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1.
Medicine (Baltimore) ; 98(40): e17127, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31577703

RESUMO

To investigate the functional connectome alterations in cerebral small-vessel disease (CSVD) patients with thalamus lacunes and its relation to cognitive impairment.This case-control study was approved by the local research ethics committee, and all participants provided informed consent. There were 14 CSVD patients with thalamus lacunes (CSVDw.), 27 without (CSVDwo.), and 34 healthy controls (HC) recruited matched for age, sex, and education to undergo a 3T resting-state functional MR examination. The whole-brain functional connectome was constructed by thresholding the Pearson correlation matrices of 90 brain regions, and the topologic properties were analyzed by using graph theory approaches. Networks were compared between CSVD patients and HC, and associations between network measures and cognitive function were tested.Compared with HC, the functional connectome in CSVDw. patients showed abnormalities at the global level and at the nodal level (P < .05, false discovery rate corrected). The network-based statistics method identified a significantly altered network consisting 6 nodes and 13 connections. Among all the 13 connections, only two connections had significant correlation with episodic memory (EM) and processing speed (PS) respectively (P < .05). The CSVDwo. patients showed no significant network alterations relative to controls (P > .05).The configurations of brain functional connectome in CSVDw. patients were perturbed but not obvious for those without, and correlated with the mild cognitive impairment, especially for EM and PS. This study suggested that lacunes on thalamus played a vital role in mediating the neural functional changes of CSVD patients.


Assuntos
Doenças de Pequenos Vasos Cerebrais/patologia , Disfunção Cognitiva/patologia , Conectoma , Leucoencefalopatias/patologia , Tálamo/patologia , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Doenças de Pequenos Vasos Cerebrais/complicações , Doenças de Pequenos Vasos Cerebrais/diagnóstico por imagem , Disfunção Cognitiva/diagnóstico por imagem , Disfunção Cognitiva/etiologia , Estudos Transversais , Escolaridade , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Leucoencefalopatias/complicações , Masculino , Pessoa de Meia-Idade , Avaliação Nutricional , Índice de Gravidade de Doença , Fatores Sexuais , Tálamo/diagnóstico por imagem
2.
Clin Neuroradiol ; 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399748

RESUMO

PURPOSE: The current study aimed to investigate the associations between diffusion dynamics of ischemic lesions and clinical functional outcome of acute and early subacute stroke. MATERIAL AND METHODS: A total of 80 patients with first ever infarcts in the territory of the middle cerebral artery underwent multi-b-values diffusion-weighted imaging and diffusion kurtosis imaging. Multiple diffusion parameters were generated in postprocessing using different diffusion models. Long-term functional outcome was evaluated with modified Rankin scale (mRS) at 6 months post-stroke. Good functional outcome was defined as mRS score ≤ 2 and poor functional outcome was defined as mRS score ≥ 3. Univariate analysis was used to compare the diffusion parameters and clinical features between patients with poor and good functional outcome. Significant parameters were further analyzed for correlations with functional outcome using partial correlation. RESULTS: In univariate analyses, standard-b-values apparent diffusion coefficient (ADCst) ratio and fractional anisotropy (FA) ratio of acute stroke, ADCst ratio and mean kurtosis (MK) ratio of early subacute stroke were statistically different between patients with poor outcome and good outcome (P < 0.05). When the potential confounding factor of lesion volume was controlled, only FA ratio of acute stroke, ADCst ratio and MK ratio of early subacute stroke remained correlated with the functional outcome (P < 0.05). CONCLUSION: Diffusion dynamics are correlated with the clinical functional outcome of ischemic stroke. This correlation is independent of the effect of lesion volume and is specific to the time period between symptom onset and imaging. More effort is needed to further investigate the predictive value of diffusion-weighted imaging.

3.
Magn Reson Imaging ; 57: 28-33, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30385381

RESUMO

OBJECTIVES: To demonstrate the feasibility of the neurite orientation dispersion and density imaging (NODDI) technique in characterizing the microstructural changes in brain tissues during ischemic stroke and to compare its sensitivity with diffusion tensor imaging (DTI) and diffusion kurtosis imaging (DKI). METHODS: Seventy-one patients with hyperacute/acute/subacute ischemic stroke were enrolled in the study. A multishell diffusion magnetic resonance imaging (dMRI) protocol was performed for each subject. Diffusion data were analyzed using the NODDI and diffusional kurtosis estimator toolboxes. Then, NODDI metrics between the lesions and the contralateral tissues were compared to evaluate their values in ischemic stroke. NODDI metrics among different stroke periods and the correlations between NODDI and the duration since stroke onset were analyzed as well. To compare the NODDI's sensitivity with established diffusion techniques, paired t-tests were performed to determine the absolute percentage changes of diffusion metrics between NODDI and DTI/DKI. RESULTS: Compared with the contralateral tissues, lesions showed significantly increased values of intracellular volume fraction (Vic) and orientation dispersion index (ODI) and decreased values of isotropic volume fraction (Viso). ODI value was significantly different among three periods and showed fair to good positive correlation with the duration since stroke onset (R = 0.450). NODDI metrics showed significantly larger absolute percentage changes than that of DTI and DKI (P < 0.05, respectively). CONCLUSION: NODDI allowed efficient evaluation of microstructural changes in brain tissues during ischemic stroke and showed increased sensitivity compared with DTI and DKI. The possible biophysical mechanisms underlying ischemia could be further elucidated using this advanced diffusion technique.


Assuntos
Isquemia Encefálica/diagnóstico por imagem , Imagem de Difusão por Ressonância Magnética , Imagem de Tensor de Difusão , Neuritos/patologia , Acidente Vascular Cerebral/diagnóstico por imagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/diagnóstico por imagem , Isquemia Encefálica/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Acidente Vascular Cerebral/patologia
4.
Eur J Radiol ; 109: 188-195, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30527302

RESUMO

PURPOSE: To compare the main parameters derived from monoexponential, biexponential and stretched-exponential diffusion-weighted imaging (DWI) and diffusion kurtosis imaging (DKI) with respect to diagnostic performance for tumor grading and proliferation assessment in diffuse astrocytic tumors (DATs). MATERIALS AND METHODS: Fifty-eight pathologically confirmed DAT patients who underwent DWI and DKI on a 3-T scanner were prospectively collected and retrospectively reviewed. Measurements including the apparent diffusion coefficient (ADC), true diffusion coefficient (D), pseudodiffusion coefficient (D*), perfusion fraction (f), distributed diffusion coefficient (DDC), heterogeneity index (α), mean diffusivity (MD), fractional anisotropy (FA), and mean kurtosis (MK) were compared between tumor grades (Ⅱ, Ⅲ, and Ⅳ) by using a Jonckheere-Terpstra test. Receiver operating characteristic (ROC) curves were used to assess the diagnostic efficacy of these parameters. Spearman's rho with the Ki-67 labeling index (LI) was calculated for each parameter. RESULTS: MK values differed significantly between all DAT subtypes and increased with grade. The ADC, D, f, DDC, α and MD values were significantly higher in grade Ⅱ tumors than in grade Ⅲ/Ⅳ tumors. D* values were significantly lower in grade Ⅱ tumors than in grade Ⅳ tumors (all P < 0.05). In discriminating between grade Ⅱ and Ⅲ tumors, α, MK, MD, D and f had significantly greater area under the ROC curve (AUC) values than D* and FA (0.927, 0.901, 0.896, 0.895, and 0.889, respectively vs 0.659 and 0.598, respectively, P < 0.05). In discriminating between grade Ⅲ and Ⅳ tumors, only MK demonstrated acceptable discrimination (AUC = 0.711). MK and D showed a strong correlation with the Ki-67 LI (ρ = 0.791 and -0.789, respectively, P < 0.001). D*, f, MD, ADC, DDC and α showed a moderate correlation (|ρ| ranged from 0.415 to 0.698, P < 0.05). CONCLUSION: MK and D have considerable potential to predict the degree of proliferation of DATs. MK could effectively characterize microstructural changes throughout the malignant transformation of DATs and provided useful complementary information for grading.


Assuntos
Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Adulto , Idoso , Anisotropia , Proliferação de Células , Imagem de Difusão por Ressonância Magnética/métodos , Imagem de Tensor de Difusão/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estudos Prospectivos , Curva ROC , Estudos Retrospectivos , Sensibilidade e Especificidade
5.
Magn Reson Imaging ; 51: 14-19, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29673894

RESUMO

BACKGROUND: Anomalous diffusion model has been introduced and shown to be beneficial in clinical applications. However, only the directionally averaged values of anomalous diffusion parameters were investigated, and the anisotropy of anomalous diffusion remains unexplored. The aim of this study was to demonstrate the feasibility of using anisotropy of anomalous diffusion for differentiating low- and high-grade cerebral gliomas. METHODS: Diffusion MRI images were acquired from brain tumor patients and analyzed using the fractional motion (FM) model. Twenty-two patients with histopathologically confirmed gliomas were selected. An anisotropy metric for the FM-related parameters, including the Noah exponent (α) and the Hurst exponent (H), was introduced and their values were statistically compared between the low- and high-grade gliomas. Additionally, multivariate logistic regression analysis was performed to assess the combination of the anisotropy metric and the directionally averaged value for each parameter. The diagnostic performances for grading gliomas were evaluated using a receiver operating characteristic (ROC) analysis. RESULTS: The Hurst exponent H was more anisotropic in high-grade than in low-grade gliomas (P = 0.015), while no significant difference was observed for the anisotropy of α. The ROC analysis revealed that larger areas under the ROC curves were produced for the combination of α (1) and the combination of H (0.813) compared with the directionally averaged α (0.979) and H (0.594), indicating an improved performance for tumor differentiation. CONCLUSION: The anisotropy of anomalous diffusion can provide distinctive information and benefit the differentiation of low- and high-grade gliomas. The utility of anisotropic anomalous diffusion may have an improved effect for investigating pathological changes in tissues.


Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/patologia , Imagem de Difusão por Ressonância Magnética/métodos , Glioma/diagnóstico por imagem , Glioma/patologia , Adulto , Idoso , Anisotropia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Gradação de Tumores
6.
Nat Prod Bioprospect ; 8(2): 91-95, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29441465

RESUMO

Three new lactones, xylanilyticolides A-C (1-3), were isolated from cultures of the actinomycete Promicromonospora xylanilytica YIM 61515. Their structures were elucidated by 1D and 2D NMR spectroscopic data in conjunction with HRESIMS analysis. Compound 1 exhibited potent cytotoxicities against five human cancer cell lines HL-60, A-549, SMMC-7721, MCF-7 and SW480 with the IC50 values of 3.9, 15.2, 11.2, 5.9, and 4.7 µM, respectively.

7.
Nat Prod Bioprospect ; 8(1): 31-35, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29230718

RESUMO

Three new podocarpane diterpenoids, namely anemhupehins A-C (1-3), together with four known analogues (4-7), have been isolated from aerial parts of Anemone hupehensis. Their structures were characterized based on extensive spectroscopic data. Compounds 1 and 4 showed certain cytotoxicities against human cancer cell lines.

8.
Magn Reson Med ; 78(5): 1944-1949, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28054416

RESUMO

PURPOSE: To demonstrate the capability of the fractional motion (FM) model for describing anomalous diffusion in cerebral gliomas and to assess the potential feasibility of FM for grading these tumors. METHODS: Diffusion MRI images were acquired from brain tumor patients using a special Stejskal-Tanner diffusion sequence with variable diffusion gradient amplitudes and separation times. Patients with histopathologically confirmed gliomas, including astrocytic and oligoastrocytic tumors, were selected. The FM-related parameters, including the Noah exponent ( α), the Hurst exponent ( H), and the memory parameter ( µ=H-1/α), were calculated and compared between low- and high-grade gliomas using a two-sample t-test. The grading performance was evaluated using the receiver operating characteristic analysis. RESULTS: Twenty-two patients were included in the present study. The calculated α, H, and µ permitted the separation of tumor lesions from surrounding normal tissues in parameter maps and helped differentiate glioma grades. Moreover, α showed greater sensitivity and specificity in distinguishing low- and high-grade gliomas compared with the apparent diffusion coefficient. CONCLUSION: The FM model could improve the diagnostic accuracy in differentiating low- and high-grade gliomas. This improved diffusion model may facilitate future studies of neuro-pathological changes in clinical populations. Magn Reson Med 78:1944-1949, 2017. © 2017 International Society for Magnetic Resonance in Medicine.


Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Imagem de Difusão por Ressonância Magnética/métodos , Glioma/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Adulto , Encéfalo/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Movimento , Curva ROC
9.
Int J Clin Exp Med ; 8(11): 21822-5, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26885149

RESUMO

PURPOSE: Pulmonary sequestration (PS) is a rare congenital lung malformation. It is characterized by an abnormal mass of dysplastic lung tissue supplied by an anomalous systemic artery and separated from normal bronchopulmonary tree. Misdiagnosis and inadequate treatment can lead to recurrent pneumonia and fatal hemoptysis. METHODS: We report a 45 years female was diagnosed PS, and performed a brief review about the clinical features, diagnostic strategies, and management options of the PS. RESULTS: Her remarkable symptoms were cough and hemoptysis, the contrast- enhanced computed tomography of the chest revealed a multiloculated cystic solid mass filled with low density lesions and a feeding artery from the descending abdominal aorta to the cystic solid mass was visualized, then the patient suffered a right lower- lobe resection, and the surgery and pathological examination all supported the diagnosis of intralobar sequestration. CONCLUSIONS: Symptomatic patients of the pulmonary sequestration should be treated by surgery to avoid the risk of death due to massive hemoptysis.

10.
J Thorac Oncol ; 9(3): 285-94, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24496003

RESUMO

INTRODUCTION: The aim of this study was to identify anaplastic lymphoma kinase (ALK) rearrangements in lung cancer patient-derived xenograft (PDX) models and to explore their responses to crizotinib. METHODS: Screening of 99 lung cancer PDX models by the NanoString ALK fusion assay identified two ALK-rearranged non-small-cell lung cancer (NSCLC) tumors, including one harboring a previously known echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion and another containing an unknown ALK fusion variant. Expression array, RNA-Seq, reverse transcription polymerase chain reaction, and direct sequencing were then conducted to confirm the rearrangements and to identify the novel fusion partner in the xenograft and/or the primary patient tumor. Finally, pharmacological studies were performed in PDX models to evaluate their responses to ALK inhibitor crizotinib. RESULTS: Two ALK-rearranged NSCLC PDX models were identified: one carried a well-known EML4-ALK variant 3a/b and the other harbored a novel huntingtin interacting protein 1 (HIP1)-ALK fusion gene. Exon 28 of the HIP1 gene located on chromosome 7 was fused to exon 20 of the ALK gene located on chromosome 2. Both cases were clinically diagnosed as squamous cell carcinoma. Compared with the other lung cancer PDX models, both ALK-rearranged models displayed elevated ALK mRNA expression. Furthermore, in vivo efficacy studies demonstrated that, similar to the EML4-ALK-positive model, the HIP1-ALK-containing PDX model was sensitive to treatment with crizotinib. CONCLUSIONS: Discovery of HIP1 as a fusion partner of ALK in NSCLC is a novel finding. In addition, the HIP1-ALK-rearranged tumor is sensitive to treatment with crizotinib in vivo, implicating HIP1-ALKas an oncogenic driver of lung tumorigenesis. Collectively, our results indicate that HIP1-ALK-positive NSCLC may benefit from clinical applications of crizotinib.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Crizotinibe , Proteínas de Ligação a DNA/antagonistas & inibidores , Feminino , Humanos , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Fusão Oncogênica/antagonistas & inibidores , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
11.
Bioorg Med Chem Lett ; 23(10): 3081-7, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23570792

RESUMO

Several potent Aurora kinase inhibitors derived from 5H-benzo[c][1,8]naphthyridin-6-one scaffold were identified. A crystal structure of Aurora kinase A in complex with an initial hit revealed a binding mode of the inhibitor within the ATP binding site and provided insight for structure-guided compound optimization. Subsequent SAR campaign provided a potent and selective pan Aurora inhibitor, which demonstrated potent target modulation and antiproliferative effects in the pancreatic cell line, MIAPaCa-2. Furthermore, this compound inhibited phosphorylation of histone H3 (pHH3) in mouse bone morrow upon oral administration, which is consistent with inhibition of Aurora kinase B activity.


Assuntos
Aurora Quinases/antagonistas & inibidores , Naftiridinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Animais , Aurora Quinases/metabolismo , Linhagem Celular Tumoral , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Histonas/antagonistas & inibidores , Histonas/metabolismo , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Naftiridinas/administração & dosagem , Naftiridinas/síntese química , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/síntese química , Relação Estrutura-Atividade
12.
PLoS One ; 3(12): e3949, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19079609

RESUMO

Prostate cancer is the second leading cause of cancer-related deaths in men. Activation of MAP kinase signaling pathway has been implicated in advanced and androgen-independent prostate cancers, although formal genetic proof has been lacking. In the course of modeling malignant melanoma in a tyrosinase promoter transgenic system, we developed a genetically-engineered mouse (GEM) model of invasive prostate cancers, whereby an activating mutation of BRAF(V600E)--a mutation found in approximately 10% of human prostate tumors--was targeted to the epithelial compartment of the prostate gland on the background of Ink4a/Arf deficiency. These GEM mice developed prostate gland hyperplasia with progression to rapidly growing invasive adenocarcinoma without evidence of AKT activation, providing genetic proof that activation of MAP kinase signaling is sufficient to drive prostate tumorigenesis. Importantly, genetic extinction of BRAF(V600E) in established prostate tumors did not lead to tumor regression, indicating that while sufficient to initiate development of invasive prostate adenocarcinoma, BRAF(V600E) is not required for its maintenance.


Assuntos
Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Androgênios , Animais , Biomarcadores Tumorais/metabolismo , Castração , Linhagem da Célula , Proliferação de Células , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Hiperplasia , Masculino , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Fosfoproteínas/metabolismo , Próstata/enzimologia , Próstata/patologia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transativadores/metabolismo , Transgenes , Urotélio/embriologia , Urotélio/patologia
13.
Cancer Res ; 67(10): 4933-9, 2007 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-17510423

RESUMO

Mutations in the BRAF and KRAS genes occur in approximately 1% to 2% and 20% to 30% of non-small-cell lung cancer patients, respectively, suggesting that the mitogen-activated protein kinase (MAPK) pathway is preferentially activated in lung cancers. Here, we show that lung-specific expression of the BRAF V600E mutant induces the activation of extracellular signal-regulated kinase (ERK)-1/2 (MAPK) pathway and the development of lung adenocarcinoma with bronchioloalveolar carcinoma features in vivo. Deinduction of transgene expression led to dramatic tumor regression, paralleled by dramatic dephosphorylation of ERK1/2, implying a dependency of BRAF-mutant lung tumors on the MAPK pathway. Accordingly, in vivo pharmacologic inhibition of MAPK/ERK kinase (MEK; MAPKK) using a specific MEK inhibitor, CI-1040, induced tumor regression associated with inhibition of cell proliferation and induction of apoptosis in these de novo lung tumors. CI-1040 treatment also led to dramatic tumor shrinkage in murine lung tumors driven by a mutant KRas allele. Thus, somatic mutations in different signaling intermediates of the same pathway induce exquisite dependency on a shared downstream effector. These results unveil a potential common vulnerability of BRAF and KRas mutant lung tumors that potentially affects rational deployment of MEK targeted therapies to non-small-cell lung cancer patients.


Assuntos
Adenocarcinoma Bronquioloalveolar/genética , Adenocarcinoma/genética , Neoplasias Pulmonares/genética , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma/enzimologia , Adenocarcinoma/metabolismo , Adenocarcinoma Bronquioloalveolar/enzimologia , Adenocarcinoma Bronquioloalveolar/metabolismo , Animais , Benzamidas/farmacologia , Modelos Animais de Doenças , Doxiciclina/farmacologia , Genes ras , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/biossíntese
14.
Genesis ; 44(5): 262-7, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16676322

RESUMO

Conditional Cre-mediated recombination has emerged as a robust method of introducing somatic genetic alterations in an organ-specific manner in the mouse. Here, we generated and characterized mice harboring a 4-hydroxytamoxifen (OHT)-inducible Cre recombinase-estrogen receptor fusion transgene under the control of the melanocyte-specific tyrosinase promoter, designated Tyr::CreER(T2). Cre-mediated recombination was induced in melanocytes in a spatially and temporally controlled manner upon administration of OHT and was documented in embryonic melanoblasts, follicular bulb melanocytes, dermal dendritic melanocytes, epidermal melanocytes of tail skin, and in putative melanocyte stem cells located within the follicular bulge. Functional evidence suggestive of recombination in follicular melanocyte stem cells included the presence of Cre-mediated recombination in follicular bulb melanocytes 1 year after topical OHT administration, by which time several hair cycles have elapsed and the melanocytes residing in this location have undergone multiple rounds of apoptosis and replenishment. These Tyr:: CreER(T2) transgenic mice represent a useful resource for the evaluation of melanocyte developmental genetics, the characterization of melanocyte stem cell function and dynamics, and the construction of refined mouse models of malignant melanoma.


Assuntos
Regulação da Expressão Gênica , Integrases , Melanócitos/fisiologia , Camundongos Transgênicos , Recombinação Genética , Animais , Sistema Nervoso Central , Embrião de Mamíferos , Elementos Facilitadores Genéticos , Feminino , Integrases/metabolismo , Melanócitos/química , Melanócitos/citologia , Camundongos , Monofenol Mono-Oxigenase/genética , Gravidez , Regiões Promotoras Genéticas , Receptores Estrogênicos/genética , Células-Tronco/fisiologia , Tamoxifeno/administração & dosagem , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia
15.
J Biol Chem ; 279(25): 26546-54, 2004 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-15024007

RESUMO

Mitchell's (Mitchell, P. (1961) Nature 191, 144-148) chemiosmotic model of energy coupling posits a bulk electrochemical proton gradient (Deltap) as the sole driving force for proton-coupled ATP synthesis via oxidative phosphorylation (OXPHOS) and for other bioenergetic work. Two properties of proton-coupled OXPHOS by alkaliphilic Bacillus species pose a challenge to this tenet: robust ATP synthesis at pH 10.5 that does not correlate with the magnitude of the Deltap and the failure of artificially imposed potentials to substitute for respiration-generated potentials in energizing ATP synthesis at high pH (Krulwich, T. (1995) Mol. Microbiol. 15, 403-410). Here we show that these properties, in alkaliphilic Bacillus pseudofirmus OF4, depend upon alkaliphile-specific features in the proton pathway through the a- and c-subunits of ATP synthase. Site-directed changes were made in six such features to the corresponding sequence in Bacillus megaterium, which reflects the consensus sequence for non-alkaliphilic Bacillus. Five of the six single mutants assembled an active ATPase/ATP synthase, and four of these mutants exhibited a specific defect in non-fermentative growth at high pH. Most of these mutants lost the ability to generate the high phosphorylation potentials at low bulk Deltap that are characteristic of alkaliphiles. The aLys(180) and aGly(212) residues that are predicted to be in the proton uptake pathway of the a-subunit were specifically implicated in pH-dependent restriction of proton flux through the ATP synthase to and from the bulk phase. The evidence included greatly enhanced ATP synthesis in response to an artificially imposed potential at high pH. The findings demonstrate that the ATP synthase of extreme alkaliphiles has special features that are required for non-fermentative growth and OXPHOS at high pH.


Assuntos
Bacillus/enzimologia , Dimaprit/análogos & derivados , Oxigênio/metabolismo , ATPases Translocadoras de Prótons/química , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Western Blotting , Divisão Celular , Membrana Celular/metabolismo , Primers do DNA/química , Dimaprit/química , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Malatos/química , Dados de Sequência Molecular , Fosforilação Oxidativa , Consumo de Oxigênio , Fosforilação , Estrutura Terciária de Proteína , ATPases Translocadoras de Prótons/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Fatores de Tempo
16.
Proc Natl Acad Sci U S A ; 100(18): 10213-8, 2003 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-12917488

RESUMO

The atp operon of alkaliphilic Bacillus pseudofirmus OF4, as in most prokaryotes, contains the eight structural genes for the F-ATPase (ATP synthase), which are preceded by an atpI gene that encodes a membrane protein of unknown function. A tenth gene, atpZ, has been found in this operon, which is upstream of and overlapping with atpI. Most Bacillus species, and some other bacteria, possess atpZ homologues. AtpZ is predicted to be a membrane protein with a hairpin topology, and was detected by Western analyses. Deletion of atpZ, atpI, or atpZI from B. pseudofirmus OF4 led to a requirement for a greatly increased concentration of Mg2+ for growth at pH 7.5. Either atpZ, atpI, or atpZI complemented the similar phenotype of a triple mutant of Salmonella typhimurium (MM281), which is deficient in Mg2+ uptake. atpZ and atpI, separately and together, increased the Mg2+-sensitive 45Ca2+ uptake by vesicles of an Escherichia coli mutant that is defective in Ca2+ and Na+ efflux. We hypothesize that AtpZ and AtpI, as homooligomers, and perhaps as heterooligomers, are Mg2+ transporter, Ca2+ transporter, or channel proteins. Such proteins could provide Mg2+, which is required by ATP synthase, and also support charge compensation, when the enzyme is functioning in the hydrolytic direction; e.g., during cytoplasmic pH regulation.


Assuntos
Bacillus/genética , Genes Bacterianos , Magnésio/metabolismo , Óperon , ATPases Translocadoras de Prótons/genética , Sequência de Aminoácidos , Bacillus/metabolismo , Sequência de Bases , Cálcio/metabolismo , Concentração de Íons de Hidrogênio , Transporte de Íons , Dados de Sequência Molecular , Fenótipo , Regiões Promotoras Genéticas , Transcrição Genética
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