RESUMEN
Recent advances in nanotechnology applied to medicine and regenerative medicine have an enormous and unexploited potential for future space and terrestrial medical applications. The Nanoparticles and Osteoporosis (NATO) project aimed to develop innovative countermeasures for secondary osteoporosis affecting astronauts after prolonged periods in space microgravity. Calcium- and Strontium-containing hydroxyapatite nanoparticles (nCa-HAP and nSr-HAP, respectively) were previously developed and chemically characterized. This study constitutes the first investigation of the effect of the exogenous addition of nCa-HAP and nSr-HAP on bone remodeling in gravity (1 g), Random Positioning Machine (RPM) and onboard International Space Station (ISS) using human bone marrow mesenchymal stem cells (hBMMSCs). In 1 g conditions, nSr-HAP accelerated and improved the commitment of cells to differentiate towards osteoblasts, as shown by the augmented alkaline phosphatase (ALP) activity and the up-regulation of the expression of bone marker genes, supporting the increased extracellular bone matrix deposition and mineralization. The nSr-HAP treatment exerted a protective effect on the microgravity-induced reduction of ALP activity in RPM samples, and a promoting effect on the deposition of hydroxyapatite crystals in either ISS or 1 g samples. The results indicate the exogenous addition of nSr-HAP could be potentially used to deliver Sr to bone tissue and promote its regeneration, as component of bone substitute synthetic materials and additive for pharmaceutical preparation or food supplementary for systemic distribution.
Asunto(s)
Nanopartículas/administración & dosificación , Nanopartículas/química , Osteoporosis/tratamiento farmacológico , Ingravidez/efectos adversos , Fosfatasa Alcalina/metabolismo , Huesos/efectos de los fármacos , Huesos/metabolismo , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Durapatita/administración & dosificación , Durapatita/química , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Osteoporosis/metabolismo , Medicina Regenerativa/métodos , Estroncio/metabolismo , Andamios del TejidoRESUMEN
The increased number of elderly people worldwide poses a major medical and socio-economic challenge: the search of strategies to combat the consequences of the aging process. Oxidative stress and inflammation have been pointed out as the leading causes of brain aging, which in turn alters the functionality of brain. In this context, decline in adult neurogenesis (AN), due to modifications in the neural progenitor stem cells (NSCs) and their microenvironment, is among the aging alterations contributing to cognitive decline. Therefore, the consumption or administration of antioxidant and anti-inflammatory molecules, such as dietary polyphenols, is under study as a potential beneficial strategy for preventing brain aging alterations including AN decline. Polyphenols, through their antioxidant and anti-inflammatory properties, modulate several cascades and effectors involved in the regulation of AN (e.g., SIRT1, Wnt, NF-κB and Nrf2, among others). This work summarizes the latest discoveries regarding the mechanisms whereby polyphenols preserve AN and counteract the cognitive decline present in aging.
Asunto(s)
Envejecimiento/fisiología , Encéfalo/efectos de los fármacos , Cognición/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Polifenoles/farmacología , Animales , Cognición/fisiología , Humanos , Neurogénesis/fisiología , Estrés Oxidativo/efectos de los fármacosRESUMEN
The mineralization process is crucial to the load-bearing characteristics of the bone extracellular matrix. In this work, we have studied the spatiotemporal dynamics of mineral deposition by human bone marrow mesenchymal stem cells differentiating toward osteoblasts promoted by the presence of exogenous hydroxyapatite nanoparticles. At the molecular level, the added nanoparticles positively modulated the expression of bone-specific markers and enhanced calcified matrix deposition during osteogenic differentiation. The nucleation, growth and spatial arrangement of newly deposited hydroxyapatite nanocrystals have been evaluated using scanning micro X-ray diffraction and scanning micro X-ray fluorescence. As leading results, we have found the emergence of a complex scenario where the spatial organization and temporal evolution of the process exhibit heterogeneous and self-organizing dynamics. At the same time the possibility of controlling the differentiation kinetics, through the addition of synthetic nanoparticles, paves the way to empower the generation of more structured bone scaffolds in tissue engineering and to design new drugs in regenerative medicine.
Asunto(s)
Matriz Ósea/crecimiento & desarrollo , Durapatita/farmacología , Células Madre Mesenquimatosas/citología , Nanopartículas , Osteogénesis , Ingeniería de Tejidos , Diferenciación Celular , Células Cultivadas , Humanos , Andamios del TejidoRESUMEN
Metabolic homeostasis of fatty acids is complex and well-regulated in all organisms. The biosynthesis of saturated fatty acids (SFA) in mammals provides substrates for ß-oxidation and ATP production. Monounsaturated fatty acids (MUFA) are products of desaturases that introduce a methylene group in cis geometry in SFA. Polyunsaturated fatty acids (n-6 and n-3 PUFA) are products of elongation and desaturation of the essential linoleic acid and α-linolenic acid, respectively. The liver processes dietary fatty acids and exports them in lipoproteins for distribution and storage in peripheral tissues. The three types of fatty acids are integrated in membrane phospholipids and determine their biophysical properties and functions. This study was aimed at investigating effects of fatty acids on membrane biophysical properties under varying nutritional and pathological conditions, by integrating lipidomic analysis of membrane phospholipids with functional two-photon microscopy (fTPM) of cellular membranes. This approach was applied to two case studies: first, pancreatic beta-cells, to investigate hormetic and detrimental effects of lipids. Second, red blood cells extracted from a genetic mouse model defective in lipoproteins, to understand the role of lipids in hepatic diseases and metabolic syndrome and their effect on circulating cells.
Asunto(s)
Ácidos Grasos/química , Fluidez de la Membrana , Humanos , Metabolismo de los LípidosRESUMEN
p66(shc), a member of the ShcA protein family, is essential for cellular response to oxidative stress, and elicits the formation of mitochondrial Reactive Oxygen Species (ROS), thus promoting vasomotor dysfunction and inflammation. Accordingly, mice lacking the p66 isoform display increased resistance to oxidative tissue damage and to cardiovascular disorders. Oxidative stress also contributes to noise-induced hearing loss (NIHL); we found that p66(shc) expression and serine phosphorylation were induced following noise exposure in the rat cochlea, together with markers of oxidative stress, inflammation and ischemia as indicated by the levels of the hypoxic inducible factor (HIF) and the vascular endothelial growth factor (VEGF) in the highly vascularised cochlear lateral region and spiral ganglion. Importantly, p66(shc) knock-out (p66 KO) 126 SvEv adult mice were less vulnerable to acoustic trauma with respect to wild type controls, as shown by preserved auditory function and by remarkably lower levels of oxidative stress and ischemia markers. Of note, decline of auditory function observed in 12 month old WT controls was markedly attenuated in p66KO mice consistent with delayed inner ear senescence. Collectively, we have identified a pivotal role for p66(shc) -induced vascular dysfunction in a common pathogenic cascade shared by noise-induced and age-related hearing loss.
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Cóclea/irrigación sanguínea , Cóclea/fisiopatología , Pérdida Auditiva Provocada por Ruido/metabolismo , Pérdida Auditiva Provocada por Ruido/fisiopatología , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/metabolismo , Animales , Cóclea/metabolismo , Inflamación/patología , Isquemia/metabolismo , Isquemia/patología , Isquemia/fisiopatología , Masculino , Ratones Noqueados , Neovascularización Fisiológica , Oxidación-Reducción , Estrés Oxidativo , Fosforilación , Ratas Wistar , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/deficienciaRESUMEN
The generation and disposal level of thermal power plant ash in India is a challenging task. The conventional mode of dilute phase ash slurry (10-20% solids by weight) transport through pipelines being practiced in majority of these plants not only consumes huge amount of precious water and pumping energy but also causes serious environmental problem at the disposal site. The present study investigates the rheological and leaching characteristics of an Indian ash samples at high solids concentrations (>50% by weight) using sodium silicate as an additive. The flow behaviour of ash slurry in the concentration range of 50-60% by weight is described by a Bingham-plastic model. It was indicated that the addition of sodium silicate (0.2-0.6% of the total solids) could able to reduce both the slurry viscosity and the yield stress. The analysis of the ash samples for the presence of heavy metals such as Fe, Cd, Ni, Pb, Zn, Cu, Co, As and Hg were carried out following Hansen and Fisher procedure. The addition of sodium silicate affected the leaching characteristics of the ash samples over a period of 300 days resulting in the reduction of leaching of heavy metals.
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Ceniza del Carbón/análisis , Contaminantes Ambientales/análisis , Metales Pesados/análisis , Ceniza del Carbón/química , Conservación de los Recursos Energéticos , Contaminantes Ambientales/química , Residuos Industriales/análisis , Metales Pesados/química , Reología , Silicatos/química , ViscosidadRESUMEN
Natural resistance against Venturia pirina and Botrytis cinerea was investigated within the Sardinian pears germplasm. The natural occurrence of V. pirina was monitored at the orchard level for 5 years, while resistance against B. cinerea was evaluated by a bioassay using methanolic extracts of the fruit rind or by artificially inoculation. Methanolic extracts of the leaves were employed for the Cladosporium bioassay on TLC plates. Among the accessions, 8 resulted sensitive to V. pirina, vegetation and fruit are severely affected every year. Seven showed an intermediate behaviour and the sole vegetation was affected slightly in two out of the 5 years. Two inhibition spots, in the methanolic extract of the leaves, were clearly evidenced in 55 accessions and a positive correlation was found between the presence of these antifungal compounds in the leaves and the resistance to V. pirina in the field. The artificial inoculation of fruit with B. cinerea evidenced a great variability in resistance, with about 12 accessions that could be considered having a good resistance. The fruit rind methanolic extracts evidenced several inhibition spots in most accessions, but no correlation could be found with fruit resistance to B. cinerea. In addition, the activity of the polygalacturonase inhibiting protein (PGIP), determined with an agarose diffusion bioassay, indicate a positive correlation between the PGIP activity evidenced in the core tissue and the infection degree by B. cinerea.
Asunto(s)
Ascomicetos/fisiología , Botrytis/fisiología , Enfermedades de las Plantas/microbiología , Pyrus/microbiología , Pyrus/fisiología , Bioensayo , Italia , Poligalacturonasa/antagonistas & inhibidoresRESUMEN
The International trade has to ensure food security and preserve both human health and the diffusion of animal and plant diseases among different countries. While organic horticultural production and trade are regulated by global guidelines, no specific restrictions relay to conventional commodities and almost free import-export occurs among UE countries. Consequently, the safety characteristics of imported fresh crops have become an important issue. On the basis of these considerations, the aim of the present study is to monitor the epiphytic microflora (particularly yeasts and fungi) carried by fresh fruit, imported from Spain to Italy. Particular attention was given to pathogenic microrganisms and to natural antagonists. In addition, the resistance to a few postharvest fungicides was determined for the isolated strains and compared to the local ones. Apples, Citrus fruits, stone fruits, and persimmon fruit were sampled at arrival of the container from Spain at the wholesale market in Sassari (Italy), this to prevent fruit contamination by local strains of microrganisms. The isolation was performed by rinsing and shaking (30 min at 100 rpm) the fruit in a beaker with 500 mL of sterile water. After concentration (7.5 mL), 100 microl of the rinse water was plated on potato dextrose agar (PDA). Isolation of pure colonies was performed by multiple streaking on plates, until unicellular cultures were obtained. Fifty three microrganisms, mainly fungi and yeasts, have been isolated and assayed in vitro and in vivo. Pathogenic behavior of isolated fungi was tested on fruits artificially wounded and only 7 strains out of 18 isolates caused decay. The resistance to different concentrations of imazalil (IMZ), tiabendazolo and azoxystrobin were tested in vitro for the pathogenic isolates. All isolates were completely inhibited with 1000 ppm IMZ evidencing that no resistant strains were present on the imported fruit. Since the baseline resistance, found for all isolates, was similar to the indigenous strains, we may conclude from this risk analysis that the import of the studied fruits produced in the 4 geographical areas of Spain does not increase the local baseline resistance.
Asunto(s)
Comercio , Frutas/microbiología , Hongos/aislamiento & purificación , Enfermedades de las Plantas/prevención & control , Hongos/efectos de los fármacos , Fungicidas Industriales/farmacología , Italia , Enfermedades de las Plantas/microbiología , Factores de Riesgo , EspañaRESUMEN
Ultrasonic technology is known for many years and is used for several purposes such as sonochemistry, extraction of natural compounds, degassing of solvent, inactivation of enzymes and microorganism. In postharvest ultrasound is applied in not destructive analysis of crop for the determination of the maturation stage. Until now, however, the potential of the sonication as a physical mean for not conventional postharvest treatment of fresh fruit has been little investigated. Here we report on the results obtained with apple fruit (Malus domestica Borkh. cv San Giovanni, from Sardinian germplasm) dipped in a solution of potassium sorbate with or without sonication. Treatment was carried out with an ultrasonic processor (1500 W, 20Khz) connected to a 25 mm phi probe immersed in 10 L of deionized water placed in a steel vessel. Fruit at different ripening stages, inoculated or not inoculated with Penicillium expansum, was sonicated before or after the inoculation. Following the treatment, fruit was left to dry, put into boxes and cold-stored. Results showed that ultrasound alone enhanced the natural resistance of ripe fruit when inoculated after sonication. No effect was observed when the ultrasound application was carried out after inoculation with P. expansum on un-ripe or ripe apples. Potassium sorbate showed to be ineffective in controlling the decay regardless of the ripening stage and the time of inoculation. On the contrary, a significant enhance in decay control was observed when the application of potassium sorbate was performed in the presence of ultrasound.
Asunto(s)
Almacenamiento de Alimentos , Malus/microbiología , Penicillium/efectos de los fármacos , Enfermedades de las Plantas/prevención & control , Ácido Sórbico/farmacología , Sonido , Frutas/crecimiento & desarrollo , Frutas/microbiología , Malus/crecimiento & desarrollo , Penicillium/crecimiento & desarrollo , Enfermedades de las Plantas/microbiología , SonicaciónRESUMEN
In the latest years, investigation on postharvest treatments has been increasingly addressed to preserve human health and environment safeguard. Several preservative compounds, physical treatments and biological control agents to restrain postharvest pathogens on horticultural products have been widely studied. Among them potassium sorbate (KS) has been generally recognized as safe for use in foods and personal care products. It acts as microbial growth inhibitor and fungistatic agent in foods, including vegetable and fruit products. The efficacy of KS, used alone or combined with heat treatments or biocontrol agents, has been demonstrated in Citrus and stone fruits. Here we report the results of 3 experiments aimed at controlling Penicillium expansum Link decay with the use of KS on a yellow ('Shiro') and a red ('Sanguigna di Bosa II', from the Sardinian germplasm) plum cultivar. An integrated approach, combining ultrasounds (US) as a physical mean and KS solutions at different concentrations, has been employed. In the first experiment, 360 fruits were wounded twice and divided into 6 sets (6 x 60), three of which were inoculated with an isolate of P. expansum (20 microl of a 10(5) cfu x mL(-1)). Then, 180 fruit (half inoculated) were treated by pipetting into each wound 20 microl of a KS solution containing 0, 1.5 or 3% (w/v), respectively. In exp. 2, all fruit (number) was wounded and inoculated, and after 24 h treated by immersion (1 min) into solutions containing 0, 1.5 or 3% (w/v) of SK, with or without the use of US. In exp. 3, wounded fruits were treated by immersion or sonication like in exp. 2, while inoculation took place after 24 h. Then, plums were kept at 25 degrees C and 75-80% RH and the infection degree was monitored after 3 and 6 days. In both cultivars, the 1.5% KS solution significantly reduced the natural infection, while the 3% KS solution resulted effective only on the red one. Moreover, the 1.5% solution was effective in controlling decay of artificially inoculated fruit, achieving a 56% reduction compared to control. Similar results were attained in exp. 2 and 3, where the combination of salt and sonication improved the efficacy, likely by increasing the salt diffusion into the wounds.
Asunto(s)
Penicillium/efectos de los fármacos , Enfermedades de las Plantas/prevención & control , Prunus/microbiología , Ácido Sórbico/farmacología , Sonido , Frutas/crecimiento & desarrollo , Frutas/microbiología , Penicillium/crecimiento & desarrollo , Enfermedades de las Plantas/microbiología , Prunus/genética , Prunus/crecimiento & desarrolloRESUMEN
To gain knowledge on the influence of postharvest treatments with ultraviolet-C light upon the keeping quality of table grapes, a trail was performed employing commercially mature 'Corina', 'Dawn Seedless', 'Centennial Seedless' and 'Gran Perlon' grape cultivars (cvs). After grading, bunches were subjected to 0.0, 0.5, 1.5 or 3 kJm(-2) and then stored at 5 degrees C and 90% relative humidity (RH) for 6 weeks followed by a 2 day shelf-life at 25 degrees C and 70% RH. A weekly inspection was performed and a visual evaluation of the appearance, treatment damage, stems browning and berry shrivelling was performed. Weight loss, decay and shatter were quantified at the end of storage and shelf-life. Regardless the cv and UV-C dose, fruit appearance was acceptable until the end of storage and shelf-life. Among the cvs, the highest score was held by 'G. Perlon'. After the fourth week of storage, the berries of 'Centennial S.' turned light brown and darkened over time when treated with 3.0 kJm(-2). Stem browning was not induced by the light treatment, but resulted cv depended and was pronounced for 'Centennial S.' and 'Dawn S.'. Berry shrivelling was insignificant, while shatter was very high in 'Corina' and did not depend upon UV-C treatment. Regarding weight loss, differences could not be attributed to the light treatment and after storage it ranged from 3 up to 5%. Decay was significantly reduced by light treatment and the efficacy increased by raising the dose. Botrytis cinerea was the main cause of decay with 'Corina' being the most jeopardized, followed by 'Dawn S.' and 'Centennial S.', whereas 'G. Perlon' resulted the less affected. In conclusion, hormetic effects of postharvest light treatment on table grapes were observed in almost all cvs with 'G. Perlon' having the best performance.
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Botrytis/efectos de la radiación , Enfermedades de las Plantas/prevención & control , Rayos Ultravioleta , Vitis/microbiología , Botrytis/crecimiento & desarrollo , Cromatografía Líquida de Alta Presión , Almacenamiento de Alimentos , Frutas/genética , Frutas/metabolismo , Frutas/microbiología , Enfermedades de las Plantas/microbiología , Resveratrol , Estilbenos/metabolismo , Vitis/genética , Vitis/metabolismoRESUMEN
Penicillium expansum and Botrytis cinerea are among the pathogens most frequently affecting apples and grapes after harvest, respectively. We studied the behaviour of these moulds when subjected to different concentrations of methanol (MeOH) and dimethyl sulfoxide (DMSO) as a alternative method to fungicides in controlling postharvest decay of horticultural products. The experiments were performed with 5 cm Petri dishes containing PDA amended with 0, 5, 10, 20, 30, 40 or 50 microL/mL of the two tested chemicals. Freshly prepared conidia of B. cinerea and P. expansum were sown onto the media and then kept into an incubation chamber at 21 degrees C up to 3 and 6 days, respectively. Daily, the colony forming units (cfu), the colony diameter and the degree of sporification were monitored. Compared to the control, both chemicals affected the growth rate of the two pathogens. The P. expansum and B. cinerea cfu value was not significantly inhibited but the colony diameter and the sporification degree decreased when concentration was raised. B. cinerea cultured on DMSO showed a significant drop of sporification up to the tested concentration of 10 microL/mL, and a complete inhibition of cfu when the concentration was higher than 20 microL/mL.
Asunto(s)
Botrytis/efectos de los fármacos , Dimetilsulfóxido/farmacología , Fungicidas Industriales/farmacología , Metanol/farmacología , Penicillium/efectos de los fármacos , Esporas Fúngicas/fisiología , Botrytis/fisiología , Relación Dosis-Respuesta a Droga , Penicillium/fisiologíaRESUMEN
The employment of biocontrol agents to restrain postharvest pathogens is an encouraging approach, although, efficacy and consistency are still below those of synthetic pesticides. Up to date, the 'integrated control strategy' seems to be the most promising way to overcome this gap. Here, we report the feasibility to control postharvest decay caused by Penicillium expansum in apples by a 2 min, single or sequential, immersion in water with an antagonistic yeast (Candida oleophila, isolate '13L'), 2% NaHCO3 (SBC) or 1% CaCl2. The treatments were carried out, on appels cv 'Miali' either un-wounded, wounded or wound-pathogen inoculated and then stored at 2 degrees C for 30 d followed by a 6 d simulated marketing period at 20 degrees C or alternatively stored only for 7 d at 20 degrees C. As a general role, the best results were attained when CaCl2 was applied with the yeast or when preceded by the SBC treatment. When the wounding and inoculation took place 24 h before the treatment, the latter application sequence of the two salts was three times more effective compared to the treatment with the sole antagonist, and one time when performed 24 h after the treatment. Interestingly, apples immersed in the sole 2% SBC solution had the highest percentage of decay during storage and when inoculated before moving to the simulated marketing period at 20 degrees C.
Asunto(s)
Cloruro de Calcio/farmacología , Candida/fisiología , Penicillium/efectos de los fármacos , Penicillium/fisiología , Enfermedades de las Plantas/prevención & control , Bicarbonato de Sodio/farmacología , Conservación de Alimentos , Malus/microbiología , Factores de TiempoRESUMEN
The activity of 2-deoxy-D-gLucose (2-DG) alone or in combination with a biocontrol yeast (Candida saitoana, strain 8C) was evaluated in vitro and in vivo against citrus green mould (Penicillium digitatum Sacc.). The in vitro assays were performed on amended potato dextrose agar (PDA) containing 0, 1.5, 3.0, 6.0, 15.0, 30.0 or 60.0 mM of 2-DG. P. digitatum conidia were sown on the amended media and growth inhibition occurred starting from 6.0 mM. A nearly total inhibition of the growth and spore germination occurred with 60.0 mM of 2-DG. The antagonist was not affected by any of the 2-DG concentrations employed and the amended plates resulted well colonized within 2 d post-treatment. In vivo assays were carried out with 'Hamlin' oranges, inoculated with P. digitatum 24 h before treating with: the antagonist; the above reported concentrations of 2-DG, or by combining the two treatments. Seven days post-treatment the inhibition activity exerted by 3.0, 6.0, 15.0, 30.0 and 60.0 mM of 2-DG combined with the yeast was 15, 37, 42, 63 and 84%, respectively. While that exerted by the antagonist was 22% and that by the different concentrations of 2-DG were 7, 11, 27, 42 and 57%, respectively. Compared to single treatments, the co-application significantly and in a synergic mode improved the control of decay. Alterations to the hyphae were observed by SEM when the pathogen was cultured on amended media and into the wounds of inoculated oranges.
Asunto(s)
Antibiosis , Candida/fisiología , Citrus/microbiología , Desoxiglucosa/farmacología , Fungicidas Industriales/farmacología , Penicillium/efectos de los fármacos , Penicillium/fisiología , Enfermedades de las Plantas/microbiología , Agricultura/métodosRESUMEN
The behaviour of Penicillium digitatum and P. italicum was investigated when subjected to different concentrations of methanol (MeOH) and dimethyl sulfoxide (DMSO). The experiments were performed in 9 cm Petri dishes containing PDA amended with 0, 5, 10, 20, 30, 40 or 50 microL/mL of each of the single or combined chemicals. Daily, the formed colonies (cfu), the colony diameter and the degree of sporification were monitored during incubation at 20 degrees C for 5 day. Additionally, the pathogen development and its performance were studied by scanning electron microscopy (SEM). According to the chemical, the mycelium growth rate was affected differently and, compared to the control, only MeOH inhibited the expansion of the colony diameter. This effect was more pronounced for P. italicum. A nearly linear drop of cfu was observed as the concentration of the two chemicals was raised, and a complete inhibition of the two pathogens was attained with 50 microL/mL MeOH. With respect to the sporification degree the two pathogens were influenced similarly, but the tested compounds had opposite effects. Indeed, with MeOH, sporification took place earlier (24-36 h postinoculation) compared to the control (60 h), while during the whole experiment, DMSO at concentrations higher than 0.5 microL/mL, drastically inhibited the sporification. SEM observations of P. digitatum growth on DMSO amended media evidenced a marked increase of mycelium branching and alterations to the conidiophore, while MeOH reduced the mycelium length and fastened the conidiophore formation. The combination of the two compounds produced a synergistic interaction reducing by 40% the concentration required to inhibit completely the germination and growth of P. digitatum.
Asunto(s)
Citrus/microbiología , Fungicidas Industriales/farmacología , Penicillium/crecimiento & desarrollo , Enfermedades de las Plantas/microbiología , Penicillium/efectos de los fármacos , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/crecimiento & desarrolloRESUMEN
Neuronal redox phenomena are involved in numerous biochemical pathways and play a key role in many pathological events and clinical situations. The oxidation/reduction (redox) state present in biological compartments is a major target for possible pharmaceutical intervention and, consequently, the processes associated with its change have attracted increased attention in recent years. Here, we analyze the redox environment and its spatial compartmentalization in differentiated neuronal phenotype of PC-12 cells using a redox-sensitive protein (i.e., a mutant of the Yellow Fluorescent protein), employed ratiometrically. Redox maps of cells were generated with an elevate spatial resolution, and the spatial distributions of highly oxidized and highly reduced regions have been determined. A quantitative analysis of redox maps allows the disclosure of a peculiar spatial organization of the redox environment.
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Técnicas Biosensibles , Compartimento Celular/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Oxidación-Reducción , Animales , Línea Celular , Células , Microscopía Confocal/métodos , Células PC12 , RatasRESUMEN
The dynamics of redox elements in biologic systems is a major challenge for redox signaling and oxidative stress research. Oxidative stress or signaling events can affect sulfur switches differently, thus creating a variation in the spatial distribution of these redox states, which therefore act simultaneously as regulators and indicators of key cellular functions in both physiological and pathological settings. A gluthatione specific redox-sensitive protein (i.e. a mutant of the Yellow Fluorescent Protein (rxYFP)) has been found to equilibrate in vivo with the gluthatione/gluthatione disulfide (GSH:GSSG) redox couple. rxYFP, employed ratiometrically, allows to generate high resolution maps of the fraction of the reduced protein (R) inside a cell. Here we developed an analytical procedure able to investigate intracellular changes in the glutathione redox-balance, which can occur in live mammalian cells, based on the deconvolution of the histogram of redox maps of 293-TPhoenix human embryonic kidney cells. The intracellular spatial distributions of oxidized and reduced elements have been discriminated. Finally, by transfecting cells with human Glutaredoxin V (GRX-V), an enzyme deputed to maintain reduced the thiol groups of their partner proteins, we can disclose that the significant shift towards more reduced state, with respect to that recovered from non-transfected cells, consists, instead, in a shift towards reduced values of the high R region (reduced), while leaving unaltered the glutathione redox-balance of the intracellular side of the plasma membrane.
Asunto(s)
Glutatión/metabolismo , Interpretación de Imagen Asistida por Computador/métodos , Riñón/metabolismo , Microscopía Fluorescente/métodos , Línea Celular , Humanos , Oxidación-ReducciónRESUMEN
BACKGROUND: Human mesenchymal stromal cells (MSCs) can be isolated from a variety of adult and perinatal tissues and exert multipotency and self renewal properties which make them suitable for cell-based therapy. Their potential plasticity extended to non-mesodermal-derived tissues has been indicated, although it is still a debated issue. In this study we have isolated MSCs from both adult and fetal tissues. Their growth, immunophenotype and multi-lineage differentiation potentials have been analyzed, focusing, in particular, on the hepatic differentiation. METHODS: Cells were isolated from bone marrow (BMSC), adipose tissue (ATSC) and second trimester amniotic fluid (AFSC), upon a written informed consent obtained from donor patients. Cells were expanded and growth kinetics was assessed by means of proliferation assay. Their immunophenotype was analyzed using cytometry and multi-lineage differentiation potential was evaluated by means of in vitro differentiation assays. Finally, the expression of tissue-specific markers was also assessed by mean of semi-quantitative PCR. RESULTS: Bipolar spindle-shaped cells were successfully isolated from all these tissues. Interestingly, ATSCs and AFSCs showed a higher proliferation potential than BMSCs. Mesodermal differentiation capacity was verified in all MSC populations, even if AFSCs were not able to undergo adipogenesis in our culture conditions. Furthermore, we showed that MSC cultured in appropriate conditions were able to induce hepatic-associated genes, such as ALB and TDO2. CONCLUSION: Taken together the data here reported suggest that MSCs from both adult and fetal tissues are capable of tissue-specific commitment along mesodermal and non-mesodermal lineages. In particular we have demonstrated that a specific hepatogenic commitment can be efficiently induced, proposing these cells as suitable tool for cell-based applications aimed at liver regeneration.
Asunto(s)
Diferenciación Celular , Hepatocitos , Células Madre Mesenquimatosas/fisiología , Células Madre Multipotentes/fisiología , Tejido Adiposo/citología , Líquido Amniótico/citología , Antígenos de Diferenciación/inmunología , Antígenos de Diferenciación/metabolismo , Células de la Médula Ósea/fisiología , Proliferación Celular , Células Cultivadas , Femenino , Hepatocitos/citología , Hepatocitos/inmunología , Hepatocitos/metabolismo , Humanos , Células Madre Mesenquimatosas/inmunología , Especificidad de Órganos , EmbarazoRESUMEN
Adrenocortical carcinoma (ACC) are highly malignant tumors with poor prognosis. To verify if it is possible to assess their differential gene expression by a cDNA macroarray analysis using RNA extracted from paraffin sections, we analyzed two different cohorts of adrenal cortical adenoma (ACA) and ACC. Paraffin sections of seven ACC and seven ACA were analyzed. Transcriptional profiles were generated by commercially available c-DNA arrays testing 82 genes. Hybridization signals were quantified by densitometry and the intensity signal was compared for each gene between ACA and ACC cohorts. RNA was successfully extracted in only four out of 14 cases. Four genes displayed a significantly different expression (ACC/ACA ratio>1.5 or<0.6). Heat shock protein 60 (HSP-60) (ratio>2), Ciclin D1 and topoisomerase I (ratio>1.5) were overexpressed in the ACC cohort, while jun proto-oncogene was down-regulated. cDNA macroarray analysis from paraffin sections of adrenal tumors is feasible, despite with a low success rate. The different expression of HSP-60, Ciclin D1, jun proto-oncogene and topoisomerase I indicates that these genes may play a role in ACC pathogenesis and could represent potential diagnostic/prognostic/therapeutic target markers. Larger series of patients are necessary to confirm the biologic, diagnostic, prognostic and therapeutic implications of these findings.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/genética , Carcinoma Corticosuprarrenal/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Regulación hacia Abajo , Humanos , Proteínas de Neoplasias/genética , Proto-Oncogenes Mas , Regulación hacia ArribaRESUMEN
Overexpression of the c-Met/hepatocyte growth factor receptor(HGF-R) proto-oncogene and abnormal generation of intracellular oxygen species (reactive oxygen species (ROS)) have been linked, by independent lines of evidence, to cell transformation and to malignant growth. By comparing two subpopulations of the B16 mouse melanoma (B16-F0 and B16-F10) endowed with different lung metastasis capacities (low and high, respectively) we found that both the expression/phosphorylation of c-Met and the steady-state levels of ROS positively correlated with metastatic growth. shRNA-mediated downregulation of c-Met in F10 cells led to a parallel decrease in the generation of oxygen species and in metastatic capacity, suggesting that oxidants may mediate the pro-metastatic activity of the HGF receptor. c-Met activation by a ligand elicits the formation of oxidant species through the oxidase-coupled small GTPase Rac-1, a relevant downstream target of the HGF-R. Moreover, cell treatment with the catalytic ROS scavengers EUK-134 and EUK-189 attenuates Met signaling to ERKs and inhibits the anchorage-independent growth of F10 cells, consistent with a critical role for oxygen species in HGF signaling and in aggressive cell behavior. Finally, genetic manipulation of the Rac-ROS cascade at different levels demonstrated its crucial role in the pro-metastatic activity of c-Met in vivo. Thus, we have outlined a novel cascade triggered by c-Met and mediated by ROS, linked to metastasis and potentially targetable by new antimetastatic, redox-based therapies.