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Most languages of the world use lexical tones to contrast words. Thus, understanding how individuals process tones when learning new words is fundamental for a better understanding of the mechanisms underlying word learning. The current study asked how tonal information is integrated during word learning. We investigated whether variability in tonal information during learning can interfere with the learning of new words and whether this is language and age dependent. Cantonese- and French-learning 30-month-olds (N = 97) and Cantonese- and French-speaking adults (N = 50) were tested with an eye-tracking task on their ability to learn phonetically different pairs of novel words in two learning conditions: a 1-tone condition in which each object was named with a single label and a 3-tone condition in which each object was named with three different labels varying in tone. We predicted learning in all groups in the 1-tone condition. For the 3-tone condition, because tones are part of the phonological system of Cantonese but not of French, we expected the Cantonese groups to either fail (toddlers) or show lower performance than in the 1-tone condition (adults), whereas the French groups might show less sensitivity to this manipulation. The results show that all participants learned in the 1-tone condition and were sensitive to tone variation to some extent. Learning in the 3-tone condition was impeded in both groups of toddlers. We argue that tonal interference in word learning likely comes from the phonological level in the Cantonese groups and from the acoustic level in the French groups.
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Percepção da Altura Sonora , Percepção da Fala , Adulto , Humanos , Idioma , Aprendizagem Verbal , LinguísticaRESUMO
BACKGROUND: Traumatic brain injury (TBI) is a leading cause of death and disability that lacks neuroprotective therapies. Following a TBI, secondary injury response pathways are activated and contribute to ongoing neurodegeneration. Microglia and astrocytes are critical neuroimmune modulators with early and persistent reactivity following a TBI. Although histologic glial reactivity is well established, a precise understanding of microglia and astrocyte function following trauma remains unknown. METHODS: Adult male C57BL/6J mice underwent either fluid percussion or sham injury. RNA sequencing of concurrently isolated microglia and astrocytes was conducted 7 days post-injury to evaluate cell-type-specific transcriptional responses to TBI. Dual in situ hybridization and immunofluorescence were used to validate the TBI-induced gene expression changes in microglia and astrocytes and to identify spatial orientation of cells expressing these genes. Comparative analysis was performed between our glial transcriptomes and those from prior reports in mild TBI and other neurologic diseases to determine if severe TBI induces unique states of microglial and astrocyte activation. RESULTS: Our findings revealed sustained, lineage-specific transcriptional changes in both microglia and astrocytes, with microglia showing a greater transcriptional response than astrocytes at this subacute time point. Microglia and astrocytes showed overlapping enrichment for genes related to type I interferon signaling and MHC class I antigen presentation. The microglia and astrocyte transcriptional response to severe TBI was distinct from prior reports in mild TBI and other neurodegenerative and neuroinflammatory diseases. CONCLUSION: Concurrent lineage-specific analysis revealed novel TBI-specific transcriptional changes; these findings highlight the importance of cell-type-specific analysis of glial reactivity following TBI and may assist with the identification of novel, targeted therapies.
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Astrócitos/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Interferon Tipo I/biossíntese , Microglia/metabolismo , Transcriptoma/fisiologia , Animais , Astrócitos/patologia , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/patologia , Interferon Tipo I/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/patologiaRESUMO
In this study, a composite multilayer film onto gold was constructed from two charged building blocks, i.e., negatively charged graphene oxide (GO) and a branched polycation (polyethylenimine, PEI) via layer-by-layer (LbL) self-assembly technology, and this process was monitored in situ with quartz crystal microbalance (QCM) under different experimental conditions. This included the differences in frequency (Δf) as well as the changes in dissipation to yield information on the absorbed mass and viscoelastic properties of the formed PEI/GO multilayer films. The experimental conditions were optimized to obtain a high amount of the adsorbed mass of the self-assembled multilayer film. The surface morphology of the PEI/GO multilayer film onto gold was studied with atomic force microscopy (AFM). It was found that the positively charged PEI chains were combined with the oppositely charged GO to form an assembled film on the QCM sensor surface, in a wrapped and curled fashion. Raman and UV-vis spectra also showed that the intensities of the GO-characteristic signals are almost linearly related to the layer number. To explore the films for their use in divalent ion detection, the frequency response of the PEI/GO multilayer-modified QCM sensor to the exposure of aqueous solutions solution of Cu2+, Ca2+, Zn2+, and Sn2+ was further studied using QCM. Based on the Sauerbrey equation and the weight of different ions, the number of metal ions adsorbed per unit area on the surface of QCM sensors was calculated. For metal ion concentrations of 40 ppm, the adsorption capacities per unit area of Cu2+, Zn2+, Sn2+, and Ca2+ were found to be 1.7, 3.2, 0.7, and 4.9 nmol/cm2, respectively. Thus, in terms of the number of adsorbed ions per unit area, the QCM sensor modified by PEI/GO multilayer film shows the largest adsorption capacity of Ca2+. This can be rationalized by the relative hydration energies.
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Mesolimbic dopamine (DA) transmission is believed to play a critical role in mediating reward responses to drugs of abuse, including alcohol (EtOH). The neurobiological mechanisms underlying EtOH-seeking behavior and dependence are not fully understood, and abstinence remains the only effective way to prevent alcohol use disorders (AUDs). Here, we developed novel RGS6fl/fl; DAT-iCreER mice to determine the role of RGS6 in DA neurons on EtOH consumption, reward, and relapse behaviors. We found that RGS6 is expressed in DA neurons in both human and mouse ventral tegmental area (VTA), and that RGS6 loss in mice upregulates DA transporter (DAT) expression in VTA DA neuron synaptic terminals. Remarkably, loss of RGS6 in DA neurons significantly reduced EtOH consumption, preference, and reward in a manner indistinguishable from that seen in RGS6-/- mice. Strikingly, RGS6 loss from DA neurons before or after EtOH behavioral reward is established significantly reduced (~ 50%) re-instatement of reward following extinguishment, demonstrating distinct roles of RGS6 in promoting reward and relapse susceptibility to EtOH. These studies identify DA neurons as the locus of RGS6 action in promoting EtOH consumption, preference, reward, and relapse. RGS6 is unique among R7 RGS proteins in promoting rather than suppressing behavioral responses to drugs of abuse and to modulate EtOH behavioral reward. This is a result of RGS6's pre-synaptic actions that we hypothesize promote VTA DA transmission by suppressing GPCR-Gαi/o-DAT signaling in VTA DA neurons. These studies identify RGS6 as a potential therapeutic target for behavioral reward and relapse to EtOH.
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Mesolimbic dopamine (DA) transmission is believed to play a critical role in mediating reward responses to drugs of abuse, including alcohol (EtOH). EtOH is the most abused substance worldwide with chronic consumption often leading to the development of dependence and abuse. Unfortunately, the neurobiological mechanisms underlying EtOH-seeking behavior and dependence are not fully understood, and abstinence remains the only effective way to prevent alcohol use disorders (AUDs). Here, we developed novel RGS6 fl/fl ; DAT-iCreER mice to determine the role of RGS6 in VTA DA neurons on EtOH consumption and reward behaviors. We found that RGS6 is expressed in DA neurons in both human and mouse VTA, and that RGS6 loss in mice upregulates DA transporter (DAT) expression in VTA DA neuron synaptic terminals. Remarkably, loss of RGS6 in VTA DA neurons significantly reduced EtOH consumption, preference, and reward in a manner indistinguishable from that seen in RGS6 -/- mice. Strikingly, RGS6 loss from VTA DA neurons before or after EtOH behavioral reward is established significantly reduced (â¼50%) re-instatement of reward following extinguishment, demonstrating distinct roles of RGS6 in promoting reward and relapse susceptibility to EtOH. These studies illuminate a critical role of RGS6 in the mesolimbic circuit in promoting EtOH seeking, reward, and reinstatement. We propose that RGS6 functions to promote DA transmission through its function as a negative modulator of GPCR-Gα i/o -DAT signaling in VTA DA neurons. These studies identify RGS6 as a potential therapeutic target for behavioral reward and relapse to EtOH.
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Accumulating evidence suggests that type I interferon (IFN-I) signaling is a key contributor to immune cell-mediated neuropathology in neurodegenerative diseases. Recently, we demonstrated a robust upregulation of type I interferon-stimulated genes in microglia and astrocytes following experimental traumatic brain injury (TBI). The specific molecular and cellular mechanisms by which IFN-I signaling impacts the neuroimmune response and neuropathology following TBI remains unknown. Using the lateral fluid percussion injury model (FPI) in adult male mice, we demonstrated that IFN α/ß receptor (IFNAR) deficiency resulted in selective and sustained blockade of type I interferon-stimulated genes following TBI as well as decreased microgliosis and monocyte infiltration. Phenotypic alteration of reactive microglia also occurred with diminished expression of molecules needed for MHC class I antigen processing and presentation following TBI. This was associated with decreased accumulation of cytotoxic T cells in the brain. The IFNAR-dependent modulation of the neuroimmune response was accompanied by protection from secondary neuronal death, white matter disruption, and neurobehavioral dysfunction. These data support further efforts to leverage the IFN-I pathway for novel, targeted therapy of TBI.
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Accumulating evidence suggests that type I interferon (IFN-I) signaling is a key contributor to immune cell-mediated neuropathology in neurodegenerative diseases. Recently, we demonstrated a robust upregulation of type I interferon-stimulated genes in microglia and astrocytes following experimental traumatic brain injury (TBI). The specific molecular and cellular mechanisms by which IFN-I signaling impacts the neuroimmune response and neuropathology following TBI remains unknown. Using the lateral fluid percussion injury model (FPI) in adult male mice, we demonstrated that IFN α/ß receptor (IFNAR) deficiency resulted in selective and sustained blockade of type I interferon-stimulated genes following TBI as well as decreased microgliosis and monocyte infiltration. Molecular alteration of reactive microglia also occurred with diminished expression of genes needed for MHC class I antigen processing and presentation following TBI. This was associated with decreased accumulation of cytotoxic T cells in the brain. The IFNAR-dependent modulation of the neuroimmune response was accompanied by protection from secondary neuronal death, white matter disruption, and neurobehavioral dysfunction. These data support further efforts to leverage the IFN-I pathway for novel, targeted therapy of TBI.
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Lesões Encefálicas Traumáticas , Interferon Tipo I , Masculino , Animais , Camundongos , Neuropatologia , Lesões Encefálicas Traumáticas/complicações , Encéfalo , AnticorposRESUMO
The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16176. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are six areas of focus: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
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Bases de Dados de Produtos Farmacêuticos , Farmacologia , Humanos , Bases de Dados Factuais , Canais Iônicos , Ligantes , Receptores Citoplasmáticos e NuclearesRESUMO
Consonants and vowels have been considered to fulfill different functions in language processing, vowels being more important for prosodic and syntactic processes and consonants for lexically related processes (Nespor, Peña, & Mehler, 2003). This C-bias hypothesis in lexical processing is supported by studies with adults and infants in many languages such as English, French, Spanish, although a few studies, on Danish and Mandarin, suggest the existence of cross-linguistic variation. The present study explores whether a C-bias exists in a tone language with a complex tone system, Cantonese, by comparing the relative weight given to consonants, vowels, and also tones during word learning. To do so, looking behaviors of Cantonese-learning 20- and 30-month-olds (24 children per age/condition, 6 groups) were recorded by an eyetracker while they watched animated cartoons in Cantonese to learn pairs of novel words. The words differed minimally by either a consonant (e.g., /tÅ6/ vs. /kÅ6/), a vowel (e.g., /khim3/ vs. /khÉm3/), or a tone (e.g., T2 vs. T5). Analyses on proportional looking times revealed significant learning in 30-month-olds only, and at that age, only for the vowel contrasts. Growth curve analyses revealed better performance for the vowel condition compared to the other two conditions. The present findings establish a V-bias in Cantonese-learning 30-month-olds, adding new evidence from that tone language that the C-bias in lexical processing is not language-general. Implications for theoretical discussions on the origins of this phonological bias, and the impact of tones in early language acquisition, are discussed.
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Fonética , Percepção da Fala , Adulto , Pré-Escolar , Humanos , Lactente , Idioma , Desenvolvimento da Linguagem , AprendizagemRESUMO
This study aimed to reveal the interaction between self-assembled multilayers and dye molecules in the environment, which is closely related to the multilayers' stable performance and service life. In this work, the pH-responsive poly (N-isopropylacrylamide-co-2-(dimethylamino) ethyl methacrylate) microgels were prepared by free-radical copolymerization and self-assembled with sodium alginate (SA) into multilayers by the layer-by-layer deposition method. Quartz crystal microbalance (QCM) and atomic force microscopy (AFM) results confirmed the construction of multilayers and the absorbed mass, resulting in a decrease in the frequency shift of the QCM sensor and the deposition of microgel particles on its surface. The interaction between the self-assembled SA/microgel multilayers and anionic dyes in the aqueous solution was further investigated by QCM, and it was found that the electrostatic attraction between dyes and microgels deposited on the QCM sensor surface was much larger than that of the microgels with SA in multilayers, leading to the release of the microgels from the self-assembled structure and a mass loss ratio of 27.6%. AFM observation of the multilayer morphology exposed to dyes showed that 29% of the microgels was peeled off, and the corresponding microgel imprints were generated on the surface. In contrast, the shape and size of the remaining self-assembled microgel particles did not change.
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The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15537. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are six areas of focus: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
Assuntos
Bases de Dados de Produtos Farmacêuticos , Farmacologia , Humanos , Canais Iônicos , Ligantes , Transporte Proteico , Receptores Citoplasmáticos e NuclearesRESUMO
Traumatic brain injury (TBI) is a leading cause of death and disability that lacks targeted therapies. Successful translation of promising neuroprotective therapies will likely require more precise identification of target populations through greater study of crucial biological factors like age and sex. A growing body of work supports the impact of these factors on response to and recovery from TBI. However, age and sex are understudied in TBI animal models. The first aim of this study was to demonstrate the feasibility of lateral fluid percussion injury (FPI) in juvenile mice as a model of pediatric TBI. Subsequently, we were interested in examining the impact of young age and sex on TBI outcome. After adapting the lateral FPI model to 21-day-old male and female mice, we characterized the molecular, histological, and functional outcomes. Whereas similar tissue injury was observed in male and female juvenile mice exposed to TBI, we observed differences in neuroinflammation and neurobehavioral function. Overall, our findings revealed less acute inflammatory cytokine expression, greater subacute microglial/macrophage accumulation, and greater neurological recovery in juvenile male mice after TBI. Given that ongoing brain development may affect progression of and recovery from TBI, juvenile models are of critical importance. The sex-dependent differences we discovered after FPI support the necessity of also including this biological variable in future TBI studies. Understanding the mechanisms underlying age- and sex-dependent differences may result in the discovery of novel therapeutic targets for TBI.
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Lesões Encefálicas/fisiopatologia , Inflamação/fisiopatologia , Macrófagos/metabolismo , Microglia/metabolismo , Recuperação de Função Fisiológica/fisiologia , Animais , Lesões Encefálicas/metabolismo , Citocinas/metabolismo , Feminino , Inflamação/metabolismo , Masculino , Camundongos , Modelos Animais , Fatores SexuaisRESUMO
Parkinson's is primarily a non-familial, age-related disorder caused by α-synuclein accumulation and the progressive loss of dopamine neurons in the substantia nigra pars compacta (SNc). G protein-coupled receptor (GPCR)-cAMP signaling has been linked to a reduction in human Parkinson's incidence and α-synuclein expression. Neuronal cAMP levels are controlled by GPCRs coupled to Gs or Gi/o, which increase or decrease cAMP, respectively. Regulator of G protein signaling 6 (RGS6) powerfully inhibits Gi/o signaling. Therefore, we hypothesized that RGS6 suppresses D2 autoreceptor- Gi/o signaling in SNc dopamine neurons promoting neuronal survival and reducing α-synuclein expression. Here we provide novel evidence that RGS6 critically suppresses late-age-onset SNc dopamine neuron loss and α-synuclein accumulation. RGS6 is restrictively expressed in human SNc dopamine neurons and, despite their loss in Parkinson's, all surviving neurons express RGS6. RGS6-/- mice exhibit hyperactive D2 autoreceptors with reduced cAMP signaling in SNc dopamine neurons. Importantly, RGS6-/- mice recapitulate key sporadic Parkinson's hallmarks, including: SNc dopamine neuron loss, reduced nigrostriatal dopamine, motor deficits, and α-synuclein accumulation. To our knowledge, Rgs6 is the only gene whose loss phenocopies these features of human Parkinson's. Therefore, RGS6 is a key regulator of D2R-Gi/o signaling in SNc dopamine neurons, protecting against Parkinson's neurodegeneration and α-synuclein accumulation.
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Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson/genética , Parte Compacta da Substância Negra/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo , Receptores de Dopamina D2/metabolismo , alfa-Sinucleína/metabolismo , Fatores Etários , Idade de Início , Animais , Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Neurônios Dopaminérgicos/patologia , Humanos , Locomoção , Camundongos , Camundongos Knockout , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Parte Compacta da Substância Negra/citologia , Parte Compacta da Substância Negra/patologia , Quimpirol/farmacologia , Transmissão SinápticaRESUMO
Bath application of GABA-A receptor agonists in neonatal rat brainstem-spinal cord preparations (BSSC) reduces respiratory frequency, an effect that is enhanced by prenatal nicotine exposure. Here we test the hypothesis that these effects can be reproduced by microinjection of GABAergic and glycinergic agonists into the pre-Botzinger complex region (PBC). We recorded the activity of phrenic motor axons from the fourth cervical ventral root in 1-3 days old BSSC that were exposed to either nicotine (6 mg/(kg day)) or saline prenatally. Microinjection of glycine or muscimol into the PBC caused abrupt, reversible apnea in all experiments. Apnea duration with glycine averaged 50.3+/-5 s in saline-exposed (N=12), and 95.7+/-9.9 s in nicotine-exposed (N=12) neonates (P<0.001). Apnea duration with muscimol averaged 51+/-5.1 s in saline-exposed (N=10), and 86+/-10.6 s in nicotine-exposed (N=12) neonates (P<0.05). These data show that prenatal nicotine exposure alters development of central ventilatory control, and that neurons in the PBC region are involved.
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Tronco Encefálico/efeitos dos fármacos , Glicina/metabolismo , Nicotina , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Respiração/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Tronco Encefálico/fisiologia , Interações Medicamentosas , Feminino , Glicina/farmacologia , Glicinérgicos/farmacologia , Técnicas In Vitro , Microinjeções , Modelos Biológicos , Gravidez , Ratos , Medula Espinal/fisiologia , Estricnina/farmacologia , Ácido gama-Aminobutírico/farmacologiaRESUMO
BACKGROUND: Congenital heart defects are the most common birth defects worldwide. Although defective Notch signaling is the major cause of mouse embryonic death from cardiovascular defects, how Notch signaling is regulated during embryonic vasculogenesis and heart development is poorly understood. METHODS AND RESULTS: Regulator of G protein signaling 6 (RGS6)-/-/Ca2+/calmodulin-dependent protein kinase II (CaMKII)VV double mutant mice were developed by crossing RGS6-/- mice with mice expressing an oxidation-resistant CaMKIIδ (CaMKIIVV), and the resulting embryonic defects/lethality were investigated using E7.5 to E15.5 embryos. While loss of either RGS6 or oxidized CaMKIIδ does not alter embryogenesis, their combined loss causes defective Notch signaling, severe cardiovascular defects, and embryonic lethality (≈E10.5-11.5). Embryos lacking RGS6 and expressing oxidation-resistant CaMKIIδ exhibit reduced myocardial wall thickness, abnormal trabeculation, and arterial specification defects. Double mutants show vascular remodeling defects, including reduced neurovascularization, delayed neural tube maturation, and small dorsal aortae. These striking cardiovascular defects were accompanied by placental and yolk sac defects in angiogenesis, hematopoiesis, and vascular remodeling similar to what is seen with defective Notch1 signaling. Double mutant hearts, embryos, and yolk sacs exhibit profound downregulation of Notch1, Jagged 1, and Notch downstream target genes Hey1, Hey2, and Hey1L as well as impaired Notch1 signaling in embryos/hearts. CONCLUSIONS: RGS6 and oxidized CaMKIIδ together function as novel critical upstream modulators of Notch signaling required for normal cardiovascular development and embryo survival. Their combined need indicates that they function in parallel pathways needed for Notch1 signaling in yolk sac, placenta and embryos. Thus, dysregulated embryonic RGS6 expression and oxidative activation of CaMKII may potentially contribute to congenital heart defects.
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Vasos Sanguíneos/enzimologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiopatias Congênitas/enzimologia , Coração , Proteínas RGS/metabolismo , Receptores Notch/metabolismo , Animais , Vasos Sanguíneos/anormalidades , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Diferenciação Celular , Células Cultivadas , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Genótipo , Idade Gestacional , Coração/embriologia , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese , Oxirredução , Fenótipo , Proteínas RGS/deficiência , Proteínas RGS/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Remodelação VascularRESUMO
Urinary bladder cancer (UBC) is largely caused by exposure to toxic chemicals including those in cigarette smoke (i.e. BBN). An activating SNP in RGS6 is associated with a pronounced reduction in UBC risk, especially among smokers. However, the mechanism underlying this reduction remains unknown. Here we demonstrate that RGS6 is robustly expressed in human urothelium, where urothelial cell carcinoma originates, and is downregulated in human UBC. Utilizing RGS6-/- mice we interrogated a possible role for RGS6 as a tumor suppressor using the BBN-induced bladder carcinogenesis model that closely recapitulates human disease. As in humans, RGS6 is robustly expressed in mouse urothelium. RGS6 loss dramatically accelerates BBN-induced bladder carcinogenesis, with RGS6-/- mice consistently displaying more advanced pathological lesions than RGS6+/+ mice. Furthermore, BBN treatment promotes urothelial RGS6 mRNA and protein downregulation. RGS6 loss impairs p53 activation and promotes aberrant accumulation of oncogenic protein DNMT1 in urothelium. Tumor suppressor RASSF1A, a DNMT1-regulated gene, is also silenced, likely via methylation of its promoter during BBN exposure. We hypothesize that this BBN-induced RGS6 loss represents a critical hit in UBC as it irrevocably impairs the anti-proliferative actions of the ATM/p53 and RASSF1A pathways. Consistent with these findings, RGS6-/- mice treated with CP-31398, a p53-stablizing agent, and/or 5-Aza, a DNMT1 inhibitor, are protected from BBN-induced tumorigenesis. Together, our data identify RGS6 as a master tumor suppressor modulating two critical signaling pathways that are often dysregulated in UBC; therefore, RGS6 represents a potential novel biomarker for UBC diagnosis/prognosis and an appealing new target in its treatment.
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DNA (Citosina-5-)-Metiltransferase 1/genética , Proteínas RGS/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Neoplasias da Bexiga Urinária/genética , Animais , Butilidroxibutilnitrosamina/toxicidade , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas RGS/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/induzido quimicamente , Neoplasias da Bexiga Urinária/metabolismo , Urotélio/efeitos dos fármacos , Urotélio/metabolismoRESUMO
We tested the hypothesis that barbiturates depress respiratory motor output by actions on the GABAA receptor. We examined the influence of pentobarbital sodium on nerve activity recorded from a fourth cervical (C4) ventral root (phrenic motoneuron output) in the in vitro brainstem-spinal cord preparation of neonatal rats aged 1-3 days. Bath application of pentobarbital slowed the respiratory rhythm but this effect could be reversed by drug washout or by simultaneous application of 8 microM bicuculline methiodide, a GABAA receptor antagonist. Pentobarbital up to a concentration of 80 microM (or 20 mg/l) did not change the magnitude of C4 nerve bursts. The GABAA receptor agonist muscimol evoked similar changes. The results support the hypothesis that respiratory depression by barbiturates is due to GABAA receptor-mediated inhibition, with the principal effects on rhythm generation. In the light of recent studies suggesting that GABAA receptors may be excitatory in the early neonatal period, we examined postnatal changes in the GABAergic slowing of respiratory rhythm. Stimulation of GABAA receptors slowed respiratory rhythm from the first postnatal day, with no change in efficacy over the first 3 days of life.
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Bicuculina/farmacologia , GABAérgicos/farmacologia , Muscimol/farmacologia , Pentobarbital/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Centro Respiratório/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Interações Medicamentosas , Feminino , Hipnóticos e Sedativos/farmacologia , Técnicas In Vitro , Ratos , Ratos Sprague-Dawley , Insuficiência Respiratória/induzido quimicamente , Insuficiência Respiratória/fisiopatologia , Mecânica Respiratória/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Raízes Nervosas Espinhais/efeitos dos fármacosRESUMO
To determine if prenatal nicotine exposure alters the postnatal development of the ventilatory pattern and the frequency and duration of apneas, we recorded respiratory airflow with head-out body plethysmography in awake neonates on postnatal days 1, 2, 6, 10, 14, and 18. Data from 12 nicotine-exposed animals were compared with data from 12 saline-exposed animals. Nicotine (6 mg/kg of nicotine tartrate per day) or saline exposure was induced by osmotic minipumps that were implanted subdermally on the fifth day of gestation in Sprague-Dawley Dams. Although both saline- and nicotine-exposed pups gained weight at the same rate throughout the studies, there were subtle differences in ventilatory indices between the two groups. Nicotine-exposed animals had a significantly higher breathing frequency on day 10, and a lower tidal volume on days 14 and 18. Although ventilation tended to be lower in the nicotine-exposed animals, the difference was not significant. There was a significantly higher frequency of apneas in the nicotine-exposed compared with the saline-exposed animals on postnatal days 1 and 2, but the apnea duration did not differ between the groups. No apneas were observed in any of the animals after the sixth postnatal day. Prenatal nicotine exposure is associated with a greater incidence of apneas on the first two postnatal days, and then an altered breathing pattern that manifests at a later stage of development.
Assuntos
Apneia/induzido quimicamente , Nicotina/efeitos adversos , Agonistas Nicotínicos/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal , Respiração/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Feminino , Pletismografia , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Infants born to mothers that smoke while pregnant have a relatively high incidence of central respiratory control abnormalities. Recent studies have shown that prenatal nicotine exposure increases GABA release and the frequency of GABAergic currents, leading to an up-regulation of GABA(A) receptors in central neurones. Activation of GABA(A) receptors inhibits ventilatory activity, with intense activation causing apnoea. These observations lead us to hypothesize that prenatal nicotine exposure alters GABAergic control of respiratory motor pattern in the early neonatal period. Osmotic minipumps were implanted in pregnant Sprague-Dawley rats on the fifth day of gestation, and filled with nicotine (6 mg kg(-1) day(-1), 2.5 microl h(-1)) or physiological saline (2.5 microl h(-1)). Brainstem-spinal cord preparations from 1- to 3-day-old neonates were studied under in vitro conditions. Electrical activity was recorded from the fourth cervical ventral root (C4 VR), which contains the axons of phrenic motoneurones. Bath application of GABA(A) receptor agonists muscimol (250 microM) or pentobarbital sodium (60 microM) to the brainstem led to consistent, reversible and significant reductions in C4 VR burst frequency. In saline-exposed animals, frequency (bursts min(-1)) fell from 6.8 +/- 0.4 to a nadir of 2.8 +/- 0.5 with muscimol, and from 6.5 +/- 0.3 to a nadir of 2.9 +/- 0.3 for pentobarbital; in nicotine-exposed animals, frequency fell from 6.3 +/- 0.4 to 1.0 +/- 0.4 with muscimol and from 6.4 +/- 0.2 to 1.7 +/- 0.4 with pentobarbital (P < 0.05 in all cases). The decrease in C4 VR frequency was significantly greater in nicotine-exposed compared to saline-exposed preparations with both muscimol and pentobarbital (P < 0.001 for both). There were no changes in the amplitude of C4 VR bursts under any condition. The GABA(A) receptor antagonist bicuculline methiodide (8 microM) did not change C4 VR frequency or amplitude in either group, although it was effective in reversing the effects of muscimol. These experiments demonstrate that prenatal nicotine exposure alters the GABAergic regulation of respiratory rhythm in a reduced preparation. The results may lead to a better understanding of the perturbed breathing pattern observed in neonates that are exposed to nicotine in utero.