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Cell cycle checkpoints, activated by stressful events, halt the cell cycle progression, and prevent the transmission of damaged DNA. These checkpoints prompt cell repair but also trigger cell death if damage persists. Decision-making between these responses is multifactorial and context-dependent, with the tumor suppressor p53 playing a central role. In many tumor cells, p53 alterations lead to G1/S checkpoint loss and the weakening of the G2 checkpoint, rendering cell viability dependent on the strength of the latter through mechanisms not fully characterized. Cells with a strong pro-survival drive can evade cell death despite substantial DNA lesions. Deciphering the integration of survival pathways with p53-dependent and -independent mechanisms governing the G2/M transition is crucial for understanding G2 arrest functionality and predicting tumor cell response to chemotherapy. The serine/threonine kinase GRK2 emerges as a signaling node in cell cycle modulation. In cycling cells, but not in G2 checkpoint-arrested cells, GRK2 protein levels decline during G2/M transition through a process triggered by CDK2-dependent phosphorylation of GRK2 at the S670 residue and Mdm2 ubiquitination. We report now that this downmodulation in G2 prevents the unscheduled activation of the PI3K/AKT pathway, allowing cells to progress into mitosis. Conversely, higher GRK2 levels lead to tyrosine phosphorylation by the kinase c-Abl, promoting the direct association of GRK2 with the p85 regulatory subunit of PI3K and AKT activation in a GRK2 catalytic-independent manner. Hyperactivation of AKT is conditioned by p53's scaffolding function, triggering FOXO3a phosphorylation, impaired Cyclin B1 accumulation, and CDK1 activation, causing a G2/M transition delay. Upon G2 checkpoint activation, GRK2 potentiates early arrest independently of p53 through AKT activation. However, its ability to overcome the G2 checkpoint in viable conditions depends on p53. Our results suggest that integrating the GRK2/PI3K/AKT axis with non-canonical functions of p53 might confer a survival advantage to tumor cells.
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Satisfaction with an intervention influences the uptake of behavior changes and the long-term efficacy of the intervention. Therefore, it is crucial to assess satisfaction by participant profile when creating and adapting behavior interventions for minoritized populations. Qualitative and quantitative data on participant trauma symptom severity and intervention satisfaction were collected through self-report surveys from 54 women. The sample was 59.3% Hispanic, with an average age of 33.21 (SD = 10.42), who were in residential treatment for substance use disorders (SUDs) and participated in a 12-session mindfulness-based intervention. Qualitative responses were coded using thematic analysis, and an integrative mixed-methods approach was used to compare qualitative theme frequency between high-trauma (N = 28) and low-trauma (N = 26) groups at session 2 and session 11. High- and low-trauma groups were determined by interquartile ranges (bottom 25% = low; top 75% = high). In session 2, the low-trauma group reported significantly higher satisfaction (M = 4.20, SD = 0.55) than the high-trauma group (M = 3.77, SD = 0.89); t(43) = 1.90, p = 0.03. In session 11, there was no significant difference between groups. The mixed-methods analysis revealed that "trouble focusing" appeared more frequently in the high-trauma group than in the low-trauma group during session 2, but the theme was not present in either group at session 11, suggesting that this might pose an initial barrier for individuals with high trauma but subsides as the intervention progresses. This speaks to the importance of retention strategies tailored for participants with SUDs and high trauma while they adjust to the intervention. Assessing initial challenges with satisfaction may help facilitators intervene to increase participant satisfaction.
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BACKGROUND: Demyelinating diseases (DD) are a group of chronic neurological diseases associated with loss and injury of brain or spinal cord regions. These conditions could trigger impairment of neurological functions and disability from earlier stages of life. Epidemiological data on DD remains insufficient for decision-making in the Mexican healthcare system. This study aims to describe the epidemiology of DD based on data from Mexico's National Registry of Demyelinating Diseases. METHODS: A cross-sectional, registry-based, observational study was performed. We analyzed 408 reports of multiple sclerosis (331, 81%), neuromyelitis optica spectrum disorder (67, 16%), chronic recurrent inflammatory optic neuropathy (5, 1%), clinically isolated syndrome (4, 0.9%), and autoimmune encephalitis (1, 0.2%) reported across 2021. RESULTS: The time from first symptoms to diagnosis of any DD was about 3 years. A treatment failure history was detected in 40% of patients. It was estimated that NMOSD accounts for 20% of all disorders. There was evidence that the use of brand-name and generic IFN drug products lead to increased therapeutic failures. CONCLUSION: Our research team suggests reinforcing educational programs and activities based on diagnosis and clinical management improvement to first-contact physicians and specialty doctors and promoting awareness in the whole population.
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Esclerose Múltipla , Neuromielite Óptica , Humanos , México/epidemiologia , Estudos Transversais , Neuromielite Óptica/complicações , Esclerose Múltipla/complicações , Inflamação/complicaçõesRESUMO
BACKGROUND: Neuromyelitis optica spectrum disorders (NMOSD) is an increasing diagnostic and therapeutic challenge in Latin America (LATAM). Despite the heterogeneity of this population, ethnic and socioeconomic commonalities exist, and epidemiologic studies from the region have had a limited geographic and population outreach. Identification of some aspects from the entire region are lacking. OBJECTIVES: To determine ethnic, clinical characteristics, and utilization of diagnostic tools and types of therapy for patients with NMOSD in the entire Latin American region. METHODS: The Latin American Committee for Treatment and Research in MS (LACTRIMS) created an exploratory investigational survey addressed by Invitation to NMOSD Latin American experts identified through diverse sources. Data input closed after 30 days from the initial invitation. The questionnaire allowed use of absolute numbers or percentages. Multiple option responses covering 25 themes included definition of type of practice; number of NMOSD cases; ethnicity; utilization of the 2015 International Panel criteria for the diagnosis of Neuromyelitis optica (IPDN); clinical phenotypes; methodology utilized for determination of anti-Aquaporin-4 (anti- AQP4) antibodies serological testing, and if this was performed locally or processed abroad; treatment of relapses, and long-term management were surveyed. RESULTS: We identified 62 investigators from 21 countries reporting information from 2154 patients (utilizing the IPDN criteria in 93.9% of cases), which were categorized in two geographical regions: North-Central, including the Caribbean (NCC), and South America (SA). Ethnic identification disclosed Mestizos 61.4% as the main group. The most common presenting symptoms were concomitant presence of optic neuritis and transverse myelitis in 31.8% (p=0.95); only optic neuritis in 31.4% (more common in SA), p<0.001); involvement of the area postrema occurred in 21.5% and brain stem in 8.3%, both were more frequent in the South American cases (p<0.001). Anti-AQP4 antibodies were positive in 63.9% and anti-Myelin Oligodendrocyte Glycoprotein (MOG) antibodies in 4.8% of total cases. The specific laboratorial method employed was not known by 23.8% of the investigators. Acute relapses were identified in 81.6% of cases, and were treated in 93.9% of them with intravenous steroids (IVS); 62.1% with plasma exchange (PE), and 40.9% with intravenous immunoglobulin-G (IVIG). Therapy was escalated in some cases due to suboptimal initial response. Respondents favored Rituximab as long-term therapy (86.3%), whereas azathioprine was also utilized on 81.8% of the cases, either agent used indistinctly by the investigators according to treatment accessibility or clinical judgement. There were no differences among the geographic regions. CONCLUSIONS: This is the first study including all countries of LATAM and the largest cohort reported from a multinational specific world area. Ethnic distributions and phenotypic features of the disease in the region, challenges in access to diagnostic tools and therapy were identified. The Latin American neurological community should play a determinant role encouraging and advising local institutions and health officials in the availability of more sensitive and modern diagnostic methodology, in facilitating the the access to licensed medications for NMOSD, and addressing concerns on education, diagnosis and management of the disease in the community.
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Neuromielite Óptica , Aquaporina 4 , Autoanticorpos , Humanos , América Latina/epidemiologia , Glicoproteína Mielina-Oligodendrócito , Recidiva Local de Neoplasia , Neuromielite Óptica/epidemiologia , Neuromielite Óptica/terapiaRESUMO
The timing of centrosome separation and the distance moved apart influence the formation of the bipolar spindle, affecting chromosome stability. Epidermal growth factor receptor (EGFR) signaling induces early centrosome separation through downstream G protein-coupled receptor kinase GRK2, which phosphorylates the Hippo pathway component MST2 (Mammalian STE20-like protein kinase 2), in turn allowing NIMA kinase Nek2A activation for centrosomal linker disassembly. However, the mechanisms that counterbalance centrosome disjunction and separation remain poorly understood. We unveil that timely degradation of GRK2 by the E3 ligase Mdm2 limits centrosome separation in the G2. Both knockout expression and catalytic inhibition of Mdm2 result in GRK2 accumulation and enhanced centrosome separation before mitosis onset. Phosphorylation of GRK2 on residue S670 enables a complex pattern of non-K48-linked polyubiquitin chains assembled by Mdm2, which correlate with kinase protein degradation. Remarkably, GRK2-S670A protein fails to phosphorylate MST2 despite overcoming Mdm2-dependent degradation, which results in defective centrosome separation, shorter spindles, and abnormal chromosome congression. Conversely, extra levels of wild-type kinase in the G2 cause increased inter-centrosome distances with longer spindles, also converging in congression issues. Our findings show that the signals enabling activity of the GRK2/MST2/Nek2A axis for separation also switches on Mdm2 degradation of GRK2 to ensure accurate centrosome dynamics and proper mitotic spindle functionality.
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Centrossomo/metabolismo , Segregação de Cromossomos , Regulação para Baixo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Animais , Fase G2 , Células HEK293 , Células HeLa , Humanos , Camundongos Knockout , Fosforilação , Fosfosserina/metabolismo , Proteólise , Fuso Acromático/metabolismo , UbiquitinaçãoRESUMO
Adaptation to hypoxia is a common feature in solid tumors orchestrated by oxygen-dependent and independent upregulation of the hypoxia-inducible factor-1α (HIF-1α). We unveiled that G protein-coupled receptor kinase (GRK2), known to be overexpressed in certain tumors, fosters this hypoxic pathway via phosphorylation of the mRNA-binding protein HuR, a central HIF-1α modulator. GRK2-mediated HuR phosphorylation increases the total levels and cytoplasmic shuttling of HuR in response to hypoxia, and GRK2-phosphodefective HuR mutants show defective cytosolic accumulation and lower binding to HIF-1α mRNA in hypoxic Hela cells. Interestingly, enhanced GRK2 and HuR expression correlate in luminal breast cancer patients. GRK2 also promotes the HuR/HIF-1α axis and VEGF-C accumulation in normoxic MCF7 breast luminal cancer cells and is required for the induction of HuR/HIF1-α in response to adrenergic stress. Our results point to a relevant role of the GRK2/HuR/HIF-1α module in the adaptation of malignant cells to tumor microenvironment-related stresses.
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OBJECTIVE: Optic Neuritis (ON) might unfold either as a single intracranial neuritis or as multiple sclerosis, a widespread demyelinating disorder. Different herpes viruses have been proposed as potential participants in the etiology of multiple sclerosis (MS). To analyze the potential presence of herpes viruses in blood and subarachnoid area at the time of ON and contrast the findings according to long-term evolution either as intracranial neuritis or as progression to multiple sclerosis. PATIENTS AND METHODS: In a prospective investigation we searched the presence of DNA from 5 herpes viruses (HSV-1, HSV-2, VZV, EBV and HHV6) in CSF and blood lymphocytes from 54 patients with ON, patients were followed 62⯱â¯3 months; those who developed MS were separated from those with ephemeral ON. Long-term prognosis of ON was related to DNA findings. RESULTS: As compared with controls, DNA from HSV-1 was significantly more frequent in CSF and blood from cases with ON; VZV and HSV-2 were found only in CSF; EBV was found only in blood samples (pâ¯<â¯0.006). CONCLUSIONS: Our results point out the potential participation of HSV, VZV and EBV in ON; suggesting the intervention of various herpes viruses as triggering agents of autoimmunity. However, the number of positive cases was minor than negative cases. Also, our results suggest that the etiological mechanisms in ON could be similar to those of neuritis of the facial nerve (Bell's palsy).
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DNA Viral/líquido cefalorraquidiano , Infecções por Herpesviridae/epidemiologia , Herpesviridae/genética , Neurite Óptica/virologia , Adulto , Paralisia de Bell/virologia , DNA Viral/sangue , Infecções por Vírus Epstein-Barr/epidemiologia , Feminino , Herpes Simples/epidemiologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Herpesvirus Humano 3/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 6/genética , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/fisiopatologia , Neurite Óptica/epidemiologia , Neurite Óptica/metabolismo , Neurite Óptica/fisiopatologia , Prognóstico , Infecções por Roseolovirus/epidemiologia , Infecção pelo Vírus da Varicela-Zoster/epidemiologia , Adulto JovemRESUMO
BACKGROUND: Retinal vasculopathy with cerebral leukodystrophy (RVCL) is an adult-onset, autosomal dominant disease involving microvessels of the brain and eye resulting in central nervous system degeneration with visual disturbances, stroke, motor impairment, and cognitive decline. Frameshift mutations at the C-terminus of TREX1 gene are the molecular cause of this disorder. OBJECTIVES: The objective of this study is to present the different clinical manifestations of RVCL in three-related patients and to investigate the presence of TREX1 mutation in the extended genealogy. METHODS: Multidisciplinary testing was performed in three related patients. Based on their family history, the study was extended to 34 relatives from the same small community. Neurological evaluation, sequencing of TREX1, and presymptomatic diagnosis were offered to all participants. RESULTS: The patients exhibited the heterozygous TREX1 mutation p.V235Gfs*6, but with phenotypic variability. In addition, 15 relatives were identified as pre-manifest mutation carriers. The remaining participants did not carry the mutation. CONCLUSIONS: This is the figrst report of a large Mexican genealogy with RVCL, where the same TREX1 mutation causes a variation in organ involvement and clinical progression. The early identification and follow-up of individuals at risk may help provide insights into the basis for this variability in presentation.
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Variação Biológica da População , Exodesoxirribonucleases/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/fisiopatologia , Fosfoproteínas/genética , Doenças Retinianas/fisiopatologia , Doenças Vasculares/fisiopatologia , Feminino , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/diagnóstico , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Heterozigoto , Humanos , Masculino , México , Pessoa de Meia-Idade , Mutação , Doenças Retinianas/diagnóstico , Doenças Retinianas/genética , Doenças Vasculares/diagnóstico , Doenças Vasculares/genéticaRESUMO
INTRODUCTION: Multiple Sclerosis (MS) is a chronic, inflammatory, neurodegenerative demyelinating disease of the central nervous system (CNS). Unfortunately, MS causes important disability in young adults and its prevalence is increasing. While the etiology of MS etiology is not completely understood, it seems to be a multifactorial entity that is influenced by both genetic and epigenetic modifications. Epigenetic mechanisms add or remove different chemical groups for the activation or inhibition of gene expression to block the production of proinflammatory proteins. It is truly important to identify the factors that can trigger epigenetic changes in MS to complement the therapeutic approach, prevent disability and improve patients quality of life. Here, we have conducted a review of external factors that influence in MS and their epigenetic mechanisms. For example, hypomethylation can promote changes in the myelin and subsequent autoimmune reactions. Therapeutic tools can be used, including the histone deacetylase inhibitor Trichostatin A, which ameliorates demyelinating diseases in rodents. However, drugs are not only the therapeutic option: recent studies have also evaluated the therapeutic potential of several bioactive dietary components in neurodegeneration and axonal dysfunction. Numerous food-derived molecules exert important metabolic actions. These molecules include plant polyphenols such as catechins and isoflavones, Ω-3 and Ω-6 polyunsaturated fatty acids, short-chain fatty acids, sulfur-containing compounds such as dally sulfide and other compounds. Antioxidant and anti-inflammatory components in the diet involve transcription factors as well. However, many external factors have shown to influence MS, although no specific epigenetic mechanisms are known. CONCLUSION: In this review, we gather both established and new evidences about the genetic, epigenetic and environmental factors influencing MS and the dietary components that could modulate MS relapse and progression.
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Antioxidantes/administração & dosagem , Epigênese Genética/fisiologia , Esclerose Múltipla/dietoterapia , Esclerose Múltipla/metabolismo , Polifenóis/administração & dosagem , Animais , Antioxidantes/metabolismo , Metilação de DNA/fisiologia , Ácidos Graxos Ômega-3/administração & dosagem , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/administração & dosagem , Ácidos Graxos Ômega-6/metabolismo , Humanos , Esclerose Múltipla/genética , Polifenóis/metabolismoRESUMO
Increasing evidences point to G protein-coupled receptor kinases (GRKs), a subfamily of protein kinase A/G/C-like kinases, as relevant players in cancer progression, in a cell-type and tumor-specific way. Alterations in the expression and/or activity of particular GRKs have been identified in several types of tumors, and demonstrated to modulate the proliferation, survival or invasive properties of tumor cells by acting as integrating signaling nodes. GRKs are able to regulate the functionality of both G protein-coupled receptors (GPCR) and growth factor receptors and to directly control cytosolic, cytoskeletal or nuclear signaling components of pathways relevant for these processes. Furthermore, many chemokines as well as angiogenic and inflammatory factors present in the tumor microenvironment act through GPCR and other GRK-modulated signaling modules. Changes in the dosage of certain GRKs in the tumor stroma can alter tumor angiogenesis and the homing of immune cells, thus putting forward these kinases as potentially relevant modulators of the carcinoma-fibroblast-endothelial-immune cell network fostering tumor development and dissemination. A better understanding of the alterations in different GRK isoforms taking place during cancer development and metastasis in specific tumors and cell types and of its impact in signaling pathways would help to design novel therapeutic strategies.
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Quinases de Receptores Acoplados a Proteína G/fisiologia , Neoplasias/patologia , Animais , Carcinogênese/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Microambiente TumoralRESUMO
Malignant features-such as sustained proliferation, refractoriness to growth suppressors, resistance to cell death or aberrant motility, and metastasis-can be triggered by a variety of mutations and signaling adaptations. Signaling nodes can act as cancer-associated factors by cooperating with oncogene-governed pathways or participating in compensatory transduction networks to strengthen tumor properties. G-protein-coupled receptor kinase 2 (GRK2) is arising as one of such nodes. Via its complex network of connections with other cellular proteins, GRK2 contributes to the modulation of basic cellular functions-such as cell proliferation, survival, or motility-and is involved in metabolic homeostasis, inflammation, or angiogenic processes. Moreover, altered GRK2 levels are starting to be reported in different tumoral contexts and shown to promote breast tumorigenesis or to trigger the tumoral angiogenic switch. The ability to modulate several of the hallmarks of cancer puts forward GRK2 as an oncomodifier, able to modulate carcinogenesis in a cell-type specific way.
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Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Neoplasias/enzimologia , Animais , Proliferação de Células , Humanos , Redes e Vias Metabólicas , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Transdução de Sinais , Microambiente TumoralRESUMO
In addition to oncogenic drivers, signaling nodes can critically modulate cancer-related cellular networks to strength tumor hallmarks. We identify G-protein-coupled receptor kinase 2 (GRK2) as a relevant player in breast cancer. GRK2 is up-regulated in breast cancer cell lines, in spontaneous tumors in mice, and in a proportion of invasive ductal carcinoma patients. Increased GRK2 functionality promotes the phosphorylation and activation of the Histone Deacetylase 6 (HDAC6) leading to de-acetylation of the Prolyl Isomerase Pin1, a central modulator of tumor progression, thereby enhancing its stability and functional interaction with key mitotic regulators. Interestingly, a correlation between GRK2 expression and Pin1 levels and de-acetylation status is detected in breast cancer patients. Activation of the HDAC6-Pin1 axis underlies the positive effects of GRK2 on promoting growth factor signaling, cellular proliferation and anchorage-independent growth in both luminal and basal breast cancer cells. Enhanced GRK2 levels promote tumor growth in mice, whereas GRK2 down-modulation sensitizes cells to therapeutic drugs and abrogates tumor formation. Our data suggest that GRK2 acts as an important onco-modulator by strengthening the functionality of key players in breast tumorigenesis such as HDAC6 and Pin1.
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Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Histona Desacetilases/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Transdução de Sinais , Acetilação , Animais , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Modelos Animais de Doenças , Feminino , Quinase 2 de Receptor Acoplado a Proteína G/genética , Expressão Gênica , Desacetilase 6 de Histona , Histona Desacetilases/genética , Humanos , Camundongos Transgênicos , Modelos Biológicos , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/genética , Carga TumoralRESUMO
BACKGROUND: Inhaled allergen challenge is a validated disease model of allergic asthma offering useful pharmacodynamic assessment of pharmacotherapeutic effects in a limited number of subjects. OBJECTIVES: To evaluate whether an RNA signature can be identified from induced sputum following an inhaled allergen challenge, whether a RNA signature could be modulated by limited doses of inhaled fluticasone, and whether these gene expression profiles would correlate with the clinical endpoints measured in this study. METHODS: Thirteen non-smoking, allergic subjects with mild-to-moderate asthma participated in a randomised, placebo-controlled, 2-period cross-over study following a single-blind placebo run-in period. Each period consisted of three consecutive days, separated by a wash-out period of at least 3 weeks. Subjects randomly received inhaled fluticasone ((FP) MDI; 500 mcg BID×5 doses in total) or placebo. On day 2, house dust mite extract was inhaled and airway response was measured by FEV1 at predefined time points until 7 h post-allergen. Sputum was induced by NaCl 4.5%, processed and analysed at 24 h pre-allergen and 7 and 24 h post-allergen. RNA was isolated from eligible sputum cell pellets (<80% squamous of 500 cells), amplified according to NuGEN technology, and profiled on Affymetrix arrays. Gene expression changes from baseline and fluticasone treatment effects were evaluated using a mixed effects ANCOVA model at 7 and at 24 h post-allergen challenge. RESULTS: Inhaled allergen-induced statistically significant gene expression changes in sputum, which were effectively blunted by fluticasone (adjusted p<0.025). Forty-seven RNA signatures were selected from these responses for correlation analyses and further validation. This included Th2 mRNA levels for cytokines, chemokines, high-affinity IgE receptor FCER1A, histamine receptor HRH4, and enzymes and receptors in the arachidonic pathway. Individual messengers from the 47 RNA signatures correlated significantly with lung function and sputum eosinophil counts. CONCLUSION: Our RNA extraction and profiling protocols allowed reproducible assessments of inflammatory signatures in sputum including quantification of drug effects on this response in allergic asthmatics. This approach offers novel possibilities for the development of pharmacodynamic (PD) biomarkers in asthma.
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BACKGROUND: Allergen-induced late airway response offers important pharmacodynamic targets, including T helper 2 (TH2) biomarkers. However, detection of inflammatory markers has been limited in dithiothreitol-processed sputum. OBJECTIVES: To test whether allergen-induced TH2 inflammatory markers can be reproducibly quantified by sensitive detection techniques in ultracentrifuged sputum and the effect of fluticasone (FP) on these endpoints. METHODS: Thirteen allergic asthmatics with dual allergen-induced airway responses, documented during a single-blind placebo run-in period, participated in a double-blind, two-period crossover study. Each period consisted of three consecutive days, separated by ≥3 weeks. Following randomization, subjects inhaled FP (500 µg bid, five doses total) or placebo. On Day 2 in each study period, allergen challenge was performed and airway response measured by forced expiratory volume in 1 sec (FEV1) until 7 h post-challenge. Sputum was induced 24 h pre-allergen and 7 and 24 h post-allergen. Sputum samples were split into two portions: TH2 biomarkers were quantified by Meso Scale multiplex platform following ultracentrifugation, and cell differentials were counted on Giemsa-May-Grünwald-stained cytospins. Allergen-induced changes in inflammatory endpoints were compared between FP and placebo using a mixed model ANCOVA. RESULTS: Inhaled allergen induced dual airway responses in all subjects during both placebo periods with reproducible late asthmatic response (LAR) and increased sputum inflammatory biomarkers (IL-2, IL-4, IL-13, and eotaxin-1) and eosinophil counts. FP effectively blunted both the LAR and the inflammatory biomarkers. CONCLUSIONS: Combining novel, sensitive quantification methods with ultracentrifugation allows reproducible quantification of sputum biomarkers following allergen challenge, reversed by FP. This approach allows non-invasive identification of pharmacodynamic targets for anti-asthma therapies.
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Downregulation of G protein-coupled receptor kinase 2 (GRK2) in endothelial cells has recently been identified as a relevant event in the tumoral angiogenic switch. Based on the effects of altering GRK2 dosage in cell and animal models, this kinase appears to act as a hub in key signaling pathways involved in vascular stabilization and remodeling. Accordingly, decreased GRK2 expression in endothelial cells accelerates tumor growth in mice by impairing the pericytes ensheathing the vessels, thereby promoting hypoxia and macrophage infiltration. These results raise new questions regarding the mechanisms by which transformed cells trigger the decrease in GRK2 observed in human breast cancer vessels and how GRK2 modulates the interactions between different cell types that occur in the tumor microenvironment.
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Tumor vessel dysfunction is a pivotal event in cancer progression. Using an in vivo neovascularization model, we identified G protein-coupled receptor kinase 2 (GRK2) as a key angiogenesis regulator. An impaired angiogenic response involving immature vessels was observed in mice hemizygous for Grk2 or in animals with endothelium-specific Grk2 silencing. ECs isolated from these animals displayed intrinsic alterations in migration, TGF-ß signaling, and formation of tubular networks. Remarkably, an altered pattern of vessel growth and maturation was detected in postnatal retinas from endothelium-specific Grk2 knockout animals. Mouse embryos with systemic or endothelium-selective Grk2 ablation had marked vascular malformations involving impaired recruitment of mural cells. Moreover, decreased endothelial Grk2 dosage accelerated tumor growth in mice, along with reduced pericyte vessel coverage and enhanced macrophage infiltration, and this transformed environment promoted decreased GRK2 in ECs and human breast cancer vessels. Our study suggests that GRK2 downregulation is a relevant event in the tumoral angiogenic switch.
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Quinase 2 de Receptor Acoplado a Proteína G/fisiologia , Neovascularização Patológica , Neovascularização Fisiológica , Receptores de Ativinas Tipo I/fisiologia , Receptores de Activinas Tipo II , Animais , Movimento Celular , Proliferação de Células , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Feminino , Quinase 2 de Receptor Acoplado a Proteína G/deficiência , Quinase 2 de Receptor Acoplado a Proteína G/genética , Hemizigoto , Humanos , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Knockout , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética , Gravidez , Proteínas Serina-Treonina Quinases/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Vasos Retinianos/anormalidades , Vasos Retinianos/embriologia , Transdução de Sinais , Fator de Crescimento Transformador beta1/fisiologiaRESUMO
Conjugate addition of thiazolidinethiones and oxazolidinethiones to N-crotonylthiazolidinethiones and -oxazolidinethiones was observed in the presence of excess triethylamine in dichloromethane. The addition takes place by the nitrogen of the heterocycle with high diastereoselectivity. It was observed that the stereoselective addition occurs on the anti-s-cis conformation of the N-enoyl sulfur-containing heterocycle.
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Tionas/química , Tionas/síntese química , Estrutura MolecularRESUMO
G protein-coupled receptor kinase 2 (GRK2) is a ubiquitous, essential protein kinase that is emerging as an integrative node in many signaling networks. Moreover, changes in GRK2 abundance and activity have been identified in several inflammatory, cardiovascular disease, and tumor contexts, suggesting that those alterations may contribute to the initiation or development of pathologies. GRKs were initially identified as key players in the desensitization and internalization of multiple G protein-coupled receptors (GPCRs), but GRK2 also phosphorylates several non-GPCR substrates and dynamically associates with a variety of proteins related to signal transduction. Ongoing research in our laboratory is aimed at understanding how specific GRK2 interactomes are orchestrated in a stimulus-, context-, or cell type-specific manner. We have recently identified an interaction between GRK2 and histone deacetylase 6 (HDAC6) that modulates cell spreading and motility. HDAC6 is a major cytoplasmic a-tubulin deacetylase that is involved in cell motility and adhesion. GRK2 dynamically and directly associates with and phosphorylates HDAC6 to stimulate its a-tubulin deacetylase activity at specific cellular localizations, such as the leading edge of migrating cells, thus promoting local tubulin deacetylation and enhanced motility. GRK2-HDAC6-mediated regulation of tubulin acetylation also modulates cellular spreading. This GRK2-HDAC6 functional interaction may have important implications in pathological contexts related to epithelial cell migration.
Assuntos
Movimento Celular/fisiologia , Quinase 2 de Receptor Acoplado a Proteína G/fisiologia , Histona Desacetilases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Acetilação , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Células HeLa , Desacetilase 6 de Histona , Humanos , Fosforilação , Ligação Proteica , Tubulina (Proteína)/metabolismoRESUMO
Precision and accuracy of the quantitative magnetic resonance (QMR) system for measuring fat in phantoms and total body fat (TBF) in humans were investigated. Measurements were made using phantoms: oil, beef with water, beef with oil, and humans with oil and water. TBF(QMR) in humans was compared with TBF by a four-compartment model (TBF(4C)). The coefficient of variation (CV) for replicate TBF(QMR) was 0.437%. QMR fat was lower at 23 °C vs. 37 °C. The fat increase in QMR phantom studies was consistent with the oil increase. When oil was added with humans, the increase in TBF(QMR) was >250 g for the initial 250 g of oil. With additional oil increments, the increase in TBF(QMR) was consistent with the amount of oil added. When water was added with humans, the TBF(QMR) increased independent of the amount of water added. TBF(QMR) was significantly less (mean ± s.e.) than TBF(4C) (females: -0.68 ± 0.27 kg, males: -4.66 ± 0.62 kg; P = 0.0001), TBF(BV) (females: -1.90 ± 0.40 kg; males: -5.68 ± 0.75 kg; P = 0.0001), and TBF(D2O) for males, but greater for females (1.19 ± 0.43 kg vs. -3.69 ± 0.81 kg for males; P = 0.0003). TBF(QMR) was lower than TBF(iDXA) with the difference greater in males (P = 0.001) and decreased with age (P = 0.011). The strong linear relationships between TBF(QMR) and TBF(4C), TBF(BV), and TBF(D2O) with slopes consistent with unity suggest that modifications are required to improve the accuracy. Should the latter be accomplished, QMR holds promise as a highly precise, rapid, and safe, noninvasive method for estimating the amount of and changes in TBF in overweight and severely obese persons.