RESUMO
OBJECTIVE: The rise in primary and revision surgeries utilizing joint replacement implants suggest the need for more reliable means of promoting implant fixation. Zoledronate-(Zol), cytochalasin-D-(cytoD), and desferrioxamine-(DFO) have been shown to enhance mesenchymal stem cell (MSC) differentiation into osteoblasts promoting bone formation. The objective was to determine whether Zol, cytoD, and DFO can improve fixation strength and enhance peri-implant bone volume about intra-medullary femoral implants. METHODS: 48 Sprague-Dawley female rats were randomized into four treatments, saline-control or experimental: Zol-(0.8 µg/µL), cytoD-(0.05 µg/µL), DFO-(0.4 µg/µL). Implants were placed bilaterally in the femoral canals following injection of treatment solution and followed for 28 days. Mechanical push-out testing and micro-CT were our primary evaluations, measuring load to failure and bone volume. Qualitative evaluation included histological assessment. Data was analyzed with a one-way ANOVA with Holm-Sidak mean comparison testing. RESULTS: Significant results included pushout tests showing an increase in maximum energy for Zol (124%) and cytoD (82%); Zol showed an increase in maximum load by 48%; Zol micro-CT showed increase in BV/TV by 35%. CONCLUSIONS: Our findings suggest that locally applied Zol and cytoD enhance implant mechanical stability. Bisphosphonates and actin regulators, like cytoD, might be further investigated as a new strategy for improving osseointegration.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Prótese Ancorada no Osso , Citocalasina D/farmacologia , Desferroxamina/farmacologia , Fêmur/diagnóstico por imagem , Ácido Zoledrônico/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Fêmur/efeitos dos fármacos , Fêmur/cirurgia , Modelos Animais , Inibidores da Síntese de Ácido Nucleico/farmacologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Sideróforos/farmacologiaRESUMO
BACKGROUND: Bone defect caused by trauma, tumor resection, infection or congenital malformation is a common clinical disease. Bone tissue engineering is regarded as a promising way of bone defect reconstruction. Thus, agents that can promote osteogenesis have received great attention. Cytochalasin D (Cyto D), a metabolite derived from molds, proves to be able to modify actin, reorganize cytoskeleton, and then promote the osteogenic differentiation. OBJECTIVE: The purpose of this study was to explore the effect and mechanism of Cyto D on osteogenic differentiation of mouse pre-osteoblast MC3T3-E1 cells. METHODS: The optimum concentration of Cyto D was explored. The osteogenic differentiation of MC3T3-E1 cells induced by Cyto D was assessed by alkaline phosphatase (ALP) staining, Alizarin Red S (ARS) staining, western blotting and quantitative real-time polymerase chain reaction (RT-qPCR). In addition, a specific pathway inhibitor was utilized to explore whether MAPK pathways were involved in this process. RESULTS: The results showed that the optimized concentration of action was 10-2µg/ml. The expression of Runx2, OCN and OSX was up-regulated by the supplement of Cyto D. ALP activity, calcium deposition, and phosphorylation level of p38 protein were also improved. Inhibition of the pathway significantly reduced the activation of p38, and the expression of osteogenic-related genes. CONCLUSION: Cyto D can promote the osteogenic differentiation of MC3T3 cells via the p38-MAPK signaling pathway, but not the ERK1/2 or JNK, and it is a potential agent to improve the osteogenesis of MC3T3 cells.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Citocalasina D/farmacologia , Osteogênese/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Células 3T3-L1 , Animais , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Camundongos , Osteogênese/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
The fungus Xylaria arbuscula was isolated as an endophyte from Cupressus lusitanica and has shown to be a prominent producer of cytochalasins, mainly cytochalasins C, D and Q. Cytochalasins comprise an important class of fungal secondary metabolites that have aroused attention due to their uncommon molecular structures and pronounced biological activities. Due to the few published studies on the ESI-MS/MS fragmentation of this important class of secondary metabolites, in the first part of our work, we studied the cytochalasin D fragmentation pathways by using an ESI-Q-ToF mass spectrometer coupled with liquid chromatography. We verified that the main fragmentation routes were generated by hydrogen and McLafferty rearrangements which provided more ions than just the ones related to the losses of H2 O and CO as reported in previous studies. We also confirmed the diagnostic ions at m/z 146 and 120 as direct precursor derived from phenylalanine. The present work also aimed the production of structurally diverse cytochalasins by varying the culture conditions used to grow the fungus X. arbuscula and further insights into the biosynthesis of cytochalasins. HPLC-MS analysis revealed no significant changes in the metabolic profile of the microorganism with the supplementation of different nitrogen sources but indicated the ability of X. arbuscula to have access to inorganic and organic nitrogen, such as nitrate, ammonium and amino acids as a primary source of nitrogen. The administration of 2-13 C-glycine showed the direct correlation of this amino acid catabolism and the biosynthesis of cytochalasin D by X. arbuscula, due to the incorporation of three labeled carbons in cytochalasin chemical structure. Copyright © 2017 John Wiley & Sons, Ltd.
Assuntos
Citocalasina D/química , Xylariales/metabolismo , Isótopos de Carbono , Cromatografia Líquida de Alta Pressão/métodos , Citocalasina D/metabolismo , Fermentação , Marcação por Isótopo , Estrutura Molecular , Peso Molecular , Isótopos de Nitrogênio , Fenilalanina/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Human dermal fibroblasts (HDFs) are typically flattened or extensible shaped and play a critical role in the metabolism of extracellular matrix components. As the properties of fibroblasts in the dermis are considered to be influenced by their morphology, we investigated the morphological changes induced in fibroblasts by ultraviolet (UV) irradiation as well as the relationship between these changes and collagen metabolism. In this study, we showed that UVA exposure induced morphological changes and reduced collagen contents in HDFs. These morphological changes were accompanied a reduction in actin filaments and upregulation of the actin filament polymerization inhibitor, capping protein muscle Z-line É1 (CAPZA1). External actin filament growth inhibitors also affected the shape of HDFs and reduced collagen levels. These results suggest that UVA exposure may inhibit the polymerization of actin filaments and induce morphological changes in skin fibroblasts. These morphological changes in fibroblasts may accelerate reductions in collagen synthesis. This mechanism may be one of the processes responsible for collagen reductions observed in photoaged skin. When natural materials that suppress these morphological changes in HDFs were evaluated, we found that an extract of Lilium 'Casa Blanca' (LCB) suppressed UVA-induced alterations in the shape of HDFs, which are typically followed by inhibition of collagen reduction. An analysis of the active compounds in LCB extract led to the identification of regaloside I, which had a structure of phenylpropanoid glycerol glucoside, as the active compound inhibiting the upregulation of CAPZA1. Therefore, inhibition of UVA-induced morphological changes in HDFs is considered to be promising way for the suppression of collagen reduction in photoaging.
Assuntos
Envelhecimento da Pele/patologia , Envelhecimento da Pele/efeitos da radiação , Pele/patologia , Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteína de Capeamento de Actina CapZ/genética , Proteína de Capeamento de Actina CapZ/metabolismo , Forma Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Citocalasina D/farmacologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Expressão Gênica/efeitos dos fármacos , Humanos , Lilium , Extratos Vegetais/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pele/metabolismo , Envelhecimento da Pele/fisiologia , Tiazolidinas/farmacologiaRESUMO
Sensitization of purinergic P2X3 receptors (P2X3Rs) contributes to the production of exaggerated nociceptive responses following inflammatory injury. We showed previously that prostaglandin E2 (PGE2) potentiates P2X3R-mediated ATP currents in dorsal root ganglion neurons isolated from both control and complete Freund's adjuvant-induced inflamed rats. PGE2 potentiation of ATP currents depends only on PKA signaling in control neurons, but it depends on both PKA and PKC signaling in inflamed neurons. We further found that inflammation evokes an increase in exchange proteins directly activated by cAMP (Epacs) in dorsal root ganglions. This increase promotes the activation of PKC to produce a much enhanced PGE2 effect on ATP currents and to elicit Epac-dependent flinch nocifensive behavioral responses in complete Freund's adjuvant rats. The link between Epac-PKC signaling and P2X3R sensitization remains unexplored. Here, we show that the activation of Epacs promotes the expression of phosphorylated PKC and leads to an increase in the cytoskeleton, F-actin, expression at the cell perimeter. Depolymerization of F-actin blocks PGE2-enhanced ATP currents and inhibits P2X3R-mediated nocifensive responses after inflammation. Thus, F-actin is dynamically involved in the Epac-PKC-dependent P2X3R sensitization. Furthermore, Epacs induce a PKC-dependent increase in the membrane expression of P2X3Rs. This increase is abolished by F-actin depolymerization, suggesting that F-actin mediates Epac-PKC signaling of P2X3R membrane expression. Thus, after inflammation, an Epac-PKC dependent increase in F-actin in dorsal root ganglion neurons enhances the membrane expression of P2X3Rs to bring about sensitization of P2X3Rs and abnormal pain behaviors.
Assuntos
Actinas/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Inflamação/patologia , Proteína Quinase C/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Citocalasina D/farmacologia , Dinoprostona/farmacologia , Adjuvante de Freund , Gânglios Espinais/efeitos dos fármacos , Hiperalgesia/patologia , Inflamação/metabolismo , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Tionucleotídeos/farmacologiaRESUMO
We hypothesized that if internalization of Staphylococcus aureus could be blocked by using cytochalasin D (an inhibitor of phagocytosis and phagolysosome fusion), then the intracellular entry and survival of the pathogen in host's phagocytic cells recruited to the inflammatory site can be restricted. At the same time, if we use antimicrobial agents (e.g., ciprofloxacin and azithromycin) having potent intracellular and extracellular microbicidal activity against the bacterium that have not entered into the phagosome and remains adhered to the phagocytic cell membrane, then they can be eradicated from the site of infection without compromising the host cell. To validate this, role of ciprofloxacin (CIP) and azithromycin (AZM) in eliminating S. aureus by suppressing the phagocytic activity of macrophages with cytochalasin D before infection was investigated. CIP and AZM were used either alone or in combination with cytochalasin D. Supernatant and lysate obtained from the culture of macrophages were used for quantification of reactive oxygen species, lysozymes, antioxidant enzymes, and cytokines produced. Azithromycin was better than ciprofloxacin in combination with cytochalasin D for eradicating S. aureus and regulating cytokine release. Further studies are required for ensuring proper delivery of this combination at the site of infection.
Assuntos
Azitromicina/uso terapêutico , Ciprofloxacina/uso terapêutico , Citocalasina D/uso terapêutico , Macrófagos/imunologia , Infecções Estafilocócicas/tratamento farmacológico , Animais , Antibacterianos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Antioxidantes/metabolismo , Quimioterapia Combinada , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Inflamação/tratamento farmacológico , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Muramidase/metabolismo , Fagocitose/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologia , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/patogenicidade , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A quantitative profile of cytochalasin D production by Xylaria arbuscula was followed by growing the fungus in rice, Czapek, Czapek enriched with yeast extract, wheat, and corn. This cytochalasin producer, X. arbuscula, was collected as an endophytic fungus from healthy tissues of Cupressus lusitanica (Cupressaceae). A new HPLC method was developed using a synthetic N-acetyl-L-phenylalanine ethyl ester as internal standard, which showed a good correlation coefficient (r2 = 0.9995). The results varied from 6.40 to 39.55 mg per 100 g of culture medium, with wheat being the best medium for cytochalasin D production. The level of any free amino acids in the medium, not necessarily phenylalanine, appeared to be an important factor to enhance cytochalasin D biosynthesis.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Citocalasina D/metabolismo , Xylariales/química , Xylariales/metabolismo , Meios de Cultura/metabolismo , Xylariales/crescimento & desenvolvimentoRESUMO
The human malaria parasite Plasmodium falciparum causes the most deadly parasitic disease worldwide, necessitating the development of interventions that block infection. Yet, preclinical assays to measure inhibition of infection date from the 1980s and are based on microscopy. Here, we describe the development of a simple flow cytometric assay that can be used to quantitatively assess P. falciparum sporozoite infection in vitro in low and medium throughput. We demonstrate the utility of this assay for assessing both drug inhibition of infection and measuring efficacy of antibodies in blocking parasite infection. This methodology will aid in assessing functional antibody responses to vaccination and novel drugs that prevent mosquito-to-man transmission of malaria.
Assuntos
Citometria de Fluxo/métodos , Plasmodium falciparum/citologia , Esporozoítos/citologia , Anticorpos Antiprotozoários/química , Antígenos de Protozoários/imunologia , Antimaláricos/farmacologia , Células Cultivadas , Citocalasina D/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Hepatócitos/citologia , Hepatócitos/parasitologia , Humanos , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Esporozoítos/efeitos dos fármacos , Esporozoítos/imunologiaRESUMO
In cardiac myocytes, cytochalasin D (CytoD) was reported to act as an actin disruptor and mechanical uncoupler. Using confocal and super-resolution STED microscopy, we show that CytoD preserves the actin filament architecture of adult rat ventricular myocytes in culture. Five hundred nanomolar CytoD was the optimal concentration to achieve both preservation of the T-tubular structure during culture periods of 3 days and conservation of major functional characteristics such as action potentials, calcium transients and, importantly, the contractile properties of single myocytes. Therefore, we conclude that the addition of CytoD to the culture of adult cardiac myocytes can indeed be used to generate a solid single-cell model that preserves both morphology and function of freshly isolated cells. Moreover, we reveal a putative link between cytoskeletal and T-tubular remodeling. In the absence of CytoD, we observed a loss of T-tubules that led to significant dyssynchronous Ca(2+)-induced Ca(2+) release (CICR), while in the presence of 0.5 µM CytoD, T-tubules and homogeneous CICR were majorly preserved. Such data suggested a possible link between the actin cytoskeleton, T-tubules and synchronous, reliable excitation-contraction-coupling. Thus, T-tubular re-organization in cell culture sheds some additional light onto similar processes found during many cardiac diseases and might link cytoskeletal alterations to changes in subcellular Ca(2+) signaling revealed under such pathophysiological conditions.
Assuntos
Citocalasina D/farmacologia , Miócitos Cardíacos/diagnóstico por imagem , Miócitos Cardíacos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Citocalasina D/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Masculino , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Ratos Wistar , UltrassonografiaRESUMO
The attachment of immortalized hypothalamic murine neurons onto the surface of an acoustic wave device yields both positive series resonant frequency (f(s)) and motional resistance (R(m)) shifts as opposed to commonly reported negative f(s) and positive R(m) shifts observed for other cell types. These unique shifts have been confirmed by a variety of experiments in order to verify the source and the validity of the signals. These studies involved monitoring responses to solution flow, the absence of serum proteins, the effect of reducing specific cell -surface interactions and the disruption of the neuronal cytoskeleton components. For the adhesion and deposition of neurons, f(s) and R(m) shifts are positively correlated to the amount of adhered neurons on the sensor surface, whereas non-adhered neurons do not produce any significant change in the monitored parameters. In the absence of serum proteins, initial cell adhesion is followed by subsequent cell death and removal from the sensor surface. The presence of the peptide, GRGDS is observed to significantly reduce cell-surface specific interactions compared to the control of SDGRG and this produces f(s) and R(m) responses that are opposite in direction to that observable for cell adhesion. Cytoskeletal studies, using the drugs nocodazole (10 µM), colchicine (1 µM), cytochalasin B (10 µM) and cytochalasin D (2 µM) all elicit neuronal responses that are validated by phalloidin actin-filament staining. These results indicate that the responses are associated with a wide range of cellular changes that can be monitored and studied using the acoustic wave method in real time, under optimal physiological conditions.
Assuntos
Adesão Celular/efeitos dos fármacos , Hipotálamo/citologia , Neurônios/citologia , Neurônios/fisiologia , Som , Animais , Células Cultivadas , Colchicina/farmacologia , Citocalasina B/farmacologia , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/fisiologia , Hipotálamo/efeitos dos fármacos , Camundongos , Neurônios/efeitos dos fármacos , Nocodazol/farmacologia , Faloidina , Propriedades de SuperfícieRESUMO
BACKGROUND: Hydroxyethyl starches (HES) could differ with regard to the origin, and the influence on the coagulation of the raw material is unknown. This study compared the effects of a new potato-derived HES with a maize-derived HES and two crystalloid solutions. METHODS: Whole blood from 10 healthy individuals was diluted by 20% and 40% using either non-balanced potato-derived HES 130/0.42/6:1, non-balanced maize-derived HES 130/0.4/9:1, isotonic saline or Ringer's lactate solution. Samples were analysed by thromboelastometry ROTEM(®) : Coagulation was initiated by acid ellagic [intrinsic thromboelastometry (INTEM)] or tissue factor (extrinsic thromboelastometry) with and without cytochalasin to determine the functional component of fibrinogen [cytochalasin-d-modified thromboelastometry (FIBTEM)]. Platelet count and fibrinogen activity were measured. RESULTS: No effect of raw material was found as no difference was detected among the HES solutions. Whatever the solution, progressive haemodilution impaired haemostasis in a dose-dependant manner: For INTEM, the clot formation time was increased up to 308% and the maximum clot firmness (MCF) was decreased down to 49%. As dilution increased, initiation of coagulation was also impaired. Thromboelastometric alterations were more severe with HES than with crystalloids, especially regarding fibrin polymerization explorations: MCF of FIBTEM was considerably reduced from 12[10-14] to 2[2-3] mm (P<0.05). Fibrinogen activity and platelet count were reduced by dilution in a dose-dependant manner and decreased similarly in all groups. CONCLUSION: Maize- and potato-derived HES have similar effects on coagulation. Both the starch preparations tested lead to more severe haemostatic defects than crystalloids, and impairment of fibrin polymerization appears to be a leading determinant of this coagulopathy.
Assuntos
Derivados de Hidroxietil Amido/química , Derivados de Hidroxietil Amido/farmacologia , Substitutos do Plasma/química , Substitutos do Plasma/farmacologia , Solanum tuberosum/química , Tromboelastografia , Zea mays/química , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Soluções Cristaloides , Citocalasina D , Ácido Elágico , Fibrinogênio/análise , Hemostasia , Humanos , Soluções Isotônicas/farmacologia , Inibidores da Síntese de Ácido Nucleico , Contagem de Plaquetas , Lactato de Ringer , Solução Salina Hipertônica , Tempo de Coagulação do Sangue TotalRESUMO
We report a drug dose-response, end-point study of intracellular filamentous actin (F-actin) by automated fluorescence microscopy, complemented with theoretical kinetic simulation of drug action. We highlight the use of an advanced orientation-sensitive image processing procedure (
Assuntos
Actinas/efeitos dos fármacos , Actinas/ultraestrutura , Citocalasina D/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/ultraestrutura , Algoritmos , Automação , Citocalasina D/química , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Cinética , Microscopia , Modelos EstatísticosRESUMO
Fine particles (10(2)- to 10(3)-nm diameter) are potentially potent adjuvants in acquired immune responses but little is known about their interaction with pathogen-associated molecular patterns (PAMPs) and impact upon innate immunity. Here we show that 200-nm-sized, food-grade titanium dioxide avidly binds lipopolysaccharide (LPS) with bridging calcium cations, and the complex induces marked proinflammatory signalling in primary human mononuclear phagocytes. In particular, caspase 1-dependent interleukin-1beta (IL-1beta) secretion was induced at levels far greater than for the sum of the individual components, and without concomitant secretion of modulatory cytokines such as interleukin-1 receptor antagonist or transforming growth factor-beta1 (TGF-beta1). Secondly, the conjugate induced apoptotic-like cell death. These responses were inhibited by blockade of both phagocytosis and scavenger receptor uptake. Specific caspase 1-facilitated IL-1beta secretion and apoptosis following phagocytosis are features of cellular responses to certain invasive, enteric pathogens, and hence induction of these events may be mimicked by fine particle-LPS conjugates. The inadvertent adsorption of PAMPs to ingested, inhaled, or "wear" fine particulate matter provides a further potential mechanism for the proinflammatory nature of fine particles.
Assuntos
Adjuvantes Imunológicos , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/imunologia , Nanopartículas/toxicidade , Titânio/química , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Adsorção , Adulto , Apoptose , Cálcio/química , Células Cultivadas , Citocalasina D/farmacologia , Humanos , Imunossupressores/farmacologia , Interleucina-1/biossíntese , Interleucina-6/biossíntese , Lipopolissacarídeos/química , Nanopartículas/química , Tamanho da Partícula , Fagocitose/efeitos dos fármacos , Receptores de Interleucina-1/antagonistas & inibidores , Titânio/imunologia , Titânio/toxicidade , Fator de Crescimento Transformador beta1/biossíntese , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Although uranium is a well-characterized nephrotoxic agent, very little is known at the cellular and molecular level about the mechanisms underlying the uptake and toxicity of this element in proximal tubule cells. The aim of this study was thus to characterize the species of uranium that are responsible for its cytotoxicity and define the mechanism which is involved in the uptake of the cytotoxic fraction of uranium using two cell lines derived from kidney proximal (LLC-PK(1)) and distal (MDCK) tubule as in vitro models. Treatment of LLC-PK(1) cells with colchicine, cytochalasin D, concanavalin A and PMA increased the sodium-dependent phosphate co-transport and the cytotoxicity of uranium. On the contrary, replacement of the extra-cellular sodium with N-methyl-D-glucamine highly reduced the transport of phosphate and the cytotoxic effect of uranium. Uranium cytotoxicity was also dependent upon the extra-cellular concentration of phosphate and decreased in a concentration-dependent manner by 0.1-10 mM phosphonoformic acid, a competitive inhibitor of phosphate uptake. Consistent with these observations, over-expression of the rat proximal tubule sodium-dependent phosphate co-transporter NaPi-IIa in stably transfected MDCK cells significantly increased the cytotoxicity of uranium, and computer modeling of uranium speciation showed that uranium cytotoxicity was directly dependent on the presence of the phosphate complexes of uranyl UO(2)(PO(4))(-) and UO(2)(HPO(4))(aq). Taken together, these data suggest that the cytotoxic fraction of uranium is a phosphate complex of uranyl whose uptake is mediated by a sodium-dependent phosphate co-transporter system.
Assuntos
Fosfatos/fisiologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/fisiologia , Urânio/toxicidade , Animais , Cádmio/toxicidade , Cloreto de Cálcio/farmacologia , Carbonatos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Colchicina/farmacologia , Simulação por Computador , Concanavalina A/farmacologia , Citocalasina D/farmacologia , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Foscarnet/farmacologia , Indóis/farmacologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Células LLC-PK1 , Maleimidas/farmacologia , Meglumina/análogos & derivados , Meglumina/farmacologia , Fosfatos/antagonistas & inibidores , Fosfatos/farmacologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismo , Suínos , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologia , Compostos de UrânioRESUMO
This work demonstrates cell swelling as a new regulatory mechanism for the cloned hyperpolarization-activated, cyclic nucleotide-gated channel 2 (HCN2). HCN2 channels were coexpressed with aquaporin1 in Xenopus laevis oocytes and currents were monitored using a two-electrode voltage-clamp. HCN2 channels were activated by hyperpolarization to -100 mV and the currents were measured before and during hypoosmotic cell swelling. Cell swelling increased HCN2 currents by 30% without changing the kinetics of the currents. Injection of 50 nl intracellular solution resulted in a current increase of 20%, indicating that an increase in cell volume also under isoosmotic conditions may lead to activation of HCN2. In the absence of aquaporin1 only negligible changes in oocyte cell volume occur during exposure to hypoosmotic media and no significant change in HCN2 channel activity was observed during perfusion with hypoosmotic media. This indicates that cell swelling and not a change in ionic strength of the media, caused the observed swelling-induced increase in current. The increase in HCN2 current induced by cell swelling could be abolished by cytochalasin D treatment, indicating that an intact F-actin cytoskeleton is a prerequisite for the swelling-induced current.
Assuntos
Canais Iônicos/fisiologia , Osmose , Actinas/química , Animais , Forma Celular , AMP Cíclico/metabolismo , Citocalasina D/farmacologia , Citoesqueleto/química , Citoesqueleto/metabolismo , DNA Complementar/metabolismo , Eletrofisiologia , Globinas/química , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais Iônicos/química , Íons , Canais de Potássio KCNQ/química , Cinética , Oócitos/metabolismo , Faloidina/farmacologia , Canais de Potássio , Fatores de Tempo , Xenopus laevis/metabolismoRESUMO
Key role in cell gravisensing is attributed to the actin cytoskeleton which acts as a mediator in signaling reactions, including graviperception. Despite of increased attention to the actin cytoskeleton, major gaps in our understanding of its functioning in plant gravisensing still remain. To fill these gaps, we propose a novel approach focused on the investigation of actin involvement in the development of columella cells and cells in the transition zone of roots submitted to clinorotation. Both statocytes and cells in the transition zone represent the postmitotic cells which take origin in root meristems and are specified into graviperceptive (root cap) and gravireacting (transition zone) root tissues. The aim of the research was to investigate and compare the microfilament arrangements in root cap statocytes and peripheral root tissues (epidermis and cortex cells) in the transition zone and to find out how the actin cytoskeleton is involved in their specification under clinostat conditions. So far, our experiments have shown that under clinorotation the cytoplasmic microfilament network in the cortex cells in the transition zone is significantly enhanced. It is suggested that more abundant cytoplasmic microfilaments could strengthen the cortical actin cytoskeleton arranged parallel with the cortical microtubules, which are found to be partially disorganized in this area. Due to microtubule disorganization, the functioning of cellulose-synthesizing machinery and proper deposition of cell wall might be affected and could cause the alterations in the growth mode. But, in our case growth of the cells in the transition zone under clinorotation was rather stable. Due to our opinion, general stability of cell growth under clinorotation is promoted by mutual functional interrelation between actin and tubulin cytoskeletons. It is suggested that a strengthened cortical actin cytoskeleton restricts the cell growth instead of disorganized microtubules.
Assuntos
Actinas/metabolismo , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Sensação Gravitacional/fisiologia , Coifa/ultraestrutura , Raízes de Plantas/ultraestrutura , Actinas/efeitos dos fármacos , Beta vulgaris/ultraestrutura , Citocalasina D/farmacologia , Citoplasma/fisiologia , Gravitação , Meristema/fisiologia , Meristema/ultraestrutura , Microscopia Confocal , Microtúbulos/fisiologia , Rotação , Plântula , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/fisiologia , Simulação de Ausência de PesoRESUMO
Actin polymerization is important in the control of pollen tube growth. Thus, treatment of pollen tubes with low concentrations of latrunculin B (Lat-B), which inhibits actin polymerization, permits streaming but reversibly blocks oscillatory growth. In the current study, we employ Jasplakinolide (Jas), a sponge cyclodepsipeptide that stabilizes actin microfilaments and promotes polymerization. Uniquely, Jas (2 microM) blocks streaming in the shank of the tube, but induces the formation of a toroidal-shaped domain in the swollen apex, of which longitudinal optical sections exhibit circles of motion. The polarity of this rotary motion is identical to that of reverse fountain motility in control pollen tubes, with the forward direction occurring at the edge of the cell and the rearward direction in the cell interior. Support for the idea that actin polymerization in the apical domain contributes to the formation of this rotary motility activity derives from the appearance therein of aggregates and flared cables of F-actin, using immunofluorescence, and by the reduction in G-actin as indicated with fluorescent DNAse. In addition, Jas reduces the tip-focused Ca2+ gradient. However, the alkaline band appears in the swollen apex and is spatially localized with the reverse fountain streaming activity. Taken together, our results support the idea that actin polymerization promotes reversal of streaming in the apex of the lily pollen tube.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Polaridade Celular/fisiologia , Corrente Citoplasmática/fisiologia , Flores/metabolismo , Lilium/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Actinas/efeitos dos fármacos , Antifúngicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Polaridade Celular/efeitos dos fármacos , Citocalasina D/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Desoxirribonucleases/metabolismo , Depsipeptídeos/farmacologia , Flores/efeitos dos fármacos , Flores/ultraestrutura , Germinação/efeitos dos fármacos , Germinação/fisiologia , Lilium/ultraestrutura , Inibidores da Síntese de Ácido Nucleico/farmacologia , Pólen/efeitos dos fármacos , Pólen/metabolismo , Pólen/ultraestrutura , Polímeros/metabolismo , Reprodução/fisiologia , Tiazóis/farmacologia , TiazolidinasRESUMO
Human MSCs have been studied to define the mechanisms involved in normal bone remodeling and the regulation of osteogenesis. During osteogenic differentiation, MSCs change from their characteristic fibroblast-like phenotype to near spherical shape. In this study, we analyzed the correlation between the organization of cytoskeleton of MSCs, changes in cell morphology, and the expression of specific markers (alkaline phosphatase activity and calcium deposition) of osteogenic differentiation. For osteoblastic differentiation, cells were cultured in a culture medium supplemented with 100 nM dexamethasone, 10 mM beta- glycerophosphate, and 50 microg/ml ascorbic acid. The organization of microfilaments and microtubules was examined by inmunofluorescence using Alexa fluor 594 phalloidin and anti alpha-tubulin monoclonal antibody. Cytochalasin D and nocodazole were used to alter reversibly the cytoskeleton dynamic. A remarkable change in cytoskeleton organization was observed in human MSCs during osteogenic differentiation. Actin cytoskeleton changed from a large number of thin, parallel microfilament bundles extending across the entire cytoplasm in undifferentiated MSCs to a few thick actin filament bundles located at the outermost periphery in differentiated cells. Under osteogenic culture conditions, a reversible reorganization of microfilaments induced by an initial treatment with cytochalasin D but not with nocodazole reduced the expression of differentiation markers, without affecting the final morphology of the cells. The results indicate that changes in the assembly and disassembly kinetics of microfilaments dynamic of actin network formation may be critical in supporting the osteogenic differentiation of human MSCs; also indicated that the organization of microtubules appears to have a regulatory role on the kinetic of this process.
Assuntos
Diferenciação Celular/fisiologia , Citoesqueleto/ultraestrutura , Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologia , Idoso , Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Feminino , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/ultraestrutura , Nocodazol/farmacologia , Pós-MenopausaRESUMO
We have investigated changes in the distribution of peroxisomes through the cell cycle in onion ( Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek ( Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the beta-oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.
Assuntos
Citoesqueleto de Actina/metabolismo , Diacetil/análogos & derivados , Miosinas/metabolismo , Cebolas/metabolismo , Peroxissomos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Diacetil/farmacologia , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Proteínas Luminescentes/metabolismo , Microscopia Imunoeletrônica , Cebolas/efeitos dos fármacos , Cebolas/crescimento & desenvolvimento , Epiderme Vegetal/efeitos dos fármacos , Epiderme Vegetal/crescimento & desenvolvimento , Epiderme Vegetal/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/ultraestrutura , Tiazóis/farmacologia , TiazolidinasRESUMO
Organic anion transport in intact renal proximal tubule cells in animal model systems is downregulated by treatments that activate protein kinase C (PKC). How this downregulation is achieved is not yet known. Stimulation of PKC with sn-1,2-dioctanoylglycerol resulted in strong inhibition of p-aminohippurate transport mediated by the cloned human organic anion transporter 1 (hOAT1) expressed in Xenopus oocytes and HEK293 cells, as well as hOAT1 internalization in both expression systems. The sn-1,2-dioctanoylglycerol-induced transport inhibition was partially prevented by staurosporine. It was independent of the conserved canonical PKC consensus sites in hOAT1, however, and was unaffected by agents that destabilize actin filaments or microtubules, which altered baseline hOAT1-mediated p-aminohippurate uptake activity in oocytes. It is concluded that PKC-induced hOAT1 downregulation is achieved through carrier retrieval from the cell membrane and does not involve phosphorylation of the predicted classic hOAT1 PKC consensus sites.