RESUMEN

The 5-lipoxygenase/leukotriene system has been implicated in both physiological and pathological states within the central nervous system. Understanding how this system interacts with the dopaminergic system could provide valuable insights into dopamine-related pathologies. This study focused on examining both motor and non-motor dopamine-related responses in 5-lipoxygenase/leukotriene-deficient mice. We used pharmacological agents such as amphetamine, apomorphine, and reserpine to challenge the dopaminergic system, evaluating their effects on prepulse inhibition reaction (PPI), general motor activity, and oral involuntary movements. Additionally, we analyzed striatal glial marker expression (GFAP and Iba-1) in reserpine-treated mice. The 5-lipoxygenase/leukotriene-deficient mice exhibited increased spontaneous locomotor activity, including both horizontal and vertical exploration, along with stereotyped behavior compared to wild-type mice. This hyperactivity was reduced by acute apomorphine treatment. Although basal PPI responses were unchanged, 5-lipoxygenase/leukotriene-deficient mice displayed a significant reduction in susceptibility to amphetamine-induced PPI disruption. Conversely, these mice were more vulnerable to reserpine-induced involuntary movements. There were no significant differences in the basal expression of striatal GFAP and Iba-1 positive cells between 5-lipoxygenase/leukotriene-deficient and wild-type mice. However, reserpine treatment significantly increased GFAP immunoreactivity in wild-type mice, an effect not observed in 5-lipoxygenase-deficient mice. Additionally, the percentage of activated microglia was significantly higher in reserpine-treated wild-type mice, an effect absents in 5-lipoxygenase/leukotriene-deficient mice. Our findings suggest that 5-lipoxygenase/leukotriene deficiency leads to a distinctive dopaminergic phenotype, indicating that leukotrienes may influence the modulation of dopamine-mediated responses.

Asunto(s)

Anfetamina , Araquidonato 5-Lipooxigenasa , Dopamina , Animales , Masculino , Ratones , Anfetamina/farmacología , Apomorfina/farmacología , Araquidonato 5-Lipooxigenasa/metabolismo , Araquidonato 5-Lipooxigenasa/deficiencia , Araquidonato 5-Lipooxigenasa/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/deficiencia , Cuerpo Estriado/metabolismo , Cuerpo Estriado/efectos de los fármacos , Dopamina/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Inhibición Prepulso/efectos de los fármacos , Inhibición Prepulso/fisiología , Reserpina/farmacología , Conducta Estereotipada/efectos de los fármacos

RESUMEN

Accidents caused by Bothrops jararaca (Bj) snakes result in several local and systemic manifestations, with pain being a fundamental characteristic. The inflammatory process responsible for hyperalgesia induced by Bj venom (Bjv) has been studied; however, the specific roles played by the peripheral and central nervous systems in this phenomenon remain unclear. To clarify this, we induced hyperalgesia in rats using Bjv and collected tissues from dorsal root ganglia (DRGs) and spinal cord (SC) at 2 and 4 h post-induction. Samples were labeled for Iba-1 (macrophage and microglia), GFAP (satellite cells and astrocytes), EGR1 (neurons), and NK1 receptors. Additionally, we investigated the impact of minocycline, an inhibitor of microglia, and GR82334 antagonist on Bjv-induced hyperalgesia. Our findings reveal an increase in Iba1 in DRG at 2 h and EGR1 at 4 h. In the SC, markers for microglia, astrocytes, neurons, and NK1 receptors exhibited increased expression after 2 h, with EGR1 continuing to rise at 4 h. Minocycline and GR82334 inhibited venom-induced hyperalgesia, highlighting the crucial roles of microglia and NK1 receptors in this phenomenon. Our results suggest that the hyperalgesic effects of Bjv involve the participation of microglial and astrocytic cells, in addition to the activation of NK1 receptors.

Asunto(s)

Bothrops , Venenos de Crotálidos , Ganglios Espinales , Hiperalgesia , Receptores de Neuroquinina-1 , Animales , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Venenos de Crotálidos/toxicidad , Masculino , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Receptores de Neuroquinina-1/metabolismo , Minociclina/farmacología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Microglía/efectos de los fármacos , Microglía/metabolismo , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Ratas , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas de Unión al Calcio/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Proteínas de Microfilamentos/metabolismo , Antagonistas del Receptor de Neuroquinina-1/farmacología , Ratas Sprague-Dawley

RESUMEN

T-cell Immunoglobulin and Mucin Domain 3 (TIM-3) is an immune checkpoint receptor known to regulate T-cell activation and has been targeted for immunotherapy in cancer and other diseases. However, its expression and function in other cell types, such as macrophages, are poorly understood. This study investigated TIM-3 expression in human macrophages polarized to M1 (stimulated with IFN-γ and LPS) and M2 (stimulated with IL-4 and IL-13) phenotypes using an in vitro model. Our results show that M1 macrophages have a lower frequency of TIM-3+ cells compared to M2 macrophages at 48 and 72 hr poststimulation. Additionally, we observed differential levels of soluble ADAM 10, an enzyme responsible for TIM-3 release, in the supernatants of M1 and M2 macrophages at 72 hr. We also found that the TIM-3 intracellular tail might associate with lymphocyte-specific protein 1 (LSP-1), a protein implicated in cell motility and podosome formation. These findings enhance our understanding of TIM-3 function in myeloid cells such as macrophages and may inform the development of immunotherapies with reduced immune-related adverse effects.

Asunto(s)

Receptor 2 Celular del Virus de la Hepatitis A , Macrófagos , Proteínas de Microfilamentos , Humanos , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Proteínas de Microfilamentos/metabolismo

RESUMEN

The master-key TP53 gene is a tumor suppressor that is mutated in more than 50% of human cancers. Some p53 mutants lose their tumor suppressor activity and acquire new oncogenic functions, known as a gain of function (GOF). Recent studies have shown that p53 mutants can exert oncogenic effects through specific miRNAs. We identified the differentially expressed miRNA profiles of the three most frequent p53 mutants (p53R273C, p53R248Q, and p53R175H) after their transfection into the Saos-2 cell line (null p53) as compared with p53WT transfected cells. The associations between these miRNAs and the signaling pathways in which they might participate were identified with miRPath Software V3.0. QRT-PCR was employed to validate the miRNA profiles. We observed that p53 mutants have an overall negative effect on miRNA expression. In the global expression profile of the human miRNome regulated by the p53R273C mutant, 72 miRNAs were underexpressed and 35 overexpressed; in the p53R175H miRNAs profile, our results showed the downregulation of 93 and upregulation of 10 miRNAs; and in the miRNAs expression profile regulated by the p53R248Q mutant, we found 167 decreased and 6 increased miRNAs compared with p53WT. However, we found overexpression of some miRNAs, like miR-182-5p, in association with processes such as cell migration and invasion. In addition, we explored whether the induction of cell migration and invasion by the p53R48Q mutant was dependent on miR-182-5p because we found overexpression of miR-182-5p, which is associated with processes such as cell migration and invasion. Inhibition of mutant p53R248Q and miR-182-5p increased FOXF2-MTSS1 levels and decreased cell migration and invasion. In summary, our results suggest that p53 mutants increase the expression of miR-182-5p, and this miRNA is necessary for the p53R248Q mutant to induce cell migration and invasion in a cancer cell model.

Asunto(s)

Genes p53 , MicroARNs , Humanos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Mutación con Ganancia de Función , Proliferación Celular , MicroARNs/metabolismo , Procesos Neoplásicos , Factores de Transcripción Forkhead/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo

RESUMEN

PURPOSE: Dishevelled-associated activator of morphogenesis 2 (DAAM2) is a formin protein and has a potential role in the tumor metastasis. The prognostic value of DAAM2 in pan-cancer is investigated in this study. METHODS: TCGA and GTEx database were downloaded to perform bioinformatics analysis and ROC curves. Then we explored protein-protein interaction and GO-KEGG enrichment to figure out the protein pathways associated with DAAM2 and studied DAAM2-related immune infiltration and methylation. Fifteen pairs of BRCA clinical samples were enrolled to determine the expression and distribution of DAAM2 in BRCA sections by immunohistochemistry. Finally, BRCA cells were transfected with siRNA targeting DAAM2 and subsequently subject to cell proliferation, migration, and invasion assays. RESULTS: DAAM2 was closely related to the diagnosis and clinical characteristics of lower grade glioma (LGG), liver hepatocellular carcinoma (LIHC), and breast cancer (BRCA). Survival curve analysis demonstrated DAAM2 served as a potential prognostic indicator of LGG and LIHC (P = 0.0029 and P = 0.025, respectively). DAAM2 was mainly participated in signaling pathways mediating cytoskeleton regulation and tumor development. The correlation of DAAM2 with tumor-infiltrating immune cells (TIICs) and methylation levels was conducive to the prediction of novel biomarkers of pan-carcinoma. DAAM2 was highly expressed in BRCA tissues than that in paracancerous tissues. The proliferation, invasion, and migration of BRCA cells were inhibited by DAAM2 siRNA. CONCLUSION: DAAM2 had a specific value in foretelling the prognosis of LGG, LIHC, and BRCA. High expression level of DAAM2 has longer survival rates in LGG and LIHC. The knockdown of DAAM2 retards the proliferation, invasion, and migration of BRCA cells. This study provides a novel sight of DAAM2 into the exploration of a potential biomarker in pan-cancer.

Asunto(s)

Neoplasias de la Mama , Carcinoma Hepatocelular , Glioma , Neoplasias Hepáticas , Humanos , Femenino , Neoplasias de la Mama/genética , Pronóstico , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/genética , Morfogénesis , Proteínas de Microfilamentos , Proteínas de Unión al GTP rho

RESUMEN

Autism spectrum disorder (ASD) is a neurodevelopmental disorder whose hallmarks are social deficits, language impairment, repetitive behaviors, and sensory alterations. It has been reported that patients with ASD show differential activity in cortical regions, for instance, increased neuronal activity in visual processing brain areas and atypical visual perception compared with healthy subjects. The causes of these alterations remain unclear, although many studies demonstrate that ASD has a strong genetic correlation. An example is Phelan-McDermid syndrome, caused by a deletion of the Shank3 gene in one allele of chromosome 22. However, the neuronal consequences relating to the haploinsufficiency of Shank3 in the brain remain unknown. Given that sensory abnormalities are often present along with the core symptoms of ASD, our goal was to study the tuning properties of the primary visual cortex to orientation and direction in awake, head-fixed Shank3+/- mice. We recorded neural activity in vivo in response to visual gratings in the primary visual cortex from a mouse model of ASD (Shank3+/- mice) using the genetically encoded calcium indicator GCaMP6f, imaged with a two-photon microscope through a cranial window. We found that Shank3+/- mice showed a higher proportion of neurons responsive to drifting gratings stimuli than wild-type mice. Shank3+/- mice also show increased responses to some specific stimuli. Furthermore, analyzing the distributions of neurons for the tuning width, we found that Shank3+/- mice have narrower tuning widths, which was corroborated by analyzing the orientation selectivity. Regarding this, Shank3+/- mice have a higher proportion of selective neurons, specifically neurons showing increased selectivity to orientation but not direction. Thus, the haploinsufficiency of Shank3 modified the neuronal response of the primary visual cortex.

Asunto(s)

Trastorno del Espectro Autista , Proteínas de Microfilamentos , Proteínas del Tejido Nervioso , Neuronas , Animales , Ratones , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/patología , Deleción Cromosómica , Haploinsuficiencia , Proteínas de Microfilamentos/genética , Proteínas del Tejido Nervioso/genética

RESUMEN

Allergies have become a rising health problem, where plentiful substances can trigger IgE-mediated allergies in humans. While profilins are considered minor allergens, these ubiquitous proteins are primary molecules involved in cross-reactivity and pollen-food allergy syndrome. Here we report the first crystal structures of murine Fab/IgE, with its chains naturally paired, in complex with the allergen profilin from Hevea brasiliensis (Hev b 8). The crystallographic models revealed that the IgE's six complementarity-determining regions (CDRs) interact with the allergen, comprising a rigid paratope-epitope surface of 926 Å2, which includes an extensive network of interactions. Interestingly, we also observed previously unreported flexibility at Fab/IgE's elbow angle, which did not influence the shape of the paratope. The Fab/IgE exhibits a high affinity for Hev b 8, even when using 1 M NaCl in BLI experiments. Finally, based on the encouraging cross-reactivity assays using two mutants of the maize profilin (Zea m 12), this antibody could be a promising tool in IgE engineering for diagnosis and research applications.

Asunto(s)

Hipersensibilidad a los Alimentos , Profilinas , Alérgenos/química , Alérgenos/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Contráctiles/metabolismo , Humanos , Inmunoglobulina E , Ratones , Proteínas de Microfilamentos/metabolismo , Profilinas/genética , Profilinas/metabolismo

RESUMEN

ER contact sites define the position of endosome bud fission during actin-dependent cargo sorting. Disrupting endosomal actin structures prevents retrograde cargo movement; however, how actin affects ER contact site formation and endosome fission is not known. Here we show that in contrast with the WASH complex, actin, its nucleator ARP2/3, and COR1C form a contained structure at the bud neck that defines the site of bud fission. We found that actin confinement is facilitated by type I coronins. Depletion of type I coronins allows actin to extend along the length of the bud in an ARP2/3-dependent manner. We demonstrate that extension of branched actin prevents ER recruitment and stalls buds before fission. Finally, our structure-function studies show that the COR1C's coiled-coil domain is sufficient to restore actin confinement, ER recruitment, and endosome fission. Together, our data reveal how the dynamics of endosomal actin and activity of actin regulators organize ER-associated bud fission.

Asunto(s)

Actinas/metabolismo , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Proteínas de Microfilamentos/metabolismo , Animales , Células COS , Chlorocebus aethiops , Humanos , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Unión Proteica , Proteínas de Unión a GTP rab7/metabolismo

RESUMEN

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental condition associated with severe social communication, interaction, and sensory processing impairments. Efforts to understand its etiology and pathophysiology are crucial for improving treatment and prevention measures. Preclinical models of ASD are essential for investigating the biological mechanisms and should present translatability potential. We aim to evaluate the consistency of the most commonly used rodent models of ASD in displaying autistic-like behavior through a systematic review and meta-analysis. METHODS: This review will focus on the most frequently used autism models, surveying studies of six genetic (Ube3a, Pten, Nlgn3, Shank3, Mecp2, and Fmr1), three chemically induced (valproic acid (VPA), lipopolysaccharide (LPS), and polyinosinic:polycytidylic acid (poly(I:C))), and one inbred model (BTBR T+ Itpr3tf/J mouse strain). Two independent reviewers will screen the records. Data extraction of behavioral outcomes and risk of bias evaluation will be performed. We will conduct a meta-analysis whenever at least five studies investigate the same model and behavioral outcome. We will also explore the heterogeneity and publication bias. Network meta-analyses are planned to compare different models. DISCUSSION: By shortening the gap between animal behavior and human endophenotypes or specific clinical symptoms, we expect to help researchers on which rodent models are adequate for research of specific behavioral manifestations of autism, which potentially require a combination of them depending on the research interest. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42021226299 .

Asunto(s)

Trastorno del Espectro Autista , Animales , Trastorno del Espectro Autista/genética , Modelos Animales de Enfermedad , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Humanos , Metaanálisis como Asunto , Ratones , Proteínas de Microfilamentos , Proteínas del Tejido Nervioso , Metaanálisis en Red , Roedores , Revisiones Sistemáticas como Asunto

RESUMEN

The Group 3 Medulloblastoma (Grp3-MB) is an aggressive molecular subtype with a high incidence of metastasis and deaths. In this study, were used an RNA sequencing data (RNA-Seq) from a Brazilian cohort of MBs to identify hub genes associated with the metastatic risk. Data validation were performed by using multiple large datasets from MBs (GSE85217, GSE37418, and EGAS00001001953). DESeq2 package in R software was used to identify the differentially expressed genes (DEGs) in our RNA-Seq data. The DEGs data were accessed to construct the modules/graphs of co-expression and to identify hub genes through Cytoscape platform. The coregulated genes were enriched by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and the protein-protein interaction (PPI) network was visualized by Cytoscape. The Kaplan-Meier plotter and ROC curves were used to validate the diagnostic and prognostic values of specific biomarkers identified through this model. We identified that inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) as a downregulated hub gene, with a high diagnostic accuracy to Grp3-MBs and associated with tumor metastasis. In addition, we identified genes significantly correlated with ITPR1 that were associated with metastasis in Grp3-MB (ATP1A2, MTTL7A, and RGL1) and worst overall survival in MBs (ANTXR1 and RGL1). Our findings suggest that the ITPR1 hub gene is potentially involved in the metastatic process for Grp3-MB. Our data also provide evidence of targets that may serve as prognostic predictors and/or regulators for the metastatic process that maybe explored for further research of individualized therapy to Grp3-MBs.

Asunto(s)

Neoplasias Cerebelosas , Meduloblastoma , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Cerebelosas/genética , Biología Computacional , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Inositol , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Meduloblastoma/genética , Proteínas de Microfilamentos/metabolismo , Pronóstico , Receptores de Superficie Celular/genética

RESUMEN

T lymphocyte activation begins with antigen/MHC recognition by the TCR/CD3 complex followed by a costimulatory signal provided by CD28. The search for novel costimulatory molecules has been extensive due to their potential use as immunotherapeutic targets. Although some molecules have been identified, they are unable to provide sustainable signaling to allow for proper T cell activation and proliferation. It has been shown that the Amaranthus leucocarpus lectin (ALL) can be used as an in vitro costimulator of CD4+ lymphocytes in the presence of anti-CD3 mAb; this lectin specifically recognizes O-glycans of the Galß1-3GalNAc-O-Ser/Thr type, including a 70-kDa moesin-like protein that has been suggested as the costimulatory molecule. However, the identity of this molecule has not been confirmed and such costimulation has not been analyzed in CD8+ lymphocytes. We show herein that the expression kinetics of the glycoproteins recognized by ALL (gpALL) is different in CD4+ and CD8+ T cells, unlike moesin expression. Results from IP experiments demonstrate that the previously described 70-kDa moesin-like protein is an O-glycosylated form of moesin (O-moesin) and that in vitro stimulation with anti-CD3 and anti-moesin mAb induces expression of the activation molecules CD69 and CD25, proliferation and IL-2 production as efficiently as cells costimulated with ALL or anti-CD28. Overall, our results demonstrate that O-moesin is expressed in CD4+ and CD8+ T lymphocytes and that moesin provides a new costimulatory activation signal in both T cell subsets.

Asunto(s)

Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Glicoproteínas/metabolismo , Activación de Linfocitos/efectos de los fármacos , Lectinas de Plantas/farmacología , Procesamiento Proteico-Postraduccional , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular/efectos de los fármacos , Glicoproteínas/farmacología , Glicosilación , Interleucina-2/metabolismo , Cinética , Masculino , Ratones Endogámicos BALB C , Proteínas de Microfilamentos/metabolismo , Transducción de Señal

RESUMEN

OBJECTIVE: Polycystic ovary syndrome can be divided into different subtypes, including insulin resistance and hyperandrogenism. The aim of this study was to investigate the relationship between serum asprosin levels and polycystic ovary syndrome subtypes. METHODS: A total of 93 women with polycystic ovary syndrome and 77 healthy women as controls were selected for this study. The clinical and laboratory data were compared between the Polycystic ovary syndrome group and the control group. The Polycystic ovary syndrome group was further divided into subgroups: (1) women with or without hyperandrogenism (polycystic ovary syndrome hyperandrogenism and Polycystic ovary syndrome none-hyperandrogenism, respectively) and (2) women with or without insulin resistance (polycystic ovary syndrome insulin resistance and Polycystic ovary syndrome none-insulin resistance, respectively). Serum asprosin was measured by using enenzyme-linked immunosorbent assay. RESULTS: Serum asprosin levels showed no significant difference between the polycystic ovary syndrome and control groups. However, it was significantly lower in the Polycystic ovary syndrome HA and insulin resistance groups compared with the respective Polycystic ovary syndrome none-hyperandrogenism and none-insulin resistance groups (p<0.05). In the Polycystic ovary syndrome group, serum asprosin was negatively correlated with body mass index, luteinizing hormone, testosterone, basal antral follicles, fasting insulin, homeostatic model assessment of insulin resistance, and triglycerides. After adjusting for body mass index, the correlations were not significant, and asprosin was only positively correlated with prolactin (prolactin; r=0.426, p<0.001). CONCLUSION: Our study shows that women with polycystic ovary syndrome hyperandrogenism or insulin resistance exhibit significantly lower serum asprosin levels compared with controls, and the lower asprosin level directly correlated with prolactin level.

Asunto(s)

Hiperandrogenismo , Resistencia a la Insulina , Hormonas Peptídicas , Síndrome del Ovario Poliquístico , Índice de Masa Corporal , Estudios Transversales , Femenino , Fibrilina-1 , Humanos , Insulina , Proteínas de Microfilamentos , Fragmentos de Péptidos , Síndrome del Ovario Poliquístico/complicaciones , Testosterona

Asunto(s)

Oftalmopatías/diagnóstico , Enfermedades Genéticas Congénitas/diagnóstico , Proteínas de Microfilamentos/genética , Adulto , Argentina , Niño , Oftalmopatías/genética , Oftalmopatías/inmunología , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/inmunología , Humanos , Leucopenia/diagnóstico , Leucopenia/genética , Leucopenia/inmunología , Masculino , Hermanos , Adulto Joven

RESUMEN

SLK (STE20-like kinase) and STK10 (serine/threonine kinase 10) are closely related kinases whose enzymatic activity is linked to the regulation of ezrin, radixin, and moesin function and to the regulation of lymphocyte migration and the cell cycle. We identified a series of 3-anilino-4-arylmaleimides as dual inhibitors of SLK and STK10 with good kinome-wide selectivity. Optimization of this series led to multiple SLK/STK10 inhibitors with nanomolar potency. Crystal structures of exemplar inhibitors bound to SLK and STK10 demonstrated the binding mode of the inhibitors and rationalized their selectivity. Cellular target engagement assays demonstrated the binding of the inhibitors to SLK and STK10 in cells. Further selectivity analyses, including analysis of activity of the reported inhibitors against off-targets in cells, identified compound 31 as the most potent and selective inhibitor of SLK and STK10 yet reported.

Asunto(s)

Compuestos de Anilina/farmacología , Maleimidas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Compuestos de Anilina/química , Compuestos de Anilina/metabolismo , Sitios de Unión , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células HEK293 , Humanos , Maleimidas/química , Maleimidas/metabolismo , Proteínas de Microfilamentos/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Fosforilación/efectos de los fármacos , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Relación Estructura-Actividad

RESUMEN

Inhibition of phosphodiesterase 4 (PDE4) is a promising pharmacological strategy for the treatment of cerebral ischemic conditions. To increase the relevance and increase the translational value of preclinical studies, it is important to conduct experiments using different animal species and strains, different animal models, and to evaluate long-term functional outcomes after cerebral ischemia. In the present study, the effects of the selective PDE4 inhibitor roflumilast were evaluated in vivo and in vitro. Balb/c mice were subjected to bilateral common carotid artery occlusion (BCCAO) and tested during 21 days in multiple behavioral tasks to investigate the long-term effects of roflumilast on functional recovery. The effects of roflumilast were also investigated on hippocampal cell loss, white matter injury, and expression of neuroinflammatory markers. Roflumilast prevented cognitive and emotional deficits induced by BCCAO in mice. Roflumilast also prevented neurodegeneration and reduced the white matter damage in the brain of ischemic animals. Besides, roflumilast decreased Iba-1 (microglia marker) levels and increased Arginase-1 (Arg-1; microglia M2 phenotype marker) levels in the hippocampus of these mice. Likewise, roflumilast suppressed inducible nitric oxide synthase (microglia M1 phenotype marker) expression and increased Arg-1 levels in a primary mouse microglia culture. These findings support evidence that PDE4 inhibition by roflumilast might be beneficial in cerebral ischemic conditions. The neuroprotective effects of roflumilast appear to be mediated by a decrease in neuroinflammation.

Asunto(s)

Aminopiridinas/farmacología , Arginasa/metabolismo , Benzamidas/farmacología , Isquemia Encefálica , Proteínas de Unión al Calcio/metabolismo , Disfunción Cognitiva , Proteínas de Microfilamentos/metabolismo , Enfermedades Neuroinflamatorias , Animales , Conducta Animal/efectos de los fármacos , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/inmunología , Isquemia Encefálica/psicología , Disfunción Cognitiva/tratamiento farmacológico , Disfunción Cognitiva/metabolismo , Ciclopropanos/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/patología , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Fármacos Neuroprotectores/farmacología , Inhibidores de Fosfodiesterasa 4/farmacología , Recuperación de la Función/efectos de los fármacos , Resultado del Tratamiento , Sustancia Blanca/efectos de los fármacos , Sustancia Blanca/metabolismo

RESUMEN

Methotrexate (MTX), an antifolate drug, is widely used in chemotherapeutic protocols for metastatic and primary brain tumors and some autoimmune diseases. Its efficacy for brain tumors is limited by the high incidence of central nervous system (CNS) complications. This investigation aimed to observe the morphological effects, including astroglial and microglial responses, following systemic short-term MTX administration in adult rats. Male Wistar rats received 5 or 10 mg/kg/day of MTX by intraperitoneal route for 4 consecutive days (respectively, MTX5 and MTX10 groups) or the same volume of 0.9% saline solution (control group). On the 5th day, brain samples were collected for hematoxylin-eosin and luxol fast blue staining techniques, as well as for immunohistochemical staining for glial fibrillary acidic protein (GFAP) expression in astrocytes and Iba1 (ionized calcium binding adaptor molecule 1) for microglia in the frontal cortex, hippocampus, hypothalamus and molecular/granular layers of the cerebellum. Morphometric analyses were performed using Image Pro-Plus software. Brain levels of the proinflammatory cytokines TNF-α and IL-1ß were determined by ELISA. No signs of neuronal loss or demyelination were observed in all groups. Increased GFAP and Iba1 expression was found in all areas from the MTX groups, although it was slightly higher in the MTX10 group compared to the MTX5. Both TNF-α and IL-1ß levels were decreased in the MTX5 group compared to controls. In the MTX10 group, TNF-α decreased, although IL-1ß was increased relative to controls. MTX administration induced microglial reaction and astrogliosis in several CNS areas. In the MTX5 group, it apparently occurred in the presence of decreased proinflammatory cytokines.

Asunto(s)

Antimetabolitos Antineoplásicos/administración & dosificación , Astrocitos/efectos de los fármacos , Proteínas de Unión al Calcio/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/fisiopatología , Metotrexato/administración & dosificación , Proteínas de Microfilamentos/metabolismo , Microglía/efectos de los fármacos , Animales , Astrocitos/metabolismo , Astrocitos/patología , Relación Dosis-Respuesta a Droga , Gliosis/inducido químicamente , Gliosis/patología , Masculino , Microglía/metabolismo , Microglía/patología , Ratas , Ratas Wistar

RESUMEN

Ezrin, radixin, and moesin (ERM) family proteins regulate cytoskeletal responses by tethering the plasma membrane to the underlying actin cortex. Mutations in ERM proteins lead to severe combined immunodeficiency, but the function of these proteins in T cells remains poorly defined. Using mice in which T cells lack all ERM proteins, we demonstrate a selective role for these proteins in facilitating S1P-dependent egress from lymphoid organs. ERM-deficient T cells display defective S1P-induced migration in vitro, despite normal responses to standard protein chemokines. Analysis of these defects revealed that S1P promotes a fundamentally different mode of migration than chemokines, characterized by intracellular pressurization and bleb-based motility. ERM proteins facilitate this process, controlling directional migration by limiting blebbing to the leading edge. We propose that the distinct modes of motility induced by S1P and chemokines are specialized to allow T cell migration across lymphatic barriers and through tissue stroma, respectively.

Asunto(s)

Movimiento Celular , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/fisiología , Citoesqueleto/fisiología , Linfocitos/metabolismo , Lisofosfolípidos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Esfingosina/análogos & derivados , Animales , Membrana Celular , Proteínas del Citoesqueleto/genética , Femenino , Linfocitos/citología , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Fosforilación , Esfingosina/metabolismo

RESUMEN

Maternal deprivation (MD) is known to be related to long-term changes that could influence the onset of psychiatric disorders. Studies have demonstrated that early life stress makes the cells in the brain more susceptible to subsequent stressors. To test it, we used an animal model of MD conducted from postnatal day (PND) 1 to 10. Deprived and non-deprived rats (control) were randomized to receive or not lipopolysaccharide (LPS) at 5 mg/kg on PND 50. The behavior and glial cells activation were evaluated in all groups from 51 to 53 PND. There was an increase in the immobility time in the MD and MD+LPS groups. The spontaneous locomotor activity was not changed between groups. We found elevated ionized calcium-binding adapter molecule 1 (Iba-1)-positive cells levels in the control+LPS and MD+LPS groups. In the MD+LPS group, it was found an increase in Iba-positive cells compared to the MD+sal group. The glial fibrillary acidic protein (GFAP)-positive cells were also increased in the MD+LPS, compared to control+sal, control+LPS, and MD+sal groups. Immune challenge by LPS in late adolescence, which was subjected to MD, did not influence the depressive-like behavior but exerted a pronounced effect in the microglial activation and astrocyte atrophy.

Asunto(s)

Conducta Animal , Inmunidad , Privación Materna , Neuroglía , Estrés Psicológico , Animales , Femenino , Ratas , Astrocitos/patología , Proteínas de Unión al Calcio/metabolismo , Depresión , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/biosíntesis , Inmunidad/fisiología , Lipopolisacáridos , Activación de Macrófagos , Proteínas de Microfilamentos/metabolismo , Actividad Motora , Neuroglía/inmunología , Ratas Wistar , Estrés Psicológico/inmunología , Natación/psicología

RESUMEN

The physiological role of the shorter isoform of with no lysine kinase (WNK)1 that is exclusively expressed in the kidney (KS-WNK1), with particular abundance in the distal convoluted tubule, remains elusive. KS-WNK1, despite lacking the kinase domain, is nevertheless capable of stimulating the NaCl cotransporter, apparently through activation of WNK4. It has recently been shown that a less severe form of familial hyperkalemic hypertension featuring only hyperkalemia is caused by missense mutations in the WNK1 acidic domain that preferentially affect cullin 3 (CUL3)-Kelch-like protein 3 (KLHL3) E3-induced degradation of KS-WNK1 rather than that of full-length WNK1. Here, we show that full-length WNK1 is indeed less impacted by the CUL3-KLHL3 E3 ligase complex compared with KS-WNK1. We demonstrated that the unique 30-amino acid NH2-terminal fragment of KS-WNK1 is essential for its activating effect on the NaCl cotransporter and recognition by KLHL3. We identified specific amino acid residues in this region critical for the functional effect of KS-WNK1 and KLHL3 sensitivity. To further explore this, we generated KLHL3-R528H knockin mice that mimic human mutations causing familial hyperkalemic hypertension. These mice revealed that the KLHL3 mutation specifically increased expression of KS-WNK1 in the kidney. We also observed that in wild-type mice, the expression of KS-WNK1 was only detectable after exposure to a low-K+ diet. These findings provide new insights into the regulation and function of KS-WNK1 by the CUL3-KLHL3 complex in the distal convoluted tubule and indicate that this pathway is regulated by dietary K+ levels.NEW & NOTEWORTHY In this work, we demonstrated that the kidney-specific isoform of with no lysine kinase 1 (KS-WNK1) in the kidney is modulated by dietary K+ and activity of the ubiquitin ligase protein Kelch-like protein 3. We analyzed the role of different amino acid residues of KS-WNK1 in its activity against the NaCl cotransporter and sensitivity to Kelch-like protein 3.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Riñón/enzimología , Proteínas de Microfilamentos/metabolismo , Potasio en la Dieta/metabolismo , Seudohipoaldosteronismo/enzimología , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Cullin/metabolismo , Estabilidad de Enzimas , Femenino , Riñón/fisiopatología , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Mutación , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Seudohipoaldosteronismo/genética , Seudohipoaldosteronismo/fisiopatología , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/deficiencia , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Xenopus laevis

RESUMEN

The 106-residue protein Q4DY78 (UniProt accession number) from Trypanosoma cruzi is highly conserved in the related kinetoplastid pathogens Trypanosoma brucei and Leishmania major. Given the essentiality of its orthologue in T. brucei, the high sequence conservation with other trypanosomatid proteins, and the low sequence similarity with mammalian proteins, Q4DY78 is an attractive protein for structural characterization. Here, we solved the structure of Q4DY78 by solution NMR and evaluated its backbone dynamics. Q4DY78 is composed of five α -helices and a small, two-stranded antiparallel ß-sheet. The backbone RMSD is 0.22 ± 0.05 Å for the representative ensemble of the 20 lowest-energy structures. Q4DY78 is overall rigid, except for N-terminal residues (V8 to I10), residues at loop 4 (K57 to G65) and residues at the C-terminus (F89 to F112). Q4DY78 has a short motif FPCAP that could potentially mediate interactions with the host cytoskeleton via interaction with EVH1 (Drosophila Enabled (Ena)/Vasodilator-stimulated phosphoprotein (VASP) homology 1) domains. Albeit Q4DY78 lacks calcium-binding motifs, its fold resembles that of eukaryotic calcium-binding proteins such as calcitracin, calmodulin, and polcacin Bet V4. We characterized this novel protein with a calcium binding fold without the capacity to bind calcium.

Asunto(s)

Proteínas Protozoarias/química , Trypanosoma cruzi/química , Secuencia de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Moléculas de Adhesión Celular/química , Dicroismo Circular , Secuencia Conservada , Motivos EF Hand , Proteínas de Microfilamentos/química , Modelos Moleculares , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Fosfoproteínas/química , Conformación Proteica en Hélice alfa , Estructura Secundaria de Proteína , Proteínas Protozoarias/metabolismo