RESUMO
Amino acid signaling mediated by the activation of mechanistic target of rapamycin complex 1 (mTORC1) is fundamental to cell growth and metabolism. However, how cells negatively regulate amino acid signaling remains largely unknown. Here, we show that interaction between 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters, and the multifunctional hub protein girders of actin filaments (Girdin) down-regulates mTORC1 activity. 4F2hc interacts with Girdin in mitogen-activated protein kinase (MAPK)- and amino acid signaling-dependent manners to translocate to the lysosome. The resultant decrease in cell surface 4F2hc leads to lowered cytoplasmic glutamine (Gln) and leucine (Leu) content, which down-regulates amino acid signaling. Consistently, Girdin depletion augments amino acid-induced mTORC1 activation and inhibits amino acid deprivation-induced autophagy. These findings uncovered the mechanism underlying negative regulation of amino acid signaling, which may play a role in tightly regulated cell growth and metabolism.
Assuntos
Cadeia Pesada da Proteína-1 Reguladora de Fusão/fisiologia , Sistema de Sinalização das MAP Quinases , Proteínas dos Microfilamentos/fisiologia , Transdução de Sinais , Proteínas de Transporte Vesicular/fisiologia , Animais , Regulação para Baixo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Camundongos , Proteínas dos Microfilamentos/metabolismo , Fosforilação , Ubiquitinação , Proteínas de Transporte Vesicular/metabolismoRESUMO
OBJECTIVE: Owing to variations in the exterior appearances of noncancerous diseases in the oral cavity, clinicians may have difficulty diagnosing oral squamous cell carcinoma (OSCC). Tissue biopsy is confirmatory, but invasive. Therefore, reliable tumor markers for OSCC are required. Here, exosomal Alix (exoAlix) levels were measured in serum/salivary samples from patients with OSCC and healthy controls (HCs). METHODS: Fifty-seven patients admitted to Nagoya University Hospital from 2017 through 2019 were enrolled, and serum samples (OSCC, n = 29; HC, n = 21) and/or saliva samples (OSCC, n = 23; HC, n = 20) were collected. Exosomal fractions were isolated using ultracentrifugation. ExoAlix levels were measured using enzyme-linked immunosorbent assay. RESULTS: Serum/salivary exoAlix levels were significantly higher in patients with OSCC than in HCs. Receiver operating characteristic analyses revealed that sensitivity, specificity, positive predictive value, and area under the curve were 0.345, 1.000, 1.000, and 0.685, respectively, for serum exoAlix and 0.348, 1.000, 1.000, and 0.712, respectively, for salivary exoAlix at optimal cut-off values (serum, 0.205; saliva, 0.193). All tested OSCC tissue sections (n = 21) were immuno-reactive for Alix. CONCLUSION: Serum and salivary exoAlix were identified as potential diagnostic OSCC biomarkers. Serum exoAlix was suitable for prediction of therapeutic responses.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Biomarcadores Tumorais , Carcinoma de Células Escamosas/diagnóstico , Humanos , Neoplasias Bucais/diagnóstico , Saliva , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
The formation and the chemical characterization of single atoms of dubnium (Db, element 105), in the form of its volatile oxychloride, was investigated using the on-line gas phase chromatography technique, in the temperature range 350-600 °C. Under the exactly same chemical conditions, comparative studies with the lighter homologues of Groupâ 5 in the Periodic Table clearly indicate the volatility sequence being NbOCl3 > TaOCl3 ≥ DbOCl3 . From the obtained experimental results, thermochemical data for DbOCl3 were derived. The present study delivers reliable experimental information for theoretical calculations on chemical properties of transactinides.
RESUMO
We report the first ionization potentials (IP1) of the heavy actinides, fermium (Fm, atomic number Z = 100), mendelevium (Md, Z = 101), nobelium (No, Z = 102), and lawrencium (Lr, Z = 103), determined using a method based on a surface ionization process coupled to an online mass separation technique in an atom-at-a-time regime. The measured IP1 values agree well with those predicted by state-of-the-art relativistic calculations performed alongside the present measurements. Similar to the well-established behavior for the lanthanides, the IP1 values of the heavy actinides up to No increase with filling up the 5f orbital, while that of Lr is the lowest among the actinides. These results clearly demonstrate that the 5f orbital is fully filled at No with the [Rn]5f147s2 configuration and that Lr has a weakly bound electron outside the No core. In analogy to the lanthanide series, the present results unequivocally verify that the actinide series ends with Lr.
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In clathrin-mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo-specific adaptors for distinct cellular functions. Here, we show that the actin-binding protein girdin is a regulator of cargo-selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase-activating protein. Interestingly, girdin depletion leads to the defect in clathrin-coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E-cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo-specific adaptor.
Assuntos
Dinamina II/metabolismo , Endocitose , Células Epiteliais/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Linhagem Celular , Membrana Celular/enzimologia , Membrana Celular/metabolismo , HumanosRESUMO
Progressive encephalopathy with oedema, hypsarrhythmia and optic atrophy (PEHO) syndrome is a rare Mendelian phenotype comprising severe retardation, early onset epileptic seizures, optic nerve/cerebellar atrophy, pedal oedema, and early death. Atypical cases are often known as PEHO-like, and there is an overlap with 'early infantile epileptic encephalopathy'. PEHO is considered to be recessive, but surprisingly since initial description in 1991, no causative recessive gene(s) have been described. Hence, we report a multiplex consanguineous family with the PEHO phenotype where affected individuals had a homozygous frame-shift deletion in CCDC88A (c.2313delT, p.Leu772*ter). Analysis of cDNA extracted from patient lymphocytes unexpectedly failed to show non-sense mediated decay, and we demonstrate that the mutation produces a truncated protein lacking the crucial C-terminal half of CCDC88A (girdin). To further investigate the possible role of CCDC88A in human neurodevelopment we re-examined the behaviour and neuroanatomy of Ccdc88a knockout pups. These mice had mesial-temporal lobe epilepsy, microcephaly and corpus callosum deficiency, and by postnatal Day 21, microcephaly; the mice died at an early age. As the mouse knockout phenotype mimics the human PEHO phenotype this suggests that loss of CCDC88A is a cause of the PEHO phenotype, and that CCDC88A is essential for multiple aspects of normal human neurodevelopment.
Assuntos
Edema Encefálico/diagnóstico , Edema Encefálico/genética , Proteínas dos Microfilamentos/genética , Mutação/genética , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/genética , Atrofia Óptica/diagnóstico , Atrofia Óptica/genética , Espasmos Infantis/diagnóstico , Espasmos Infantis/genética , Proteínas de Transporte Vesicular/genética , Animais , Encéfalo/patologia , Criança , Feminino , Humanos , Lactente , Masculino , Camundongos , Camundongos Knockout , LinhagemRESUMO
In gastric cancer, the non-canonical Wnt signaling pathway is activated by Wnt5a, which has a critical role in disease outcome. Previous studies have shown that Wnt5a mediates the expression of the extracellular matrix protein laminin γ2 through Rac and JNK activation to promote gastric cancer progression. However, the mechanism of this regulatory pathway has not been completely addressed. The scaffold protein Dvl is a major component of the Wnt signaling pathway. Here, we show that Dvl-associating protein with a high frequency of leucine residues (Daple) mediates Wnt5a-induced laminin γ2 expression. Immunohistochemical analysis showed marked expression of Daple in advanced clinical stages of gastric cancer, where it highly correlated with Wnt5a/b and laminin γ2 expression, the depth of wall invasion, and the frequency of lymph node metastasis. In cultured cancer cells, Daple depletion led to the suppression of Wnt5a-induced Rac and JNK activation, laminin γ2 expression, and cell migration and invasion. Accordingly, Daple depletion also suppressed liver metastasis in a mouse xenograft model of gastric cancer. These results suggest that the non-canonical Wnt signaling pathway contributes to gastric cancer progression at least in part via Daple, which provides a new therapeutic opportunity for the treatment of the disease.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neoplasias Gástricas/patologia , Via de Sinalização Wnt/fisiologia , Animais , Western Blotting , Movimento Celular , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Synaptic plasticity in hippocampal neurons has been thought to represent a variety of memories. Although accumulating evidence indicates a crucial role of BDNF/TrkB/Akt signaling in the synaptic plasticity of the hippocampus, the mechanism by which Akt, a serine/threonine kinase, controls activity-dependent neuronal plasticity remains unclear. Girdin (also known as APE, GIV, and HkRP1), an actin-binding protein involved both in the remodeling of the actin cytoskeleton and in cell migration, has been identified as a substrate of Akt. Previous studies have demonstrated that deficit of neuronal migration in the hippocampus of Girdin-deficient (Girdin(-/-)) mice is independent on serine phosphorylation of Girdin at S1416 (Girdin S1416) by Akt. In the present study, we focused on the role of Girdin S1416 phosphorylation in BDNF/TrkB/Akt signaling associated with synaptic plasticity. We found that Girdin in the hippocampus was phosphorylated at S1416 in an activity-dependent manner. Phosphorylation-deficient knock-in mice (Girdin(SA/SA) mice), in which S1416 is replaced with alanine, exhibited shrinkage of spines, deficit of hippocampal long-term potentiation, and memory impairment. These phenotypes of Girdin(SA/SA) mice resembled those of Girdin(+/-) mice, which have 50% loss of Girdin expression. Furthermore, Girdin interacted with Src kinase and NR2B subunit of NMDA receptor, leading to phosphorylation of the NR2B subunit and NMDA receptor activation. Our findings suggest that Girdin has two different functions in the hippocampus: Akt-independent neuronal migration and Akt-dependent NR2B phosphorylation through the interaction with Src, which is associated with synaptic plasticity in the hippocampus underlying memory formation.
Assuntos
Potenciação de Longa Duração , Memória , Proteínas dos Microfilamentos/metabolismo , Neurônios/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Espinhas Dendríticas/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/fisiologia , Camundongos , Proteínas dos Microfilamentos/genética , Neurônios/citologia , Neurônios/fisiologia , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor trkB/metabolismo , Proteínas de Transporte Vesicular/genética , Quinases da Família src/metabolismoRESUMO
Girdin is an actin-binding protein that has multiple functions in postnatal neural development and cancer progression. We previously showed that Girdin is a regulator of migration for neuroblasts born from neural stem cells in the subventricular zone (SVZ) and the dentate gyrus of the hippocampus in the postnatal brain. Despite a growing list of Girdin-interacting proteins, the mechanism of Girdin-mediated migration has not been fully elucidated. Girdin interacts with Disrupted-In-Schizophrenia 1 and partitioning-defective 3, both of which have been shown to interact with the kinesin microtubule motor proteins. Based on this, we have identified that Girdin also interacts with kinesin-1, a member of neuronal kinesin proteins. Although a direct interaction of Girdin and kinesin-1 has not been determined, it is of interest to find that Girdin loss-of-function mutant mice with the mutation of a basic amino acid residue-rich region (Basic mut mice) exhibit limited interaction with kinesin-1. Furthermore, expression of a kinesin-1 mutant with motor defects, leads to Girdin mislocalization. Finally, consistent with previous studies on the role of kinesin proteins in trafficking a cell-cell adhesion molecule N-cadherin, Basic mut mice showed an aberrant expression pattern of N-cadherin in migrating SVZ neuroblasts. These findings suggest a potential role of Girdin/kinesin-1 interaction in the regulation of neuroblast migration in the postnatal brain.
Assuntos
Cinesinas/fisiologia , Proteínas dos Microfilamentos/metabolismo , Frações Subcelulares/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Caderinas/metabolismo , Cinesinas/metabolismo , Ventrículos Laterais/metabolismo , Camundongos , Camundongos MutantesRESUMO
The mammalian protein Girdin interacts with several key molecules such as actin, and it functions as a regulator of the cytoskeleton. Silencing of Girdin mRNA results in defective migration in a variety of cultured cells. Moreover, knockout of Girdin causes phenotypes related to defective migration, including hypoplasia of olfactory bulbs and a widened rostral migratory stream (RMS) in mice. To elucidate the molecular basis underlying cellular migration, we generated site- and phosphorylation state-specific antibodies against human Girdin peptides carrying four putative phosphorylation sites (serine1386 [S1386], S1416, tyrosine1764 [Y1764] and Y1798) that had been identified by mutagenesis analyses or mass spectrometric studies. We found that these residues were phosphorylated in an epidermal growth factor (EGF)-dependent manner. Among the four antibodies we developed, the antibody that targeted Girdin when phosphorylated at Y1798 (pY1798) worked well for immunohistochemistry of paraffin-embedded tissues as well as for cultured cells. Immunocytochemistry of HEK293FT cells transfected with an EGF receptor expression plasmid exhibited punctate signals with pY1798. These signals colocalized with those of endocytosed EGF receptors after EGF stimulation. Signals from pY1798 were also observed on lamellipodia, filopodia, focal adhesion and stress fibers in NIH3T3 cells under conventional culture conditions. Immunohistochemistry of paraffin-embedded mouse brain at P14 using anti-pY1798 antibody displayed signals at the hilum-side (internal side) of the dentate gyrus of the hippocampus, the RMS, the accessory olfactory bulb and the olfactory bulb in which Girdin expression was detected. Primary culture of RMS neurons showed punctate signals of pY1798 at the tips of leading processes as well as in the cytoplasm, whereas no signals were observed when neurons were treated with Src inhibitor, PP2. Our data revealed the changes in the phosphorylation status of Y1798 in Girdin when it associated with migration-related structures in vitro and in vivo.
Assuntos
Movimento Celular , Proteínas dos Microfilamentos/metabolismo , Tirosina/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Animais , Química Encefálica , Adesões Focais/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular , Células NIH 3T3 , Fosforilação , Tirosina/análise , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/genéticaRESUMO
Fetal brain injury is often related to prenatal inflammation; however, there is a lack of effective therapy. Recently, molecular hydrogen (H2), a specific antioxidant to hydroxyl radical and peroxynitrite, has been reported to have anti-inflammatory properties. The aim of this study was to investigate whether maternal H2 administration could protect the fetal brain against inflammation. Pregnant C3H/HeN mice received an intraperitoneal injection of lipopolysaccharide (LPS) on gestational day 15.5 and were provided with H2 water for 24 h prior to LPS injection. Pup brain samples were collected on gestational day 16.5, and the levels of apoptosis and oxidative damage were evaluated using immunohistochemistry. Interleukin-6 (IL-6) levels were examined using real-time PCR. The levels of apoptosis and oxidative damage, as well as the levels of IL-6 mRNA, increased significantly when the mother was injected with LPS than that in the control group. However, these levels were significantly reduced when H2 was administered prior to the LPS-injection. Our results suggest that LPS-induced apoptosis, oxidative damage and inflammation in the fetal brain were ameliorated by maternal H2 administration. Antenatal H2 administration might protect the premature brain against maternal inflammation.
RESUMO
REV7 (also known as MAD2L2 and MAD2B) is involved in DNA repair, cell cycle regulation, gene expression, and carcinogenesis. In vitro studies show that REV7 interacts with several proteins and regulates their function. It has been reported that human REV7 is highly expressed in the adult testis by Northern blot analysis. However, the significance of REV7 in mammalian development has not been elucidated. Here, we present analyses of REV7-deficient (Rev7(-/-)) mice to clarify the significance of Rev7 in mouse development. In WT mice (Rev7(+/+)), Rev7 expression was ubiquitously observed in the embryo and confined to germ cells in the testes after birth. Rev7(-/-) mice exhibited growth retardation and a partial embryonic lethal phenotype. Mice that survived to adulthood were infertile in both sexes and showed germ cell aplasia in the testes and ovaries. Analyses of Rev7(-/-) embryos revealed that primordial germ cells (PGCs) were present at embryonic day 8.5 (E8.5). However, progressive loss of PGCs was observed during migration, and PGCs were absent in the genital ridges at E13.5. An increase of apoptotic cells was detected not only among PGCs but also in the forebrain of the Rev7(-/-) embryo, whereas cell proliferation was unaffected. Moreover, DNA damage accumulation and increased levels of histone methylation were detected in Rev7(-/-) embryos, and expression of Oct4 and Nanog was deregulated by REV7 deficiency at E8.5. These findings indicate that Rev7 is essential for PGC maintenance by prevention of apoptotic cell death in the mouse.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Células Germinativas/metabolismo , Proteínas Nucleares/metabolismo , Animais , Apoptose/genética , Proteínas de Ciclo Celular/genética , Movimento Celular/fisiologia , Dano ao DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Células Germinativas/citologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas Mad2 , Masculino , Camundongos , Camundongos Knockout , Proteína Homeobox Nanog , Proteínas Nucleares/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismoRESUMO
Human REV7 (also known as MAD2L2 and MAD2B) is involved in DNA repair, cell cycle regulation, gene transcription, and carcinogenesis. In this study, we evaluated the expression of REV7 in epithelial ovarian cancer (EOC) and analyzed the association between its expression and chemosensitivity in ovarian clear cell carcinoma (CCC) cells. Expression of REV7 in human EOC tissues was assessed by immunohistochemical staining. Expression was detected in the majority of EOCs (92.0%) with especially high levels of expression frequently observed in CCCs (73.5%) compared with that of non-CCCs (53.4%). Enhanced immunoreactivity to REV7 was associated with poor prognosis represented by reduced progression-free survival in advanced stage (stage II-IV) EOC as assessed using Kaplan-Meier curves and log-rank tests. The effects of REV7 knockdown on cell proliferation and chemosensitivity in CCC cells were also analyzed in vitro and in vivo. Knockdown of REV7 in CCC cells decreased cell proliferation without affecting cell cycle distribution. Additionally, the number of apoptotic cells and DNA damaged cells were increased after cisplatin treatment. In a nude mouse tumor xenograft model, inoculated REV7-knockdown tumors showed significantly reduced tumor volumes after cisplatin treatment compared with those of the control group. These findings indicate that depletion of REV7 enhances sensitivity to cisplatin treatment in CCC, suggesting that REV7 is a candidate molecular target in CCC management.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Mad2/genética , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/genética , Carcinoma Epitelial do Ovário , Ciclo Celular/genética , Proliferação de Células , Cisplatino/uso terapêutico , Reparo do DNA/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Interferência de RNA , RNA Interferente Pequeno , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Vascular endothelial growth factor (VEGF) is recognized as a principal mediator of vessel growth. VEGF regulates various endothelial cellular processes, including cell migration, proliferation, and survival, through the serine threonine protein kinase Akt. The Akt substrate girdin, an actin-binding protein, is known to regulate VEGF-mediated postnatal angiogenesis. However, the role of girdin and its phosphorylation in neonatal retinal vascular development and ocular pathological neovascularization in vivo has not been elucidated. In the present study, therefore, we investigated these processes using Girdin(+/-) mice lacking one copy of the girdin gene and girdin S1416A knockin (Girdin-KI(SA/SA)) mice in which the phosphorylation site of girdin is completely disrupted. We used three mouse models of pathological ocular neovascularization: oxygen-induced retinopathy (a mouse model of ischemic retinopathies), laser-induced choroidal neovascularization, and a human VEGF transgenic mouse that overexpresses human VEGF specifically in photoreceptor cells and generates pathological neovascularization in the retina. Neonatal vascular development was delayed and pathological neovascularization was decreased in both Girdin(+/-) mice and Girdin-KI(SA/SA) mice. These results demonstrate that girdin and its phosphorylation play an important role in neonatal vascular development and in pathological neovascularization in the retina.
Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Proteínas dos Microfilamentos/metabolismo , Neovascularização Fisiológica , Neovascularização Retiniana/patologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Animais Recém-Nascidos , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Proteínas dos Microfilamentos/genética , Morfolinas/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Neovascularização Retiniana/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
Stroke is a leading cause of death and disability worldwide. Tissue plasminogen activator (tPA) is currently the most effective medicine for stroke; however, it has a narrow therapeutic time window (4.5 h after symptom onset). We demonstrated that nestorone, a progesterone (P4) receptor agonist, exerted neuroprotective effects against transient focal cerebral ischemia 6 h post-ischemic administration in adult male rats. This study examines its effects on permanent focal cerebral ischemia in adult and aged male rats, which are better models for evaluating treatment outcomes in typical stroke patients. Adult (6-month-old) or aged (18-month-old) male rats subjected to permanent middle cerebral artery occlusion (pMCAO) were continuously administered nestorone (10µg/day) or its vehicle (30% hydroxypropyl-ß-cyclodextrin) for 7 days via an osmotic pump subcutaneously implanted, starting at 18 h post-pMCAO. Nestorone-treated adult male rats showed marked improvements in behavioral outcomes in the adhesive removal and rotarod tests and a significant reduction in infarct size compared to vehicle-treated rats 9 and 30 days post-pMCAO. The same administration of nestorone resulted in apparently comparable neuroprotective effects in aged male rats. The inflammatory mediator NF-κB/p65 was increased in Iba-1 positive cells 24 h post-pMCAO, but was significantly suppressed by subcutaneous injection of nestorone. These results suggested that nestorone exerts long-term neuroprotective effects against permanent focal cerebral ischemia in adult and aged male rats. Nestorone is thus a promising agent for post-stroke treatment owing to its wide age-independent therapeutic time window (18 h after symptom onset), which is longer than that of tPA therapy.
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CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in several types of human cancer tissues, in particular, squamous cell carcinomas. In normal human tissues, human CD109 expression is limited to certain cell types including myoepithelial cells of the mammary, lacrimal, salivary, and bronchial glands and basal cells of the prostate and bronchial epithelium. Although CD109 has been reported to negatively regulate transforming growth factor-ß signaling in keratinocytes in vitro, its physiologic role in vivo remains largely unknown. To investigate the function of CD109 in vivo, we generated CD109-deficient (CD109(-/-)) mice. Although CD109(-/-) mice were born normally, transient impairment of hair growth was observed. At histologic analysis, kinked hair shafts, ectatic hair follicles with an accumulation of sebum, and persistent hyperplasia of the epidermis and sebaceous glands were observed in CD109(-/-) mice. Immunohistochemical analysis revealed thickening of the basal and suprabasal layers in the epidermis of CD109(-/-) mice, which is where endogenous CD109 is expressed in wild-type mice. Although CD109 was reported to negatively regulate transforming growth factor-ß signaling, no significant difference in levels of Smad2 phosphorylation was observed in the epidermis between wild-type and CD109(-/-) mice. Instead, Stat3 phosphorylation levels were significantly elevated in the epidermis of CD109(-/-) mice compared with wild-type mice. These results suggest that CD109 regulates differentiation of keratinocytes via a signaling pathway involving Stat3.
Assuntos
Antígenos CD/metabolismo , Epiderme/anormalidades , Epiderme/patologia , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/metabolismo , Animais , Epiderme/metabolismo , Técnicas de Introdução de Genes , Marcação de Genes , Cabelo/crescimento & desenvolvimento , Cabelo/metabolismo , Humanos , Hiperplasia , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Testículo/metabolismo , Fator de Crescimento Transformador beta/metabolismo , beta-Galactosidase/metabolismoRESUMO
The reduction behavior of mendelevium (Md) was studied using a flow electrolytic chromatography apparatus. By application of the appropriate potentials on the chromatography column, the more stable Md(3+) is reduced to Md(2+). The reduction potential of the Md(3+) + e(-) â Md(2+) couple was determined to be -0.16 ± 0.05 V versus a normal hydrogen electrode.
RESUMO
22q11.2 deletion syndrome (22q11.2DS) is associated with a high risk of developing various psychiatric and developmental disorders, including schizophrenia and early-onset Parkinson's disease. Recently, a mouse model of this disease, Del(3.0Mb)/+, mimicking the 3.0 Mb deletion which is most frequently found in patients with 22q11.2DS, was generated. The behavior of this mouse model was extensively studied and several abnormalities related to the symptoms of 22q11.2DS were found. However, the histological features of their brains have been little addressed. Here we describe the cytoarchitectures of the brains of Del(3.0Mb)/+ mice. First, we investigated the overall histology of the embryonic and adult cerebral cortices, but they were indistinguishable from the wild type. However, the morphologies of individual neurons were slightly but significantly changed from the wild type counterparts in a region-specific manner. The dendritic branches and/or dendritic spine densities of neurons in the medial prefrontal cortex, nucleus accumbens, and primary somatosensory cortex were reduced. We also observed reduced axon innervation of dopaminergic neurons into the prefrontal cortex. Given these affected neurons function together as the dopamine system to control animal behaviors, the impairment we observed may explain a part of the abnormal behaviors of Del(3.0Mb)/+ mice and the psychiatric symptoms of 22q11.2DS.
Assuntos
Síndrome de DiGeorge , Doença de Parkinson , Esquizofrenia , Animais , Camundongos , Síndrome de DiGeorge/genética , Síndrome de DiGeorge/complicações , Síndrome de DiGeorge/diagnóstico , Esquizofrenia/patologia , Encéfalo/patologia , Doença de Parkinson/patologia , Córtex Pré-FrontalRESUMO
In postnatally developing and adult brains, interneurons of the olfactory bulb (OB) are continuously generated at the subventricular zone of the forebrain. The newborn neuroblasts migrate tangentially to the OB through a well defined pathway, the rostral migratory stream (RMS), where the neuroblasts undergo collective migration termed "chain migration." The cell-intrinsic regulatory mechanism of neuroblast chain migration, however, has not been uncovered. Here we show that mice lacking the actin-binding Akt substrate Girdin (a protein that interacts with Disrupted-In-Schizophrenia 1 to regulate neurogenesis in the dentate gyrus) have profound defects in neuroblast chain migration along the RMS. Analysis of two gene knock-in mice harboring Girdin mutants identified unique amino acid residues in Girdin's C-terminal domain that are responsible for the regulation of neuroblast chain migration but revealed no apparent requirement of Girdin phosphorylation by Akt. Electron microscopic analyses demonstrated the involvement of Girdin in neuroblast cell-cell interactions. These findings suggest that Girdin is an important intrinsic factor that specifically governs neuroblast chain migration along the RMS.
Assuntos
Encéfalo/fisiologia , Movimento Celular/fisiologia , Proteínas dos Microfilamentos/fisiologia , Células-Tronco Neurais/fisiologia , Bulbo Olfatório/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Animais , Encéfalo/anatomia & histologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Movimento Celular/genética , Células Cultivadas , Técnicas de Introdução de Genes/métodos , Junções Intercelulares/genética , Junções Intercelulares/ultraestrutura , Interneurônios/metabolismo , Interneurônios/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/ultraestrutura , Bulbo Olfatório/anatomia & histologia , Bulbo Olfatório/crescimento & desenvolvimento , Bulbo Olfatório/metabolismo , Bulbo Olfatório/ultraestrutura , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
The glial cell line-derived neurotrophic factor (GDNF)/RET tyrosine kinase signaling pathway plays crucial roles in the development of the enteric nervous system (ENS) and the kidney. Tyrosine 1062 (Y1062) in RET is an autophosphorylation residue that is responsible for the activation of the PI3K/AKT and RAS/MAPK signaling pathways. Mice lacking signaling via Ret Y1062 show renal hypoplasia and hypoganglionosis of the ENS although the phenotype is milder than the Gdnf- or Ret-deficient mice. Sprouty2 (Spry2) was found to be an antagonist for fibroblast growth factor receptor (FGFR) and acts as an inhibitory regulator of ERK activation. Spry2-deficient mice exhibit hearing loss and enteric nerve hyperplasia. In the present study, we generated Spry2-deficient and Ret Y1062F knock-in (tyrosine 1062 is replaced with phenylalanine) double mutant mice to see if abnormalities of the ENS and kidney, caused by loss of signaling via Ret Y1062, are rescued by a deficiency of Spry2. Double mutant mice showed significant recovery of ureteric bud branching and ENS development in the stomach. These results indicate that Spry2 regulates downstream signaling mediated by GDNF/RET signaling complex in vivo.