RESUMEN
Quiescin/sulfhydryl oxidase (QSOX1) is a secreted flavoprotein that modulates cellular proliferation, migration and adhesion, roles attributed to its ability to organize the extracellular matrix. We previously showed that exogenously added QSOX1b induces smooth muscle cells migration in a process that depends on its enzymatic activity and that is mediated by hydrogen peroxide derived from Nox1, a catalytic subunit of NAD(P)H oxidases. Here, we report that exogenous QSOX1b also stimulates the migration of L929 fibroblasts and that this effect is regulated by its endocytosis. The use of endocytosis inhibitors and caveolin 1-knockdown demonstrated that this endocytic pathway is caveola-mediated. QSOX1b colocalized with Nox1 in intracellular vesicles, as detected by confocal fluorescence, suggesting that extracellular QSOX1b is endocytosed with the transmembrane Nox1. These results reveal that endosomal QSOX1b is a novel intracellular redox regulator of cell migration.
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Caveolas , NADPH Oxidasas , Fibroblastos , Endocitosis , Proliferación CelularRESUMEN
Loss of the tumor suppressor gene Pten in murine prostate recapitulates human carcinogenesis and causes stromal proliferation surrounding murine prostate intraepithelial neoplasia (mPIN), which is reactive to microinvasion. In turn, invasion has been shown to be regulated in part by de novo fatty acid synthesis in prostate cancer. We therefore investigated the effects of genetic ablation of Fasn on invasive potential in prostate-specific Pten knockout mice. Combined genetic ablation of Fasn and Pten reduced the weight and volume of all the prostate lobes when compared to single knockouts. The stromal reaction to microinvasion and the cell proliferation that typically occurs in Pten knockout were largely abolished by Fasn knockout. To verify that Fasn knockout indeed results in decreased invasive potential, we show that genetic ablation and pharmacologic inhibition of FASN in prostate cancer cells significantly inhibit cellular motility and invasion. Finally, combined loss of PTEN with FASN overexpression was associated with lethality as assessed in 660 prostate cancer patients with 14.2 years of median follow-up. Taken together, these findings show that de novo lipogenesis contributes to the aggressive phenotype induced by Pten loss in murine prostate and targeting Fasn may reduce the invasive potential of prostate cancer driven by Pten loss. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
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Acido Graso Sintasa Tipo I/genética , Fosfohidrolasa PTEN/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Movimiento Celular/genética , Acido Graso Sintasa Tipo I/metabolismo , Humanos , Lipogénesis/fisiología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Invasividad Neoplásica/genética , Neoplasias de la Próstata/patologíaRESUMEN
A Disintegrin And Metalloprotease 23 (ADAM23) is a member of the ADAMs family of transmembrane proteins, mostly expressed in nervous system, and involved in traffic and stabilization of Kv1-potassium channels, synaptic transmission, neurite outgrowth, neuronal morphology and cell adhesion. Also, ADAM23 has been linked to human pathological conditions, such as epilepsy, cancer metastasis and cardiomyopathy. ADAM23 functionality depends on the molecule presence at the cell surface and along the secretory pathway, as expected for a cell surface receptor. Because endocytosis is an important functional regulatory mechanism of plasma membrane receptors and no information is available about the traffic or turnover of non-catalytic ADAMs, we investigated ADAM23 internalization, recycling and half-life properties. Here, we show that ADAM23 undergoes constitutive internalization from the plasma membrane, a process that depends on lipid raft integrity, and is redistributed to intracellular vesicles, especially early and recycling endosomes. Furthermore, we observed that ADAM23 is recycled from intracellular compartments back to the plasma membrane and thus has longer half-life and higher cell surface stability compared with other ADAMs. Our findings suggest that regulation of ADAM23 endocytosis/stability could be exploited therapeutically in diseases in which ADAM23 is directly involved, such as epilepsy, cancer progression and cardiac hypertrophy.
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Proteínas ADAM/metabolismo , Endocitosis , Membrana Celular/metabolismo , Células Cultivadas , Endosomas/metabolismo , Semivida , Humanos , Microdominios de Membrana/metabolismoRESUMEN
Apoptosis-inducing factor (AIF) is a flavoprotein and essential partner of the CHCHD4 redox protein during the mitochondrial intermembrane space import machinery. Mammalian AIF has three cysteine residues, which have received little attention. Previous reports have evidenced a redox interaction between AIF and thioredoxin 1 (Trx1), particularly after oxidant conditions. Therefore, we asked whether the cysteine residues of the human AIF could be oxidized. Our data showed that endogenous AIF could be oxidized to disulfide-linked conjugates (DLC). Overexpressed WT AIF in HEK293T cells, as well as recombinant WT AIF, formed DLC. Expression of C256S, C317S or C441S AIF mutants severely inhibited DLC formation in cells exposed to oxidants. In vitro, DLC formation was completely precluded with C256S and C441S AIF mutants and partially inhibited with the C317S mutant. DLC was shown to enhance cellular susceptibility to apoptosis induced by staurosporine, likely by preventing AIF to maintain mitochondrial oxidative phosphorylation. Cells with decreased expression of Trx1 produced more AIF DLC than those with normal Trx1 levels, and in vitro, Trx1 was able to decrease the amount of AIF DLC. Finally, confocal analysis, as well as immunoblotting of mitochondrial fraction, indicated that a fraction of Trx1 is present in mitochondria. Overall, these data provide evidence that all three cysteine residues of AIF can be oxidized to DLC, which can be disrupted by mitochondrial Trx1.
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Factor Inductor de la Apoptosis , Apoptosis , Disulfuros , Sustitución de Aminoácidos , Factor Inductor de la Apoptosis/química , Factor Inductor de la Apoptosis/genética , Factor Inductor de la Apoptosis/metabolismo , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Disulfuros/química , Disulfuros/metabolismo , Células HEK293 , Células HeLa , Humanos , Mutación Missense , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estaurosporina/farmacologíaRESUMEN
Quiescent and contractile VSMC can switch to proliferative and migratory phenotype in response to growth factors and cytokines, an effect underscored by Nox family NADPH oxidases, particularly Nox1. We previously showed that quiescin/sulfhydryl oxidase 1 (QSOX1) has a role in neointima formation in balloon-injured rat carotid. Here, we investigated the intracellular redox mechanisms underlying these effects in primary VSMC. Our results show that exogenous incubation with wild type QSOX1b (wt QSOX), or with secreted QSOX1, but not with the inactive C452S QSOX 1b (C452S QSOX) or secreted inactive C455S QSOX1, induces VSMC migration and chemotaxis. PEG-catalase (PEG-CAT) prevented, while PEG-superoxide dismutase (PEG-SOD) increased migration induced by wt QSOX. Moreover, wt QSOX-induced migration was abrogated in NOX1-null VSMC. In contrast, both wt QSOX and C452S QSOX, and both secreted QSOX1 and C455S QSOX1, induce cell proliferation. Such effect was unaltered by PEG-CAT, while being inhibited by PEG-SOD. However, QSOX1-induced proliferation was not significantly affected in NOX1-null VSMC, compared with WT VSMC. These results indicate that hydrogen peroxide and superoxide mediate, respectively, migration and proliferation. However, Nox1 was required only for QSOX1-induced migration. In parallel, QSOX1-induced proliferation was independent of its redox activity, although mediated by intracellular superoxide.
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Movimiento Celular , Músculo Liso Vascular/citología , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Animales , Proliferación Celular , Células HEK293 , Humanos , Peróxido de Hidrógeno/metabolismo , Espacio Intracelular/metabolismo , Ratones , NADPH Oxidasa 1/metabolismo , Oxidación-Reducción/efectos de los fármacos , Superóxidos/metabolismoRESUMEN
Quiescin sulfhydryl oxidase 1 (QSOX1) is a flavoenzyme largely present in the extracellular milieu whose physiological functions and substrates are not known. QSOX1 has been implicated in the regulation of tumor cell survival, proliferation and migration, in addition to extracellular matrix (ECM) remodeling. However, data regarding other pathophysiological conditions are still lacking. Arterial injury by balloon catheter is an established model of post-angioplasty restenosis. This technique induces neointima formation due to migration and proliferation of vascular smooth muscle cells (VSMC), followed by ECM synthesis and remodeling. Here, we show that QSOX1 knockdown inhibited VSMC migration and proliferation in vitro. In contrast, QSOX1 overexpression stimulated these processes. While migration could be induced by the incubation of cells with the active recombinant QSOX1, proliferation was induced by addition of the active and also of an inactive mutant QSOX1 protein. The proliferation induced by both recombinants was independent of intracellular hydrogen peroxide and dependent of the MEK/ERK pathway. To recapitulate in vivo VSMC pathophysiology, balloon-induced arterial injury was performed. The expression of QSOX1 in the neointimal layer of balloon-injured rat carotids was high and peaked at 14 days post-injury. In vivo QSOX1 knockdown led to a significant decrease in PCNA expression at day 14 post-injury and a decreased intima/media area ratio at day 21 post-injury, compared with scrambled siRNA transfection. In summary, our findings demonstrate that QSOX1 induces VSMC migration and proliferation in vitro and contributes to neointima thickening in balloon-injured rat carotids.
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Movimiento Celular , Proliferación Celular , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima/metabolismo , Tiorredoxinas/metabolismo , Animales , Arterias Carótidas/patología , Arterias Carótidas/cirugía , Células Cultivadas , Peróxido de Hidrógeno/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/fisiología , Ratas , Ratas Wistar , Tiorredoxinas/genéticaRESUMEN
Herbaspirillum bacteria are best known as plant growth-promoting rhizobacteria but have also been recovered from clinical samples. Here, biochemical tests, matrix-assisted laser deionization-time of flight (MALDI-TOF) mass spectrometry, adherence, and cytotoxicity to eukaryotic cells were used to compare clinical and environmental isolates of Herbaspirillum spp. Discrete biochemical differences were observed between human and environmental strains. All strains adhered to HeLa cells at low densities, and cytotoxic effects were discrete, supporting the view that Herbaspirillum bacteria are opportunists with low virulence potential.
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Adhesión Bacteriana/fisiología , Microbiología Ambiental , Infecciones por Bacterias Gramnegativas/microbiología , Herbaspirillum/fisiología , Herbaspirillum/patogenicidad , Supervivencia Celular , Células HeLa , Herbaspirillum/química , Herbaspirillum/clasificación , Humanos , Filogenia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Depression is increasingly present in the population, and its pathophysiology and treatment have been investigated with several animal models, including olfactory bulbectomy (Obx). Fish oil (FO) supplementation during the prenatal and postnatal periods decreases depression-like and anxiety-like behaviors. The present study evaluated the effect of FO supplementation on Obx-induced depressive-like behavior and cognitive impairment. Female rats received supplementation with FO during habituation, mating, gestation, and lactation, and their pups were subjected to Obx in adulthood; after the recovery period, the adult offspring were subjected to behavioral tests, and the hippocampal levels of brain-derived neurotrophic factor (BDNF), serotonin (5-HT) and the metabolite 5-hydroxyindoleacetic (5-HIAA) were determined. Obx led to increased anxiety-like and depressive-like behaviors, and impairment in the object location task. All behavioral changes were reversed by FO supplementation. Obx caused reductions in the levels of hippocampal BDNF and 5-HT, whereas FO supplementation restored these levels to normal values. In control rats, FO increased the hippocampal level of 5-HT and reduced that of 5-HIAA, indicating low 5-HT metabolism in this brain region. The present results indicate that FO supplementation during critical periods of brain development attenuated anxiety-like and depressive-like behaviors and cognitive dysfunction induced by Obx. These results may be explained by increased levels of hippocampal BDNF and 5-HT, two major regulators of neuronal survival and long-term plasticity in this brain structure.
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Trastornos de Ansiedad/tratamiento farmacológico , Fármacos del Sistema Nervioso Central/uso terapéutico , Trastornos del Conocimiento/tratamiento farmacológico , Trastorno Depresivo/tratamiento farmacológico , Aceites de Pescado/uso terapéutico , Animales , Trastornos de Ansiedad/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Trastornos del Conocimiento/metabolismo , Trastorno Depresivo/metabolismo , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Ácido Hidroxiindolacético/metabolismo , Masculino , Pruebas Neuropsicológicas , Bulbo Olfatorio/fisiología , Bulbo Olfatorio/cirugía , Embarazo , Efectos Tardíos de la Exposición Prenatal , Ratas , Ratas Wistar , Serotonina/metabolismoRESUMEN
Melatonin is a hormone secreted by the pineal gland, it can be associated with circadian rhythms, aging and neuroprotection. Melatonin levels are decreased in sporadic Alzheimer's disease (sAD) patients, which suggests a relationship between the melatonergic system and sAD. Melatonin may reduce inflammation, oxidative stress, TAU protein hyperphosphorylation, and the formation of ß-amyloid (Aß) aggregates. Therefore, the objective of this work was to investigate the impact of treatment with 10 mg/kg of melatonin (i.p) in the animal model of sAD induced by the intracerebroventricular (ICV) infusion of 3 mg/kg of streptozotocin (STZ). ICV-STZ causes changes in the brain of rats similar to those found in patients with sAD. These changes include; progressive memory decline, the formation of neurofibrillary tangles, senile plaques, disturbances in glucose metabolism, insulin resistance and even reactive astrogliosis characterized by the upregulation of glucose levels and glial fibrillary acidic protein (GFAP). The results show that ICV-STZ caused short-term spatial memory impairment in rats after 30 days of STZ infusion without locomotor impairment which was evaluated on day 27 post-injury. Furthermore, we observed that a prolonged 30-day treatment with melatonin can improve the cognitive impairment of animals in the Y-maze test, but not in the object location test. Finally, we demonstrated that animals receiving ICV-STZ have high levels of Aß and GFAP in the hippocampus and that treatment with melatonin reduces Aß levels but does not reduce GFAP levels, concluding that melatonin may be useful to control the progression of amyloid pathology in the brain.
RESUMEN
BACKGROUND: Celiac disease (CD) is an autoimmune disorder triggered by an abnormal immunological response to gluten ingestion and is associated with deregulated expression of cellular microRNAs (miRNAs) of the gut mucosa. It is frequently misdiagnosed as lactose intolerance (LI) due to symptom resemblance. Microvilli loss may be counteracted by a rigorous gluten-free diet (GFD). AIMS: To identify altered extracellular vesicle miRNAs from plasma among CD patients on GFD (n=34), lactose intolerant individuals on restrictive diet (n=14) and controls (n=23), and to predict biological pathways in which these altered miRNAs may play a part. METHODS: Five different small RNA samples of each group were pooled twice and then screened by new-generation sequencing. Four miRNAs were selected to be quantified by RT-qPCR in the entire sample. RESULTS: The levels of four miRNAs - miR-99b-3p, miR-197-3p, miR-223-3p, and miR-374b-5p - differed between CD patients and controls (P<0.05). Apart from miR-223-3p, all these miRNAs tended to have altered levels also between LI and controls (P<0.10). The results for miR-99b-3p and miR-197-3p between CD and controls were confirmed by RT-qPCR, which also indicated different levels of miR-99b-3p and miR-374b-5p between CD-associated LI and LI (P<0.05). CONCLUSIONS: These miRNAs may have targets that affect cell death, cell communication, adhesion, and inflammation modulation pathways. Hence, altered miRNA levels could be associated with CD-related aspects and gut mucosa recovery.
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AIM: To test the hypothesis that the antidepressant-like effect of omega-3 polyunsaturated fatty acids is related to the Indoleamine-2,3-Dioxygenase (IDO) inhibition. METHODS: Animals were supplemented for 50 days with 3.0 g/kg of Fish Oil (FO) or received water (Control group - C), via gavage. At the end of this period, both groups were injected with LPS 24 h before the modified forced swim test (MFST) and the open field. To assess the possible involvement of IDO in the FO effects, we performed two independent experiments, using two IDO inhibitors: the direct inhibitor 1-methyl-DL-tryptophan (1-MT) and the anti-inflammatory drug minocycline (MINO), administered 23 h, 5 h and 1 h before the tests. After the tests, the animals' hippocampi were removed for quantification of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) by HPLC, and for IDO expression by western blot. RESULTS: LPS induced a depressive-like state in the animals, and this effect was blocked by 1-MT, MINO and FO. Regardless of IDO inhibition, FO supplemented animals displayed an antidepressant-like response by increasing swimming and decreasing immobility frequencies in the MFST when compared to the control group. The immune challenge induced an over-expression of IDO and reduced hippocampal 5-HT levels, both of which were reversed by MINO and FO. CONCLUSION: FO induced a pronounced antidepressant-like effect and prevented LPS-induced depressive-like behavior, and this effect was related to decreased IDO expression and increased 5-HT levels in the hippocampus.
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Antiinflamatorios/farmacología , Antidepresivos/farmacología , Depresión/metabolismo , Depresión/prevención & control , Aceites de Pescado/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa , Minociclina/farmacología , Serotonina/metabolismo , Triptófano/farmacología , Animales , Antiinflamatorios/administración & dosificación , Antidepresivos/administración & dosificación , Conducta Animal/efectos de los fármacos , Depresión/inducido químicamente , Suplementos Dietéticos , Ácidos Grasos Omega-3/farmacología , Aceites de Pescado/administración & dosificación , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Indolamina-Pirrol 2,3,-Dioxigenasa/efectos de los fármacos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Lipopolisacáridos/farmacología , Masculino , Minociclina/administración & dosificación , Ratas , Ratas Wistar , Triptófano/administración & dosificaciónRESUMEN
MicroRNAs derived from extracellular vesicles (EV-miRNAs) are circulating miRNAs considered as potential new diagnostic markers for cancer that can be easily detected in liquid biopsies. In this study, we performed RNA sequencing analysis as a screening strategy to identify EV-miRNAs derived from serum of clinically well-annotated breast cancer (BC) patients from the south of Brazil. EVs from three groups of samples (healthy controls (CT), luminal A (LA), and triple-negative (TNBC)) were isolated from serum using a precipitation method and analyzed by RNA-seq (screening phase). Subsequently, four EV-miRNAs (miR-142-5p, miR-150-5p, miR-320a, and miR-4433b-5p) were selected to be quantified by quantitative real-time PCR (RT-qPCR) in individual samples (test phase). A panel composed of miR-142-5p, miR-320a, and miR-4433b-5p distinguished BC patients from CT with an area under the curve (AUC) of 0.8387 (93.33% sensitivity, 68.75% specificity). The combination of miR-142-5p and miR-320a distinguished LA patients from CT with an AUC of 0.9410 (100% sensitivity, 93.80% specificity). Interestingly, decreased expression of miR-142-5p and miR-150-5p were significantly associated with more advanced tumor grades (grade III), while the decreased expression of miR-142-5p and miR-320a was associated with a larger tumor size. These results provide insights into the potential application of EVs-miRNAs from serum as novel specific markers for early diagnosis of BC.
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Neoplasias de la Mama/genética , Vesículas Extracelulares/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Adulto , Anciano , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Neoplasias de la Mama/sangre , Femenino , Perfilación de la Expresión Génica , Humanos , MicroARNs/sangre , Persona de Mediana Edad , Neoplasias de la Mama Triple Negativas/sangre , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
BACKGROUND: ADAM33 protein is a member of the family of transmembrane glycoproteins composed of multidomains. ADAM family members have different activities, such as proteolysis and adhesion, making them good candidates to mediate the extracellular matrix remodelling and changes in cellular adhesion that characterise certain pathologies and cancer development. It was reported that one family member, ADAM23, is down-regulated by promoter hypermethylation. This seems to correlate with tumour progression and metastasis in breast cancer. In this study, we explored the involvement of ADAM33, another ADAM family member, in breast cancer. METHODS: First, we analysed ADAM33 expression in breast tumour cell lines by RT-PCR and western blotting. We also used 5-aza-2'-deoxycytidine (5azadCR) treatment and DNA bisulphite sequencing to study the promoter methylation of ADAM33 in breast tumour cell lines. We evaluated ADAM33 methylation in primary tumour samples by methylation specific PCR (MSP). Finally, ADAM33 promoter hypermethylation was correlated with clinicopathological data using the chi-square test and Fisher's exact test. RESULTS: The expression analysis of ADAM33 in breast tumour cell lines by RT-PCR revealed gene silencing in 65% of tumour cell lines. The corresponding lack of ADAM33 protein was confirmed by western blotting. We also used 5-aza-2'-deoxycytidine (5-aza-dCR) demethylation and bisulphite sequencing methodologies to confirm that gene silencing is due to ADAM33 promoter hypermethylation. Using MSP, we detected ADAM33 promoter hypermethylation in 40% of primary breast tumour samples. The correlation between methylation pattern and patient's clinicopathological data was not significantly associated with histological grade; tumour stage (TNM); tumour size; ER, PR or ERBB2 status; lymph node status; metastasis or recurrence. Methylation frequency in invasive lobular carcinoma (ILC) was 76.2% compared with 25.5% in invasive ductal carcinoma (IDC), and this difference was statistically significant (p = 0.0002). CONCLUSION: ADAM33 gene silencing may be related to the discohesive histological appearance of ILCs. We suggest that ADAM33 promoter methylation may be a useful molecular marker for differentiating ILC and IDC.
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Proteínas ADAM/genética , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Carcinoma Lobular/genética , Silenciador del Gen , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/patología , Línea Celular Tumoral , Metilación de ADN , Femenino , Expresión Génica/genética , Humanos , Persona de Mediana Edad , Regiones Promotoras Genéticas/genéticaRESUMEN
Quiescin Q6/sulfhydryl oxidases (QSOX) are revisited thiol oxidases considered to be involved in the oxidative protein folding, cell cycle control and extracellular matrix remodeling. They contain thioredoxin domains and introduce disulfide bonds into proteins and peptides, with the concomitant hydrogen peroxide formation, likely altering the redox environment. Since it is known that several developmental processes are regulated by the redox state, here we assessed if QSOX could have a role during mouse fetal development. For this purpose, an anti-recombinant mouse QSOX antibody was produced and characterized. In E(13.5), E(16.5) fetal tissues, QSOX immunostaining was confined to mesoderm- and ectoderm-derived tissues, while in P1 neonatal tissues it was slightly extended to some endoderm-derived tissues. QSOX expression, particularly by epithelial tissues, seemed to be developmentally-regulated, increasing with tissue maturation. QSOX was observed in loose connective tissues in all stages analyzed, intra and possibly extracellularly, in agreement with its putative role in oxidative folding and extracellular matrix remodeling. In conclusion, QSOX is expressed in several tissues during mouse development, but preferentially in those derived from mesoderm and ectoderm, suggesting it could be of relevance during developmental processes.
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Feto/enzimología , Oxidorreductasas/metabolismo , Animales , Animales Recién Nacidos , Especificidad de Anticuerpos , Inmunohistoquímica , Masculino , Ratones , Oxidorreductasas/análisis , Oxidorreductasas/inmunología , Conejos , Ratas , Ratas Wistar , Distribución TisularRESUMEN
Dopamine (DA) has, as of late, become singled out from the profusion of other neurotransmitters as what could be called a key substance, in the regulation of the sleep-wake states. We have hypothesized that dopaminergic D(2) receptor blockage induced by haloperidol could generate a reduction or even an ablation of rapid eye movement (REM) sleep. Otherwise, the use of the selective D(2) agonist, piribedil, could potentiate REM sleep. Electrophysiological findings demonstrate that D(2) blockage produced a dramatic reduction of REM sleep during the rebound (REB) period after 96 h of REM sleep deprivation (RSD). This reduction of REM sleep was accompanied by an increment in SWS, which is possibly accounted for the observed increase in the sleep efficiency. Conversely, our findings also demonstrate that the administration of piribedil did not generate additional increase of REM sleep. Additionally, D(2) receptors were found down-regulated, in the haloperidol group, after RSD, and subsequently up-regulated after REB group, contrasting to the D(1) down-regulation at the same period. In this sense, the current data indicate a participation of the D(2) receptor for REM sleep regulation and consequently in the REM sleep/SWS balance. Herein, we propose that the mechanism underlying the striatal D(2) up-regulation is due to an effect as consequence of RSD which originally produces selective D(2) supersensitivity, and after its period probably generates a surge in D(2) expression. In conclusion we report a particular action of the dopaminergic neurotransmission in REM sleep relying on D(2) activation.
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Receptores de Dopamina D2/fisiología , Privación de Sueño/fisiopatología , Sueño REM/fisiología , Sueño/fisiología , Análisis de Varianza , Animales , Conducta Animal , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Haloperidol/farmacología , Masculino , Piribedil/farmacología , Ratas , Receptores de Dopamina D1/metabolismo , Sueño/efectos de los fármacos , Sueño REM/efectos de los fármacos , Tirosina 3-Monooxigenasa/metabolismo , Vigilia/efectos de los fármacos , Vigilia/fisiologíaRESUMEN
Malignant melanoma is one of the most lethal cancers. Nowadays, several anti-melanoma therapies have been employed. However, the poor prognosis and/or the increased toxicity of those treatments clearly demonstrate the requirement of searching for new drugs or novel combined chemotherapeutic protocols, contemplating both effectiveness and low toxicity. Guanosine (Guo) has been used in combination with acriflavina to potentiate the latter's antitumor activity, through still unknown mechanisms. Here, we show that Guo induces B16F10 melanoma cell differentiation, attested by growth arrest, dendrite-like outgrowth and increased melanogenesis, and also reduced motility. A sustained ERK 1/2 phosphorylation was observed after Guo treatment and ERK inhibition led to blockage of dendritogenesis. Intracellular cyclic AMP was not involved in ERK activation, since its levels remained unchanged. Protein kinase C (PKC), in contrast to phospholipase C (PLC), inhibition completely prevented ERK activation. While the classical melanoma differentiation agent forskolin activates cAMP-PKA-Raf-MEK-ERK pathway in B16F10 cells, here we suggest that a cAMP-independent, PKC-ERK axis is involved in Guo-induced B16F10 differentiation. Altogether, our results show that Guo acts as a differentiating agent, with cytostatic rather than cytotoxic properties, leading to a decreased melanoma malignancy. Thus, we propose that Guo may be envisaged in combination with lower doses of conventional anti-melanoma drugs, in an attempt to prevent or diminish their adverse effects.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Guanosina/farmacología , Melanoma/tratamiento farmacológico , Proteína Quinasa C/metabolismo , Western Blotting , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Humanos , Melaninas/inmunología , Melanoma/enzimología , Melanoma/patología , Microscopía Electrónica de Transmisión , Fosforilación/efectos de los fármacosRESUMEN
Early impairments in cerebral glucose metabolism and insulin signaling pathways may participate in the pathogenesis of the sporadic form of Alzheimer's disease (sAD). Intracerebroventricular (ICV) injections of low doses of streptozotocin (STZ) are used to mimic sAD and study these alterations in rodents. Streptozotocin causes impairments in insulin signaling and has been reported to trigger several alterations in the brain, such as oxidative stress, neuroinflammation, and dysfunctions in adult neurogenesis, which may be involved in cognitive decline and are features of human AD. The aim of the present study was to assess the influence of neuroinflammation on the process of adult neurogenesis and consequent cognitive deficits in the STZ-ICV model of sAD in Wistar rats. Streptozotocin caused an acute and persistent neuroinflammatory response, reflected by reactive microgliosis and astrogliosis in periventricular areas and the dorsal hippocampus, accompanied by a marked reduction of the proliferation of neural stem cells in the dentate gyrus of the hippocampus and subventricular zone. Streptozotocin also reduced the survival, differentiation, and maturation of newborn neurons, resulting in impairments in short-term and long-term spatial memory. These results support the hypothesis that neuroinflammation has a detrimental effect on neurogenesis, and both neuroinflammation and impairments in neurogenesis contribute to cognitive deficits in the STZ-ICV model of sAD.
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Envejecimiento/patología , Enfermedad de Alzheimer/fisiopatología , Encéfalo/patología , Encéfalo/fisiopatología , Inflamación/patología , Trastornos de la Memoria/fisiopatología , Neurogénesis , Memoria Espacial , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Astrocitos/patología , Conducta Animal , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Proteínas de Dominio Doblecortina , Miedo , Inyecciones Intraventriculares , Antígeno Ki-67/metabolismo , Masculino , Aprendizaje por Laberinto , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/patología , Microglía/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Neuropéptidos/metabolismo , Ratas Wistar , EstreptozocinaRESUMEN
A disintegrin and metalloprotease protein 23 (ADAM23) is a transmembrane type I glycoprotein involved with the development and maintenance of the nervous system, including neurite outgrowth, neuronal adhesion and differentiation and regulation of synaptic transmission. In addition, ADAM23 seems to participate in immune response and tumor establishment through interaction with different members of integrin receptors. Here, we describe a novel monoclonal antibody (DL11C8) that specifically recognizes the cysteine-rich domain of both pre-protein (100â¯kDa) and mature (70â¯kDa) forms of ADAM23 from different species, including human, rodents and avian orthologs. Using this antibody, we detected both forms of ADAM23 on the cell surface of three neuronal cell lineages (Neuro-2a, SH-SY5Y and CHLA-20), with a higher relative content of ADAM23100â¯kDa. Furthermore, we demonstrate for the first time that a catalytically inactive member of the ADAM family is present in the membrane signaling platforms, namely lipid rafts. Indeed, the mature ADAM2370â¯kDa partitions between raft and non-raft membrane domains, while the pro-protein ADAM23100â¯kDa is mainly expressed in non-raft domains. These membranous distributions were observed in both different brain regions homogenates and primary cultured neurons lysates from mouse cortex and cerebellum. Taken together, these findings point out ADAM23 as a lipid raft molecular component.
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Proteínas ADAM/metabolismo , Microdominios de Membrana/metabolismo , Animales , Anticuerpos Monoclonales , Línea Celular Tumoral , Humanos , RatonesRESUMEN
Diet and exercise are known to affect learning and memory. However, the effects of these interventions in the brain under development remains to be better investigated as the effects of high-intensity exercise. Moreover, it is still unclear how long the influence of diet and exercise lasts after the interventions are ceased. To investigate this, juvenile Wistar rats (30â¯days old) were supplemented with fish oil rich in polyunsaturated fatty acids (PUFAs) and performed swimming training for 50â¯days, 45â¯min per day, 5 times/week. The animals were assessed for locomotor activity with the open field test and for spatial memory with the object location task. To investigate neurochemical parameters such as fatty acids incorporation within the plasma membrane and brain-derived neurotrophic factor (BDNF) levels, the animals were euthanized, and the hippocampus dissected. These investigations were made at the end of the supplementation and exercise protocols and 21â¯days after the protocol has ended. Results indicate that high-intensity exercise impaired the spatial memory and decreased the levels of BDNF. Although supplementation led to PUFAs incorporation in plasma membrane, it did not prevent the harmful effect of exercise on memory. After 21â¯days of interruption, we observed that the supplementation reversed not only the deleterious effect of exercise on memory but also increased the BDNF levels. These results point to a complex influence of diet and exercise on spatial memory of juvenile rats, persisting after 21â¯days of interruption.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Ácidos Grasos Insaturados/metabolismo , Aceites de Pescado/uso terapéutico , Trastornos de la Memoria/dietoterapia , Natación/fisiología , Natación/psicología , Animales , Membrana Celular/metabolismo , Suplementos Dietéticos , Aceites de Pescado/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Locomoción/fisiología , Masculino , Condicionamiento Físico Animal/fisiología , Ratas , Memoria Espacial/efectos de los fármacosRESUMEN
The present study investigated the effects of the selective cyclooxygenase-2 (COX-2) inhibitor parecoxib (Bextratrade mark) in the prevention of motor and cognitive impairments observed in rats after an intranigral infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a model of the early phase of Parkinson's disease. The treatment with parecoxib (10 mg/kg) administered prior to the surgery and daily (2 mg/kg) for the subsequent 21 days, prevented the MPTP-treated rats from presenting decreased locomotor and exploratory behavior, increased immobility, and impairment while performing the cued version of the Morris water maze. Furthermore, parecoxib treatment also significantly prevented the reduction of tyrosine hydroxylase protein expression in the substantia nigra (7, 14 and 21 days after surgery), and in the striatum (14 and 21 days after surgery) as immunodetected by western blotting. These results strongly suggest that parecoxib exerts a neuroprotective effect on motor, tyrosine hydroxylase expression, and cognitive functions as it prevents their impairments within the confines of this animal model of the early phase of Parkinson's disease.