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1.
Biol Chem ; 403(3): 345-360, 2022 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-34883001

RESUMEN

The activity and interacting ability of a polyamidoamine (PAMAM) dendrimer modified with 4-N-methylpiperazine-1,8-naphthalimide units (termed D) and complexed by Cu(ii) ions, towards healthy and cancer cells were studied. Comparative electron paramagnetic resonance (EPR) studies of the Cu(ii)-D complex are presented: coordination mode, chemical structure, flexibility and stability of these complexes, in the absence and presence of myeloid cancer cells and peripheral blood mononuclear cells (PBMC). The interactions of Cu(ii) ions in the biological media at different equilibrium times were studied, highlighting different stability and interacting conditions with the cells. Furthermore, flow cytometry and confocal analysis, trace the peculiar properties of the dendrimers in PBMC and U937 cells. Indeed, a new probe (Fly) was used as a potential fluorescent tool for biological imaging of Cu(ii). The study highlights that dendrimer and, mainly, the Cu(ii) metallodendrimer are cytotoxic agents for the cells, specifically for U937 tumor cells, inducing mitochondrial dysfunction, ROS increase and lysosome involvement. The metallodendrimer shows antitumor selectivity, fewer affecting healthy PBMC, inducing a massive apoptotic cell death on U937 cells, in line with the high stability of this complex, as verified by EPR studies. The results underline the potentiality of this metallodendrimer to be used as anticancer drug.


Asunto(s)
Antineoplásicos , Dendrímeros , Neoplasias , Antineoplásicos/química , Antineoplásicos/farmacología , Dendrímeros/química , Dendrímeros/metabolismo , Dendrímeros/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Leucocitos Mononucleares , Naftalimidas/farmacología , Poliaminas
2.
Int J Mol Sci ; 24(1)2022 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-36613943

RESUMEN

Cytolethal distending toxin (CDT) is produced by a range of Gram-negative pathogenic bacteria such as Campylobacter jejuni. CDT represents an important virulence factor that is a heterotrimeric complex composed of CdtA, CdtB, and CdtC. CdtA and CdtC constitute regulatory subunits whilst CdtB acts as the catalytic subunit exhibiting phosphatase and DNase activities, resulting in cell cycle arrest and cell death. Extracellular vesicle (EV) secretion is an evolutionarily conserved process that is present throughout all kingdoms. Mammalian EVs play important roles in regular cell-to-cell communications but can also spread pathogen- and host-derived molecules during infections to alter immune responses. Here, we demonstrate that CDT targets the endo-lysosomal compartment, partially evading lysosomal degradation and exploiting unconventional secretion (EV release), which is largely involved in bacterial infections. CDT-like effects are transferred by Caco-2 cells to uninfected heterologous U937 and homologous Caco-2 cells. The journey of EVs derived from CDT-treated Caco-2 cells is associated with both intestinal and myeloid tumour cells. EV release represents the primary route of CDT dissemination, revealing an active toxin as part of the cargo. We demonstrated that bacterial toxins could represent suitable tools in cancer therapy, highlighting both the benefits and limitations. The global cell response involves a moderate induction of apoptosis and autophagic features may play a protective role against toxin-induced cell death. EVs from CDT-treated Caco-2 cells represent reliable CDT carriers, potentially suitable in colorectal cancer treatments. Our data present a potential bacterial-related biotherapeutic supporting a multidrug anticancer protocol.


Asunto(s)
Toxinas Bacterianas , Campylobacter jejuni , Humanos , Toxinas Bacterianas/farmacología , Toxinas Bacterianas/metabolismo , Células CACO-2 , Campylobacter jejuni/metabolismo , Proliferación Celular , Bacterias Gramnegativas/metabolismo , Células U937
3.
Int J Mol Sci ; 23(6)2022 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-35328491

RESUMEN

Fluorescent silica nanoparticles (SiNPs) appear to be a promising imaging platform, showing a specific subcellular localization. In the present study, we first investigated their preferential mitochondrial targeting in myeloid cells, by flow cytometry, confocal microscopy and TEM on both cells and isolated mitochondria, to acquire knowledge in imaging combined with therapeutic applications. Then, we conjugated SiNPs to one of the most used anticancer drugs, doxorubicin (DOX). As an anticancer agent, DOX has high efficacy but also an elevated systemic toxicity, causing multiple side effects. Nanostructures are usually employed to increase the drug circulation time and accumulation in target tissues, reducing undesired cytotoxicity. We tested these functionalized SiNPs (DOX-NPs) on breast cancer cell line MCF-7. We evaluated DOX-NP cytotoxicity, the effect on the cell cycle and on the expression of CD44 antigen, a molecule involved in adhesion and in tumor invasion, comparing DOX-NP to free DOX and stand-alone SiNPs. We found a specific ability to release a minor amount of CD44+ extracellular vesicles (EVs), from both CD81 negative and CD81 positive pools. Modulating the levels of CD44 at the cell surface in cancer cells is thus of great importance for disrupting the signaling pathways that favor tumor progression.


Asunto(s)
Antineoplásicos , Neoplasias de la Mama , Nanopartículas , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Doxorrubicina/química , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Femenino , Humanos , Mitocondrias , Células Mieloides , Nanopartículas/química , Dióxido de Silicio/química
4.
Biol Chem ; 402(10): 1225-1237, 2021 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-34090314

RESUMEN

The fluorescent probes represent an important tool in the biological study, in fact characterization of cellular structures and organelles are an important tool-target for understanding the mechanisms regulating most biological processes. Recently, a series of polyamino-macrocycles based on 1,4,7,10-tetraazacyclododecane was synthesized, bearing one or two NBD units (AJ2NBD·4HCl) useful as sensors for metal cations and halides able to target and to detect apolar environment, as lipid membranes. In this paper, we firstly illustrate the chemical synthesis of the AJ2NBD probe, its electronic absorption spectra and its behavior regarding pH of the environment. Lack of any cellular toxicity and an efficient labelling on fresh, living cells was demonstrated, allowing the use of AJ2NBD in biological studies. In particular, this green fluorescent probe may represent a potential dye for the compartments involved in the endosomal/autophagic pathway. This research's field should benefit from the use of AJ2NBD as a vesicular tracer, however, to ensure the precise nature of vesicles/vacuoles traced by this new probe, other more specific tests are needed.


Asunto(s)
Colorantes Fluorescentes , Lisosomas , Autofagia , Endosomas
5.
Int J Mol Sci ; 21(15)2020 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-32751833

RESUMEN

: Experimental evidence highlights the involvement of the endoplasmic reticulum (ER)-mediated Ca2+ signals in modulating synaptic plasticity and spatial memory formation in the hippocampus. Ca2+ release from the ER mainly occurs through two classes of Ca2+ channels, inositol 1,4,5-trisphosphate receptors (InsP3Rs) and ryanodine receptors (RyRs). Calsequestrin (CASQ) and calreticulin (CR) are the most abundant Ca2+-binding proteins allowing ER Ca2+ storage. The hippocampus is one of the brain regions expressing CASQ, but its role in neuronal activity, plasticity, and the learning processes is poorly investigated. Here, we used knockout mice lacking both CASQ type-1 and type-2 isoforms (double (d)CASQ-null mice) to: a) evaluate in adulthood the neuronal electrophysiological properties and synaptic plasticity in the hippocampal Cornu Ammonis 1 (CA1) field and b) study the performance of knockout mice in spatial learning tasks. The ablation of CASQ increased the CA1 neuron excitability and improved the long-term potentiation (LTP) maintenance. Consistently, (d)CASQ-null mice performed significantly better than controls in the Morris Water Maze task, needing a shorter time to develop a spatial preference for the goal. The Ca2+ handling analysis in CA1 pyramidal cells showed a decrement of Ca2+ transient amplitude in (d)CASQ-null mouse neurons, which is consistent with a decrease in afterhyperpolarization improving LTP. Altogether, our findings suggest that CASQ deletion affects activity-dependent ER Ca2+ release, thus facilitating synaptic plasticity and spatial learning in post-natal development.


Asunto(s)
Región CA1 Hipocampal/metabolismo , Proteínas de Unión al Calcio/fisiología , Calsecuestrina/fisiología , Plasticidad Neuronal , Aprendizaje Espacial , Animales , Región CA1 Hipocampal/citología , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Calsecuestrina/genética , Retículo Endoplásmico/metabolismo , Técnicas de Inactivación de Genes , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Piramidales/citología , Células Piramidales/metabolismo
6.
Fish Shellfish Immunol ; 73: 66-74, 2018 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-29208501

RESUMEN

Autophagy is a highly conserved and regulated catabolic process involved in maintaining cell homeostasis in response to different stressors. The autophagic machinery is also used as an innate immune mechanism against microbial infection. In invertebrates, that lack acquired immunity, autophagy may thus play a key role in the protection against potential pathogens. In aquatic molluscs, evidence has been provided for induction of autophagy by starvation and different environmental stressors; however, no information is available on autophagic pathways in the immune cells, the hemocytes. In this work, the autophagic processes were investigated in the hemocytes of the marine bivalve, the mussel Mytilus galloprovincialis. The effects of classical inducers/inhibitors of mammalian autophagy were first tested. Rapamycin induced a decrease in lysosomal membrane stability-LMS that was prevented by the autophagy inhibitor Wortmannin. Increased MDC fluorescence and expression of LC3-II were also observed. Moreover, responses to in vitro challenge with the bivalve pathogen Vibrio tapetis were evaluated. Mussel hemocytes were unable to activate the immune response towards V. tapetis; however, bacterial challenge induced a moderate decrease in LMS, corresponding to lysosomal activation but no cytotoxicity; the effect was prevented by Wortmannin. TEM observations showed that V. tapetis resulted in rapid formation of autophagosomes and autolysosomes. Accordingly, increased LC3-II expression, decreased levels of phosphorylated mTor and of p62 were observed. The results represent the first evidence for autophagic processes in bivalve hemocytes in response to bacterial challenge, and underline the protective role of autophagy towards potential pathogenic vibrios.


Asunto(s)
Autofagia , Hemocitos/fisiología , Mytilus/fisiología , Vibrio/fisiología , Animales , Western Blotting , Electroforesis , Hemocitos/inmunología , Lisosomas/fisiología , Microscopía Confocal , Microscopía Electrónica de Transmisión , Mytilus/inmunología
7.
Biochim Biophys Acta ; 1848(6): 1393-401, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25786874

RESUMEN

We recently reported that U937 cell mitochondria express a functional Na+-dependent ascorbic acid (AA) transporter recognised by anti-SVCT2 antibodies. The present study confirms and extends these observations by showing that this transporter is characterised by a Km and a pH-dependence comparable with that reported for the plasma membrane SVCT2. In isolated mitochondria, Na+ increased AA transport rate in a cooperative manner, revealed by a sigmoid curve and a Hill coefficient of 2, as also observed in intact Raw 264.7 cells (uniquely expressing SVCT2). There was however a striking difference on the Na+ concentrations necessary to reach saturation, i.e., 1 or 100 mM for the mitochondrial and plasma membrane transporters, respectively. Furthermore the mitochondrial, unlike the plasma membrane, transporter was fully active also in the absence of added Ca++ and/or Mg++. Taken together, the results presented in this study indicate that the U937 cell mitochondrial transporter of AA, because of its very low requirement for Na+ and independence for Ca++ and Mg++, displays kinetic characteristics surprisingly similar with those of the plasma membrane SVCT2.


Asunto(s)
Ácido Ascórbico/metabolismo , Calcio/farmacología , Magnesio/farmacología , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Sodio/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Humanos , Cinética , Ratones , Mitocondrias/efectos de los fármacos , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Células U937
8.
Environ Res ; 150: 73-81, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27257827

RESUMEN

The bivalve Mytilus galloprovincialis has proven as a suitable model invertebrate for evaluating the potential impact of nanoparticles (NPs) in the marine environment. In particular, in mussels, the immune system represents a sensitive target for different types of NPs. In environmental conditions, both NP intrinsic properties and those of the receiving medium will affect particle behavior and consequent bioavailability/uptake/toxicity. However, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment. In mammalian systems, different NPs associate with serum soluble components, organized into a "protein corona", which affects particle interactions with target cells. However, no information is available so far on the interactions of NPs with biological fluids of aquatic organisms. In this work, the influence of hemolymph serum (HS) on the in vitro effects of amino modified polystyrene NPs (PS-NH2) on Mytilus hemocytes was investigated. Hemocytes were incubated with PS-NH2 suspensions in HS (1, 5 and 50µg/mL) and the results were compared with those obtained in ASW medium. Cell functional parameters (lysosomal membrane stability, oxyradical production, phagocytosis) were evaluated, and morphological changes were investigated by TEM. The activation state of the signalling components involved in Mytilus immune response (p38 MAPK and PKC) was determined. The results show that in the presence of HS, PS-NH2 increased cellular damage and ROS production with respect to ASW medium. The effects were apparently mediated by disregulation of p38 MAPK signalling. The formation of a PS-NH2-protein corona in HS was investigated by centrifugation, and 1D- gel electrophoresis and nano-HPLC-ESI-MS/MS. The results identified the Putative C1q domain containing protein (MgC1q6) as the only component of the PS-NH2 hard protein corona in Mytilus hemolymph. These data represent the first evidence for the formation of a NP bio-corona in aquatic organisms and underline the importance of the recognizable biological identity of NPs in physiological exposure medium when testing their potential impact environmental model organisms. Although the results obtained in vitro do not entirely reflect a realistic exposure scenario and the more complex formation of a bio-corona that is likely to occur in vivo, these data will contribute to a better understanding of the effects of NPs in marine invertebrates.


Asunto(s)
Hemocitos/efectos de los fármacos , Mytilus/efectos de los fármacos , Nanopartículas/toxicidad , Poliestirenos/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Cationes/toxicidad , Hemocitos/metabolismo , Hemolinfa/efectos de los fármacos , Hemolinfa/metabolismo , Mytilus/metabolismo , Proteínas/metabolismo
9.
Fungal Genet Biol ; 81: 52-61, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26021705

RESUMEN

In a natural forest ecosystem, ectomycorrhiza formation is a way for soil fungi to obtain carbohydrates from their host plants. However, our knowledge of sugar transporters in ectomycorrhizal ascomycetous fungi is limited. To bridge this gap we used data obtained from the sequenced genome of the ectomycorrhizal fungus Tuber melanosporum Vittad. to search for sugar transporters. Twenty-three potential hexose transporters were found, and three of them (Tmelhxt1, Tmel2281 and Tmel131), differentially expressed during the fungus life cycle, were investigated. The heterologous expression of Tmelhxt1 and Tmel2281 in an hxt-null Saccharomyces cerevisiae strain restores the growth in glucose and fructose. The functional characterization and expression profiles of Tmelhxt1 and Tmel2281 in the symbiotic phase suggest that they are high affinity hexose transporters at the plant-fungus interface. On the contrary, Tmel131 is preferentially expressed in the fruiting body and its inability to restore the S. cerevisiae mutant strain growth led us to hypothesize that it could be involved in the transport of alternative carbon sources important for a hypothetical saprophytic strategy for the complete maturation of the carpophore.


Asunto(s)
Ascomicetos/genética , Ascomicetos/metabolismo , Transporte Biológico , Metabolismo de los Hidratos de Carbono , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Clonación Molecular , Biología Computacional , Medios de Cultivo/química , Fructosa/metabolismo , Expresión Génica , Glucosa/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo
10.
Environ Sci Pollut Res Int ; 31(6): 9745-9763, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38194171

RESUMEN

Several studies have reported the high bioindication capacity of Isopoda (Crustacea, Oniscidea), which is related to their important ability to accumulate contaminants, usefulness in soil ecotoxicology and bioindication activities. Any change in the isopod population, diversity and life cycle can indicate relevant pollution levels. The analysis of target tissues, such as the hepatopancreas, is another emerging approach (from a cytologic/histological level) to detect contaminant accumulation from different sources. In this study, tissue disaggregation procedures were optimised in the hepatopancreas, and flow cytometry (FC) was applied to detect cell viability and several cell functions. After disaggregation, two hepatopancreatic cell types, small (S) and big (B), were still recognisable: they differed in morphology and behaviour. The analyses were conducted for the first time on isopods from sites under different conditions of ecological disturbance through cytometric re-interpretation of ecological-environmental parameters. Significant differences in cell functional parameters were found, highlighting that isopod hepatopancreatic cells can be efficiently analysed by FC and represent standardisable, early biological indicators, tracing environmental-induced stress through cytologic/histologic analyses.


Asunto(s)
Isópodos , Animales , Isópodos/metabolismo , Biomarcadores Ambientales , Citometría de Flujo , Hepatopáncreas/metabolismo , Contaminación Ambiental
11.
Life (Basel) ; 13(4)2023 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-37109392

RESUMEN

Environmental disturbances resulting from anthropogenic energy pollution are intensely growing and represent a concern for the marine environment. Benthic organisms are the significant fauna exposed to this kind of pollution; among them, foraminifera are largely used as pollution bioindicators in marine environments, but studies on the effects induced by electrical stimulation are not documented. In the present research, we evaluated the effects of short-term different electric current densities on the viability of benthic foraminiferal species Amphistegina lessonii by checking the pseudopodial activity and defined the threshold electrical density range. After 3 days of treatment, A. lessonii stimulated with a constant current showed pseudopodial activity at a lower electric current density (0.29, 0.86 µA/cm2) up to 24 h. With increasing stimulation time, the percentages of pseudopodial activity decreased. The pseudopodial activity was absent at high current densities (5.71, 8.57 µA/cm2). The viability of A. lessonii exposed to a pulsed current was higher at a low and middle electric current density (from 0.29 to 5.71 µA/cm2) than at a high electric current density (from 11.43 to 20 µA/cm2). Based on these preliminary results, the selected benthic foraminiferal species seems to better stand pulsed currents than constant ones. These first experiments might provide useful information for the definition of the appropriate electrical density threshold to avoid side effects on a part of the benthic community.

12.
Environ Pollut ; 330: 121538, 2023 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-37011780

RESUMEN

Titanium dioxide nanoparticles (NPs) have numerous applications, and their demands have increased as an alternative for banned sunscreen filters. However, the underlying mechanisms of their toxicity, remain largely unknown. Here, we investigate the mechanism of TiO2 NP cytotoxicity and detoxification through time-course experiments (1, 6, and 24 h) based on cellular observations and single-cell transcriptome analyses in a marine benthic foraminifer strain, derived from a common unicellular eukaryotic organism worldwide. After exposure for 1 h, cells enhanced the production of reactive oxygen species (ROS) in acidic endosomes containing TiO2 NPs as well as in mitochondria. In acidic endosomes, ROS were produced through the Fenton reaction on the surface of charged TiO2 NPs. In mitochondria, ROS were associated with porphyrin synthesis that chelated metal ions. Glutathione peroxide and neutral lipids acted as a sink for free radicals, whereas lipid peroxides were excreted to prevent further radical chain reactions. By 24 h, aggregated TiO2 NPs were encapsulated in organic compounds, possibly ceramide, and excreted as mucus, thereby preventing their further uptake. Thus, we reveal that foraminifers can tolerate the toxicity of TiO2 NPs and even prevent their further phagocytosis and uptake by trapping TiO2 NPs inside mucus. This previously unknown strategy could be applied in bioremediation to sequester NPs from the marine environment and can guide management of TiO2 pollution.


Asunto(s)
Contaminantes Ambientales , Nanopartículas del Metal , Nanopartículas , Especies Reactivas de Oxígeno , Nanopartículas/toxicidad , Titanio/toxicidad , Nanopartículas del Metal/toxicidad
13.
Int J Biochem Mol Biol ; 14(5): 87-100, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38020445

RESUMEN

BACKGROUND: Phospholipids are highly diverse molecules with pleiotropic biological roles, from membrane components and signaling molecules, whose composition can change in response to both endogenous and external stimuli. Recent lipidomic studies on edible bivalve mollusks were focused on lipid nutritional value and growth requirements. However, no data are available on phospholipid profiles during bivalve larval development. In the model marine bivalve Mytilus galloprovincialis, early larvae (up to 48 hours post fertilization-hpf) undergo dramatic molecular and functional changes, including shell biogenesis and neurogenesis, that are sustained by egg lipid reserves. Changes in phospholipid composition may also occur participating in the complex processes of early development. OBJECTIVE: The lipidome of M. galloprovincialis eggs and early larval stages (24 and 48 hpf) was investigated in order to identify possible changes in phospholipid classes and related metabolic pathways that may play a role in key steps of development. MATERIALS AND METHODS: Lipidomic analysis were performed by NMR spectroscopy and liquid chromatography-mass spectrometry (LC-MS), with focus on phospholipids. Shifts in relative species composition of phosphatidylcholine, phosphatidylethanolamine, plasmalogen, and ceramide aminoethylphosphonate-CAEP, the bivalve analogue of the main mammalian ceramide sphingomyelin, were observed. Expression of genes involved in ceramide homeostasis was also modulated from eggs to early larval stages. RESULTS: The results represent the first data on changes in phospholipid composition in bivalve larvae and suggest a functional role of phospholipids in mussel early development. CONCLUSION: The results underline the importance of lipidomic studies in bivalve larvae, in both physiological conditions and in response to environmental stress.

14.
Biology (Basel) ; 11(7)2022 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-36101341

RESUMEN

The evaluation of the effects of pollution (e.g., Hg pollution) is a difficult task and relies mostly on biomonitoring based on bioindicators. The application of biomarkers may represent a complementary or alternative approach in environmental biomonitoring. Mercury is known to pose a significant health hazard due to its ability to cross cellular membranes, bioaccumulate, and biomagnify. In the present research, the effects of short-term (i.e., 24 h) Hg exposure in the symbiont-bearing benthic foraminiferal species Amphistegina lessonii are evaluated using several biomarkers (i.e., proteins and enzymes). Mercury leads to significant changes in the biochemistry of cells. Its effects are mainly associated with oxidative stress (i.e., production of reactive oxygen species: ROS), depletion of glutathione (GSH), and alteration of protein synthesis. Specifically, our findings reveal that exposure to Hg leads to the consumption of GSH by GPx and GST for the scavenging of ROS and the activation of antioxidant-related enzymes, including SOD and GSH-enzymes (GST, GSR, GPx, and Se-GPx), that are directly related to a defense mechanism against ROS. The Hg exposure also activates the MAPK (e.g., p-p38) and HSP (e.g., HSP 70) pathways. The observed biochemical alterations associated with Hg exposure may represent effective and reliable proxies (i.e., biomarkers) for the evaluation of stress in A. lessonii and lead to a possible application for the detection of early warning signs of environmental stress in biomonitoring.

15.
Biomolecules ; 12(5)2022 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-35625628

RESUMEN

The first step to obtain a cellular suspension from tissues is the disaggregation procedure. The cell suspension method has to provide a representative sample of the different cellular subpopulations and to maximize the number of viable functional cells. Here, we analyzed specific cell functions in cell suspensions from several rat tissues obtained by two different methods, automated-mechanical and enzymatic disaggregation. Flow cytometric, confocal, and ultrastructural (TEM) analyses were applied to the spleen, testis, liver and other tissues. Samples were treated by an enzymatic trypsin solution or processed by the Medimachine II (MMII). The automated-mechanical and enzymatic disaggregation procedures have shown to work similarly in some tissues, which displayed comparable amounts of apoptotic/necrotic cells. However, cells obtained by the enzyme-free Medimachine II protocols show a better preservation lysosome and mitochondria labeling, whereas the enzymatic gentle dissociation appears to constantly induce a lower amount of intracellular ROS; nevertheless, lightly increased ROS can be recognized as a complimentary signal to promote cell survival. Therefore, MMII represents a simple, fast, and standardized method for tissue processing, which allows to minimize bias arising from the operator's ability. Our study points out technical issues to be adopted for specific organs and tissues to obtain functional cells.


Asunto(s)
Testículo , Animales , Recuento de Células , Supervivencia Celular , Citometría de Flujo/métodos , Masculino , Ratas , Especies Reactivas de Oxígeno
16.
Front Mol Biosci ; 9: 902706, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35693554

RESUMEN

Ostreolysin A6 (OlyA6) is a 15 kDa protein produced by the oyster mushroom (Pleurotus ostreatus). It belongs to the aegerolysin family of proteins and binds with high affinity to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with its partnering protein with the membrane-attack-complex/perforin domain, pleurotolysin B (PlyB), OlyA6 can form bicomponent 13-meric transmembrane pores in artificial and biological membranes containing the aegerolysin lipid receptor, CPE. This pore formation is the main underlying molecular mechanism of potent and selective insecticidal activity of OlyA6/PlyB complexes against two economically important coleopteran plant pests: the western corn rootworm and the Colorado potato beetle. In contrast to insects, the main sphingolipid in cell membranes of marine invertebrates (i.e., molluscs and cnidarians) is ceramide aminoethylphosphonate (CAEP), a CPE analogue built on a phosphono rather than the usual phosphate group in its polar head. Our targeted lipidomic analyses of the immune cells (hemocytes) of the marine bivalve, the mussel Mytilus galloprovincialis, confirmed the presence of 29.0 mol% CAEP followed by 36.4 mol% of phosphatidylcholine and 34.6 mol% of phosphatidylethanolamine. Further experiments showed the potent binding of OlyA6 to artificial lipid vesicles supplemented with mussel CAEP, and strong lysis of these vesicles by the OlyA6/PlyB mixture. In Mytilus haemocytes, short term exposure (max. 1 h) to the OlyA6/PlyB mixture induced lysosomal membrane destabilization, decreased phagocytic activity, increased Annexin V binding and oxyradical production, and decreased levels of reduced glutathione, indicating rapid damage of endo-lysosomal and plasma membranes and oxidative stress. Our data suggest CAEP as a novel high-affinity receptor for OlyA6 and a target for cytolytic OlyA6/PlyB complexes.

17.
Life (Basel) ; 11(5)2021 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-34063137

RESUMEN

Proteins are essential to life, and the evaluation of their content, identification, and modification represents a fundamental assay in biochemistry research. Different analytical techniques and protocols have been specifically designed but have rarely been compared. Here, we test and compare a variety of methodologies and treatments for the quantification of proteins in Amphistegina lessonii, a larger symbiont-bearing benthic foraminiferal species. These analyses specifically include (a) lysis buffer (homemade vs. RIPA), (b) protein assays (Lowry, BCA, and Bradford), (c) ultrasonic bath treatment, and (d) protein staining (silver staining vs. Coomassie blue). On the basis of the comparative outcome, we suggest using the homemade lysis buffer, Lowry or BCA assays, ultrasonic bath treatment, and silver stain to maximize the extraction and characterization of protein for A. lessonii. This protocol might be suitable and extended to other benthic foraminiferal species, including the smaller ones.

18.
Front Immunol ; 12: 618726, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33679759

RESUMEN

The increasing number of data studies on the biological impact of anthropogenic chemicals in the marine environment, together with the great development of invertebrate immunology, has identified marine bivalves as a key invertebrate group for studies on immunological responses to pollutant exposure. Available data on the effects of contaminants on bivalve immunity, evaluated with different functional and molecular endpoints, underline that individual functional parameters (cellular or humoral) and the expression of selected immune-related genes can distinctly react to different chemicals depending on the conditions of exposure. Therefore, the measurement of a suite of immune biomarkers in hemocytes and hemolymph is needed for the correct evaluation of the overall impact of contaminant exposure on the organism's immunocompetence. Recent advances in -omics technologies are revealing the complexity of the molecular players in the immune response of different bivalve species. Although different -omics represent extremely powerful tools in understanding the impact of pollutants on a key physiological function such as immune defense, the -omics approach has only been utilized in this area of investigation in the last few years. In this work, available information obtained from the application of -omics to evaluate the effects of pollutants on bivalve immunity is summarized. The data shows that the overall knowledge on this subject is still quite limited and that to understand the environmental relevance of any change in immune homeostasis induced by exposure to contaminants, a combination of both functional assays and cutting-edge technology (transcriptomics, proteomics, and metabolomics) is required. In addition, the utilization of metagenomics may explain how the complex interplay between the immune system of bivalves and its associated bacterial communities can be modulated by pollutants, and how this may in turn affect homeostatic processes of the host, host-pathogen interactions, and the increased susceptibility to disease. Integrating different approaches will contribute to knowledge on the mechanism responsible for immune dysfunction induced by pollutants in ecologically and economically relevant bivalve species and further explain their sensitivity to multiple stressors, thus resulting in health or disease.


Asunto(s)
Adaptación Biológica/inmunología , Organismos Acuáticos/inmunología , Exposición a Riesgos Ambientales , Mytilus/inmunología , Contaminación del Agua , Adaptación Biológica/genética , Animales , Organismos Acuáticos/genética , Organismos Acuáticos/metabolismo , Genómica , Metabolómica , Mytilus/genética , Mytilus/metabolismo , Proteómica , Transcriptoma , Contaminación del Agua/efectos adversos
19.
Eur J Med Chem ; 215: 113292, 2021 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-33631696

RESUMEN

Iminopyridine-decorated carbosilane metallodendrimers have recently emerged as a promising strategy in the treatment of cancer diseases. Their unique features such as the nanometric size, the multivalent nature and the structural perfection offer an extraordinary platform to explore structure-to-property relationships. Herein, we showcase the outstanding impact on the antitumor activity of a parameter not explored before: the iminopyridine substituents in meta position. New Cu(II) carbosilane metallodendrimers, bearing methyl or methoxy substituents in the pyridine ring, were synthesized and thoroughly characterized. Electron Paramagnetic Resonance (EPR) was exploited to unveil the properties of the metallodendrimers. This study confirmed the presence of different coordination modes of the Cu(II) ion (Cu-N2O2, Cu-N4 and Cu-O4), whose ratios were determined by the structural features of the dendritic molecules. These metallodendrimers exhibited IC50 values in the low micromolar range (<6 µM) in tumor cell lines such as HeLa and MCF-7. The subsequent in vitro assays on both healthy (PBMC) and tumor (U937) myeloid cells revealed two key facts which improved the cytotoxicity and selectivity of the metallodrug: First, maximizing the Cu-N2O2 coordination mode; second, adequately selecting the pair ring-substituent/metal-counterion. The most promising candidates, G1(-CH3)Cl (8) and G1(-OCH3)NO3(17), exhibited a substantial increase in the antitumor activity in U937 tumor cells, compared to the non-substituted counterparts, probably through two different ROS-production pathways.


Asunto(s)
Antineoplásicos/farmacología , Complejos de Coordinación/farmacología , Dendrímeros/farmacología , Piridinas/farmacología , Silanos/farmacología , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Complejos de Coordinación/síntesis química , Cobre/química , Dendrímeros/síntesis química , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Piridinas/síntesis química , Especies Reactivas de Oxígeno/metabolismo , Silanos/síntesis química
20.
J Invertebr Pathol ; 105(1): 49-55, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20493193

RESUMEN

In mussel (Mytilus sp.) hemocytes, differential functional responses to injection with different types of live and heat-killed Vibrio species have been recently demonstrated. In this work, responses of Mytilus hemocytes to heat-killed Vibrio splendidus LGP32 and the mechanisms involved were investigated in vitro and the results were compared with those obtained with Vibrio anguillarum (ATCC 19264). Adhesion of hemocytes after incubation with bacteria was evaluated by flow cytometry: both total hemocyte counts (THC) and percentage of hemocyte sub-populations were determined in non-adherent cells. Functional parameters such as lysosomal membrane stability, lysozyme release, extracellular ROS production and NO production were evaluated, as well as the phosphorylation state of the stress-activated p38 MAPK and PKC. Neither Vibrio affected total hemocyte adhesion, while both induced similar lysosomal destabilization and NO production. However, V. splendidus decreased adhesion of large granulocytes, induced rapid and persistent lysozyme release and stimulated extracellular ROS production: these effects were associated with persistent activation of p38 MAPK and PKC. In contrast, V. anguillarum decreased adhesion of large semigranular hemocytes and increased that of hyalinocytes, had no effect on the extracellular ROS production, and induced significantly lower lysozyme release and phosphorylation of p-38 MAPK and PKC than V. splendidus. These data reinforced the existence of specific interactions between mussel hemocytes and V. splendidus LGP32 and suggest that this Vibrio strain affects bivalve hemocytes through disregulation of immune signaling. The results support the hypothesis that responses of bivalve hemocytes to different bacterial stimuli may depend not only on the nature of the stimulus, but also on the cell subtype, thus leading to differential activation of signaling components.


Asunto(s)
Hemocitos/metabolismo , Mytilus/inmunología , Mytilus/microbiología , Proteína Quinasa C/metabolismo , Vibrio/clasificación , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Adhesión Celular/fisiología , Hemocitos/citología , Muramidasa/metabolismo , Mytilus/metabolismo , Óxido Nítrico/metabolismo , Fosforilación/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Especificidad de la Especie , Vibrio/patogenicidad
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