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1.
Gastroenterology ; 160(4): 1315-1329.e13, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33227282

RESUMO

BACKGROUND & AIMS: Hepatic encephalopathy (HE) is a serious neurologic complication in patients with liver cirrhosis. Very little is known about the role of the meningeal lymphatic system in HE. We tested our hypothesis that enhancement of meningeal lymphatic drainage could decrease neuroinflammation and ameliorate HE. METHODS: A 4-week bile duct ligation model was used to develop cirrhosis with HE in rats. Brain inflammation in patients with HE was evaluated by using archived GSE41919. The motor function of rats was assessed by the rotarod test. Adeno-associated virus 8-vascular endothelial growth factor C (AAV8-VEGF-C) was injected into the cisterna magna of HE rats 1 day after surgery to induce meningeal lymphangiogenesis. RESULTS: Cirrhotic rats with HE showed significantly increased microglia activation in the middle region of the cortex (P < .001) as well as increased neuroinflammation, as indicated by significant increases in interleukin 1ß, interferon γ, tumor necrosis factor α, and ionized calcium binding adaptor molecule 1 (Iba1) expression levels in at least 1 of the 3 regions of the cortex. Motor function was also impaired in rats with HE (P < .05). Human brains of patients with cirrhosis with HE also exhibited up-regulation of proinflammatory genes (NFKB1, IbA1, TNF-α, and IL1ß) (n = 6). AAV8-VEGF-C injection significantly increased meningeal lymphangiogenesis (P = .035) and tracer dye uptake in the anterior and middle regions of the cortex (P = .006 and .003, respectively), their corresponding meninges (P = .086 and .006, respectively), and the draining lymph nodes (P = .02). Furthermore, AAV8-VEGF-C decreased microglia activation (P < .001) and neuroinflammation and ameliorated motor dysfunction (P = .024). CONCLUSIONS: Promoting meningeal lymphatic drainage and enhancing waste clearance improves HE. Manipulation of meningeal lymphangiogenesis could be a new therapeutic strategy for the treatment of HE.


Assuntos
Sistema Glinfático/patologia , Encefalopatia Hepática/imunologia , Cirrose Hepática/complicações , Transtornos Motores/imunologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Córtex Cerebral/imunologia , Córtex Cerebral/patologia , Cisterna Magna/imunologia , Cisterna Magna/patologia , Dependovirus/genética , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Sistema Glinfático/imunologia , Encefalopatia Hepática/patologia , Humanos , Cirrose Hepática/imunologia , Linfangiogênese/imunologia , Masculino , Microglia/imunologia , Microglia/patologia , Transtornos Motores/patologia , Ratos , Fator C de Crescimento do Endotélio Vascular/genética
2.
J Neurophysiol ; 123(2): 645-657, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31851560

RESUMO

Gain-of-function variants in voltage-gated sodium channel NaV1.7 that increase firing frequency and spontaneous firing of dorsal root ganglion (DRG) neurons have recently been identified in 5-10% of patients with idiopathic small fiber neuropathy (I-SFN). Our previous in vitro observations suggest that enhanced sodium channel activity can contribute to a decrease in length of peripheral sensory axons. We have hypothesized that sustained sodium influx due to the expression of SFN-associated sodium channel variants may trigger an energetic deficit in neurons that contributes to degeneration and loss of nerve fibers in SFN. Using an ATP FRET biosensor, we now demonstrate reduced steady-state levels of ATP and markedly faster ATP decay in response to membrane depolarization in cultured DRG neurons expressing an SFN-associated variant NaV1.7, I228M, compared with wild-type neurons. We also observed that I228M neurons show a significant reduction in mitochondrial density and size, indicating dysfunctional mitochondria and a reduced bioenergetic capacity. Finally, we report that exposure to dexpramipexole, a drug that improves mitochondrial energy metabolism, increases the neurite length of I228M-expressing neurons. Our data suggest that expression of gain-of-function variants of NaV1.7 can damage mitochondria and compromise cellular capacity for ATP production. The resulting bioenergetic crisis can consequently contribute to loss of axons in SFN. We suggest that, in addition to interventions that reduce ionic disturbance caused by mutant NaV1.7 channels, an alternative therapeutic strategy might target the bioenergetic burden and mitochondrial damage that occur in SFN associated with NaV1.7 gain-of-function mutations.NEW & NOTEWORTHY Sodium channel NaV1.7 mutations that increase dorsal root ganglion (DRG) neuron excitability have been identified in small fiber neuropathy (SFN). We demonstrate reduced steady-state ATP levels, faster depolarization-evoked ATP decay, and reduced mitochondrial density and size in cultured DRG neurons expressing SFN-associated variant NaV1.7 I228M. Dexpramipexole, which improves mitochondrial energy metabolism, has a protective effect. Because gain-of-function NaV1.7 variants can compromise bioenergetics, therapeutic strategies that target bioenergetic burden and mitochondrial damage merit study in SFN.


Assuntos
Trifosfato de Adenosina/metabolismo , Gânglios Espinais , Mitocôndrias , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Neuritos , Neurônios , Fármacos Neuroprotetores/farmacologia , Pramipexol/farmacologia , Neuropatia de Pequenas Fibras/metabolismo , Animais , Técnicas Biossensoriais , Células Cultivadas , Mutação com Ganho de Função , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo
3.
Opt Express ; 24(17): 19607-15, 2016 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-27557239

RESUMO

Stereoscopic images could have asymmetric distortions caused by image processing in capture, synthesis, and compression of them. In 3D perception in stereoscopic display, the visibility threshold of the asymmetric distortions in the left and right images is important, which is tolerable to the human visual system. In this paper, we investigate the effect of the binocular disparity on the visibility threshold of asymmetric noises in stereoscopic images via subjective assessments. Existing just-noticeable-difference (JND) models for stereoscopic images have not taken into account the effect of the disparity in stereoscopic viewing. In this paper, we subjectively assessed the visibility threshold of asymmetric noises in stereoscopic images according to the disparity. Subjective evaluations showed that large disparity magnitudes could make more tolerable to perceive the asymmetric noises in the stereoscopic viewing.

4.
J Neurosci ; 34(49): 16369-84, 2014 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-25471575

RESUMO

NG2 is purportedly one of the most growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) produced after spinal cord injury. Nonetheless, once the severed axon tips dieback from the lesion core into the penumbra they closely associate with NG2+ cells. We asked if proteoglycans play a role in this tight cell-cell interaction and whether overadhesion upon these cells might participate in regeneration failure in rodents. Studies using varying ratios of CSPGs and adhesion molecules along with chondroitinase ABC, as well as purified adult cord-derived NG2 glia, demonstrate that CSPGs are involved in entrapping neurons. Once dystrophic axons become stabilized upon NG2+ cells, they form synaptic-like connections both in vitro and in vivo. In NG2 knock-out mice, sensory axons in the dorsal columns dieback further than their control counterparts. When axons are double conditioned to enhance their growth potential, some traverse the lesion core and express reduced amounts of synaptic proteins. Our studies suggest that proteoglycan-mediated entrapment upon NG2+ cells is an additional obstacle to CNS axon regeneration.


Assuntos
Antígenos/fisiologia , Axônios/fisiologia , Comunicação Celular/fisiologia , Regeneração Nervosa/fisiologia , Proteoglicanas/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Sinapses/fisiologia , Animais , Antígenos/genética , Axônios/ultraestrutura , Rastreamento de Células , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/fisiologia , Fibronectinas/fisiologia , Gânglios Espinais/fisiopatologia , Gânglios Espinais/ultraestrutura , Integrina beta1/fisiologia , Laminina/fisiologia , Camundongos , Camundongos Knockout , Degeneração Neural/fisiopatologia , Proteoglicanas/genética
5.
J Neurosci ; 33(6): 2541-54, 2013 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-23392682

RESUMO

When the CNS is injured, damaged axons do not regenerate. This failure is due in part to the growth-inhibitory environment that forms at the injury site. Myelin-associated molecules, repulsive axon guidance molecules, and extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs) found within the glial scar inhibit axon regeneration but the intracellular signaling mechanisms triggered by these diverse molecules remain largely unknown. Here we provide biochemical and functional evidence that atypical protein kinase C (PKCζ) and polarity (Par) complex proteins mediate axon growth inhibition. Treatment of postnatal rat neurons in vitro with the NG2 CSPG, a major component of the glial scar, activates PKCζ, and this activation is both necessary and sufficient to inhibit axonal growth. NG2 treatment also activates Cdc42, increases the association of Par6 with PKCζ, and leads to a Par3-dependent activation of Rac1. Transfection of neurons with kinase-dead forms of PKCζ, dominant-negative forms of Cdc42, or mutant forms of Par6 that do not bind to Cdc42 prevent NG2-induced growth inhibition. Similarly, transfection with either a phosphomutant Par3 (S824A) or dominant-negative Rac1 prevent inhibition, whereas expression of constitutively active Rac1 inhibits axon growth on control surfaces. These results suggest a model in which NG2 binding to neurons activates PKCζ and modifies Par complex function. They also identify the Par complex as a novel therapeutic target for promoting axon regeneration after CNS injury.


Assuntos
Axônios/fisiologia , Moléculas de Adesão Celular/fisiologia , Inibição Neural/fisiologia , Proteína Quinase C/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos/farmacologia , Axônios/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular Transformada , Células Cultivadas , Galinhas , Cicatriz/metabolismo , Feminino , Humanos , Masculino , Camundongos , Inibição Neural/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Proteoglicanas/farmacologia , Ratos , Ratos Sprague-Dawley
6.
Nat Cell Biol ; 9(3): 316-23, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17277771

RESUMO

Insulin stimulates protein synthesis and cell growth by activation of the protein kinases Akt (also known as protein kinase B, PKB) and mammalian target of rapamycin (mTOR). It was reported that Akt activates mTOR by phosphorylation and inhibition of tuberous sclerosis complex 2 (TSC2). However, in recent studies the physiological requirement of Akt phosphorylation of TSC2 for mTOR activation has been questioned. Here, we identify PRAS40 (proline-rich Akt/PKB substrate 40 kDa) as a novel mTOR binding partner that mediates Akt signals to mTOR. PRAS40 binds the mTOR kinase domain and its interaction with mTOR is induced under conditions that inhibit mTOR signalling, such as nutrient or serum deprivation or mitochondrial metabolic inhibition. Binding of PRAS40 inhibits mTOR activity and suppresses constitutive activation of mTOR in cells lacking TSC2. PRAS40 silencing inactivates insulin-receptor substrate-1 (IRS-1) and Akt, and uncouples the response of mTOR to Akt signals. Furthermore, PRAS40 phosphorylation by Akt and association with 14-3-3, a cytosolic anchor protein, are crucial for insulin to stimulate mTOR. These findings identify PRAS40 as an important regulator of insulin sensitivity of the Akt-mTOR pathway and a potential target for the treatment of cancers, insulin resistance and hamartoma syndromes.


Assuntos
Insulina/fisiologia , Fosfoproteínas/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Células 3T3 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Desoxiglucose/farmacologia , Humanos , Imunoprecipitação , Insulina/farmacologia , Camundongos , Modelos Biológicos , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Antissenso/genética , Proteína Regulatória Associada a mTOR , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR , Espectrometria de Massas em Tandem , Transfecção
7.
Cell Rep ; 38(11): 110483, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35294878

RESUMO

The development of the apical dendrite from the leading process of the bipolar pyramidal neuron might be directed by spatially organized extrinsic cues acting on localized intrinsic determinants. The extracellular cues regulating apical dendrite polarization remain elusive. We show that leading process and apical dendrite development are directed by class III Semaphorins and mediated by a localized cGMP-synthesizing complex. The scaffolding protein Scribble that associates with the cGMP-synthesizing enzyme soluble guanylate cyclase (sGC) also associates with the Semaphorin3A (Sema3A) co-receptor PlexinA3. Deletion or knockdown of PlexinA3 and Sema3A or disruption of PlexinA3-Scribble association prevents Sema3A-mediated cGMP increase and causes defects in apical dendrite development. These manipulations also impair bipolar polarity and leading process establishment. Local cGMP elevation or sGC expression rescues the effects of PlexinA3 knockdown or PlexinA3-Scribble complex disruption. During neuronal polarization, leading process and apical dendrite development are directed by a scaffold that links Semaphorin cue to cGMP increase.


Assuntos
Semaforina-3A , Semaforinas , Células Cultivadas , GMP Cíclico/metabolismo , Dendritos/metabolismo , Neurogênese , Semaforina-3A/metabolismo , Semaforina-3A/farmacologia , Semaforinas/metabolismo
8.
Opt Express ; 19(8): 7325-38, 2011 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-21503043

RESUMO

Human perception becomes difficult in the event of binocular color fusion when the color difference presented for the left and right eyes exceeds a certain threshold value, known as the binocular color fusion limit. This paper discusses the binocular color fusion limit for non-spectral colors within the color gamut of a conventional LCD 3DTV. We performed experiments to measure the color fusion limit for eight chromaticity points sampled from the CIE 1976 chromaticity diagram. A total of 2480 trials were recorded for a single observer. By analyzing the results, the color fusion limit was quantified by ellipses in the chromaticity diagram. The semi-minor axis of the ellipses ranges from 0.0415 to 0.0923 in terms of the Euclidean distance in the u'v´ chromaticity diagram and the semi-major axis ranges from 0.0640 to 0.1560. These eight ellipses are drawn on the chromaticity diagram.


Assuntos
Percepção de Cores/fisiologia , Óptica e Fotônica , Visão Binocular/fisiologia , Visão Ocular/fisiologia , Algoritmos , Cor , Desenho de Equipamento , Humanos , Imageamento Tridimensional , Masculino , Modelos Estatísticos , Modelos Teóricos , Análise de Regressão , Reprodutibilidade dos Testes , Espectrofotometria/métodos
9.
J Hazard Mater ; 417: 126071, 2021 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-34229387

RESUMO

This study is to propose a pyro-hydrometallurgical process for recovering Pb, Sn, and Sb from lead dross (LD), incorporating stages of roasting, leaching, and precipitation. The LD, containing 67.2% of Pb, 4.0% of Sn, and 1.4% of Sb, was first roasted at 750 °C for 2 h to oxidise the sulphide metals. Approximately 90% of Pb was oxidised from the first roasting. The LD was second roasted by mixing with 95% H2SO4 for sulphatising at 300 °C for 3 h to break the complex oxide structure of the oxyplumboromeite (Pb2Sb2O7). After the two-step roasting process, over 99% of Pb was oxidised and Sb was separated. The calcine obtained was desulphurised by 2 M Na2CO3 solution for insoluble PbSO4 to PbCO3 for selective leaching. The residue was then leached in 2.5 M HNO3 at 50 °C for 3 h and over 99% of Pb dissolved into the solution while Sn and Sb remained in the solid residue. The Pb containing solution was neutralised at pH 8 using 2 M Na2CO3 and over 99% Pb was precipitated as PbCO3 and Pb hydroxides. A residue containing Sn and Sb was leached in 7 M NaOH at 95 for 1 h and over 99% Sn was leached selectively. Sn in the solution was precipitated at pH 7 using 2 M H2SO4 as SnO2. Sb was recovered as Sb2O3 in solid reside from Sn leaching. The overall recovery rates of Pb, Sn, and Sb from the LD were 99.5%, 95.4%, and 86.3%, respectively. The proposed process is expected to contribute to recycling Pb and other metal values from LD by minimising hazardous waste emissions.

10.
Pain ; 162(6): 1758-1770, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33323889

RESUMO

ABSTRACT: Small-fiber neuropathy (SFN), characterized by distal unmyelinated or thinly myelinated fiber loss, produces a combination of sensory dysfunction and neuropathic pain. Gain-of-function variants in the sodium channel Nav1.7 that produce dorsal root ganglion (DRG) neuron hyperexcitability are present in 5% to 10% of patients with idiopathic painful SFN. We created 2 independent knock-in mouse lines carrying the Nav1.7 I228M gain-of-function variant, found in idiopathic SFN. Whole-cell patch-clamp and multielectrode array recordings show that Nav1.7 I228M knock-in DRG neurons are hyperexcitable compared with wild-type littermate-control neurons, but despite this, Nav1.7 I228M mice do not display mechanical or thermal hyperalgesia or intraepidermal nerve fiber loss in vivo. Therefore, although these 2 Nav1.7 I228M knock-in mouse lines recapitulate the DRG neuron hyperexcitability associated with gain-of-function mutations in Nav1.7, they do not recapitulate the pain or neuropathy phenotypes seen in patients. We suggest that the relationship between hyperexcitability in sensory neurons and the pain experienced by these patients may be more complex than previously appreciated and highlights the challenges in modelling channelopathy pain disorders in mice.


Assuntos
Gânglios Espinais , Canal de Sódio Disparado por Voltagem NAV1.7 , Animais , Mutação com Ganho de Função , Humanos , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Fenótipo , Células Receptoras Sensoriais
11.
Cell Rep ; 31(2): 107519, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32294442

RESUMO

Studies in cultured neurons have established that axon specification instructs neuronal polarization and is necessary for dendrite development. However, dendrite formation in vivo occurs when axon formation is prevented. The mechanisms promoting dendrite development remain elusive. We find that apical dendrite development is directed by a localized cyclic guanosine monophosphate (cGMP)-synthesizing complex. We show that the scaffolding protein Scribble associates with cGMP-synthesizing enzymes soluble-guanylate-cyclase (sGC) and neuronal nitric oxide synthase (nNOS). The Scribble scaffold is preferentially localized to and mediates cGMP increase in dendrites. These events are regulated by kinesin KifC2. Knockdown of Scribble, sGC-ß1, or KifC2 or disrupting their associations prevents cGMP increase in dendrites and causes severe defects in apical dendrite development. Local cGMP elevation or sGC expression rescues the effects of Scribble knockdown on dendrite development, indicating that Scribble is an upstream regulator of cGMP. During neuronal polarization, dendrite development is directed by the Scribble scaffold that might link extracellular cues to localized cGMP increase.


Assuntos
Técnicas de Cultura de Células/métodos , GMP Cíclico/farmacologia , Dendritos/metabolismo , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Células Cultivadas , GMP Cíclico/metabolismo , Feminino , Guanilato Ciclase/metabolismo , Hipocampo/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos , Neurogênese/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Alicerces Teciduais/química , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/fisiologia
12.
Nat Biotechnol ; 21(10): 1208-14, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12960965

RESUMO

We have developed a method in which randomized libraries of zinc finger-containing artificial transcription factors are used to induce phenotypic variations in yeast and mammalian cells. By linking multiple zinc-finger domains together, we constructed more than 100,000 zinc-finger proteins with diverse DNA-binding specificities and fused each of them to either a transcription activation or repression domain. The resulting transcriptional regulatory proteins were expressed individually in cells, and the transfected cells were screened for various phenotypic changes, such as drug resistance, thermotolerance or osmotolerance in yeast, and differentiation in mammalian cells. Genes associated with the selected phenotypes were also identified. Our results show that randomized libraries of artificial transcription factors are useful tools for functional genomics and phenotypic engineering.


Assuntos
Clonagem Molecular/métodos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Biblioteca de Peptídeos , Fenótipo , Engenharia de Proteínas/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco/genética , Animais , Células Cultivadas , Células Eucarióticas/metabolismo , Regulação da Expressão Gênica/genética , Variação Genética , Camundongos , Mutagênese Sítio-Dirigida/genética , Mioblastos/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Biomaterials ; 112: 176-191, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27768972

RESUMO

During mammalian embryonic development, neurons polarize to create distinct cellular compartments of axon and dendrite that inherently differ in form and function, providing the foundation for directional signaling in the nervous system. Polarization results from spatio-temporal segregation of specific proteins' activities to discrete regions of the neuron to dictate axonal vs. dendritic fate. We aim to manipulate axon formation by directed subcellular localization of crucial intracellular protein function. Here we report critical steps toward the development of a nanotechnology for localized subcellular introduction and retention of an intracellular kinase, LKB1, crucial regulator of axon formation. This nanotechnology will spatially manipulate LKB1-linked biomagnetic nanocomplexes (LKB1-NCs) in developing rodent neurons in culture and in vivo. We created a supramolecular assembly for LKB1 rapid neuronal uptake and prolonged cytoplasmic stability. LKB1-NCs retained kinase activity and phosphorylated downstream targets. NCs were successfully delivered to cultured embryonic hippocampal neurons, and were stable in the cytoplasm for 2 days, sufficient time for axon formation. Importantly, LKB1-NCs promoted axon formation in these neurons, representing unique proof of concept for the sufficiency of intracellular protein function in dictating a central developmental event. Lastly, we established NC delivery into cortical progenitors in live rat embryonic brain in utero. Our nanotechnology provides a viable platform for spatial manipulation of intracellular protein-activity, to dictate central events during neuronal development.


Assuntos
Orientação de Axônios/fisiologia , Preparações de Ação Retardada/administração & dosagem , Nanopartículas de Magnetita/administração & dosagem , Nanocápsulas/administração & dosagem , Neurônios/fisiologia , Proteínas Serina-Treonina Quinases/administração & dosagem , Quinases Proteína-Quinases Ativadas por AMP , Animais , Orientação de Axônios/efeitos dos fármacos , Preparações de Ação Retardada/química , Preparações de Ação Retardada/efeitos da radiação , Campos Magnéticos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/efeitos da radiação , Micelas , Nanocápsulas/química , Nanocápsulas/efeitos da radiação , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
14.
Integr Biol (Camb) ; 8(10): 1067-1078, 2016 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-27713970

RESUMO

Although much is known about chemotaxis- induced by gradients of soluble chemical cues - the molecular mechanisms involved in haptotaxis (migration induced by substrate-bound protein gradients) are largely unknown. We used micropatterning to produce discontinuous gradients consisting of µm-sized fibronectin-dots arranged at constant lateral but continuously decreasing axial spacing. Parameters like gradient slope, protein concentration and size or shape of the fibronectin dots were modified to determine optimal conditions for directional cell migration in gradient patterns. We demonstrate that fibroblasts predominantly migrate uphill towards a higher fibronectin density in gradients with a dot size of 2 × 2 µm, a 2% and 6% slope, and a low fibronectin concentration of 1 µg ml-1. Increasing dot size to 3.5 × 3.5 µm resulted in stationary cells, whereas rectangular dots (2 × 3 µm) orientated perpendicular to the gradient axis preferentially induce lateral migration. During haptotaxis, the Golgi apparatus reorients to a posterior position between the nucleus and the trailing edge. Using pharmacological inhibitors, we demonstrate that actomyosin contractility and microtubule dynamics are a prerequisite for gradient recognition indicating that asymmetric intracellular forces are necessary to read the axis of adhesive gradients. In the haptotaxis signalling cascade, RhoA and Cdc42, and the atypical protein kinase C zeta (aPKCζ), but not Rac, are located upstream of actomyosin contractility.


Assuntos
Actomiosina/fisiologia , Polaridade Celular , Quimiotaxia/fisiologia , Fibroblastos/fisiologia , Fibronectinas/metabolismo , Mecanotransdução Celular/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Células Cultivadas , Embrião de Galinha , Matriz Extracelular/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Fibroblastos/citologia , Contração Muscular/fisiologia , Estresse Mecânico
15.
Biotechnol Prog ; 21(3): 664-70, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15932240

RESUMO

Randomized ZFP-TF libraries could induce a specific phenotype without detailed knowledge about the phenotype of interest because, theoretically, the libraries could modulate any gene in the target organism. We have developed a novel method for enhancing the efficiency of recombinant protein production in mammalian and microbial cells using combinatorial libraries of zinc finger protein transcription factors. To this end, we constructed tens of thousands of zinc finger proteins (ZFPs) with distinct DNA-binding specificities and fused these ZFPs to either a transcriptional activation or repression domain to make transcriptional activators or repressors, respectively. Expression vectors that encode these artificial transcription factors were delivered into Saccharomyces cerevisiae or HEK 293 cells along with reporter plasmids that code for human growth hormone (hGH) or SEAP (secreted alkaline phosphatase) (for yeast or HEK, respectively). Expression of the reporter genes was driven by either the cytomegalovirus (CMV) or SV40 virus promoters. After transfection, we screened the cells for increased synthesis of the reporter proteins. From these cells, we then isolated several ZFP-transcription factors (ZFP-TFs) that significantly increased hGH or SEAP synthesis and subjected these regulatory proteins to further characterization. Our results show that randomized ZFP-TF libraries are useful tools for improving the yield of heterologous recombinant protein both in yeast and mammalian cells.


Assuntos
Fosfatase Alcalina/biossíntese , Hormônio do Crescimento Humano/biossíntese , Rim/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco/genética , Fosfatase Alcalina/genética , Linhagem Celular , Melhoramento Genético/métodos , Hormônio do Crescimento Humano/genética , Humanos , Biblioteca de Peptídeos , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
16.
J Biol Chem ; 282(35): 25604-12, 2007 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-17599906

RESUMO

The protein kinase mammalian target of rapamycin (mTOR) plays an important role in the coordinate regulation of cellular responses to nutritional and growth factor conditions. mTOR achieves these roles through interacting with raptor and rictor to form two distinct protein complexes, mTORC1 and mTORC2. Previous studies have been focused on mTORC1 to elucidate the central roles of the complex in mediating nutritional and growth factor signals to the protein synthesis machinery. Functions of mTORC2, relative to mTORC1, have remained little understood. Here we report identification of a novel component of mTORC2 named PRR5 (PRoline-Rich protein 5), a protein encoded by a gene located on a chromosomal region frequently deleted during breast and colorectal carcinogenesis (Johnstone, C. N., Castellvi-Bel, S., Chang, L. M., Sung, R. K., Bowser, M. J., Pique, J. M., Castells, A., and Rustgi, A. K. (2005) Genomics 85, 338-351). PRR5 interacts with rictor, but not raptor, and the interaction is independent of mTOR and not disturbed under conditions that disrupt the mTOR-rictor interaction. PRR5, unlike Sin1, another component of mTORC2, is not important for the mTOR-rictor interaction and mTOR activity toward Akt phosphorylation. Despite no significant effect of PRR5 on mTORC2-mediated Akt phosphorylation, PRR5 silencing inhibits Akt and S6K1 phosphorylation and reduces cell proliferation rates, a result consistent with PRR5 roles in cell growth and tumorigenesis. The inhibition of Akt and S6K1 phosphorylation by PRR5 knock down correlates with reduction in the expression level of platelet-derived growth factor receptor beta (PDGFRbeta). PRR5 silencing impairs PDGF-stimulated phosphorylation of S6K1 and Akt but moderately reduces epidermal growth factor- and insulin-stimulated phosphorylation. These findings propose a potential role of mTORC2 in the cross-talk with the cellular machinery that regulates PDGFRbeta expression and signaling.


Assuntos
Proteínas de Transporte/metabolismo , Proliferação de Células , Regulação da Expressão Gênica/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas Quinases/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/biossíntese , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Transporte/genética , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Células HeLa , Humanos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/genética , Fosforilação , Proteínas Quinases/genética , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Proteína Regulatória Associada a mTOR , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Deleção de Sequência , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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