RESUMEN
Inhalation is a major route by which human exposure to substances can occur. Resources have therefore been dedicated to optimize human-relevant in vitro approaches that can accurately and efficiently predict the toxicity of inhaled chemicals for robust risk assessment and management. In this study-the IN vitro Systems to PredIct REspiratory toxicity Initiative-2 cell-based systems were used to predict the ability of chemicals to cause portal-of-entry effects on the human respiratory tract. A human bronchial epithelial cell line (BEAS-2B) and a reconstructed human tissue model (MucilAir, Epithelix) were exposed to triethoxysilane (TES) and trimethoxysilane (TMS) as vapor (mixed with N2 gas) at the air-liquid interface. Cell viability, cytotoxicity, and secretion of inflammatory markers were assessed in both cell systems and, for MucilAir tissues, morphology, barrier integrity, cilia beating frequency, and recovery after 7 days were also examined. The results show that both cell systems provide valuable information; the BEAS-2B cells were more sensitive in terms of cell viability and inflammatory markers, whereas MucilAir tissues allowed for the assessment of additional cellular effects and time points. As a proof of concept, the data were also used to calculate human equivalent concentrations. As expected, based on chemical properties and existing data, the silanes demonstrated toxicity in both systems with TMS being generally more toxic than TES. Overall, the results demonstrate that these in vitro test systems can provide valuable information relevant to predicting the likelihood of toxicity following inhalation exposure to chemicals in humans.
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Células Epiteliales , Silanos , Humanos , Silanos/toxicidad , Silanos/metabolismo , Línea Celular , BronquiosRESUMEN
The application of siRNA in gene therapy is mainly limited because of the problems with its transport into cells. Utilization of cationic dendrimers as siRNA carriers seems to be a promising solution in overcoming these issues, due to their positive charge and ability to penetrate cell membranes. The following two types of carbosilane dendrimers were examined: CBD-1 and CBD-2. Dendrimers were complexed with pro-apoptotic siRNA (Mcl-1 and Bcl-2) and the complexes were characterized by measuring their zeta potential, circular dichroism and fluorescence of ethidium bromide associated with dendrimers. CBD-2/siRNA complexes were also examined by agarose gel electrophoresis. Both dendrimers form complexes with siRNA. Moreover, the cellular uptake and influence on the cell viability of the dendrimers and dendriplexes were evaluated using microscopic methods and XTT assay on MCF-7 cells. Microscopy showed that both dendrimers can transport siRNA into cells; however, a cytotoxicity assay showed differences in the toxicity of these dendrimers.
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ARN Interferente Pequeño/uso terapéutico , Silanos/farmacología , Cationes , Supervivencia Celular , Dicroismo Circular , Dendrímeros/química , Dendrímeros/farmacología , Terapia Genética/métodos , Humanos , Células MCF-7 , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Tamaño de la Partícula , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Bicatenario/genética , ARN Interferente Pequeño/genética , Silanos/química , Silanos/metabolismoRESUMEN
To determine whether quaternary ammonium (k21) binds to Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) spike protein via computational molecular docking simulations, the crystal structure of the SARS-CoV-2 spike receptor-binding domain complexed with ACE-2 (PDB ID: 6LZG) was downloaded from RCSB PD and prepared using Schrodinger 2019-4. The entry of SARS-CoV-2 inside humans is through lung tissues with a pH of 7.38-7.42. A two-dimensional structure of k-21 was drawn using the 2D-sketcher of Maestro 12.2 and trimmed of C18 alkyl chains from all four arms with the assumption that the core moiety k-21 was without C18. The immunogenic potential of k21/QA was conducted using the C-ImmSim server for a position-specific scoring matrix analyzing the human host immune system response. Therapeutic probability was shown using prediction models with negative and positive control drugs. Negative scores show that the binding of a quaternary ammonium compound with the spike protein's binding site is favorable. The drug molecule has a large Root Mean Square Deviation fluctuation due to the less complex geometry of the drug molecule, which is suggestive of a profound impact on the regular geometry of a viral protein. There is high concentration of Immunoglobulin M/Immunoglobulin G, which is concomitant of virus reduction. The proposed drug formulation based on quaternary ammonium to characterize affinity to the SARS-CoV-2 spike protein using simulation and computational immunological methods has shown promising findings.
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Descubrimiento de Drogas , Compuestos de Amonio Cuaternario/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Sitios de Unión , COVID-19/patología , COVID-19/virología , Humanos , Simulación del Acoplamiento Molecular , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Compuestos de Amonio Cuaternario/química , SARS-CoV-2/aislamiento & purificación , Silanos/química , Silanos/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Tratamiento Farmacológico de COVID-19RESUMEN
We disclose our studies on a copper-mediated reaction of alkynes with trimethylsilyl azide to afford nitriles, and proposed a reaction mechanism, which involves an iodoalkyne and an iodotriazole as intermediates.
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Alquinos/química , Cobre/química , Nitrilos/química , Triazoles/química , Azidas/química , Azidas/metabolismo , Catálisis , Reacción de Cicloadición , Silanos/metabolismo , Solventes/químicaRESUMEN
In the frame of developing sustainable, eco-friendly and high performance materials, microcrystalline cellulose modified through silane coupling agent (MCC Si) is used as a reinforcing agent of benzoxazine resin to manufacture composites at different loadings of 5, 10, 15, 20 wt%. The structural, morphological and crystallinity characterizations of the modified MCC were initially performed to scrutinize the changes and confirm the modification. Then, an investigation on the crosslinking process of the prepared composites was held through curing kinetic study employing isoconversional methods. The kinetic data revealed a decrease in the average values of activation energy and the pre-exponential factor, particularly for composite supplemented with 10% MCC Si, whereas all samples disclosed a tendency of an autocatalytic curing mechanism. Furthermore, the study of the dynamic mechanical properties and degradation features of the cured specimens, respectively, indicated a superior stiffness attributable to the good interaction between BA-a and MCC Si, and enhanced thermal stability for the composites compared to pristine resin.
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Benzoxazinas/química , Benzoxazinas/metabolismo , Celulosa/química , Celulosa/metabolismo , Resinas de Plantas/química , Resinas de Plantas/metabolismo , Silanos/química , Silanos/metabolismo , Temperatura , Resinas Compuestas/síntesis química , Reactivos de Enlaces Cruzados/química , Cristalización , Enlace de Hidrógeno , Cinética , Estructura MolecularRESUMEN
Mesoporous silica particles of controlled size and shape are potentially beneficial for many applications, but their usage may be limited by the complex procedure of fabrication. Biotemplating provides a facile approach to synthesize materials with desired shapes. Herein, a bioinspired design principle is adopted through displaying silaffin-derived 5R5 proteins on the surface of Escherichia coli by genetic manipulations. The genetically modified Escherichia coli provides a three-dimensional template to regulate the synthesis of rod-shaped silica. The silicification is initiated on the cell surface under the functionality of 5R5 proteins and subsequentially the inner space is gradually filled. Density functional theory simulation reveals the interfacial interactions between silica precursors and R5 peptides at the atomic scale. There is a large conformation change of this protein during biosilicification. Electrostatic interactions contribute to the high affinity between positively charged residues (Lys4, Arg16, Arg17) and negatively charged tetraethyl orthosilicate. Hydrogen bonds develop between Arg16 (OH), Arg17 (OH and NH), Leu19 (OH) residues and the forming silica agglomerates. In addition, the resulting rod-shaped silica copy of the bacteria can transform into mesoporous SiOx nanorods composed of carbon-coated nanoparticles after carbonization, which is shown to allow superior lithium storage performance.
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Escherichia coli/metabolismo , Nanopartículas/metabolismo , Dióxido de Silicio/metabolismo , Biomineralización , Carbono/química , Escherichia coli/genética , Enlace de Hidrógeno , Litio/química , Nanopartículas/química , Nanotubos/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Porosidad , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Silanos/química , Silanos/metabolismo , Dióxido de Silicio/química , Electricidad EstáticaRESUMEN
Organisms belonging to Synechococcus sp. genera are observed in all freshwater, brackish, and marine waters of the world. They play a relevant role in these ecosystems, since they are one of the main primary producers, especially in open ocean. Eventually, they form mass blooms in coastal areas, which are potentially dangerous for the functioning of marine ecosystems. Allelopathy could be an important factor promoting the proliferation of these organisms. According to the authors' best knowledge, there is no information on the allelopathic activity and allelopathic compounds exhibited by different Synechococcus sp. phenotypes. Therefore, the research conducted here aimed to study the bioactivity of compounds produced by three phenotypes of Synechococcus sp. by studying their influence on the growth, chlorophyll fluorescence, and photosynthetic pigments of eighteen cyanobacteria and microalgae species. We demonstrated that three different Synechococcus sp. phenotypes, including a phycocyanin (PC)-rich strain (Type 1; green strain) and phycoerythrin (PE)-rich strains containing phycoerythrobilin (PEB) and phycocyanobilin (PCB) (Type 2; red strain and Type 3a; brown strain), had a significant allelopathic effect on the selected species of cyanobacteria, diatoms, and green algae. For all green algae, a decrease in cell abundance under the influence of phenotypes of donor cyanobacteria was shown, whereas, among some target cyanobacteria and diatom species, the cell-free filtrate was observed to have a stimulatory effect. Our estimates of the stress on photosystem II (Fv/Fm) showed a similar pattern, although for some diatoms, there was an effect of stress on photosynthesis, while a stimulatory effect on growth was also displayed. The pigment content was affected by allelopathy in most cases, particularly for chlorophyll a, whilst it was a bit less significant for carotenoids. Our results showed that Synechococcus sp. Type 3a had the strongest effect on target species, while Synechococcus sp. Type 1 had the weakest allelopathic effect. Furthermore, GC-MS analysis produced different biochemical profiles for the Synechococcus strains. For every phenotype, the most abundant compound was different, with oxime-, methoxy-phenyl- being the most abundant substance for Synechococcus Type 1, eicosane for Synechococcus Type 2, and silanediol for Synechococcus Type 3a.
Asunto(s)
Floraciones de Algas Nocivas/fisiología , Feromonas/metabolismo , Fitoplancton/fisiología , Synechococcus/fisiología , Microbiología del Agua , Alelopatía/fisiología , Proliferación Celular/fisiología , Feromonas/química , Fotosíntesis , Ficobilinas/metabolismo , Ficocianina/metabolismo , Ficoeritrina/metabolismo , Fitoplancton/química , Silanos/metabolismo , Synechococcus/químicaRESUMEN
Hydroxytyrosol (HT) is a diphenolic compound prevalent mainly in olives with pronounced antioxidant activity and proven benefits for human health. Current production limitations have motivated studies concerning the hydroxylation of tyrosol to HT with tyrosinase; however, accumulation of the diphenol is restricted due to its rapid subsequent oxidation to 3,4-quinone-phenylethanol. In this study, a continuous two-enzyme reaction system of sol-gel-immobilized tyrosinase and glucose dehydrogenase (GDH) was developed for the synthesis of HT. Purified tyrosinase from Bacillus megaterium (TyrBm) and E. coli cell extract expressing GDH from B. megaterium were encapsulated in a sol-gel matrix based on triethoxysilane precursors. While tyrosinase oxidized tyrosol to 3,4-quinone-phenylethanol, GDH catalyzed the simultaneous reduction of the cofactor NAD+ to NADH, which was the reducing agent enabling the accumulation of HT. Using 50 mM tyrosol, the immobilized system under optimized conditions, enabled a final HT yield of 7.68 g/L with productivity of 2.30 mg HT/mg TyrBm beads. Furthermore, the immobilized bi-enzyme system showed the feasibility for HT production from 1 mM tyrosol using a 0.5-L bioreactor as well as stable activity over 8 repeated cycles. The production of other diphenols with commercial importance such as L-dopa (3,4-dihydroxyphenylalanine) or piceatannol may be synthesized with this efficient approach.
Asunto(s)
Bacillus megaterium/enzimología , Biocatálisis , Glucosa 1-Deshidrogenasa/metabolismo , Monofenol Monooxigenasa/metabolismo , Alcohol Feniletílico/análogos & derivados , Reactores Biológicos , Enzimas Inmovilizadas/metabolismo , Escherichia coli/enzimología , NAD/metabolismo , Oxidación-Reducción , Alcohol Feniletílico/metabolismo , Silanos/metabolismoRESUMEN
Bioorthogonal chemistry capable of operating in live animals is needed to investigate biological processes such as cell death and immunity. Recent studies have identified a gasdermin family of pore-forming proteins that executes inflammasome-dependent and -independent pyroptosis1-5. Pyroptosis is proinflammatory, but its effect on antitumour immunity is unknown. Here we establish a bioorthogonal chemical system, in which a cancer-imaging probe phenylalanine trifluoroborate (Phe-BF3) that can enter cells desilylates and 'cleaves' a designed linker that contains a silyl ether. This system enabled the controlled release of a drug from an antibody-drug conjugate in mice. When combined with nanoparticle-mediated delivery, desilylation catalysed by Phe-BF3 could release a client protein-including an active gasdermin-from a nanoparticle conjugate, selectively into tumour cells in mice. We applied this bioorthogonal system to gasdermin, which revealed that pyroptosis of less than 15% of tumour cells was sufficient to clear the entire 4T1 mammary tumour graft. The tumour regression was absent in immune-deficient mice or upon T cell depletion, and was correlated with augmented antitumour immune responses. The injection of a reduced, ineffective dose of nanoparticle-conjugated gasdermin along with Phe-BF3 sensitized 4T1 tumours to anti-PD1 therapy. Our bioorthogonal system based on Phe-BF3 desilylation is therefore a powerful tool for chemical biology; our application of this system suggests that pyroptosis-induced inflammation triggers robust antitumour immunity and can synergize with checkpoint blockade.
Asunto(s)
Preparaciones de Acción Retardada/administración & dosificación , Neoplasias Mamarias Experimentales/inmunología , Piroptosis/inmunología , Animales , Cumarinas/administración & dosificación , Cumarinas/química , Cumarinas/metabolismo , Cumarinas/farmacocinética , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/metabolismo , Preparaciones de Acción Retardada/farmacocinética , Femenino , Proteínas Fluorescentes Verdes/administración & dosificación , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/farmacocinética , Células HeLa , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/química , Inmunoconjugados/metabolismo , Inmunoconjugados/farmacocinética , Inflamasomas/inmunología , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/administración & dosificación , Oligopéptidos/química , Oligopéptidos/metabolismo , Oligopéptidos/farmacocinética , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Proteínas/administración & dosificación , Proteínas/química , Proteínas/metabolismo , Proteínas/farmacocinética , Silanos/administración & dosificación , Silanos/química , Silanos/metabolismo , Silanos/farmacocinética , Linfocitos T/inmunología , Trastuzumab/administración & dosificación , Trastuzumab/química , Trastuzumab/metabolismo , Trastuzumab/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Compared to the biological world's rich chemistry for functionalizing carbon, enzymatic transformations of the heavier homologue silicon are rare. We report that a wild-type cytochromeâ P450 monooxygenase (P450BM3 from Bacillus megaterium, CYP102A1) has promiscuous activity for oxidation of hydrosilanes to give silanols. Directed evolution was applied to enhance this non-native activity and create a highly efficient catalyst for selective silane oxidation under mild conditions with oxygen as the terminal oxidant. The evolved enzyme leaves C-H bonds present in the silane substrates untouched, and this biotransformation does not lead to disiloxane formation, a common problem in silanol syntheses. Computational studies reveal that catalysis proceeds through hydrogen atom abstraction followed by radical rebound, as observed in the native C-H hydroxylation mechanism of the P450 enzyme. This enzymatic silane oxidation extends nature's impressive catalytic repertoire.
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Bacillus megaterium/enzimología , Sistema Enzimático del Citocromo P-450/metabolismo , Silanos/metabolismo , Biocatálisis , Modelos Moleculares , Oxidación-Reducción , Silanos/químicaRESUMEN
The organization of virus-like particles (VLPs) on surfaces is a relevant matter for both fundamental and biomedical sciences. In this work, the authors have tailored surfaces with different surface tension components aiming at finding a relationship with the affinity of the different geometric/surface features of icosahedral P22 VLPs. The surfaces have been prepared by titanate assisted organosilanization with glycidyloxy, amino, and perfluoro silanes. Vibrational and photoelectron spectroscopies have allowed identifying the different functional groups of the organosilanes on the surfaces. Atomic force microscopy (AFM) showed that, irrespective of the organosilane used, the final root mean square roughness remains below 1 nm. Contact angle analyses confirm the effective formation of a set of surface chemistries exhibiting different balance among surface tension components. The study of the adsorption of P22 VLPs has involved the analysis of the dynamics of virus immobilization by fluorescence microscopy and the interpretation of the final VLP orientation by AFM. These analyses give rise to statistical distributions pointing to a higher affinity of VLPs toward perfluorinated surfaces, with a dominant fivefold conformation on this hydrophobic surface, but threefold and twofold symmetries dominating on hydrophilic surfaces. These results can be explained in terms of a reinforced hydrophobic interaction between the perfluorinated surface and the dominating hydrophobic residues present at the P22 pentons.
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Adsorción , Bacteriófago P22/metabolismo , Silanos/metabolismo , Virosomas/metabolismo , Microscopía de Fuerza Atómica , Análisis EspectralRESUMEN
Pregnane X receptor (PXR) is a ligand-dependent transcription factor that is considered to be a potential therapeutic target for multiple diseases. Herein, we report the development and structure-activity relationship studies of a new series of hPXR agonists. Focusing on our recently developed silanol-sulfonamide scaffold, we developed the potent hPXR agonist 28, which shows good selectivity over hLXRα and ß, hFXR, and hRORα and γ. Examination of the structure-activity relationship suggested a possible strategy to manipulate the selectivity. Docking simulation indicated the presence of an additional binding cavity and polar contacts in the ligand-binding pocket of hPXR. This information should be helpful for the future development of more potent and selective hPXR ligands.
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Receptor X de Pregnano/agonistas , Silanos/química , Sitios de Unión , Diseño de Fármacos , Humanos , Receptores X del Hígado/agonistas , Receptores X del Hígado/metabolismo , Simulación del Acoplamiento Molecular , Receptores Nucleares Huérfanos/antagonistas & inhibidores , Receptores Nucleares Huérfanos/metabolismo , Receptor X de Pregnano/metabolismo , Estructura Terciaria de Proteína , Silanos/síntesis química , Silanos/metabolismo , Relación Estructura-ActividadRESUMEN
Isomeric O- and N-glucuronides are common drug metabolites produced in phase II of drug metabolism. Distinguishing these isomers by using common analytical techniques has proven challenging. A tandem mass spectrometric method based on gas-phase ion/molecule reactions of deprotonated glucuronide drug metabolites with trichlorosilane (HSiCl3) in a linear quadrupole ion trap mass spectrometer is reported here to readily enable differentiation of the O- and N-isomers. The major product ion observed upon reactions of HSiCl3 with deprotonated N-glucuronides is a diagnostic HSiCl3 adduct that has lost two HCl molecules ([M - H + HSiCl3 - 2HCl]-). This product ion was not observed for deprotonated O-glucuronides. Reaction mechanisms were explored with quantum chemical calculations at the M06-2X/6-311++G(d,p) level of theory.
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Glucurónidos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Espectrometría de Masas en Tándem/métodos , Cromatografía Líquida de Alta Presión/métodos , Glucurónidos/química , Isomerismo , Preparaciones Farmacéuticas/química , Protones , Silanos/química , Silanos/metabolismoRESUMEN
The aggregation processes of magnetic nanoparticles in biosystems are analysed by comparing the magnetic properties of three systems with different spatial distributions of the nanoparticles. The first one is iron oxide nanoparticles (NPs) of 14 nm synthesized by coprecipitation with two coatings, (3-aminopropyl)trimethoxysilane (APS) and dimercaptosuccinic acid (DMSA). The second one is liposomes with encapsulated nanoparticles, which have different configurations depending on the NP coating (NPs attached to the liposome surface or encapsulated in its aqueous volume). The last system consists of two cell lines (Pan02 and Jurkat) incubated with the NPs. Dynamic magnetic behaviour (AC) was analysed in liquid samples, maintaining their colloidal properties, while quasi-static (DC) magnetic measurements were performed on lyophilised samples. AC measurements provide a direct method for determining the effect of the environment on the magnetization relaxation of nanoparticles. Thus, the imaginary (χ'') component shifts to lower frequencies as the aggregation state increases from free nanoparticles to those attached or embedded into liposomes in cell culture media and more pronounced when internalized by the cells. DC magnetization curves show no degradation of the NPs after interaction with biosystems in the analysed timescale. However, the blocking temperature is shifted to higher temperatures for the nanoparticles in contact with the cells, regardless of the location, the incubation time, the cell line and the nanoparticle coating, supporting AC susceptibility data. These results indicate that the simple fact of being in contact with the cells makes the nanoparticles aggregate in a non-controlled way, which is not the same kind of aggregation caused by the contact with the cell medium nor inside liposomes.
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Portadores de Fármacos/química , Liposomas/química , Fenómenos Magnéticos , Nanopartículas de Magnetita/química , 1,2-Dipalmitoilfosfatidilcolina/química , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Portadores de Fármacos/toxicidad , Endocitosis , Humanos , Liposomas/toxicidad , Nanopartículas de Magnetita/toxicidad , Ratones , Tamaño de la Partícula , Propilaminas/química , Propilaminas/metabolismo , Propilaminas/toxicidad , Silanos/química , Silanos/metabolismo , Silanos/toxicidad , Succímero/química , Succímero/metabolismo , Succímero/toxicidad , TemperaturaRESUMEN
BACKGROUND/AIM: Failure of cancer chemotherapy caused by multidrug resistance (MDR) of tumor cells is mediated by ABC transporters that reduce the uptake of cytotoxic agents. Similar transporters are responsible for amyloid clearance in nerve cells in Alzheimer's disease (AD). The aim of this study was to compare the biological effects of amyloid complexes of some known ABC transporter inhibitors e.g. disiloxanes. One of the most active fragments of the pathological "endogen" substrate responsible for AD was investigated in the presence of amyloid-beta fragment on the reversal of multidrug resistance and apoptosis induction on multidrug-resistant tumor cells in model experiments. MATERIALS AND METHODS: The efflux pump activity of the cells treated with amyloid-beta complexes was studied by Rhodamin-123 accumulation. Apoptosis induction was measured by staining of treated cells by Annexin-V and propidium iodine. The fluorescent activity FL-1 and FL-2 of the cells was measured and analyzed on a PARTEC FACScan instrument. RESULTS: The resistance modifiers: disiloxanes and memantine complexed with amyloid-beta 1-42 reduced the activity of ABC transporter in MDR tumor cells. Early apoptosis was moderately increased by amyloid-beta complexes. Late apoptosis and the number of total viable cells were not changed. CONCLUSION: Amyloid-beta and its complexes inactivate the efflux pump of tumor cells resulting in accumulation of amyloid. It is supposed that reduced membrane transport can explain the lower incidence of cancer in AD.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Péptidos beta-Amiloides/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Memantina/farmacología , Silanos/farmacología , Péptidos beta-Amiloides/metabolismo , Animales , Antiparkinsonianos/metabolismo , Antiparkinsonianos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Memantina/metabolismo , Ratones , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Unión Proteica , Silanos/metabolismoRESUMEN
Exploratory studies using human fetal tissue have suggested that intrastriatal transplantation of dopaminergic neurons may become a future treatment for patients with Parkinson's disease. However, the use of human fetal tissue is compromised by ethical, regulatory and practical concerns. Human stem cells constitute an alternative source of cells for transplantation in Parkinson's disease, but efficient protocols for controlled dopaminergic differentiation need to be developed. Short-term, low-level carbon monoxide (CO) exposure has been shown to affect signaling in several tissues, resulting in both protection and generation of reactive oxygen species. The present study investigated the effect of CO produced by a novel CO-releasing molecule on dopaminergic differentiation of human neural stem cells. Short-term exposure to 25 ppm CO at days 0 and 4 significantly increased the relative content of ß-tubulin III-immunoreactive immature neurons and tyrosine hydroxylase expressing catecholaminergic neurons, as assessed 6 days after differentiation. Also the number of microtubule associated protein 2-positive mature neurons had increased significantly. Moreover, the content of apoptotic cells (Caspase3) was reduced, whereas the expression of a cell proliferation marker (Ki67) was left unchanged. Increased expression of hypoxia inducible factor-1α and production of reactive oxygen species (ROS) in cultures exposed to CO may suggest a mechanism involving mitochondrial alterations and generation of ROS. In conclusion, the present procedure using controlled, short-term CO exposure allows efficient dopaminergic differentiation of human neural stem cells at low cost and may as such be useful for derivation of cells for experimental studies and future development of donor cells for transplantation in Parkinson's disease.
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Monóxido de Carbono/administración & dosificación , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Apoptosis/efectos de los fármacos , Monóxido de Carbono/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células-Madre Neurales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Silanos/administración & dosificación , Silanos/metabolismoRESUMEN
Nanodiamonds have many attractive properties that make them suitable for a range of biological applications, but their practical use has been limited because nanodiamond conjugates tend to aggregate in solution during or after functionalisation. Here we demonstrate the production of DNA-detonation nanodiamond (DNA-DND) conjugates with high dispersion and solubility using an ultrasonic, mixed-silanization chemistry protocol based on the in situ Bead-Assisted Sonication Disintegration (BASD) silanization method. We use two silanes to achieve these properties: (1) 3-(trihydroxysilyl)propyl methylphosphonate (THPMP); a negatively charged silane that imparts high zeta potential and solubility in solution; and (2) (3-aminopropyl)triethoxysilane (APTES); a commonly used functional silane that contributes an amino group for subsequent bioconjugation. We target these amino groups for covalent conjugation to thiolated, single-stranded DNA oligomers using the heterobifunctional crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The resulting DNA-DND conjugates are the smallest reported to date, as determined by Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). The functionalisation method we describe is versatile and can be used to produce a wide variety of soluble DND-biomolecule conjugates.
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Fenómenos Químicos , ADN/metabolismo , Nanodiamantes/química , Silanos/metabolismo , Dispersión Dinámica de Luz , Microscopía de Fuerza Atómica , Solubilidad , SonicaciónRESUMEN
Dendrimers, which are considered as one of the most promising tools in the field of nanobiotechnology due to their structural organization, showed a great potential in gene therapy, drug delivery, medical imaging and as antimicrobial and antiviral agents. This article is devoted to study interactions between new carbosilane-based metallodendrimers containing ruthenium and anti-cancer small interfering RNA (siRNA). Formation of complexes between anti-cancer siRNAs and Ru-based carbosilane dendrimers was evaluated by transmission electron microscopy, circular dichroism and fluorescence. The zeta-potential and the size of dendriplexes were determined by dynamic light scattering. The internalization of dendriplexes were estimated using HL-60 cells. Results show that ruthenium dendrimers associated with anticancer siRNA have the ability to deliver siRNA as non-viral vectors into the cancer cells. Moreover, dendrimers can protect siRNA against nuclease degradation. Nevertheless, further research need to be performed to examine the therapeutic potential of ruthenium dendrimers as well as dendrimers complexed with siRNA and anticancer drugs towards cancer cells.
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Antineoplásicos/metabolismo , Dendrímeros/metabolismo , Sustancias Intercalantes/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Modelos Moleculares , ARN Interferente Pequeño/metabolismo , Rutenio/metabolismo , Absorción Fisiológica , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Dicroismo Circular , Dendrímeros/administración & dosificación , Dendrímeros/química , Estabilidad de Medicamentos , Dispersión Dinámica de Luz , Células HL-60 , Humanos , Sustancias Intercalantes/administración & dosificación , Sustancias Intercalantes/química , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/patología , Microscopía Electrónica de Transmisión , Conformación Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Tamaño de la Partícula , Interferencia de ARN , Estabilidad del ARN , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , ARN Interferente Pequeño/ultraestructura , Rutenio/administración & dosificación , Rutenio/química , Silanos/química , Silanos/metabolismo , Propiedades de SuperficieRESUMEN
Tailor-made: Discussed herein is the ability to adapt biology's mechanisms for innovation and optimization to solving problems in chemistry and engineering. The evolution of nature's enzymes can lead to the discovery of new reactivity, transformations not known in biology, and reactivity inaccessible by small-molecule catalysts.
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Boranos/metabolismo , Citocromos c/metabolismo , Evolución Molecular Dirigida , Ingeniería de Proteínas , Silanos/metabolismo , Biocatálisis , Boranos/química , Citocromos c/química , Ensayos Analíticos de Alto Rendimiento , Rhodothermus/enzimología , Silanos/químicaRESUMEN
The interaction of a double stranded small interference RNA (siRNA Nef) with cationic carbosilane dendrimers of generations 1-3 with two different ammonium functions at the periphery ([-NMe2R]+, R=Me, (CH2)2OH) has been studied by experimental techniques (zeta potential, electrophoresis, single molecule pulling experiments) and molecular dynamic calculations. These studies state the presence of different forces on dendriplex formation, depending on generation and type of ammonium group. Whilst for higher dendrimers electrostatic forces mainly drive the stability of dendriplexes, first generation compounds can penetrate into siRNA strands due to the establishment of hydrophobic interactions. Finally, in the particular case of first generation dendrimer [G1O3(NMe2(CH2)2OH))6]6+; the presence of hydroxyl groups reinforces dendriplex stability by hydrogen bonds formation. However, since these small dendrimers do not cover the RNA, only higher generation derivatives protect RNA from degradation.