Assuntos
Babesia/efeitos dos fármacos , Insulina/fisiologia , Putrescina/farmacologia , Ácido Selenioso/farmacologia , Transferrina/farmacologia , Animais , Babesia/crescimento & desenvolvimento , Babesia/fisiologia , Babesiose/parasitologia , Reatores Biológicos/parasitologia , Bovinos , Meios de Cultura Livres de Soro , Eritrócitos/parasitologia , Técnicas In VitroRESUMO
This study evaluated the effect of the protocatechuic acid (PCA) as the sole antioxidant in the base medium for in vitro culture of ovine secondary follicles. Secondary follicles (200-230 µm) were isolated and cultured in α-minimal essential medium supplemented with BSA, insulin, glutamine and hypoxanthine (α-MEM: antioxidant-free medium) or α-MEM also added by transferrin, selenium and ascorbic acid (α-MEM+: with antioxidant) or α-MEM added by PCA (56.25; 112.5; 225; 450; or 900 µg/ml). Moreover, after culture, oocytes were matured and the chromatin configuration and DNA fragmentation were evaluated. After 12 days, the treatment containing 56.25 µg/ml PCA showed higher percentage of normal follicles than control medium or the other treatments (p < .05), except for 900 µg/ml PCA (p > .05). The antrum formation was significantly higher in treatments containing 56.25, 112.5 or 900 µg/ml PCA, compared to the α-MEM and similar (p > .05) to the other treatments. The rates of fully grown oocytes (≥110 µm) were similar (p > .05) among all treatments containing PCA and α-MEM+, and those were superior (p < .05) than α-MEM, except for 450 µg/ml PCA (p > .05). GSH levels and mitochondrial activity were higher (p < .05) in α-MEM+ than in α-MEM and similar (p > .05) to all PCA treatments. The rates of meiotic resumption and DNA fragmentation were similar (p > .05) among α-MEM+ and 56.25 µg/ml PCA. In conclusion, PCA at 56.25 µg/ml as the sole antioxidant added to the medium for ovine isolated secondary follicle culture maintains follicular survival, GSH and active mitochondria levels, meiotic developmental competence and DNA integrity of cultured oocytes.
Assuntos
Antioxidantes/farmacologia , Hidroxibenzoatos/farmacologia , Folículo Ovariano/efeitos dos fármacos , Carneiro Doméstico , Animais , Ácido Ascórbico/farmacologia , Técnicas de Cultura de Células/veterinária , Meios de Cultura , Fragmentação do DNA/efeitos dos fármacos , Feminino , Glutationa/metabolismo , Mitocôndrias , Oogênese/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Selênio/farmacologia , Transferrina/farmacologiaRESUMO
The present study evaluated the effect of addition of rutin alone or combined with other antioxidants (transferrin, selenium and ascorbic acid) present in the culture medium on the in vitro development of ovine isolated secondary follicles. After collection of the sheep ovaries, secondary follicles (200-230 µm) were isolated and cultured for 12 days in α-Minimal Essential Medium (α-MEM) supplemented with BSA, insulin, glutamine and hypoxanthine (α-MEM: antioxidant free-medium) or in this medium also added by transferrin, selenium and ascorbic acid (AO: base medium with antioxidants). Moreover, different concentrations of rutin (0.1; 1 or 10 µg/mL) were added to the different base media (α-MEM or AO). The parameters analyzed were morphology, antrum formation, extrusion rate, follicular diameter, growth and fully-grown oocytes (oocytes ≥ 110 µm) rates. In treatments that had the best results of morphology, follicular viability, apoptosis, glutathione (GSH), reactive oxygen species (ROS) levels and mitochondrial activity were also analyzed. After 12 days, the percentage of normal follicles was higher (P < 0.05) in α-MEM + 0.1 µg/mL rutin than the other treatments, except compared to AO medium (P > 0.05). There is no difference (P > 0.05) in the diameter and growth rate among treatments. Moreover, AO medium and α-MEM + 0.1 µg/mL rutin showed similar (P > 0.05) percentages of follicular viability, antrum formation, extruded follicles, fully-grown oocytes, levels of ROS and active mitochondria. However, α-MEM + 0.1 µg/mL rutin treatment showed higher (P > 0.05) GSH levels than AO medium. In conclusion, 0.1 µg/mL rutin can be used as the single antioxidant present in the base medium, replacing the addition of transferrin, selenium and ascorbic acid during in vitro culture of ovine secondary follicles, maintaining follicular viability and increasing GSH levels.
Assuntos
Antioxidantes/farmacologia , Folículo Ovariano/efeitos dos fármacos , Rutina/farmacologia , Ovinos , Animais , Apoptose , Ácido Ascórbico/farmacologia , Técnicas de Cultura de Células/veterinária , Sobrevivência Celular , Meios de Cultura , Feminino , Glutationa Peroxidase/metabolismo , Marcação In Situ das Extremidades Cortadas , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Folículo Ovariano/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Selênio/farmacologia , Transferrina/farmacologiaRESUMO
A lot of effort has been devoted to achieving active targeting for cancer therapy in order to reach the right cells. Hence, increasingly it is being realized that active-targeted nanocarriers notably reduce off-target effects, mainly because of targeted localization in tumors and active cellular uptake. In this context, by taking advantage of the overexpression of transferrin receptors on the surface of tumor cells, transferrin-conjugated nanodevices have been designed, in hope that the biomarker grafting would help to maximize the therapeutic benefit and to minimize the side effects. Notably, active targeting nanoparticles have shown improved therapeutic performances in different tumor models as compared to their passive targeting counterparts. In this review, current development of nano-based devices conjugated with transferrin for active tumor-targeting drug delivery are highlighted and discussed. The main objective of this review is to provide a summary of the vast types of nanomaterials that have been used to deliver different chemotherapeutics into tumor cells, and to ultimately evaluate the progression on the strategies for cancer therapy in view of the future research.
Assuntos
Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Neoplasias/tratamento farmacológico , Receptores da Transferrina/antagonistas & inibidores , Transferrina/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Portadores de Fármacos/química , Humanos , Nanotecnologia , Neoplasias/metabolismo , Neoplasias/patologia , Receptores da Transferrina/biossíntese , Transferrina/síntese química , Transferrina/químicaRESUMO
The aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal-Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1-3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.
Assuntos
Ácido Ascórbico/farmacologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Insulina/farmacologia , Selênio/farmacologia , Transferrina/farmacologia , Animais , Blastocisto/citologia , Blastocisto/fisiologia , Bovinos , Meios de Cultura/química , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária/métodos , Feminino , Fertilização in vitro , Masculino , Oócitos/efeitos dos fármacos , Oócitos/fisiologiaRESUMO
Thyroid hormones (THs) and transferrin (Tf) are factors capable of favoring myelination due to their positive effects on oligodendroglial cell (OLG) differentiation. The first notion of a combined effect of apotransferrin (aTf) and TH emerged from experiments conducted in young hyperthyroid animals, which showed a seven-fold increase in the expression of Tf mRNA and precocious myelination when compared with control animals. The mechanism underlying this phenomenon in young hyperthyroid rats could consist of an increase in Tf synthesis, which in the CNS is almost exclusively produced by OLG. Overall, our results show that, during the initial stages of OLG differentiation, Tf synthesis triggers thyroid hormone receptor alpha 1 (TRα1) expression in the subventricular zone (SVZ) and promotes proliferating cells to become responsive to this trophic factor. Exposure to TH could then regulate Tf expression through TRα1 and promote the induction of thyroid hormone receptor beta (TRß) expression, which mediates TH effects on myelination through the activation of final OLG differentiation. This regulation of the combined effects of Tf and THs implies that both factors are fundamental actors during oligodendrogenesis. GLIA 2016;64:1879-1891.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Oligodendroglia/fisiologia , Transferrina/metabolismo , Transferrina/farmacologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/fisiologia , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Ventrículos Laterais/citologia , Proteína Básica da Mielina/metabolismo , Oligodendroglia/efeitos dos fármacos , Ratos , Ratos Wistar , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Células-Tronco/efeitos dos fármacos , Hormônios Tireóideos , Transferrina/genéticaRESUMO
The antioxidant properties of Amburana cearensis extract may be a useful substitute for standard cell culture medium. Thus, the aim of this study was to evaluate the effect of this extract, with or without supplementation, on in vitro survival and development of sheep isolated secondary follicles. After collection of the ovaries, secondary follicles were isolated and cultured for 18 days in α-MEM+ supplemented with bovine serum albumin, insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (control medium) or into medium composed of different concentrations of A. cearensis extract without supplements (Amb 0.1; 0.2 or 0.4 mg/ml) or A. cearensis extract supplemented with the same substances described above for α-MEM+ supplementation. The A. cearensis supplemented medium was named Amb 0.1+; 0.2+ or 0.4+ mg/ml. There were more morphologically normal follicles in Amb 0.1 or Amb 0.4 mg/ml than in the control medium (α-MEM+) after 18 days of culture. Moreover, the percentage of antrum formation was significantly higher in Amb 0.1 or Amb 0.2 mg/ml than in α-MEM+ and Amb 0.1+ mg/ml, and similar to the other treatments. All A. cearensis extract media induced a progressive and significant increase in follicular diameter throughout the culture period. In conclusion, this study showed that 0.1 mg/ml of this extract, without supplementation, maintains follicular survival and promotes the development of ovine isolated secondary follicles in vitro. This extract can be an alternative culture medium for preantral follicle development.
Assuntos
Fabaceae/química , Folículo Ovariano/efeitos dos fármacos , Extratos Vegetais/farmacologia , Folhas de Planta/química , Animais , Ácido Ascórbico/farmacologia , Bovinos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Glutamina/farmacologia , Hipoxantina/farmacologia , Insulina/farmacologia , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Selênio/farmacologia , Soroalbumina Bovina/farmacologia , Ovinos , Transferrina/farmacologiaRESUMO
This study aims to evaluate if a pre-maturation culture (PMC) using cilostamide as a meiotic inhibitor in combination with insulin, transferrin and selenium (ITS) for 8 or 24 h increases in vitro embryo production. To evaluate the effects of PMC on embryo development, cleavage rate, blastocyst rate, embryo size and total cell number were determined. When cilostamide (20 µM) was used in PMC for 8 or 24 h, 98% of oocytes were maintained in germinal vesicles. Although the majority of oocytes resumed meiosis after meiotic arrest, the cleavage and blastocyst rates were lower than the control (P 0.05) to the control. The deleterious effect of 20 µM cilostamide treatment for 24 h on a PMC was confirmed by lower cumulus cell viability, determined by trypan blue staining, in that group compared with the other groups. A lower concentration (10 µM) and shorter exposure time (8 h) minimized that effect but did not improve embryo production. More studies should be performed to determine the best concentration and the arresting period to increase oocyte competence and embryo development.
Assuntos
Insulina/farmacologia , Oócitos/efeitos dos fármacos , Inibidores da Fosfodiesterase 3/farmacologia , Selênio/farmacologia , Transferrina/farmacologia , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/fisiologia , Bovinos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células do Cúmulo/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Feminino , Fertilização in vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Masculino , Meiose/efeitos dos fármacos , Oócitos/citologia , Oócitos/fisiologia , Quinolonas/farmacologia , Fatores de TempoRESUMO
Oligodendroglial damage and demyelination are common pathological features characterizing white matter and neurodegenerative disorders. Identifying the signaling pathways involved in myelin repair through oligodendroglial progenitor maturation is essential for the development of new therapies. This article investigated the role of the Notch signaling pathway in CNS demyelination and apotransferrin-induced remyelination in a focal lysolecithin-induced demyelination model in rats. Notch was found activated in Nestin-expressing neural progenitor cells and in NG2-expressing oligodendroglial precursor cells in the subventricular zone and corpus callosum of lysolecithin-demyelinated rats. Notch activation seemed to be driven by Jagged1, which led to a high expression of downstream gene Hes5 in the subventricular zone of demyelinated rats. Apotransferrin injection induced remyelination, while the injection of the γ-secretase inhibitor reversed this effect. In addition, 24 h after apotransferrin injection, evidence showed Notch activation concomitantly with an increase in F3/contactin levels and the up-regulation of the myelin-associated glycoprotein gene in the subventricular zone and corpus callosum of demyelinated rats. Collected evidence supports the participation of both canonical and non-canonical Notch signaling pathways in demyelination/remyelination. Notch activation was found to trigger Hes5 expression as a consequence of focal demyelination, which might promote oligodendroglial precursor cell proliferation. During apotransferrin-induced remyelination, Notch activation seemed to be mediated by the expression of F3/contactin, which might induce apotransferrin-mediated oligodendroglial maturation. Evidence of the participation of Notch signaling in the demyelination/remyelination process will help further understand demyelinating disorders such as Multiple Sclerosis and the use of aTf should be taken into consideration as a possible therapeutic intervention.
Assuntos
Apoproteínas/farmacologia , Encéfalo/metabolismo , Doenças Desmielinizantes/metabolismo , Bainha de Mielina/metabolismo , Receptores Notch/metabolismo , Transferrina/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Feminino , Lisofosfatidilcolinas , Masculino , Células-Tronco Neurais/patologia , Oligodendroglia/patologia , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
NSC (neural stem cells)/NPC (neural progenitor cells) are multipotent and self-renew throughout adulthood in the SVZ (subventricular zone) of the mammalian CNS (central nervous system). These cells are considered interesting targets for CNS neurodegenerative disorder cell therapies, and understanding their behaviour in vitro is crucial if they are to be cultured prior to transplantation. We cultured the SVZ tissue belonging to newborn rats under the form of NS (neurospheres) to evaluate the effects of Tf (transferrin) on cell proliferation. The NS were heterogeneous in terms of the NSC/NPC markers GFAP (glial fibrillary acidic protein), Nestin and Sox2 and the OL (oligodendrocyte) progenitor markers NG2 (nerve/glia antigen 2) and PDGFRα (platelet-derived growth factor receptor α). The results of this study indicate that aTf (apoTransferrin) is able to increase cell proliferation of SVZ-derived cells in vitro, and that these effects were mediated at least in part by the TfRc1 (Tf receptor 1). Since OPCs (oligodendrocyte progenitor cells) represent a significant proportion of the proliferating cells in the SVZ-derived primary cultures, we used the immature OL cell line N20.1 to show that Tf was able to augment the proliferation rate of OPC, either by adding aTf to the culture medium or by overexpressing rat Tf in situ. The culture medium supplemented with ferric iron, together with aTf, increased the DNA content, while ferrous iron did not. The present work provides data that could have a potential application in human cell replacement therapies for neurodegenerative disease and/or CNS injury that require the use of in vitro amplified NPCs.
Assuntos
Proliferação de Células/efeitos dos fármacos , Ventrículos Laterais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Transferrina/farmacologia , Animais , Animais Recém-Nascidos , Antígenos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bromodesoxiuridina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Ferro/administração & dosagem , Ventrículos Laterais/crescimento & desenvolvimento , Masculino , Proteínas do Tecido Nervoso/metabolismo , Nestina , Fator de Transcrição 2 de Oligodendrócitos , Proteoglicanas/metabolismo , Ratos , Ratos Wistar , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transferrina/metabolismoRESUMO
Clathrin-mediated endocytosis plays an important role in the maintenance of neuronal integrity in the synaptic terminals. Here we studied the effect of anomalous polyglutamine expansion in huntingtin on the interaction of coat proteins with membranes, in areas of mouse brain or in cultured striatal cells. We observed that this anomaly induces a redistribution of AP-2, but not other coat proteins, from the membrane to the cytosol in the striatum, and in the cultured striatal cells. It was also noted that huntingtin associates with AP-2, and that this association decreases due to the mutation in huntingtin. This decreased receptor-mediated endocytosis, measured by the internalization of transferrin in the mutated cells. It was also confirmed that huntingtin mutation made the cells more vulnerable to the action of quinolinic acid, with an increasing degradation of the AP-2 alpha subunits. On the basis of these results, we conclude that abnormal polyglutamine expansion in huntingtin affects clathrin-mediated endocytosis, and may be one of the pathogenic mechanisms of neurodegeneration.
Assuntos
Corpo Estriado/citologia , Endocitose/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Proteínas Nucleares/genética , Fator de Transcrição AP-2/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Clatrina/farmacologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Proteína Huntingtina , Imunoprecipitação , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Ácido Quinolínico/farmacologia , Estatísticas não Paramétricas , Fatores de Tempo , Transferrina/farmacologiaRESUMO
Neural stem and progenitor cells (NSC/NPCs) are multipotent self-renewing cells that are able to generate neurons, astrocytes and oligodendrocytes (OLs) within the adult central nervous system. We cultured NSC/NPCs from the rat subventricular zone as neurospheres (NS) and studied apoTransferrin (aTf) effects on oligodendroglial specification and maturation. Our findings suggest that aTf acts at different stages during progression from NSC to mature oligodendrocytes. On the one hand, an early event associated with the activation of NSC/NPCs proliferation and commitment toward the oligodendroglial fate, as indicated by increased BrdU incorporation, larger neurospheres production, and higher ability to generate OL precursors (OPCs) from undifferentiated cultures. On the other hand, aTf exposure during differentiating conditions favours OL maturation from OPCs by promoting OL morphological development. This evidence supports a key role of Tf on the generation of OL from NSC/NPCs and highlights its potential in demyelinating disorder treatment.
Assuntos
Apoproteínas/metabolismo , Plexo Corióideo/metabolismo , Células-Tronco Neurais/metabolismo , Transferrina/metabolismo , Animais , Antígenos/metabolismo , Apoproteínas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Plexo Corióideo/citologia , Feminino , Masculino , Modelos Biológicos , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Cultura Primária de Células , Proteoglicanas/metabolismo , Ratos , Ratos Wistar , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores da Transferrina/imunologia , Receptores da Transferrina/metabolismo , Transferrina/farmacologiaRESUMO
Mechanisms that regulate oligodendroglial cell (OLGc) differentiation are the focus of intensive research in the field of cellular and molecular neurobiology. We have previously shown that the addition of apotransferrin (aTf) to primary OLGc cultures accelerates their differentiation and induces an increase in the expression of different components of the myelin cytoskeleton (CSK) such as actin, tubulin, and some of the microtubule-associated proteins, particularly the stable tubulin only peptide (STOP). Fyn protein-tyrosine kinase (Fyn kinase), a member of the Src family, participates in signalling pathways that regulate OLGs/myelin cytoskeletal reorganization. It is essential for myelin development in the central nervous system (CNS), and its absence results in hypomyelination. In the present study, we used both primary cell and N19 cell line cultures to investigate further the mechanisms of action involved in the accelerated differentiation of OLGcs induced by aTf. In particular, we were interested in studying the participation of Fyn kinase in the different pathways involved in the reorganization of the OLGc/myelin cytoskeleton. In agreement with results already published, we found that in OLGcs, Fyn kinase is associated with Tau and tubulin. Using a dominant-negative of Tau in which the Fyn-Tau-microtubules (MTs) interaction is blocked, we found that aTf was unable to induce OLGc morphological differentiation. It was also observed that aTf decreases the activated RhoA content in coincidence with a redistribution of actin immunoreactivity. These results give support to our hypothesis that Fyn kinase plays a key role in the differentiation process of OLGcs promoted by aTf.
Assuntos
Apoproteínas/farmacologia , Citoesqueleto/metabolismo , Oligodendroglia/enzimologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Células-Tronco/enzimologia , Transferrina/farmacologia , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Apoproteínas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/enzimologia , Bainha de Mielina/ultraestrutura , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Ratos , Ratos Wistar , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Transferrina/metabolismo , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Proteína rhoA de Ligação ao GTP/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas tau/efeitos dos fármacos , Proteínas tau/metabolismoRESUMO
We have used a model of iron deficiency in the rat to analyze the effects of a disruption in iron availability on oligodendroglial cell (OLGc) maturation and myelinogenesis and to explore the possible beneficial influence of an intracranial injection (ICI) of apotransferrin (aTf) at 3 days of age on this process. Studies carried out on postnatal days 17 and 24 showed that iron deficiency produced a decrease in myelin proteins and lipids at 24 days of age. Immunohistochemistry showed that in untreated iron-deficient (ID) rats, the immunoreactivity of anti-adenomatous polyposis coli (APC) and anti-MBP antibodies decreased markedly with reference to normal controls, whereas in ID rats treated with an ICI of aTf, the immunoreactivity of these markers increased. A similar situation occurred with the immunoreactivity of H-ferritin. In primary OLGc cultures from ID rats, there was a high number of cells positive to the antibody against the polysialylated form of the cell surface glycoprotein NCAM (PSA-NCAM) compared with in OLGc cultures prepared from normal controls or from ID animals treated with aTf. The number of MBP+ cells in cultures from ID rats increased after treatment with aTf. The presence of lipid rafts evaluated with a specific anti-protein prion cellular (PrPc) antibody showed a smaller number of PrPc-positive structures in ID rat cultures. Treatment of the ID animals with a single ICI of aTf stimulated myelination, producing a significant correction in the different biochemical parameters affected by ID.
Assuntos
Anemia Ferropriva/tratamento farmacológico , Anemia Ferropriva/patologia , Apoproteínas/uso terapêutico , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/patologia , Fibras Nervosas Mielinizadas/patologia , Transferrina/uso terapêutico , Anemia Ferropriva/sangue , Animais , Animais Recém-Nascidos , Apoproteínas/farmacologia , Células Cultivadas , Doenças Desmielinizantes/sangue , Modelos Animais de Doenças , Feminino , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Gravidez , Ratos , Ratos Wistar , Transferrina/farmacologiaRESUMO
Iron is an essential growth element of virtually all microorganisms and its restriction is one of the mechanisms used by macrophages to control microbial multiplication. Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America, is inhibited in its conidia-to-yeast conversion in the absence of iron. We studied the participation of iron in the nitric oxide (NO)-mediated fungicidal mechanism against conidia. Peritoneal murine macrophages activated with 50 U/mL of IFN-gamma or treated with 35 microM Deferoxamine (DEX) and infected with P. brasiliensis conidia, were co-cultured and incubated for 96 h in the presence of different concentrations of holotransferrin (HOLO) and FeS04. The supernatants were withdrawn in order to assess NO2 production by the Griess method. The monolayers were fixed, stained and observed microscopically. The percentage of the conidia-to-yeast transition was estimated by counting 200 intracellular propagules. IFN-gamma-activated or DEX-treated Mthetas presented marked inhibition of the conidia-to-yeast conversion (19 and 56%, respectively) in comparison with non-activated or untreated Mthetas (80%). IFN-gamma-activated macrophages produced high NO levels in comparison with the controls. Additionally, when the activated or treated-macrophages were supplemented with iron donors (HOLO or FeSO4), the inhibitory action was reversed, although NO production remained intact. These results suggest that the NO-mediated fungicidal mechanism exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of an iron interaction.
Assuntos
Compostos Ferrosos/farmacologia , Interferon gama/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Óxido Nítrico/biossíntese , Paracoccidioides/crescimento & desenvolvimento , Animais , Desferroxamina/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioides/efeitos dos fármacos , Transferrina/farmacologiaRESUMO
Iron is an essential growth element of virtually all microorganisms and its restriction is one of the mechanisms used by macrophages to control microbial multiplication. Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America, is inhibited in its conidia-to-yeast conversion in the absence of iron. We studied the participation of iron in the nitric oxide (NO)-mediated fungicidal mechanism against conidia. Peritoneal murine macrophages activated with 50U/mL of IFN-gamma or treated with 35 æM Deferoxamine (DEX) and infected with P. brasiliensis conidia, were co-cultured and incubated for 96 h in the presence of different concentrations of holotransferrin (HOLO) and FeS0(4). The supernatants were withdrawn in order to assess NO2 production by the Griess method. The monolayers were fixed, stained and observed microscopically. The percentage of the conidia-to-yeast transition was estimated by counting 200 intracellular propagules. IFN-gamma-activated or DEX-treated Mthetas presented marked inhibition of the conidia-to-yeast conversion (19 and 56 percent, respectively) in comparison with non-activated or untreated Mthetas (80 percent). IFN-gamma-activated macrophages produced high NO levels in comparison with the controls. Additionally, when the activated or treated-macrophages were supplemented with iron donors (HOLO or FeSO4), the inhibitory action was reversed, although NO production remained intact. These results suggest that the NO-mediated fungicidal mechanism exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of an iron interaction.
O ferro é elemento essencial para o crescimento de microrganismos e sua limitação é um dos mecanismos usados por macrófagos para controlar a multiplicação microbiana. Paracoccidioides brasiliensis, o agente da paracoccidioidomicose, uma das micoses sistêmicas mais importantes na América Latina, é inibido em sua conversão de conídia-à-levedura na ausência do ferro. Estudamos a participação do ferro no mecanismo fungicida mediado pelo óxido nítrico (NO) na sua interação com as conídias do fungo. Macrófagos peritoneais murinos ativados com 50U/mL de IFN-gama ou tratados com 35 æM Deferoxamina (DEX) e infectados com conídias do P. brasiliensis foram co-cultivados e incubados por 96 h na presença de concentrações diferentes de holotransferrina (HOLO) e FeS0(4). Os sobrenadantes foram retirados a fim de avaliar a produção de NO2 pelo método de Griess. Os macrófagos eram fixados, corados e observados ao microscópio. A porcentagem da transição de conídia-à-levedura foi estimada contando 200 propágulos intracelulares. Os macrófagos ativados com citocina ou tratados com DEX apresentaram inibição marcada da conversão de conídia-à-levedura (19 e 56 por cento, respectivamente) em comparação com macrófagos controle (80 por cento). Os macrófagos ativados com IFN-gama produziram elevação nos níveis de NO em comparação com macrófagos não-tratados ou não-activados. Adicionalmente, quando as monocapas ativadas ou tratadas foram suplementadas com doadores do ferro (HOLO ou FeSO4), a ação inibitória foi revertida embora a produção de NO permanecesse intacto. Estes resultados sugerem que o mecanismo fungicida mediado pelo NO exercido por macrófagos ativados com IFN-gama contra conídias do P. brasiliensis é dependente de uma interação do ferro.
Assuntos
Animais , Masculino , Camundongos , Desferroxamina/farmacologia , Interferon gama/farmacologia , Ferro/farmacologia , Macrófagos Peritoneais/microbiologia , Óxido Nítrico Sintase/efeitos dos fármacos , Paracoccidioides/crescimento & desenvolvimento , Transferrina/farmacologia , Camundongos Endogâmicos BALB C , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/imunologiaRESUMO
In this work, we have immunohistochemically analyzed the effects of single injections of apotransferrin (aTf) on the expression of myelin (myelin basic proteins [MBPs]) and axonal (protein gene product 9.5 [PGP 9.5] and beta(III)-tubulin [beta(III)-tub]) proteins in colchicine-injected and crushed sciatic nerves of adult rats. A protein redistribution was seen in the distal stump of injured nerves, with the appearance of MBP- and PGP 9.5-immunoreactive (IR) clusters which occurred earlier in crushed nerves (3 days post-injury [PI]) as compared to colchicine-injected nerves (7 days PI). beta(III)-tub-IR clusters appeared at 1 day PI preceding the PGP 9.5- and MBP-IR clusters in colchicine-injected nerves. With image analysis, the peak of clustering formation was found at 14 days PI for MBP and at 3 days PI for beta(III)-tub in colchicine-injected nerves. At 28 days of survival, the protein distribution patterns were almost normal. The intraneural application of aTf, at different concentrations (0.0005 mg/ml, 0.005 mg/ml, 0.05 mg/ml, 0.5 mg/ml), prevented nerve degeneration produced by colchicine, with the appearance of only a small number of MBP- and beta(III)-tub-IR clusters. However, aTf was not able to prevent clustering formation when the nerve was crushed, a kind of injury that also involves necrosis and blood flow alterations. The results suggest that aTf could prevent the colchicine effects by stabilizing the cytoskeleton proteins of the nerve fibers, avoiding the disruption of the axonal transport and thus the myelin degeneration. Transferrin is proposed as a complementary therapeutic avenue for treatment of cytotoxic nerve injuries.
Assuntos
Apoproteínas/farmacologia , Axônios/efeitos dos fármacos , Proteínas do Tecido Nervoso/efeitos dos fármacos , Neuropatia Ciática/tratamento farmacológico , Transferrina/farmacologia , Degeneração Walleriana/tratamento farmacológico , Degeneração Walleriana/prevenção & controle , Animais , Transporte Axonal/efeitos dos fármacos , Transporte Axonal/fisiologia , Axônios/metabolismo , Colchicina/antagonistas & inibidores , Colchicina/toxicidade , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/patologia , Proteína Básica da Mielina/efeitos dos fármacos , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Proteínas do Tecido Nervoso/metabolismo , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/toxicidade , Ratos , Ratos Wistar , Neuropatia Ciática/metabolismo , Neuropatia Ciática/fisiopatologia , Resultado do Tratamento , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Ubiquitina Tiolesterase/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Degeneração Walleriana/fisiopatologiaRESUMO
In the CNS, transferrin (Tf) is expressed by the oligodendroglial cells (OLGcs) and is essential for their development. We have previously shown that apotransferrin (aTf) accelerates maturation of OLGcs in vivo as well as in vitro. The mechanisms involved in this action appear to be complex and have not been completely elucidated. The aim of this study was to investigate if Tf participates in the regulation of the cell cycle of oligodendroglial progenitor cells (OPcs). Primary cultures of OPcs were treated with aTf and/or with different combinations of mitogenic factors. Cell cycle progression was studied by BrdU incorporation, flow cytometry and by the expression of cell cycle regulatory proteins. Apotransferrin decreased the number of BrdU+ cells, increasing the cell cycle time and decreasing the number of cells in S phase. The cell cycle inhibitors p27kip1, p21cip1 and p53 were increased, and in agreement with these results, the activity of the complexes involved in G1-S progression (cyclin D/CDK4, cyclin E/CDK2), was dramatically decreased. Apotransferrin also inhibited the mitogenic effects of PDGF and PDGF/IGF on OPcs, but did not affect their proliferation rate in the presence of bFGF, bFGF/PDGF or bFGF/IGF. Our results indicate that inhibition of the progression of the cell cycle of OPcs by aTf, even in the presence of PDGF, leads to an early beginning of the differentiation program, evaluated by different maturation markers (O4, GC and MBP) and by morphological criteria. The modulation by aTf of the response of OPcs to PDGF supports the idea that this glycoprotein might act as a key regulator of the OLGc lineage progression.
Assuntos
Apoproteínas/farmacologia , Ciclo Celular/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Fator de Crescimento Derivado de Plaquetas/farmacologia , Células-Tronco/efeitos dos fármacos , Transferrina/farmacologia , Animais , Antimetabólitos , Western Blotting , Bromodesoxiuridina , Diferenciação Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , DNA/biossíntese , Depressão Química , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Fase G1 , Imuno-Histoquímica , Oligodendroglia/ultraestrutura , Ratos , Fase S , Células-Tronco/ultraestrutura , Sais de Tetrazólio , TiazóisRESUMO
Twenty-one-day-old Wistar rats were fed a diet containing 0.6% cuprizone for 2 weeks. Studies carried out after withdrawal of cuprizone showed histological evidences of marked demyelination in the corpus callosum. Biochemical studies of isolated myelin showed a marked decrease in myelin proteins, phospholipids, and galactocerebrosides as well as a marked decrease in myelin yield. Treatment of these animals with a single intracranial injection of 350 ng of apotransferrin at the time of withdrawal of cuprizone induced a marked increase in myelin deposition resulting in a significantly improved remyelination, evaluated by histological, immunocytochemical, and biochemical parameters, in comparison to what was observed in spontaneous recovery. Immunocytochemical studies of cryotome sections to analyze developmental parameters of the oligodendroglial cell population at the time of termination of cuprizone and at different times thereafter showed that in the untreated animals, there was a marked increase in the number of NG2-BrdU-positive precursor cells together with a marked decrease in MBP expression at the peak of cuprizone-induced demyelination. As expected, the amount of precursor cells decreased markedly during spontaneous remyelination and was accompanied by an increase in MBP reactivity. In the apotransferrin-treated animals, these phenomena occurred much faster, and remyelination was much more efficient than in the untreated controls. The results of this study suggest that apotransferrin is a very active promyelinating agent which could be important for the treatment of certain demyelinating conditions.
Assuntos
Apoproteínas/uso terapêutico , Cuprizona , Doenças Desmielinizantes/tratamento farmacológico , Recuperação de Função Fisiológica/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Transferrina/uso terapêutico , Análise de Variância , Animais , Animais Recém-Nascidos , Antígenos/metabolismo , Apoproteínas/farmacologia , Encéfalo/patologia , Bromodesoxiuridina/farmacocinética , Antígeno CD11b/metabolismo , Contagem de Células/métodos , Proteínas do Citoesqueleto/metabolismo , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/fisiopatologia , Interações Medicamentosas , Galactolipídeos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica/métodos , Indóis , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Proteoglicanas/metabolismo , Ratos , Ratos Wistar , Regeneração/fisiologia , Fatores de Tempo , Transferrina/farmacologiaRESUMO
The mechanisms used by Paracoccidioides brasiliensis to survive into phagocytic cells are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Thus, the objective of this paper was to investigate the role of intracellular iron in regulating the capacity of P. brasiliensis yeast cells to survive within human monocytes. Treatment of monocytes with deferoxamine, an iron chelator, suppressed the survival of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by nonsaturated transferrin (apotransferrin). These results strongly suggest that P. brasiliensis survival in human monocytes is iron dependent.