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Lung endothelial cell proliferation with decreased shear stress is mediated by reactive oxygen species 总被引:4,自引:0,他引:4
Milovanova T Chatterjee S Manevich Y Kotelnikova I Debolt K Madesh M Moore JS Fisher AB 《American journal of physiology. Cell physiology》2006,290(1):C66-C76
Acute cessation of flow (ischemia) leads to depolarization of the endothelial cell (EC) membrane mediated by KATP channels and followed by production of reactive oxygen species (ROS) from NADPH oxidase. We postulated that ROS are a signal for initiating EC proliferation associated with the loss of shear stress. Flow cytometry was used to identify proliferating CD31-positive pulmonary microvascular endothelial cells (mPMVECs) from wild-type, Kir6.2/, and gp91phox/ mice. mPMVECs were labeled with PKH26 and cultured in artificial capillaries for 72 h at 5 dyn/cm2 (flow adaptation), followed by 24 h of stop flow or continued flow. ROS production during the first hour of ischemia was markedly diminished compared with wild-type mice in both types of gene-targeted mPMVECs. Cell proliferation was defined as the proliferation index (PI). After 72 h of flow, >98% of PKH26-labeled wild-type mPMVECs were at a single peak (PI 1.0) and the proportion of cells in the S+G2/M phases were at 5.8% on the basis of cell cycle analysis. With ischemia (24 h), PI increased to 2.5 and the ratio of cells in S+G2/M phases were at 35%. Catalase, diphenyleneiodonium, and cromakalim markedly inhibited ROS production and cell proliferation in flow-adapted wild-type mPMVECs. Significant effects of ischemia were not observed in Kir6.2/ and gp91phox/ cells. ANG II activation of NADPH oxidase was unaffected by KATP gene deletion. Thus loss of shear stress in flow-adapted mPMVECs results in cell division associated with ROS generated by NADPH oxidase. This effect requires a functioning cell membrane KATP channel. cell signaling; ischemia; mechanotransduction; KATP channels; NADPH oxidase 相似文献
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Milovanova T Chatterjee S Hawkins BJ Hong N Sorokina EM Debolt K Moore JS Madesh M Fisher AB 《Biochimica et biophysica acta》2008,1783(10):1866-1875
Abrupt cessation of flow representing the acute loss of shear stress (simulated ischemia) to flow-adapted pulmonary microvascular endothelial cells (PMVEC) leads to reactive oxygen species (ROS) generation that signals for EC proliferation. We evaluated the role of caveolin-1 on this cellular response with mouse PMVEC that were preconditioned for 72 h to laminar flow at 5 dyn/cm(2) followed by stop of flow ("ischemia"). Preconditioning resulted in a 2.7-fold increase in cellular expression of K(ATP) (K(IR) 6.2) channels but no change in expression level of caveolin-1, gp91(phox), or MAP kinases. The initial response to ischemia in wild type cells was cell membrane depolarization that was abolished by gene targeting of K(IR) 6.2. The subsequent response was increased ROS production associated with activation of NADPH oxidase (NOX2) and then phosphorylation of MAP kinases (Erk, JNK). After 24 h of ischemia in wild type cells, the cell proliferation index increased 2.5 fold and the % of cells in S+G(2)/M phases increased 6-fold. This signaling cascade (cell membrane depolarization, ROS production, MAP kinase activation and cell proliferation) was abrogated in caveolin-1 null PMVEC or by treatment of wild type cells with filipin. These studies indicate that caveolin-1 functions as a shear sensor in flow-adapted EC resulting in ROS-mediated cell signaling and endothelial cell proliferation following the abrupt reduction in flow. 相似文献
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Jonni Koia Richard Moyle Caroline Hendry Lionel Lim José Ramón Botella 《Plant molecular biology》2013,81(4-5):327-336
The availability of a variety of promoter sequences is necessary for the genetic engineering of plants, in basic research studies and for the development of transgenic crops. In this study, the promoter and 5′ untranslated regions of the evolutionally conserved protein translation factor SUI1 gene and ribosomal protein L36 gene were isolated from pineapple and sequenced. Each promoter was translationally fused to the GUS reporter gene and transformed into the heterologous plant system Arabidopsis thaliana. Both the pineapple SUI1 and L36 promoters drove GUS expression in all tissues of Arabidopsis at levels comparable to the CaMV35S promoter. Transient assays determined that the pineapple SUI1 promoter also drove GUS expression in a variety of climacteric and non-climacteric fruit species. Thus the pineapple SUI1 and L36 promoters demonstrate the potential for using translation factor and ribosomal protein genes as a source of promoter sequences that can drive constitutive transgene expression patterns. 相似文献
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Edward A Stadtmauer Donald E Tsai Cheryl J Sickles Rosemarie Mick Selina M Luger David L Porter Patricia A Mangan Lynn M Schuchter Stephen J Schuster Elwyn Y Loh Deborah A Magee Robert A Sachs Mark E Wall Jonni Moore Gordon P Buzby Ellen Zaleta Malek Kamoun Leslie E Silberstein 《Cancer immunology, immunotherapy : CII》1999,16(4):279-288
The purpose of this study was to determine the efficacy, engraftment kinetics, effect of bone marrow tumor contamination,
and safety of high-dose therapy and granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood progenitor cell
(PBPC) support for patients with responding metastatic breast cancer. Forty two patients underwent G-CSF (10 μg/kg) stimulated
PBPC harvest. PBPC and bone marrow aspirates were analyzed by histologic and immunocytochemical methods for tumor contamination.
Thirty-seven patients received high-dose therapy consisting of cyclophosphamide 6 g/m2, thiotepa 500 mg/m2, and carboplatin 800 mg/m2 (CTCb) given as an infusion over 4 d followed by PBPC reinfusion and G-CSF (5 μg/kg) support. No transplant related deaths
or grade 4 toxicity was recorded. CD34+ cells/kg infused was predictive of neutrophil and platelet recovery. With a median
follow-up of 38 months, three year survival was 44% with relapse-free survival of 19%. Histological bone marrow involvement,
found in 10 patients, was a negative prognostic factor and was associated with a median relapse-free survival of 3.5 months.
Tumor contamination of PBPC by immunohistochemical staining was present in 22.5% of patients and found not to be correlated
with decreased survival. G-CSF stimulated PBPC collection followed by a single course of high dose chemotherapy and stem cell
infusion with G-CSF stimulated marrow recovery leads to rapid, reliable engraftment with low toxicity and promising outcome
in women with responding metastatic breast cancer. 相似文献
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Joseph D. Tario Jr. Kristen Humphrey Andrew D. Bantly Katharine A. Muirhead Jonni S. Moore Paul K. Wallace 《Journal of visualized experiments : JoVE》2012,(70)
Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types
in vitro
and
in vivo
.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
- stem and progenitor cell quiescence, proliferation and/or differentiation6-8
- antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and
- immune regulatory and effector cell function1,18-21.
- Failure to achieve bright, uniform, reproducible labeling . This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.
- Suboptimal fluorochrome combinations and/or failure to include critical compensation controls . Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.
- Failure to obtain a good fit with peak modeling software . Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.
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Richard L. Moyle Jonni H. Koia Julia Vrebalov James Giovannoni Jose R. Botella 《Plant molecular biology》2014,86(4-5):395-407
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PineappleDB: An online pineapple bioinformatics resource 总被引:1,自引:0,他引:1
Richard?L?Moyle Mark?L?Crowe Jonni?Ripi-Koia David?J?Fairbairn José?R?BotellaEmail author 《BMC plant biology》2005,5(1):21
Background
A world first pineapple EST sequencing program has been undertaken to investigate genes expressed during non-climacteric fruit ripening and the nematode-plant interaction during root infection. Very little is known of how non-climacteric fruit ripening is controlled or of the molecular basis of the nematode-plant interaction. PineappleDB was developed to provide the research community with access to a curated bioinformatics resource housing the fruit, root and nematode infected gall expressed sequences. 相似文献10.
Mohler ER Fang Y Shaffer RG Moore J Wilensky RL Parmacek M Levitan I 《Biochemical and biophysical research communications》2007,358(1):317-324
OBJECTIVE: Inwardly-rectifying K(+) (Kir) channels are responsible for maintaining membrane potentials in a variety of cell types including endothelial cells where they modulate endothelium-dependent vasorelaxation. The goal of this study is to determine the functional expression of Kir channels in porcine bone marrow-derived side population (BM-SP) cells that demonstrate phenotypes of endothelial progenitor cells (EPCs). We further asses the hypercholesterolemia sensitivity of Kir channels in BM-SP cells, which may play a key role in hypercholesterolemia-mediated regulation of EPCs. METHODS: To assess the effect of hypercholesterolemia on Kir channels in BM-SP, Kir currents were recorded in SP cells sorted from the bone marrow of healthy or hypercholesterolemic animals. RESULTS: We found Kir channels constitute the major conductance in porcine bone marrow-derived side population (BM-SP) cells. These cells are defined by their efficiency of Hoechst dye efflux and have been reported to differentiate into multiple cell lineages including endothelium in vivo. We demonstrate here that porcine BM-SP cells differentiate to an endothelial lineage (CD31(+), vWF(+)) supporting the hypothesis that these cells are endothelial progenitor cells. Also, BM-SP cells express Kir with biophysical properties recapitulating those in mature endothelial cells, but with a much higher current density. Flow cytometric (FACS) analysis indicated that the number of SP cells was unaffected by hypercholesterolemia. However, hypercholesterolemia significantly inhibited Kir channels in BM-SP cells. CONCLUSIONS: We successfully demonstrate that BM side population cells represent an origin of endothelial progenitor cells. This study further shows, for the fist time, that the functional expression of Kir channels in bone marrow (BM)-derived SP. Moreover, we demonstrate that hypercholesterolemia condition significantly suppresses the Kir channels in BM-SP cells, suggesting that hypercholesterolemia-mediated regulation of Kir channels may be an important factor not only in dysfunction of mature endothelium but also in dysfunction of BM-SP cells. 相似文献