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1.
Nat Commun ; 12(1): 4718, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354069

RESUMO

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.


Assuntos
Fosfatidato Fosfatase/química , Células 3T3-L1 , Adipogenia , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Células HEK293 , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Genética
2.
Gastroenterology ; 154(5): 1494-1508.e13, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29248441

RESUMO

BACKGROUND & AIMS: Activating mutations in KRAS are detected in most pancreatic ductal adenocarcinomas (PDACs). Expression of an activated form of KRAS (KrasG12D) in pancreata of mice is sufficient to induce formation of pancreatic intraepithelial neoplasia (PanINs)-a precursor of PDAC. Pancreatitis increases formation of PanINs in mice that express KrasG12D by promoting acinar-to-ductal metaplasia (ADM). We investigated the role of the transcription factor Krüppel-like factor 5 (KLF5) in ADM and KRAS-mediated formation of PanINs. METHODS: We performed studies in adult mice with conditional disruption of Klf5 (Klf5fl/fl) and/or expression of KrasG12D (LSL-KrasG12D) via CreERTM recombinase regulated by an acinar cell-specific promoter (Ptf1a). Activation of KrasG12D and loss of KLF5 was achieved by administration of tamoxifen. Pancreatitis was induced in mice by administration of cerulein; pancreatic tissues were collected, analyzed by histology and immunohistochemistry, and transcriptomes were compared between mice that did or did not express KLF5. We performed immunohistochemical analyses of human tissue microarrays, comparing levels of KLF5 among 96 human samples of PDAC. UN-KC-6141 cells (pancreatic cancer cells derived from Pdx1-Cre;LSL-KrasG12D mice) were incubated with inhibitors of different kinases and analyzed in proliferation assays and by immunoblots. Expression of KLF5 was knocked down with small hairpin RNAs or CRISPR/Cas9 strategies; cells were analyzed in proliferation and gene expression assays, and compared with cells expressing control vectors. Cells were subcutaneously injected into flanks of syngeneic mice and tumor growth was assessed. RESULTS: Of the 96 PDAC samples analyzed, 73% were positive for KLF5 (defined as nuclear staining in more than 5% of tumor cells). Pancreata from Ptf1a-CreERTM;LSL-KrasG12D mice contained ADM and PanIN lesions, which contained high levels of nuclear KLF5 within these structures. In contrast, Ptf1a-CreERTM;LSL-KrasG12D;Klf5fl/fl mice formed fewer PanINs. After cerulein administration, Ptf1a-CreERTM;LSL-KrasG12D mice formed more extensive ADM than Ptf1a-CreERTM;LSL-KrasG12D;Klf5fl/fl mice. Pancreata from Ptf1a-CreERTM;LSL-KrasG12D;Klf5fl/fl mice had increased expression of the tumor suppressor NDRG2 and reduced phosphorylation (activation) of STAT3, compared with Ptf1a-CreERTM;LSL-KrasG12D mice. In UN-KC-6141 cells, PI3K and MEK signaling increased expression of KLF5; a high level of KLF5 increased proliferation. Cells with knockdown of Klf5 had reduced proliferation, compared with control cells, had reduced expression of ductal markers, and formed smaller tumors (71.61 ± 30.79 mm3 vs 121.44 ± 34.90 mm3 from control cells) in flanks of mice. CONCLUSION: Levels of KLF5 are increased in human PDAC samples and in PanINs of Ptf1a-CreERTM;LSL-KrasG12D mice, compared with controls. KLF5 disruption increases expression of NDRG2 and reduces activation of STAT3 and reduces ADM and PanINs formation in mice. Strategies to reduce KLF5 activity might reduce progression of acinar cells from ADM to PanIN and pancreatic tumorigenesis.


Assuntos
Carcinoma in Situ/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Carcinoma in Situ/genética , Carcinoma in Situ/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Ceruletídeo , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Genes ras , Humanos , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Pancreatite/induzido quimicamente , Pancreatite/genética , Pancreatite/metabolismo , Pancreatite/patologia , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Carga Tumoral
3.
Opt Express ; 24(10): 10547-55, 2016 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-27409877

RESUMO

We present a field-calibrated electro-optic sensing system for measurement of the electric field radiating from a high-power vacuum oscillator at ~95 GHz. The intense electric field is measured in absolute scale via two probe-calibration steps, associated with a photonic heterodyne scheme. First, a micro-electro-optic probe, fabricated to less than one-tenth the oscillation wavelength scale to minimize field-perturbation due to the probe, is placed on the aperture of a field-calculable WR-10 waveguide to calibrate the probe in V/m scale. Then, using this arrangement as a calibrated reference probe at the first-tier position, another probe-bulkier, and thus more robust and sensitive but not accessible to the aperture-is calibrated at the second-tier position away from the waveguide aperture. This two-tier calibrated probe was utilized to diagnose the sub-MV/m scale of intense electric fields and emissions from a high-power W-band gyrotron. The experimental results obtained proved consistent with calculated analytical results-verifying the efficacy of the developed system.

4.
Clin Exp Otorhinolaryngol ; 1(1): 20-3, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19434257

RESUMO

OBJECTIVES: Cross-hatching incisions have been considered mandatory for correcting cartilaginous septal deviation. We evaluated the clinical outcome of septoplasty without cross-hatching incisions to determine the necessity for making septal cartilage incisions. METHODS: THE RECONSTRUCTED SEPTAL COMPONENTS DURING SEPTOPLASTY WERE CATEGORIZED INTO FOUR ANATOMICAL AREAS: vomer, maxillary crest, perpendicular plate of ethmoid (PPE) and septal cartilage (the area for cross-hatching incisions). During septoplasty, we attempted to complete the surgery only by removing or fracturing the bony part of the septum without cross-hatching incisions on the cartilage. Only in the cases that the deviation was not immediately corrected, the cross-hatching incisions were made onto the cartilage at the end of the procedure. We analyzed the frequency of manipulating the septal components. The changes of symptoms were evaluated using a modified nasal obstruction symptom evaluation (NOSE) scale and a visual analog scale (VAS) preoperatively, 1 and 3 months after the surgery. RESULTS: Seventy five percents of the deviated septums were immediately corrected only by removing or fracturing of the bony septal components. In decreasing order of frequency, the sepal components for correcting septal deviation were the vomer (59%), maxillary crest (49%), septal cartilage (cross-hatching only: 25%) and PPE (15%). The modified NOSE scale and the VAS demonstrated significant improvement of the nasal symptoms postoperatively (P<0.05). CONCLUSION: Most of septal deviations could be corrected by manipulating only the bony septum. The results of this procedure were not different from conventional septoplasty with cross-hatching incisions. Our data suggest cross-hatching incisions during septoplasty might have been overemphasized and that the main cause for cartilaginous deviation may be the extrinsic forces that are generated by the neighboring bony structures.

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