultivo de Órganos , Propidio/metabolismo , Antagonistas de Receptores Purinérgicos P1 , Ratas , Ratas Wistar , Receptores Purinérgicos P1/efectos de los fármacos , Receptores Purinérgicos P1/metabolismo , Sales de Tetrazolio , TiazolesRESUMEN
UNLABELLED: In the present study we have analyzed the viability and metabolic competence of isolated rat hepatocytes subjected first, to subzero nonfreezing storage (up to 120 h at -4 degrees C) in modified University of Wisconsin (UW) solution with 8% 1,4-butanediol, and then to a normothermic rewarming step (KHR media, 37 degrees C, up to 120 min, carbogen atmosphere). Results were compared with hepatocytes stored up to 120 h at 0 degrees C in modified UW solution and with freshly isolated hepatic cells. We have found that only cell suspensions stored in subzero nonfreezing conditions were able to finish the rewarming period with a viability comparable with the control group. Also, we have investigated the enzyme activities and the relative expression at messenger RNAs levels of two of the Urea cycle (UC) enzymes: Carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), during 60 min of rewarming. Results were compared with the ammonium removal efficiency of the three groups. IN CONCLUSION: These data indicated that hepatocytes preserved under cold or subzero conditions up to 120 h followed by 60 min of rewarming, maintain UC enzymes at levels similar to freshly isolated hepatocytes, allowing their use in bioartificial liver devices.
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Butileno Glicoles/farmacología , Criopreservación/métodos , Crioprotectores/farmacología , Hepatocitos/efectos de los fármacos , Soluciones Preservantes de Órganos/farmacología , Urea/metabolismo , Adenosina/farmacología , Alopurinol/farmacología , Animales , Carbamoil-Fosfato Sintasa (Amoniaco)/genética , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Supervivencia Celular/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutatión/farmacología , Hepatocitos/enzimología , Insulina/farmacología , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ornitina Carbamoiltransferasa/genética , Ornitina Carbamoiltransferasa/metabolismo , Propidio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , ARN Mensajero/metabolismo , Rafinosa/farmacología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Evaluation of apoptosis by flow cytometry is generally accomplished by methods that use annexin V-FITC as vital dye, which access phosphatidylserine exposed on the external membrane at the beginning of this process. In addition, the concomitant use of propidium iodide makes possible to verify the characteristic nuclear alterations in the late stages of apoptosis, as a consequence of the increase in membrane permeability. On the other hand, the use of calcein-AM in association with ethidium homodimer (EthD-1) allows the evaluation of cell apoptosis through detection of esterase activity and cellular membrane physical and chemical alterations. The aim of this study was to compare the sensibility of calcein-AM and EthD-1 with annexin V-FITC and propidium iodide for early apoptosis evaluation in peripheral blood mononuclear cell culture, obtained from HIV-infected patients. Apoptosis and cellular viability were detected and quantified by flow cytometry after 24 and 48 hours incubation times. Our results showed that calcein-AM/EthD-1 was more sensitive for apoptotic cell quantification in both incubation times than annexin V-FITC/propidium iodide (mean of 46.95% +/- 3.56, p < 0.0001, for 24 hours and mean of 37.67% +/- 2.47, p < 0.0014 for 48 hours), besides allowing to clearly define viable, apoptotic and dead cell populations.
Asunto(s)
Anexina A5/metabolismo , Apoptosis , Citometría de Flujo/métodos , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Infecciones por VIH/patología , Linfocitos/fisiología , Biomarcadores/metabolismo , Permeabilidad de la Membrana Celular , Etidio/análogos & derivados , Etidio/metabolismo , Humanos , Propidio/metabolismo , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Evaluation of apoptosis by flow cytometry is generally accomplished by methods that use annexin V-FITC as vital dye, which access phosphatidylserine exposed on the external membrane at the beginning of this process. In addition, the concomitant use of propidium iodide makes possible to verify the characteristic nuclear alterations in the late stages of apoptosis, as a consequence of the increase in membrane permeability. On the other hand, the use of calcein-AM in association with ethidium homodimer (EthD-1) allows the evaluation of cell apoptosis through detection of esterase activity and cellular membrane physical and chemical alterations. The aim of this study was to compare the sensibility of calcein-AM and EthD-1 with annexin V-FITC and propidium iodide for early apoptosis evaluation in peripheral blood mononuclear cell culture, obtained from HIV-infected patients. Apoptosis and cellular viability were detected and quantified by flow cytometry after 24 and 48 hours incubation times. Our results showed that calcein-AM/EthD-1 was more sensitive for apoptotic cell quantification in both incubation times than annexin V-FITC/propidium iodide (mean of 46.95 percent ± 3.56, p < 0.0001, for 24 hours and mean of 37.67 percent ± 2.47, p < 0.0014 for 48 hours), besides allowing to clearly define viable, apoptotic and dead cell populations.