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1.
Chin J Physiol ; 66(2): 65-72, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37082994

RESUMO

Prenatal opioid exposure may impede the development of adaptive responses to environmental stimuli by altering the stress-sensitive brain circuitry located at the paraventricular nucleus of the hypothalamus (PVH) and locus coeruleus (LC). Corticotropin-releasing factor (CRF) released from neurons in the PVH has emerged as a key molecule to initiate and integrate the stress response. Methadone (Meth) and buprenorphine (Bu) are two major types of synthetic opioid agonists for first-line medication-assisted treatment of opioid (e.g., morphine, Mor) use disorder in pregnant women. No studies have compared the detrimental effects of prenatal exposure to Meth versus Bu on the stress response of their offspring upon reaching adulthood. In this study, we aimed to compare stress-related neuronal activation in the PVH and LC induced by restraint (RST) stress in adult male rat offspring with prenatal exposure to the vehicle (Veh), Bu, Meth, or Mor. CFos-immunoreactive cells were used as an indicator for neuronal activation. We found that RST induced less neuronal activation in the Meth or Mor exposure groups compared with that in the Bu or Veh groups; no significant difference was detected between the Bu and Veh exposure groups. RST-induced neuronal activation was completely prevented by central administration of a CRF receptor antagonist (α-helical CRF9-41, 10 µg/3 µL) in all exposure groups, suggesting the crucial role of CRF in this stress response. In offspring without RST, central administration of CRF (0.5 µg/3 µL)-induced neuronal activation in the PVH and LC. CRF-induced neuronal activation was lessened in the Meth or Mor exposure groups compared with that in the Bu or Veh groups; no significant difference was detected between the Bu and Veh exposure groups. Moreover, RST- or CRF-induced neuronal activation in the Meth exposure group was comparable with that in the Mor exposure group. Further immunohistochemical analysis revealed that the Meth and Mor exposure groups displayed less CRF neurons in the PVH of offspring with or without RST compared with the Bu or Veh groups. Thus, stress-induced neuronal activation in the PVH and LC was well preserved in adult male rat offspring with prenatal exposure to Bu, but it was substantially lessened in those with prenatal exposure to Meth or Mor. Lowered neuronal activation found in the Meth or Mor exposure groups may be, at least in part, due to the reduction in the density of CRF neurons in the PVH.


Assuntos
Buprenorfina , Efeitos Tardios da Exposição Pré-Natal , Ratos , Masculino , Feminino , Gravidez , Humanos , Animais , Morfina/farmacologia , Metadona/farmacologia , Hormônio Liberador da Corticotropina/farmacologia , Hormônio Liberador da Corticotropina/fisiologia , Buprenorfina/farmacologia , Analgésicos Opioides/farmacologia , Ratos Sprague-Dawley , Neurônios
2.
Stress Health ; 36(4): 496-506, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32343480

RESUMO

Although researchers have confirmed the relationship between positive leadership styles and work-family enrichment, benevolent leadership has received little attention. Drawing from the concept of mood contagion, this study explores the underlying mechanism between benevolent leadership and work-family enrichment. Using a survey of 459 employees, across 36 supervisors and their work groups, and multilevel structural equation modelling, this study revealed that benevolent leadership is positively associated with work-family enrichment via cross-level paths. The results indicated that benevolent leadership is associated with positive group affective tone, which further predicts subordinates' work-family enrichment. Moreover, work engagement mediates the relationship between positive group affective tone and work-family enrichment. Theoretical and practical implications are discussed.


Assuntos
Emprego , Liderança , Equilíbrio Trabalho-Vida , Afeto , Emprego/organização & administração , Humanos , Inquéritos e Questionários
3.
J Food Drug Anal ; 27(4): 860-868, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31590757

RESUMO

The continuous re-isolation of the known and non-applicable compounds that is time-consuming and wasting resources is still a critical problem in the discovery of bioactive entities from natural resources. To efficiently address the problem, high performance liquid chromatography-diode array detector-microfractionation (HPLC-DAD-microfractionation) guided by disk agar diffusion assay was developed, and the active compounds were further identified using the tandem mass spectrometry (MS/MS)-based molecular networking. Of 150 fungal strains screened, the methanolic extracts of Phoma herbarum PPM7487, Cryptosporiopsis ericae PPM7405, and Albifimbria verrucaria PPM945 exhibited potent antimicrobial activity against Candida albicans SC5314 and Cryptococcus neoformans H99 in the preliminary agar diffusion assay. The concept of OSMAC (one strain many compounds) was employed in the fungal cultures in order to enrich the diversity of the 2nd metabolites in this study. HPLC coupled with off-line bioactivity-directed profiling of the extracts enabled a precise localization of the compounds responsible for the conspicuous antimicrobial activity. The purified active compounds were identified based mainly on MS/MS database, and further supported by 13C nuclear magnetic resonance (NMR) spectral data compared to the literatures. In addition to nineteen known compounds, a new trichothecene derivative 1, namely trichoverrin D, was isolated and identified through this protocol. The antifungal activities of all the pure isolates were evaluated, and the structure activity relationships were also inferred. This report has demonstrated the combination of HPLC microfractination and MS/MS coupled by NMR spectral dereplication for speeding up the antimicrobial natural products discovery process.


Assuntos
Antifúngicos/análise , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Espectrometria de Massas em Tandem
4.
Toxicology ; 408: 39-45, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29935984

RESUMO

Toluene, a commonly used organic solvent, produces a variety of behavioral disturbances in both humans and animals comparable to noncompetitive N-methyl-D-aspartate receptor (NMDARs) antagonists, such as phencyclidine (PCP). N-acetylcysteine (NAC) is capable of reversing the psychotomimetic effects of PCP via activation of cystine-glutamate antiporters (xCT). The present study examined whether NAC is capable of attenuating the toluene-induced brain stimulation reward enhancement and behavioral manifestations. Male mice received various doses of NAC prior to toluene exposure for assessment of intracranial self-stimulation (ICSS) thresholds, rotarod test, novel object recognition task and social interaction test. NAC ameliorated the lowering of ICSS thresholds, motor incoordination, object recognition memory impairments and social withdrawal induced by toluene. Furthermore, the capacity of NAC to ameliorate acute toluene-induced deficits in object recognition and social interaction was blocked by the xCT inhibitor (S)-4-carboxyphenylglycine and the mGluR2/3 antagonist LY341495. These results indicate that NAC could prevent toluene-induced reward facilitation and behavioral disturbances and its beneficial effects, at least for cognitive function and social interaction, are associated with activation of the xCT and mGluR2/3. These findings show the potential promise for NAC to treat toluene dependence and to prevent toluene intoxication caused by unintentional or deliberate inhalation.


Assuntos
Acetilcisteína/farmacologia , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Fármacos Neuroprotetores/farmacologia , Recompensa , Solventes/toxicidade , Tolueno/toxicidade , Sistema y+ de Transporte de Aminoácidos/agonistas , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Comportamento Exploratório/efeitos dos fármacos , Relações Interpessoais , Masculino , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/metabolismo , Reconhecimento Psicológico/efeitos dos fármacos
5.
PLoS One ; 13(4): e0196520, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29698535

RESUMO

Potato common scab, which is caused by soil-borne Streptomyces species, is a severe plant disease that results in a significant reduction in the economic value of potatoes worldwide. Due to the lack of efficacious pesticides, crop rotations, and resistant potato cultivars against the disease, we investigated whether biological control can serve as an alternative approach. In this study, multiple Bacillus species were isolated from healthy potato tubers, and Bacillus amyloliquefaciens Ba01 was chosen for further analyses based on its potency against the potato common scab pathogen Streptomyces scabies. Ba01 inhibited the growth and sporulation of S. scabies and secreted secondary metabolites such as surfactin, iturin A, and fengycin with potential activity against S. scabies as determined by imaging mass spectrometry. In pot assays, the disease severity of potato common scab decreased from 55.6 ± 11.1% (inoculated with S. scabies only) to 4.2 ± 1.4% (inoculated with S. scabies and Ba01). In the field trial, the disease severity of potato common scab was reduced from 14.4 ± 2.9% (naturally occurring) to 5.6 ± 1.1% after Ba01 treatment, representing evidence that Bacillus species control potato common scab in nature.


Assuntos
Bacillus amyloliquefaciens/metabolismo , Agentes de Controle Biológico/metabolismo , Doenças das Plantas/prevenção & controle , Solanum tuberosum/microbiologia , Bacillus amyloliquefaciens/classificação , Bacillus amyloliquefaciens/genética , Agentes de Controle Biológico/química , Agentes de Controle Biológico/farmacologia , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Lipopeptídeos/química , Lipopeptídeos/metabolismo , Lipopeptídeos/farmacologia , Espectrometria de Massas , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Filogenia , Doenças das Plantas/microbiologia , RNA Ribossômico 16S/classificação , RNA Ribossômico 16S/metabolismo , Solanum tuberosum/crescimento & desenvolvimento , Streptomyces/efeitos dos fármacos , Streptomyces/crescimento & desenvolvimento
6.
eNeuro ; 5(6)2018.
Artigo em Inglês | MEDLINE | ID: mdl-30627660

RESUMO

Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by degeneration of spinal motor neurons resulting in variable degrees of muscular wasting and weakness. It is caused by a loss-of-function mutation in the survival motor neuron (SMN1) gene. Caenorhabditis elegans mutants lacking SMN recapitulate several aspects of the disease including impaired movement and shorted life span. We examined whether genes previously implicated in life span extension conferred benefits to C. elegans lacking SMN. We find that reducing daf-2/insulin receptor signaling activity promotes survival and improves locomotor behavior in this C. elegans model of SMA. The locomotor dysfunction in C. elegans lacking SMN correlated with structural and functional abnormalities in GABAergic neuromuscular junctions (NMJs). Moreover, we demonstrated that reduction in daf-2 signaling reversed these abnormalities. Remarkably, enhancing GABAergic neurotransmission alone was able to correct the locomotor dysfunction. Our work indicated that an imbalance of excitatory/inhibitory activity within motor circuits and underlies motor system dysfunction in this SMA model. Interventions aimed at restoring the balance of excitatory/inhibitory activity in motor circuits could be of benefit to individuals with SMA.


Assuntos
Transtornos Neurológicos da Marcha/etiologia , Transtornos Neurológicos da Marcha/terapia , Atrofia Muscular Espinal/complicações , Ácido gama-Aminobutírico/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Animais Geneticamente Modificados , Fenômenos Biomecânicos/efeitos dos fármacos , Fenômenos Biomecânicos/genética , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Inibidores da Colinesterase/farmacologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Transtornos Neurológicos da Marcha/patologia , Levamisol/farmacologia , Longevidade/efeitos dos fármacos , Longevidade/genética , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/terapia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/patologia , Brometo de Piridostigmina/farmacologia , Interferência de RNA/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Análise de Sobrevida , Proteína 1 de Sobrevivência do Neurônio Motor/genética
7.
Virulence ; 9(1): 331-347, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29254431

RESUMO

Candida tropicalis is one of the most important human fungal pathogens causing superficial infections in locations such as the oral mucosa and genital tract, as well as systemic infections with high mortality. In its sister species Candida albicans, the cyclic AMP/protein kinase A (cAMP/PKA) pathway regulates fungal adhesion and dimorphism, both of which correlate closely with virulence. CaTpk1 and CaTpk2, the catalytic subunits of PKA, not only share redundant functions in hyphal growth, adhesion, and biofilm formation, but also have distinct roles in stress responses and pathogenesis, respectively. However, studies on PKA in the emerging fungal pathogen C. tropicalis are limited. Our results suggest that Tpk1 is involved in cell wall integrity and drug tolerance. The tpk2/tpk2 mutants, which have no protein kinase A activity, have reduced hyphal growth and adhesion. In addition, the tpk1/tpk1 tpk2/tpk2 double deletion mutant demonstrated delayed growth and impaired hyphal formation. In a murine model of systemic infection, both TPK1 and TPK2 were required for full virulence. We further found that EFG1 and HWP1 expression is regulated by PKA, while BCR1, FLO8, GAL4, and RIM101 are upregulated in the tpk1/tpk1 tpk2/tpk2 mutant. This study demonstrates that Tpk1 is involved in drug tolerance and cell wall integrity, while Tpk2 serves as a key regulator in dimorphism and adhesion. Both Tpk1 and Tpk2 are required for growth and full virulence in C. tropicalis.


Assuntos
Candida tropicalis/enzimologia , Candida tropicalis/crescimento & desenvolvimento , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fatores de Virulência/metabolismo , Animais , Antifúngicos/metabolismo , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/patogenicidade , Candidíase/microbiologia , Candidíase/patologia , Adesão Celular , Parede Celular/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/genética , Modelos Animais de Doenças , Tolerância a Medicamentos , Deleção de Genes , Humanos , Hifas/crescimento & desenvolvimento , Camundongos , Virulência
8.
J Neurosci ; 35(42): 14286-306, 2015 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-26490867

RESUMO

Misfolded proteins accumulate and aggregate in neurodegenerative disease. The existence of these deposits reflects a derangement in the protein homeostasis machinery. Using a candidate gene screen, we report that loss of RAD-23 protects against the toxicity of proteins known to aggregate in amyotrophic lateral sclerosis. Loss of RAD-23 suppresses the locomotor deficit of Caenorhabditis elegans engineered to express mutTDP-43 or mutSOD1 and also protects against aging and proteotoxic insults. Knockdown of RAD-23 is further neuroprotective against the toxicity of SOD1 and TDP-43 expression in mammalian neurons. Biochemical investigation indicates that RAD-23 modifies mutTDP-43 and mutSOD1 abundance, solubility, and turnover in association with altering the ubiquitination status of these substrates. In human amyotrophic lateral sclerosis spinal cord, we find that RAD-23 abundance is increased and RAD-23 is mislocalized within motor neurons. We propose a novel pathophysiological function for RAD-23 in the stabilization of mutated proteins that cause neurodegeneration. SIGNIFICANCE STATEMENT: In this work, we identify RAD-23, a component of the protein homeostasis network and nucleotide excision repair pathway, as a modifier of the toxicity of two disease-causing, misfolding-prone proteins, SOD1 and TDP-43. Reducing the abundance of RAD-23 accelerates the degradation of mutant SOD1 and TDP-43 and reduces the cellular content of the toxic species. The existence of endogenous proteins that act as "anti-chaperones" uncovers new and general targets for therapeutic intervention.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Doença dos Neurônios Motores/genética , Mutação/genética , Interferência de RNA/fisiologia , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Camundongos , Atividade Motora/genética , Fotodegradação , Ratos , Ratos Sprague-Dawley
9.
J Appl Toxicol ; 35(3): 273-9, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25092119

RESUMO

Hair cells are highly sensitive to environmental insults and other therapeutic drugs. The adverse effects of drugs such as aminoglycosides can cause hair cell death and lead to hearing loss and imbalance. The objective of the present study was to evaluate the protective activity of L-ascorbic acid, N-acetylcysteine (NAC) and apocynin on neomycin-induced hair cell damage in zebrafish (Danio rerio) larvae at 5 days post fertilization (dpf). Results showed that the loss of hair cells within the neuromasts of the lateral lines after neomycin exposure was evidenced by a significantly lower number of neuromasts labeled with fluorescent dye FM1-43FX observed under a microscope. Co-administration with L-ascorbic acid, NAC and apocynin protected neomycin-induced hair cell loss within the neuromasts. Moreover, these three compounds reduced the production of reactive oxygen species (ROS) in neuromasts exposed to neomycin, indicating that their antioxidant action is involved. In contrast, the neuromasts were labeled with specific fluorescent dye Texas-red conjugated with neomycin to detect neomycin uptake. Interestingly, the uptake of neomycin into hair cells was not influenced by these three antioxidant compounds. These data imply that prevention of hair cell damage against neomycin by L-ascorbic acid, NAC and apocynin might be associated with inhibition of excessive ROS production, but not related to modulating neomycin uptake. Our findings conclude that L-ascorbic acid, NAC and apocynin could be used as therapeutic drugs to protect aminoglycoside-induced listening impairment after further confirmatory studies.


Assuntos
Antibacterianos/toxicidade , Antioxidantes/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Neomicina/toxicidade , Peixe-Zebra , Acetofenonas/farmacologia , Acetilcisteína/farmacologia , Alternativas ao Uso de Animais , Animais , Ácido Ascórbico/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/patologia , Mecanorreceptores/efeitos dos fármacos , Mecanorreceptores/metabolismo , Mecanorreceptores/patologia , Microscopia Confocal , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Peixe-Zebra/embriologia
10.
Gastroenterology ; 147(6): 1405-16.e7, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25311989

RESUMO

BACKGROUND & AIMS: New drug targets are urgently needed for the treatment of patients with pancreatic ductal adenocarcinoma (PDA). Nearly all PDAs contain oncogenic mutations in the KRAS gene. Pharmacological inhibition of KRAS has been unsuccessful, leading to a focus on downstream effectors that are more easily targeted with small molecule inhibitors. We investigated the contributions of phosphoinositide 3-kinase (PI3K) to KRAS-initiated tumorigenesis. METHODS: Tumorigenesis was measured in the Kras(G12D/+);Ptf1a(Cre/+) mouse model of PDA; these mice were crossed with mice with pancreas-specific disruption of genes encoding PI3K p110α (Pik3ca), p110ß (Pik3cb), or RAC1 (Rac1). Pancreatitis was induced with 5 daily intraperitoneal injections of cerulein. Pancreata and primary acinar cells were isolated; acinar cells were incubated with an inhibitor of p110α (PIK75) followed by a broad-spectrum PI3K inhibitor (GDC0941). PDA cell lines (NB490 and MiaPaCa2) were incubated with PIK75 followed by GDC0941. Tissues and cells were analyzed by histology, immunohistochemistry, quantitative reverse-transcription polymerase chain reaction, and immunofluorescence analyses for factors involved in the PI3K signaling pathway. We also examined human pancreas tissue microarrays for levels of p110α and other PI3K pathway components. RESULTS: Pancreas-specific disruption of Pik3ca or Rac1, but not Pik3cb, prevented the development of pancreatic tumors in Kras(G12D/+);Ptf1a(Cre/+) mice. Loss of transformation was independent of AKT regulation. Preneoplastic ductal metaplasia developed in mice lacking pancreatic p110α but regressed. Levels of activated and total RAC1 were higher in pancreatic tissues from Kras(G12D/+);Ptf1a(Cre/+) mice compared with controls. Loss of p110α reduced RAC1 activity and expression in these tissues. p110α was required for the up-regulation and activity of RAC guanine exchange factors during tumorigenesis. Levels of p110α and RAC1 were increased in human pancreatic intraepithelial neoplasias and PDAs compared with healthy pancreata. CONCLUSIONS: KRAS signaling, via p110α to activate RAC1, is required for transformation in Kras(G12D/+);Ptf1a(Cre/+) mice.


Assuntos
Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Neuropeptídeos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Células Acinares/citologia , Células Acinares/metabolismo , Adenocarcinoma/genética , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinoma Ductal Pancreático/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Classe I de Fosfatidilinositol 3-Quinases , Citoesqueleto/metabolismo , Feminino , Humanos , Masculino , Camundongos Mutantes , Neuropeptídeos/genética , Fosfatidilinositol 3-Quinases/genética , Cultura Primária de Células , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/fisiologia , Transcriptoma , Proteínas rac1 de Ligação ao GTP/genética
11.
J Am Heart Assoc ; 3(3): e000527, 2014 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-24958777

RESUMO

BACKGROUND: A highly organized transverse tubule (T-tubule) network is necessary for efficient Ca(2+)-induced Ca(2+) release and synchronized contraction of ventricular myocytes. Increasing evidence suggests that T-tubule remodeling due to junctophilin-2 (JP-2) downregulation plays a critical role in the progression of heart failure. However, the mechanisms underlying JP-2 dysregulation remain incompletely understood. METHODS AND RESULTS: A mouse model of reversible heart failure that is driven by conditional activation of the heterotrimeric G protein Gαq in cardiac myocytes was used in this study. Mice with activated Gαq exhibited disruption of the T-tubule network and defects in Ca(2+) handling that culminated in heart failure compared with wild-type mice. Activation of Gαq/phospholipase Cß signaling increased the activity of the Ca(2+)-dependent protease calpain, leading to the proteolytic cleavage of JP-2. A novel calpain cleavage fragment of JP-2 is detected only in hearts with constitutive Gαq signaling to phospholipase Cß. Termination of the Gαq signal was followed by normalization of the JP-2 protein level, repair of the T-tubule network, improvements in Ca(2+) handling, and reversal of heart failure. Treatment of mice with a calpain inhibitor prevented Gαq-dependent JP-2 cleavage, T-tubule disruption, and the development of heart failure. CONCLUSIONS: Disruption of the T-tubule network in heart failure is a reversible process. Gαq-dependent activation of calpain and subsequent proteolysis of JP-2 appear to be the molecular mechanism that leads to T-tubule remodeling, Ca(2+) handling dysfunction, and progression to heart failure in this mouse model.


Assuntos
Calpaína/fisiologia , Insuficiência Cardíaca/fisiopatologia , Proteínas de Membrana/fisiologia , Proteínas Musculares/fisiologia , Animais , Cálcio/metabolismo , Notificação de Doenças , Regulação para Baixo/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Musculares/fisiologia , Proteínas Musculares/metabolismo , Proteólise , Transdução de Sinais/fisiologia
12.
Diabetes ; 62(12): 4257-65, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23974924

RESUMO

Diabetes is an independent risk factor for sudden cardiac death and ventricular arrhythmia complications of acute coronary syndrome. Prolongation of the QT interval on the electrocardiogram is also a risk factor for arrhythmias and sudden death, and the increased prevalence of QT prolongation is an independent risk factor for cardiovascular death in diabetic patients. The pathophysiological mechanisms responsible for this lethal complication are poorly understood. Diabetes is associated with a reduction in phosphoinositide 3-kinase (PI3K) signaling, which regulates the action potential duration (APD) of individual myocytes and thus the QT interval by altering multiple ion currents, including the persistent sodium current INaP. Here, we report a mechanism for diabetes-induced QT prolongation that involves an increase in INaP caused by defective PI3K signaling. Cardiac myocytes of mice with type 1 or type 2 diabetes exhibited an increase in APD that was reversed by expression of constitutively active PI3K or intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate (PIP3), the second messenger produced by PI3K. The diabetic myocytes also showed an increase in INaP that was reversed by activated PI3K or PIP3. The increases in APD and INaP in myocytes translated into QT interval prolongation for both types of diabetic mice. The long QT interval of type 1 diabetic hearts was shortened by insulin treatment ex vivo, and this effect was blocked by a PI3K inhibitor. Treatment of both types of diabetic mouse hearts with an INaP blocker also shortened the QT interval. These results indicate that downregulation of cardiac PI3K signaling in diabetes prolongs the QT interval at least in part by causing an increase in INaP. This mechanism may explain why the diabetic population has an increased risk of life-threatening arrhythmias.


Assuntos
Arritmias Cardíacas/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Sistema de Condução Cardíaco/anormalidades , Fosfatidilinositol 3-Quinases/metabolismo , Sódio/fisiologia , Potenciais de Ação/fisiologia , Animais , Arritmias Cardíacas/fisiopatologia , Síndrome de Brugada , Doença do Sistema de Condução Cardíaco , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Camundongos , Miócitos Cardíacos/metabolismo , Fosforilação
13.
Pharmacol Biochem Behav ; 103(2): 418-24, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23026060

RESUMO

Toluene, a widely used and commonly abused organic solvent, produces various behavioral disturbances in both humans and animals. Blockade of N-methyl-d-aspartate (NMDA) receptors has been suggested to play a critical role in acute toluene-induced behavioral manifestations. Activation of type 5 metabotropic glutamate receptors (mGluR5) attenuates behavioral responses induced by NMDA receptor blockade. The present study elucidated the role of mGluR5 on toluene-induced behavioral and hypothermic responses. Male Sprague-Dawley rats received the mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) or antagonist 6-methyl-2-[phenylethynyl]-pyridine (MPEP) prior to toluene administration. Rotarod test, step-down inhibitory avoidance learning task, and rectal temperature were monitored. Pretreatment of CHPG and MPEP attenuated and potentiated these toluene-induced responses, respectively. In addition, the inhibitory effects of CHPG on toluene-induced motor incoordination, learning impairment, and hypothermia were reversed by the protein kinase C (PKC) inhibitor chelerythrine chloride. These findings suggest that mGluR5 may modulate the neural circuits responsible for motor incoordination, learning impairment, and hypothermic action of toluene through a PKC-dependent signal transduction pathway.


Assuntos
Comportamento Animal , Hipotermia/induzido quimicamente , Receptores de Glutamato Metabotrópico/fisiologia , Tolueno/toxicidade , Animais , Hipotermia/fisiopatologia , Masculino , Ratos , Ratos Sprague-Dawley , Receptor de Glutamato Metabotrópico 5 , Teste de Desempenho do Rota-Rod
14.
J Vis Exp ; (64): e3813, 2012 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-22711008

RESUMO

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb9. Using this robust protocol, we achieved 51 ± 0.8% of differentiation efficiency (n = 3; p < 0.01, Student's t-test). Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.


Assuntos
Técnicas Citológicas/métodos , Células-Tronco Embrionárias/citologia , Neurônios Motores/citologia , Animais , Proteínas de Transporte/farmacologia , Diferenciação Celular/efeitos dos fármacos , Colina O-Acetiltransferase/biossíntese , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 8 de Crescimento de Fibroblasto/farmacologia , Imunofluorescência , Proteínas de Fluorescência Verde/análise , Proteínas Hedgehog/antagonistas & inibidores , Proteínas com Homeodomínio LIM/biossíntese , Camundongos , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Fatores de Transcrição/biossíntese , Tretinoína/farmacologia
15.
Sci Transl Med ; 4(131): 131ra50, 2012 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-22539774

RESUMO

Many drugs, including some commonly used medications, can cause abnormal heart rhythms and sudden death, as manifest by a prolonged QT interval in the electrocardiogram. Cardiac arrhythmias caused by drug-induced long QT syndrome are thought to result mainly from reductions in the delayed rectifier potassium ion (K(+)) current I(Kr). Here, we report a mechanism for drug-induced QT prolongation that involves changes in multiple ion currents caused by a decrease in phosphoinositide 3-kinase (PI3K) signaling. Treatment of canine cardiac myocytes with inhibitors of tyrosine kinases or PI3Ks caused an increase in action potential duration that was reversed by intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate. The inhibitors decreased the delayed rectifier K(+) currents I(Kr) and I(Ks), the L-type calcium ion (Ca(2+)) current I(Ca,L), and the peak sodium ion (Na(+)) current I(Na) and increased the persistent Na(+) current I(NaP). Computer modeling of the canine ventricular action potential showed that the drug-induced change in any one current accounted for less than 50% of the increase in action potential duration. Mouse hearts lacking the PI3K p110α catalytic subunit exhibited a prolonged action potential and QT interval that were at least partly a result of an increase in I(NaP). These results indicate that down-regulation of PI3K signaling directly or indirectly via tyrosine kinase inhibition prolongs the QT interval by affecting multiple ion channels. This mechanism may explain why some tyrosine kinase inhibitors in clinical use are associated with increased risk of life-threatening arrhythmias.


Assuntos
Síndrome do QT Longo/induzido quimicamente , Miócitos Cardíacos/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/toxicidade , Transdução de Sinais/efeitos dos fármacos , Potenciais de Ação , Animais , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Classe I de Fosfatidilinositol 3-Quinases , Simulação por Computador , Canais de Potássio de Retificação Tardia/efeitos dos fármacos , Canais de Potássio de Retificação Tardia/metabolismo , Cães , Eletrocardiografia , Feminino , Síndrome do QT Longo/enzimologia , Síndrome do QT Longo/genética , Síndrome do QT Longo/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Modelos Cardiovasculares , Miócitos Cardíacos/enzimologia , Fosfatidilinositol 3-Quinases/deficiência , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Medição de Risco , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/metabolismo , Fatores de Tempo
16.
Neuroreport ; 22(16): 809-13, 2011 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-21934539

RESUMO

Acetylcholine release at motor neuron synapses has been long established; however, recent discoveries indicate that synaptic transmission by motor neurons is more complex than previously thought. Using whole-cell patch clamp, we show that spontaneous excitatory postsynaptic currents of rat motor neurons in primary ventral horn cultures are entirely glutamatergic, although the cells respond to exogenous acetylcholine. Motor neurons in cultures express the vesicular glutamate transporter VGlut2, and culturing motor neurons for weeks with glutamate receptors blocked upregulates glutamate signaling without increasing cholinergic signaling. In spinal cord slices, motor neurons showed no decrease in spontaneous excitatory synaptic potentials after blocking acetylcholine receptors. Our results suggest that motor neuron synapses formed on other neurons are largely glutamatergic in culture and the spinal cord.


Assuntos
Axônios/fisiologia , Ácido Glutâmico/fisiologia , Interneurônios/fisiologia , Neurônios Motores/fisiologia , Sinapses/fisiologia , Animais , Técnicas de Cultura de Órgãos , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Medula Espinal/fisiologia
17.
PLoS One ; 6(9): e24404, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21912691

RESUMO

BACKGROUND: Phosphoinositide 3-kinases (PI3Ks) regulate numerous physiological processes including some aspects of cardiac function. Although regulation of cardiac contraction by individual PI3K isoforms has been studied, little is known about the cardiac consequences of downregulating multiple PI3Ks concurrently. METHODS AND RESULTS: Genetic ablation of both p110α and p110ß in cardiac myocytes throughout development or in adult mice caused heart failure and death. Ventricular myocytes from double knockout animals showed transverse tubule (T-tubule) loss and disorganization, misalignment of L-type Ca(2+) channels in the T-tubules with ryanodine receptors in the sarcoplasmic reticulum, and reduced Ca(2+) transients and contractility. Junctophilin-2, which is thought to tether T-tubules to the sarcoplasmic reticulum, was mislocalized in the double PI3K-null myocytes without a change in expression level. CONCLUSIONS: PI3K p110α and p110ß are required to maintain the organized network of T-tubules that is vital for efficient Ca(2+)-induced Ca(2+) release and ventricular contraction. PI3Ks maintain T-tubule organization by regulating junctophilin-2 localization. These results could have important medical implications because several PI3K inhibitors that target both isoforms are being used to treat cancer patients in clinical trials.


Assuntos
Miócitos Cardíacos/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Sarcolema/metabolismo , Animais , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/genética , Deleção de Genes , Técnicas de Inativação de Genes , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Contração Muscular/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosfatidilinositol 3-Quinases/deficiência , Fosfatidilinositol 3-Quinases/genética , Transporte Proteico , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sarcolema/patologia
18.
Biochim Biophys Acta ; 1814(9): 1134-9, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21609790

RESUMO

The survival motor neuron (SMN) protein plays an essential role in the assembly of uridine-rich small nuclear ribonuclear protein complexes. Phosphorylation of SMN can regulate its function, stability, and sub-cellular localization. This study shows that protein kinase A (PKA) phosphorylates SMN both in vitro and in vivo. Bioinformatic analysis predicts 12 potential PKA phosphorylation sites in human SMN. Mass spectrometric analysis of a tryptic digest of SMN after PKA phosphorylation identified five distinct phosphorylation sites in SMN (serines 4, 5, 8, 187 and threonine 85). Mutagenesis of this subset of PKA-phosphorylated sites in SMN affects association of SMN with Gemin2 and Gemin8. This result indicates that phosphorylation of SMN by PKA may play a role in regulation of the in vivo function of SMN.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Proteínas do Complexo SMN/química , Sequência de Aminoácidos , Biologia Computacional , Células HEK293 , Humanos , Dados de Sequência Molecular , Fosforilação , Proteínas do Complexo SMN/fisiologia
19.
BMC Neurosci ; 12: 25, 2011 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-21385431

RESUMO

BACKGROUND: Deletion or mutation(s) of the survival motor neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA), a neuromuscular disease characterized by spinal motor neuron death and muscle paralysis. Complete loss of the SMN protein is embryonically lethal, yet reduced levels of this protein result in selective death of motor neurons. Why motor neurons are specifically targeted by SMN deficiency remains to be determined. In this study, embryonic stem (ES) cells derived from a severe SMA mouse model were differentiated into motor neurons in vitro by addition of retinoic acid and sonic hedgehog agonist. Proteomic and western blot analyses were used to probe protein expression alterations in this cell-culture model of SMA that could be relevant to the disease. RESULTS: When ES cells were primed with Noggin/fibroblast growth factors (bFGF and FGF-8) in a more robust neural differentiation medium for 2 days before differentiation induction, the efficiency of in vitro motor neuron differentiation was improved from ~25% to ~50%. The differentiated ES cells expressed a pan-neuronal marker (neurofilament) and motor neuron markers (Hb9, Islet-1, and ChAT). Even though SMN-deficient ES cells had marked reduced levels of SMN (~20% of that in control ES cells), the morphology and differentiation efficiency for these cells are comparable to those for control samples. However, proteomics in conjunction with western blot analyses revealed 6 down-regulated and 14 up-regulated proteins with most of them involved in energy metabolism, cell stress-response, protein degradation, and cytoskeleton stability. Some of these activated cellular pathways showed specificity for either undifferentiated or differentiated cells. Increased p21 protein expression indicated that SMA ES cells were responding to cellular stress. Up-regulation of p21 was confirmed in spinal cord tissues from the same SMA mouse model from which the ES cells were derived. CONCLUSION: SMN-deficient ES cells provide a cell-culture model for SMA. SMN deficiency activates cellular stress pathways, causing a dysregulation of energy metabolism, protein degradation, and cytoskeleton stability.


Assuntos
Regulação da Expressão Gênica/fisiologia , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/patologia , Proteoma/metabolismo , Proteômica/métodos , Medula Espinal/patologia , Animais , Anticorpos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Modelos Animais de Doenças , Embrião de Mamíferos , Fator 8 de Crescimento de Fibroblasto/farmacologia , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteoma/análise , Proteoma/imunologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo
20.
Toxicology ; 265(1-2): 34-40, 2009 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-19765629

RESUMO

Toluene, a widely used and commonly abused organic solvent, causes a variety of behavioral disturbances in both humans and animals. In this study, the effects of toluene on locomotor activity, motor coordination, and passive avoidance learning, along with the possible mechanism underlying these toluene-induced behavioral manifestations, were investigated. Sprague-Dawley rats were tested in the open field test, rotarod test, and step-through avoidance learning task after receiving toluene (250-750 mg/kg, i.p.). Toluene dose-dependently produced locomotor hyperactivity, motor incoordination, and memory impairment. In order to determine the possible roles of dopamine and NMDA receptors in these behavioral responses to toluene, dopamine D1 receptor antagonist SCH23390, D2 receptor antagonist raclopride, D3 receptor antagonist nafadotride, or d-serine, a co-agonist at the glycine binding site of NMDA receptors, were given prior to toluene administration. SCH23390, raclopride, and nafadotride attenuated locomotor hyperactivity, but not motor incoordination and memory impairment in response to toluene, whereas d-serine reduced all the toluene-induced behavioral alterations. These findings suggest that blockade of NMDA receptors may play a critical role in acute toluene-induced locomotor hyperactivity, motor incoordination, and memory impairment, and that dopamine neurotransmission may be specifically involved in locomotor hyperactivity.


Assuntos
Comportamento Animal/efeitos dos fármacos , Receptores Dopaminérgicos/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Tolueno/toxicidade , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Benzazepinas/farmacologia , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Masculino , Atividade Motora/efeitos dos fármacos , Naftalenos/farmacologia , Equilíbrio Postural/efeitos dos fármacos , Pirrolidinas/farmacologia , Racloprida/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Dopaminérgicos/efeitos dos fármacos , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D3/antagonistas & inibidores , Receptores de Dopamina D3/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Serina/farmacologia
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