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1.
Exp Eye Res ; 213: 108859, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34822854

RESUMO

Proliferative vitreoretinopathy (PVR) is the main cause of retinal detachment surgery failure. The epithelial-mesenchymal transition (EMT) induced by transforming growth factor (TGF-ß2) plays an important role in the development of PVR. Artesunate has been widely studied as a treatment for ophthalmic diseases because of its antioxidant, anti-inflammatory, antiapoptotic and antiproliferative properties. The purpose of this study was to investigate the effects of artesunate on the TGF-ß2-induced EMT in ARPE-19 cells and PVR development. We found that artesunate inhibited the proliferation and contraction of ARPE-19 cells after the EMT and the autocrine effects of TGF-ß2 on ARPE-19 cells. Additionally, the levels of Smad3 and p-Smad3 were increased in clinical samples, and artesunate decreased the levels of Smad3 and p-Smad3 in ARPE-19 cells treated with TGF-ß2. Artesunate also inhibited the occurrence and development of PVR in vivo. In summary, artesunate inhibits the occurrence and development of PVR by inhibiting the EMT in ARPE-19 cells.


Assuntos
Antimaláricos/uso terapêutico , Artesunato/uso terapêutico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Proteína Smad3/antagonistas & inibidores , Fator de Crescimento Transformador beta2/antagonistas & inibidores , Vitreorretinopatia Proliferativa/tratamento farmacológico , Animais , Antimaláricos/farmacologia , Artesunato/farmacologia , Western Blotting , Ciclo Celular/fisiologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Injeções Intravítreas , Coelhos , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Vitreorretinopatia Proliferativa/metabolismo
2.
FASEB J ; 34(6): 7234-7246, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32347987

RESUMO

There is an unmet need for treatments for diseases associated with aging. The antiaging, life-extending, and cognition-enhancing protein Klotho is neuroprotective due to its anti-inflammatory, antioxidative, and pro-myelinating effects. In addition, Klotho is also a tumor suppressor and has beneficial roles in multiple organs. Klotho is downregulated as part of the aging process. Thus, upregulating Klotho in the brain may lead to novel therapeutics to people suffering or at risk for neurodegenerative diseases such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis, and demyelinating diseases such as multiple sclerosis. We attempted to upregulate Klotho for its beneficial effects in the brain and elsewhere. Here, we describe a method to specifically activate Klotho gene expression. To accomplish this task, we designed zinc finger proteins (ZFPs) targeting within -300 bps of the human Klotho promoter. We designed the ZPF constructs either de novo from modular building blocks, or modified sequences from the natural endogenous Egr1 transcription factor backbone structure. Egr1 is known to upregulate Klotho expression. We tested the transcriptional activation effects of these ZFPs in a dual luciferase coincidence reporter system under the control of 4-kb promoter of human Klotho in stable HEK293 cells and in HK-2 cells that express Klotho protein endogenously. We found that the best ZFPs are the de novo designed ones targeting -250 bps of Klotho promoter and one of the Egr1-binding sites. We further enhanced Klotho's activation using p65-Rta transcriptional activation domains in addition to VP64. These upregulation approaches could be useful for studying Klotho's protective effects and designing Klotho boosting therapeutics for future in vivo experiments.


Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Glucuronidase/genética , Regiões Promotoras Genéticas/genética , Dedos de Zinco/genética , Envelhecimento/genética , Sítios de Ligação/genética , Encéfalo/metabolismo , Linhagem Celular , Cognição/fisiologia , Expressão Gênica/genética , Células HEK293 , Humanos , Proteínas Klotho , Luciferases/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Ativação Transcricional/genética , Regulação para Cima/genética
3.
FASEB J ; 34(2): 2087-2104, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907991

RESUMO

Proteinuria is associated with renal function decline and cardiovascular mortality. This association may be attributed in part to alterations of Klotho expression induced by albuminuria, yet the underlying mechanisms are unclear. The presence of albumin decreased Klotho expression in the POD-ATTAC mouse model of proteinuric kidney disease as well as in kidney epithelial cell lines. This downregulation was related to both decreased Klotho transcription and diminished protein half-life, whereas cleavage by ADAM proteases was not modified. The regulation was albumin specific since it was neither observed in the analbuminemic Col4α3-/- Alport mice nor induced by exposure of kidney epithelial cells to purified immunoglobulins. Albumin induced features of ER stress in renal tubular cells with ATF3/ATF4 activation. ATF3 and ATF4 induction downregulated Klotho through altered transcription mediated by their binding on the Klotho promoter. Inhibiting ER stress with 4-PBA decreased the effect of albumin on Klotho protein levels without altering mRNA levels, thus mainly abrogating the increased protein degradation. Taken together, albuminuria decreases Klotho expression through increased protein degradation and decreased transcription mediated by ER stress induction. This implies that modulating ER stress may improve proteinuria-induced alterations of Klotho expression, and hence renal and extrarenal complications associated with Klotho loss.


Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Albuminúria/metabolismo , Regulação para Baixo , Estresse do Retículo Endoplasmático , Glucuronidase/biossíntese , Túbulos Renais/metabolismo , Transcrição Gênica , Fator 3 Ativador da Transcrição/genética , Albuminúria/genética , Albuminúria/patologia , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Glucuronidase/genética , Humanos , Túbulos Renais/patologia , Proteínas Klotho , Camundongos , Camundongos Knockout
4.
Hepatobiliary Pancreat Dis Int ; 20(3): 251-261, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33129711

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most highly malignant tumors. Liver tumor-initiating cells (LTICs) have been considered to contribute to HCC progression and metastasis. ATP-citrate lyase (ACLY), as a key enzyme for de novo lipogenesis, has been reported to be upregulated in various tumors. However, its expression and role in HCC and LTICs remain unknown. METHODS: The expressions of ACLY in HCC tissues were detected by quantitative real-time PCR (qRT-PCR), Western blotting and immunohistochemistry. Kaplan-Meier curves and Chi-square test were used to determine the clinical significance of ACLY expression in HCC patients. A series of assays were performed to determine the function of ACLY on stemness, migration and invasion of HCC cells. Luciferase reporter assay, Western blotting and immunoprecipitation were used to study the regulation of the Wnt/ß-catenin signaling by ACLY. Rescue experiments were performed to investigate whether ß-catenin was the mediator of ACLY-regulated stemness and migration in HCC cells. RESULTS: ACLY was highly expressed in HCC tissues and LTICs. Overexpression of ACLY was significantly correlated with poor prognosis, progression and metastasis of HCC patients. Knockdown of ACLY remarkably suppressed stemness properties, migration and invasion in HCC cells. Mechanistically, ACLY could regulate the canonical Wnt pathway by affecting the stability of ß-catenin, and Lys49 acetylation of ß-catenin might mediate ACLY-regulated ß-catenin level in HCC cells. CONCLUSIONS: ACLY is a potent regulator of Wnt/ß-catenin signaling in modulating LTICs stemness and metastasis in HCC. ACLY may serve as a new target for the diagnosis and treatment of HCC.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , ATP Citrato (pro-S)-Liase/genética , Trifosfato de Adenosina , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Complexos Multienzimáticos , Oxo-Ácido-Liases , Via de Sinalização Wnt , beta Catenina/genética
5.
Chem Res Toxicol ; 33(8): 2208-2217, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32633501

RESUMO

Nomilin is a furan-containing triterpenoid isolated from the medicinal plants of citrus. The aim of this study was to investigate the in vitro and in vivo bioactivation of nomilin and the role in nomilin-induced hepatotoxicity. Microsomal incubations of nomilin supplemented with NADPH and GSH or NAL resulted in the detection of six conjugates (M1-M6). The structures of the metabolites were characterized based on LC-HRMS and NMR. Nomilin was bioactivated to a reactive cis-butene-dial (BDA) intermediate dependent on NADPH, and this intermediate suffered from the reaction with the nucleophiles (GSH and NAL) to form stable adducts. M1-M4 were identified as pyrrole derivatives, and M5 and M6 were pyrrolinone derivatives. M1 was further chemically synthesized and characterized by 13C NMR spectroscopy. M1 was the major metabolite detected in mice bile. Pretreatment with ketoconazole significantly reduced the formation of M1 in mice bile, while pretreatment with rifampicin significantly increased the formation of M1. Chemical inhibition together with recombinant human CYP450 phenotyping demonstrated that CYP3A4 was the major enzyme contributing to the bioactivation of nomilin. Toxicity study suggested that nomilin displayed dose-dependent liver injury in mice, while tetrahydro-nomilin was found to be nonhepatotoxic. Pretreatment with ketoconazole prevented mice from nomilin-induced liver injury. The liver injury induced by nomilin was deteriorated when the mice were pretreated with rifampicin. These findings provide evidence that CYP3A4-mediated bioactivation was indispensable in nomilin-induced hepatotoxicity.


Assuntos
Benzoxepinas/toxicidade , Citocromo P-450 CYP3A/metabolismo , Limoninas/toxicidade , Fígado/efeitos dos fármacos , Administração Oral , Animais , Benzoxepinas/administração & dosagem , Feminino , Humanos , Limoninas/administração & dosagem , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo
6.
Sensors (Basel) ; 17(12)2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29215559

RESUMO

The spectral linewidth from two cross-correlated fiber Bragg gratings (FBGs) are interrogated and characterized using a delayed self-homodyne method for fiber strain sensing. This approach employs a common higher frequency resolution instead of wavelength. A sensitivity and resolution of 166 MHz/µÎµ and 50 nε were demonstrated from 4 GHz spectral linewidth characterization on the electric spectrum analyzer. A 10 nε higher resolution can be expected through random noise analyses when the spectral linewidth from two FBG correlations is reduced to 1 GHz. Moreover, the FBG spectrum is broadened during strain and experimentally shows a 0.44 pm/µÎµ sensitivity, which is mainly caused by the photo elastic effect from the fiber grating period stretch.

7.
Zhongguo Zhong Xi Yi Jie He Za Zhi ; 37(3): 338-344, 2017 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-30650486

RESUMO

Objective To observe the effects of Feiliuping Ointment (FLP) containing serum on A549 cell epithelial-mesenchymal transition (EMT) related mRNA and protein expressions under macro- phage co-culture conditions. Methods FLP containing serum was prepared. A co-culture system of A549 cells and macrophages was established. A549 cells were divided into 3 groups, i.e., the blank serum group (A549 +NRS) , the co-culture cells + blank serum group (co-culture + NRS) , the co-culture cells with FLP containing serum group (co-culture + FLP). The effects of FLP on A549 cell EMT related gene (SNAIL1, SNAIL2, ZEB1, CDH1, CDH2, VIM, TJP1, CLDN1, CTNNB1, FLRT1) and proteins (E-cadherin, N-cadher- in, ZO-1, Vimentin) expressions were observed under co-culture conditions by fluorescent quantitative PCR. Results A549 cells developed into mesenchymal-like cells in co-culture conditions, which could been blocked by FLP containing serum in part. Compared with the A549 +NRS group, mRNA expressions of SNAIL1, ZEB1, CTNNB1, FLRT1, CDH2, and VIM were up-regulated (P <0. 05), but the expression of TJP1 was down-regulated in the co-culture + NRS group (all P <0. 05). Compared with the co-culture + NRS group, mRNA expressions of SNAIL2, TJP1, CLDN1, CDH1, and VIM were up-regulated, but mRNA ex- pressions of CTNNB1, FLRT1, and CDH2 were down-regulated in the co-culture + FLP group (all P <0. 05). Immunofluorescent results showed that E-cadherin expressed on cell membrane and inside cytoplasm, and most expressed on cell membrane. N-cadherin expressed on cell membrane and inside cytoplasm, and most expressed inside cytoplasm. Vimentin expressed within the cytoplasm. ZO-1 expressed mainly in cell junction. Parts of the cell membrane were positively stained. Compared with the A549 +NRS group, mRNA expression of E-cadherin was down-regulated in A549 cells, and mRNA expressions of N-cadherin and Vi- mentin were up-regulated in the co-culture +NRP group. However, E-cadherin was up-regulated and protein expressions of N-cadherin and Vimentin were down-regulated after intervention of FLP containing serum. (all P <0. 05). Conclusion FLP could inhibit the EMT of A549 cells under co-culture conditions.


Assuntos
Medicamentos de Ervas Chinesas , Transição Epitelial-Mesenquimal , Macrófagos , RNA Mensageiro , Células A549 , Caderinas , Linhagem Celular Tumoral , Técnicas de Cocultura , Medicamentos de Ervas Chinesas/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , RNA Mensageiro/metabolismo
8.
J Biol Chem ; 289(35): 24700-15, 2014 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-25037225

RESUMO

Generation of reactive oxygen species (ROS), leading to oxidative damage and neuronal cell death, plays an important role in the pathogenesis of neurodegenerative disorders, including Alzheimer disease. The present study aimed to examine the mechanism by which the anti-aging protein Klotho exerts neuroprotective effects against neuronal damage associated with neurodegeneration and oxidative stress. Pretreatment of rat primary hippocampal neurons and mouse hippocampal neuronal cell line HT22 with recombinant Klotho protected these cells from glutamate and oligomeric amyloid ß (oAß)-induced cytotoxicity. In addition, primary hippocampal neurons obtained from Klotho-overexpressing mouse embryos were more resistant to both cytotoxic insults, glutamate and oAß, compared with neurons from wild-type littermates. An antioxidative stress array analysis of neurons treated with Klotho revealed that Klotho significantly enhances the expression of the thioredoxin/peroxiredoxin (Trx/Prx) system with the greatest effect on the induction of Prx-2, an antioxidant enzyme, whose increase was confirmed at the mRNA and protein levels. Klotho-induced phosphorylation of the PI3K/Akt pathway, a pathway important in apoptosis and longevity, was associated with sustained inhibitory phosphorylation of the transcription factor forkhead box O3a (FoxO3a) and was essential for the induction of Prx-2. Down-regulation of Prx-2 expression using a lentivirus harboring shRNA almost completely abolished the ability of Klotho to rescue neurons from glutamate-induced death and significantly, but not completely, inhibited cell death mediated by oAß, suggesting that Prx-2 is a key modulator of neuroprotection. Thus, our results demonstrate, for the first time, the neuroprotective role of Klotho and reveal a novel mechanism underlying this effect.


Assuntos
Glucuronidase/fisiologia , Neurônios/fisiologia , Animais , Feminino , Proteínas Klotho , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Oxirredução , Estresse Oxidativo , Gravidez , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo
9.
Osteoporos Int ; 26(3): 875-83, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25477231

RESUMO

UNLABELLED: Participants with physical limitation and high degree of pain had poor mental and physical health-related quality of life. In addition, the more support and exercise that the participants had, the more likely they were to report better health-related quality of life. INTRODUCTION: Osteoporosis is a public health threat worldwide. The aim of this study is to examine the effects of individual demographics, disease characteristics, and social support on health-related quality of life (HrQoL) of adults with osteoporosis. Most psychosocial studies focused on the relationships but not the specific construct of social support on HrQoL. METHODS: In a correlational design, face-to-face, structured interviews were employed to collect information. Study questionnaires included a demographic sheet, the modified Social Support Inventory, and the Short-Form 36 scales on a convenience sample of 161 individuals recruited from four outpatient centers. Using the structural equation modeling approach, all relationships among factors, mediators, and HrQoL were analyzed. RESULTS: The mean duration of osteoporosis was longer than 5 years. Participants who exercised more than three times per week had greater HrQoL than individuals who exercised less frequently. Participants with physical limitation and high degree of pain had poor mental and physical HrQoL. The more support that the participants perceived, the more likely they were to report better HrQoL. The best fitted structural equation modeling (SEM) model included individual demographics and physical function, and social support as significant predictors on HrQoL, with informational support and physical function acting as mediators in those relationships. Moreover, this structural model explained 35, 42, and 40 % of the variance on activity of daily living (ADL), physical, and mental health-related quality of life. CONCLUSIONS: The more informational support that individuals have, the more likely they were to report better HrQoL. Individuals with osteoporosis who have lower pain and more exercise are considered having better HrQoL. Further longitudinal research will help clarify the direction of these relationships.


Assuntos
Modelos Teóricos , Osteoporose/reabilitação , Qualidade de Vida , Atividades Cotidianas , Idoso , Exercício Físico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoporose/fisiopatologia , Osteoporose/psicologia , Psicometria , Apoio Social , Fatores Socioeconômicos
10.
Clin Sci (Lond) ; 128(10): 665-78, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25511237

RESUMO

The Ankrd1 (ankyrin repeat domain 1) gene is known to be up-regulated in heart failure and acts as a co-activator of p53, modulating its transcriptional activity, but it remains inconclusive whether this gene promotes or inhibits cell apoptosis. In the present study, we attempted to investigate the role of Ankrd1 on AngII (angiotensin II)- or pressure-overload-induced cardiomyocyte apoptosis. In the failing hearts of mice with pressure overload, the protein expression of Ankrd1-encoded CARP (cardiac ankyrin repeat protein) was significantly increased. In NRCs (neonatal rat cardiomyocytes), AngII increased the expression of Ankrd1 and CARP. In the presence of AngII in NRCs, infection with a recombinant adenovirus containing rat Ankrd1 cDNA (Ad-Ankrd1) enhanced the mitochondrial translocation of Bax and phosphorylated p53, increased mitochondrial permeability and cardiomyocyte apoptosis, and reduced cell viability, whereas these effects were antagonized by silencing of Ankrd1. Intra-myocardial injection of Ad-Ankrd1 in mice with TAC (transverse aortic constriction) markedly exacerbated cardiac dysfunction with an increase in the lung weight/body weight ratio and a decrease in left ventricular fractional shortening. Cardiomyocyte apoptosis and the expression of phosphorylated p53 were also significantly increased in Ad-Ankrd1-infected TAC mice, whereas knockdown of Ankrd1 significantly inhibited the apoptotic signal pathway as well as cardiomyocyte apoptosis in pressure-overload mice. These findings indicate that overexpression of Ankrd1 exacerbates pathological cardiac dysfunction through enhancement of cardiomyocyte apoptosis mediated by the up-regulation of p53.


Assuntos
Apoptose/fisiologia , Regulação da Expressão Gênica/fisiologia , Insuficiência Cardíaca/metabolismo , Doenças Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Miócitos Cardíacos/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Análise de Variância , Animais , Pressão Sanguínea , Western Blotting , Primers do DNA/genética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Doenças Mitocondriais/etiologia , Miócitos Cardíacos/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Proteína X Associada a bcl-2/metabolismo
11.
Spinal Cord ; 53(5): 413-6, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25266700

RESUMO

STUDY DESIGN: Cross-sectional, correlational design. OBJECTIVES: To examine the effects of individual demographics, activities of daily living, social support, and self-concept on depressive symptoms in people with spinal cord injury (SCI). SETTING: A convenience sample of 135 adults with SCI was recruited from medical and rehabilitation centres in Taiwan. METHODS: Face-to-face, structured interviews were employed to collect information. Study questionnaires included a demographic sheet, the Barthel scale, the modified Social Support Inventory, the Huang self-concept scale and the Beck Depression Inventory. Data were analysed by structural equation modelling (SEM). RESULTS: The average age of the participants was 43.3 years (±11.98), the mean duration of injury was 114 months (±93.78), and most were males. Emotional support (r=-0.173, P<0.05) and appraisal support (r=-0.261, P<0.01) were negatively correlated with depressive symptoms. The best fitted SEM model included individual demographics and physical function, social support and self-concept as significant predictors of depressive symptoms, with self-concept acting as a mediator in this relationship. Participants' characteristics and social support both contributed substantial indirect effects on depressive symptoms via self-concept. Self-concept also mediated the relationship between education, income, physical functioning and participants' depressive symptoms. CONCLUSION: For this sample, the more negative that individuals perceived themselves, the more likely they were to report worsening depressive symptoms. The more social support that individuals have, the more likely they were to report less depressive symptoms. Further longitudinal research will help clarify the direction of these relationships.


Assuntos
Depressão/etiologia , Depressão/reabilitação , Autoimagem , Apoio Social , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/psicologia , Atividades Cotidianas , Adulto , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Escalas de Graduação Psiquiátrica , Inquéritos e Questionários , Taiwan
12.
J Neurosci ; 33(5): 1927-39, 2013 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-23365232

RESUMO

We have previously shown that myelin abnormalities characterize the normal aging process of the brain and that an age-associated reduction in Klotho is conserved across species. Predominantly generated in brain and kidney, Klotho overexpression extends life span, whereas loss of Klotho accelerates the development of aging-like phenotypes. Although the function of Klotho in brain is unknown, loss of Klotho expression leads to cognitive deficits. We found significant effects of Klotho on oligodendrocyte functions, including induced maturation of rat primary oligodendrocytic progenitor cells (OPCs) in vitro and myelination. Phosphoprotein analysis indicated that Klotho's downstream effects involve Akt and ERK signal pathways. Klotho increased OPC maturation, and inhibition of Akt or ERK function blocked this effect on OPCs. In vivo studies of Klotho knock-out mice and control littermates revealed that knock-out mice have a significant reduction in major myelin protein and gene expression. By immunohistochemistry, the number of total and mature oligodendrocytes was significantly lower in Klotho knock-out mice. Strikingly, at the ultrastructural level, Klotho knock-out mice exhibited significantly impaired myelination of the optic nerve and corpus callosum. These mice also displayed severe abnormalities at the nodes of Ranvier. To decipher the mechanisms by which Klotho affects oligodendrocytes, we used luciferase pathway reporters to identify the transcription factors involved. Together, these studies provide novel evidence for Klotho as a key player in myelin biology, which may thus be a useful therapeutic target in efforts to protect brain myelin against age-dependent changes and promote repair in multiple sclerosis.


Assuntos
Encéfalo/metabolismo , Glucuronidase/metabolismo , Bainha de Mielina/metabolismo , Fibras Nervosas Mielinizadas/metabolismo , Oligodendroglia/metabolismo , Animais , Contagem de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Corpo Caloso/metabolismo , Feminino , Glucuronidase/genética , Proteínas Klotho , Camundongos , Camundongos Knockout , Proteína Básica da Mielina/metabolismo , Células-Tronco Neurais/metabolismo , Nervo Óptico/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT1/fisiologia
13.
Biochemistry ; 53(34): 5579-87, 2014 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-25110992

RESUMO

Membrane protein shedding is a critical step in many normal and pathological processes. The anti-aging protein klotho (KL), mainly expressed in kidney and brain, is secreted into the serum and CSF, respectively. KL is proteolytically released, or shed, from the cell surface by ADAM10 and ADAM17, which are the α-secretases that also cleave the amyloid precursor protein and other proteins. The transmembrane KL is a coreceptor with the FGF receptor for FGF23, whereas the shed form acts as a circulating hormone. However, the precise cleavage sites in KL are unknown. KL contains two major cleavage sites: one close to the juxtamembrane region and another between the KL1 and KL2 domains. We identified the cleavage site involved in KL release by mutating potential sheddase(s) recognition sequences and examining the production of the KL extracellular fragments in transfected COS-7 cells. Deletion of amino acids T958 and L959 results in a 50-60% reduction in KL shedding, and an additional P954E mutation results in further reduction of KL shedding by 70-80%. Deletion of amino acids 954-962 resulted in a 94% reduction in KL shedding. This mutant also had moderately decreased cell surface expression, yet had overall similar subcellular localization as that of WT KL, as demonstrated by immunofluorescence. Cleavage-resistant mutants could function as a FGFR coreceptor for FGF23, but they lost activity as a soluble form of KL in proliferation and transcriptional reporter assays. Cleavage between the KL1 and KL2 domains is dependent on juxtamembrane cleavage. Our results shed light onto mechanisms underlying KL release from the cell membrane and provide a target for potential pharmacologic interventions aimed at regulating KL secretion.


Assuntos
Glucuronidase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Primers do DNA , Glucuronidase/química , Glucuronidase/genética , Proteínas Klotho , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Frações Subcelulares/metabolismo
14.
IEEE Trans Cybern ; PP2024 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-39037879

RESUMO

Policy iteration (PI), an iterative method in reinforcement learning, has the merit of interactions with a little-known environment to learn a decision law through policy evaluation and improvement. However, the existing PI-based results for output-feedback (OPFB) continuous-time systems relied heavily on an initial stabilizing full state-feedback (FSFB) policy. It thus raises the question of violating the OPFB principle. This article addresses such a question and establishes the PI under an initial stabilizing OPFB policy. We prove that an off-policy Bellman equation can transform any OPFB policy into an FSFB policy. Based on this transformation property, we revise the traditional PI by appending an additional iteration, which turns out to be efficient in approximating the optimal control under the initial OPFB policy. We show the effectiveness of the proposed learning methods through theoretical analysis and a case study.

15.
Mol Pharmacol ; 83(4): 814-21, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23340494

RESUMO

The study of complex heterodimeric peptide ligands has been hampered by a paucity of pharmacological tools. To facilitate such investigations, we have explored the utility of membrane tethered ligands (MTLs). Feasibility of this recombinant approach was explored with a focus on Drosophila bursicon, a heterodimeric cystine-knot protein that activates the G protein-coupled receptor rickets (rk). Rk/bursicon signaling is an evolutionarily conserved pathway in insects required for wing expansion, cuticle hardening, and melanization during development. We initially engineered two distinct MTL constructs, each composed of a type II transmembrane domain, a peptide linker, and a C terminal extracellular ligand that corresponded to either the α or ß bursicon subunit. Coexpression of the two complementary bursicon MTLs triggered rk-mediated signaling in vitro. We were then able to generate functionally active bursicon MTLs in which the two subunits were fused into a single heterodimeric peptide, oriented as either α-ß or ß-α. Carboxy-terminal deletion of 32 amino acids in the ß-α MTL construct resulted in loss of agonist activity. Coexpression of this construct with rk inhibited receptor-mediated signaling by soluble bursicon. We have thus generated membrane-anchored bursicon constructs that can activate or inhibit rk signaling. These probes can be used in future studies to explore the tissue and/or developmental stage-dependent effects of bursicon in the genetically tractable Drosophila model organism. In addition, our success in generating functionally diverse bursicon MTLs offers promise that such technology can be broadly applied to other complex ligands, including the family of mammalian cystine-knot proteins.


Assuntos
Proteínas de Drosophila/fisiologia , Hormônios de Invertebrado/fisiologia , Multimerização Proteica , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células HEK293 , Humanos , Hormônios de Invertebrado/química , Hormônios de Invertebrado/genética , Dados de Sequência Molecular , Ligação Proteica/genética , Multimerização Proteica/genética , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética
16.
Parasite Immunol ; 35(1): 1-10, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23009341

RESUMO

Cardiac inflammation that develops during infection with Trypanosoma cruzi may result in part from autoimmunity, which may occur after bystander activation, after parasite-induced cardiomyocyte damage, or molecular mimicry. A/J mice infected with T. cruzi or immunized with heat-killed T. cruzi (HKTC) develop strong autoimmunity accompanied by cardiac damage. To determine whether this cardiac damage occurs via an antibody-dependent mechanism, we analysed T. cruzi-infected and HKTC-immunized mice for the presence of autoantibodies, cardiac antibody deposition, and serum cardiac troponin I as a measure of cardiac damage. We also performed a serum transfer experiment in which sera from T. cruzi-infected and T. cruzi-immunized mice (and controls) were transferred into naïve recipients, which were then analysed for the presence of antibodies and serum troponin. Unlike T. cruzi-infected mice, T. cruzi-immunized mice did not show significant antibody deposition in the myocardium. These results indicate that antibody deposition does not precede cardiac damage and inflammation in mice immunized with or infected with T. cruzi. Serum adoptive transfer did not induce cardiac damage in any recipients. Based on these findings, we conclude that the cardiac damage induced by immunization with HKTC is not mediated by antibodies.


Assuntos
Doença de Chagas/imunologia , Trypanosoma cruzi/imunologia , Animais , Anticorpos/sangue , Autoimunidade , Doença de Chagas/parasitologia , Doença de Chagas/patologia , Masculino , Camundongos , Miocárdio/imunologia , Miocárdio/patologia
17.
J Fluoresc ; 23(4): 725-32, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23494166

RESUMO

As the hardware of FLIM technique becomes mature, the most important criterion for FLIM application is the correct interpretation of its data. In this research, first of all, a more orthogonal phasor approach, called as Modified Phasor Approach (MPA), is put forward. It is a way to calculate the lifetime of the complex fluorescent process, and a rule to measure how much the fluorescence process deviates from single exponential decay. Secondly, MPA is used to analysis the time-resolved fluorescence processes of the transfected CHO-K1 Cell lines expressing adenosine receptor A1R tagged by CYP and YFP, measured in the channel of the acceptor. The image of the fluorescence lifetime and the multiplication of the fluorescence lifetime and deviation from single exponential decay reveal the details of the Homo-FRET. In one word, MPA provides the physical meaning in its whole modified phasor space, and broadens the way for the application of the fluorescence lifetime imaging.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Processamento de Imagem Assistida por Computador/métodos , Imagem Óptica/métodos , Animais , Células CHO , Cricetinae , Cricetulus , Fatores de Tempo
18.
Int J Clin Pract ; 67(10): 1015-33, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24073974

RESUMO

AIMS: Studies on the burden and comorbidities associated with urgency urinary incontinence (UUI) are difficult to compare, partly because of the evolution of definitions for lower urinary tract symptoms and the various instruments used to assess health-related quality of life (HRQL). This article summarises published evidence on comorbidities and the personal burden associated specifically with UUI to provide clinicians with a clear perspective on the impact of UUI on patients. METHODS: A PubMed search was conducted using the terms: (urgency urinary incontinence OR urge incontinence OR mixed incontinence OR overactive bladder) AND (burden OR quality of life OR well-being OR depression OR mental health OR sexual health OR comorbid), with limits for English-language articles published between 1991 and 2011. RESULTS: Of 1364 identified articles, data from 70 retained articles indicate that UUI is a bothersome condition that has a marked negative impact on HRQL, with the severity of UUI a predictor of HRQL. UUI is significantly associated with falls in elderly individuals, depression, urinary tract infections, increased body mass index, diabetes and deaths. The burden of UUI appears to be greater than that of stress urinary incontinence or overactive bladder symptoms without UUI. UUI adversely impacts physical and mental health, sexual function and work productivity. CONCLUSIONS: UUI is associated with numerous comorbid conditions and inflicts a substantial personal burden on many aspects of patients' lives. Healthcare providers should discuss UUI with patients and be aware of the impact of UUI and its associated comorbidities on patients' lives.


Assuntos
Incontinência Urinária/complicações , Acidentes por Quedas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Ansiedade/etiologia , Efeitos Psicossociais da Doença , Depressão/etiologia , Complicações do Diabetes/complicações , Eficiência , Feminino , Fraturas Ósseas/etiologia , Nível de Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Qualidade de Vida , Disfunções Sexuais Fisiológicas/etiologia , Incontinência Urinária/mortalidade , Incontinência Urinária/psicologia , Infecções Urinárias/etiologia , Adulto Jovem
19.
Sci Rep ; 13(1): 5331, 2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-37005450

RESUMO

GDP-amylose transporter protein 1 (SLC35C1) plays an important role in many types of cancer. Therefore, it is clinically important to further investigate the expression profile of SLC35C1 in human tumors to provide new molecular clues for the pathogenesis of glioma. In this study, we performed a comprehensive pan-cancer analysis of SLC35C1 using a series of bioinformatics approaches and validated its differential tissue expression and biological function. The results showed that SLC35C1 was aberrantly expressed in different types of tumors and significantly correlated with overall survival (OS) and progression-free interval (PFI). More importantly, the expression level of SLC35C1 was closely correlated with Tumor Microenvironment (TME), immune infiltration and immune-related genes. In addition, we found that SLC35C1 expression was also closely related to Tumor Mutation Burden (TMB), Microsatellite Instability (MSI) and antitumor drug sensitivity in various cancer types. Functional bioinformatics analysis indicated that SLC35C1 may be involved in multiple signaling pathways and biological processes in glioma. Based on SLC35C1 expression, a risk factor model was found to predict OS of glioma. In addition, in vitro experiments showed that SLC35C1 knockdown significantly inhibited the proliferation, migration and invasive ability of glioma cells, while SLC35C1 overexpression promoted proliferation, migration, invasion and colony formation of glioma cells. Finally, quantitative real-time PCR confirmed that SLC35C1 was highly expressed in gliomas.


Assuntos
Glioma , Humanos , Prognóstico , Glioma/genética , Amilose , Biologia Computacional , Biomarcadores , Microambiente Tumoral/genética , Proteínas de Transporte de Monossacarídeos
20.
Crit Care Med ; 40(11): 3026-33, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22926332

RESUMO

OBJECTIVES: To test the hypothesis that resveratrol would improve cardiac remodeling by inhibiting the detrimental effects of fractalkine. We previously reported that fractalkine exacerbates heart failure. Furthermore, this study sought to determine whether resveratrol targets fractalkine to improve myocardial ischemia and cardiac remodeling. DESIGN: Randomized and controlled laboratory investigation. SETTING: Research laboratory. SUBJECTS: Neonatal rat cardiac cells and C57BL/6 mice. INTERVENTIONS: Cardiac cells were treated with recombinant mouse soluble fractalkine for 24 hrs or pretreated with 25 µM resveratrol. Cardiomyocytes were exposed to anoxia/reoxygenation, H2O2, or pretreatment with resveratrol. Ex vivo murine hearts were perfusioned with soluble fractalkine or pretreated with resveratrol after global ischemia. Mice were subjected to the left coronary artery ligation to induce myocardial infarction and randomized to treatment with resveratrol or vehicle alone for 42 days. MEASUREMENTS AND MAIN RESULTS: In a murine myocardial infarction model, we found that resveratrol increased survival and delayed the progression of cardiac remodeling evaluated by serial echocardiography. At 6 wks, the heart weight/body weight ratio, lung weight/body weight ratio, and old infarct size were significantly smaller in resveratrol-treated mice than in untreated myocardial infarction mice. In cultures of neonatal rat cells, exposure to soluble fractalkine increased the atrial natriuretic peptide expression by cardiomyocytes, matrix metalloproteinase-9 and procollagen expression by fibroblasts, and intercellular adhesion molecule-1 expression by microvascular endothelial cells, while it decreased autophagy in cardiomyocytes. All these effects were blocked by coculture with resveratrol. The methyl thiazolyl tetrazolium assay showed that soluble fractalkine reduced the viability of cultured cardiomyocytes during exposure to anoxia/reoxygenation or H2O2, while pretreatment with resveratrol blocked this effect. Perfusion of ex vivo murine hearts with soluble fractalkine after global ischemia led to an increase of infarct size, which was prevented by pretreatment with resveratrol. CONCLUSION: Resveratrol alleviates the deleterious effects of fractalkine on myocardial ischemia and thus reduces subsequent cardiac remodeling.


Assuntos
Quimiocina CX3CL1/efeitos adversos , Insuficiência Cardíaca/tratamento farmacológico , Isquemia Miocárdica/tratamento farmacológico , Inibidores da Agregação Plaquetária/farmacologia , Estilbenos/uso terapêutico , Animais , Animais Recém-Nascidos , Quimiocina CX3CL1/antagonistas & inibidores , Modelos Animais de Doenças , Eletrocardiografia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Isquemia Miocárdica/fisiopatologia , Distribuição Aleatória , Ratos , Resveratrol , Remodelação Ventricular/efeitos dos fármacos
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