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Cell Treatment (cell + treatment)
Selected AbstractsRhoA/ROCK and Cdc42 regulate cell-cell contact and N-cadherin protein level during neurodetermination of P19 embryonal stem cellsDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2004Isabel Laplante Abstract RhoGTPases regulate actin-based signaling cascades and cellular contacts. In neurogenesis, their action modulates cell migration, neuritogenesis, and synaptogenesis. Murine P19 embryonal stem cells differentiate to neurons upon aggregation in the presence of retinoic acid, and we previously showed that RhoA and Cdc42 RhoGTPases are sequentially up-regulated during neuroinduction, suggesting a role at this very early developmental stage. In this work, incubation of differentiating P19 cells with C3 toxin resulted in decreased aggregate cohesion and cadherin protein level. In contrast, C3 effects were not observed in cells overexpressing recombinant dominant active RhoA. On the other hand, C3 did not affect cadherin in uninduced cells and their postmitotic neuronal derivatives, respectively expressing E- and N-cadherin. RhoA is thus influential on cell aggregation and cadherin expression during a sensitive time window that corresponds to the switch of E- to N-cadherin. Cell treatment with Y27632 inhibitor of Rho-associated-kinase ROCK, or advanced overexpression of Cdc42 by gene transfer of a constitutively active form of the protein reproduced C3 effects. RhoA-antisense RNA also reduced cadherin level and the size of cell aggregates, and increased the generation of fibroblast-like cells relative to neurons following neuroinduction. Colchicin, a microtubule disrupter, but not cytochalasin B actin poison, importantly decreased cadherin in neurodifferentiating cells. Overall, our results indicate that the RhoA/ROCK pathway regulates cadherin protein level and cell-cell interactions during neurodetermination, with an impact on the efficiency of the process. The effect on cadherin seems to involve microtubules. The importance of correct timing of RhoA and Cdc42 functional expression in neurogenesis is also raised. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 289,307, 2004 [source] Removal of benzo(a)pyrene diol epoxide (BPDE)-DNA adducts as a measure of DNA repair capacity in lymphoblastoid cell lines from sisters discordant for breast cancerENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2002Grazyna Motykiewicz Abstract The mutagen sensitivity assay is one of the approaches used to investigate individual DNA repair capacity. This method is based on the premise that after in vitro treatment with a test mutagen, DNA from subjects with defective repair will be more damaged than DNA from those with an efficient repair system. However, very little is known about unmeasured processes that occur between cell treatment and final assessment of DNA damage. To develop a more precise assay, we modified the traditional mutagen sensitivity assay to also include measurement of DNA damage after culturing cells in the absence of mutagen. First, we treated apparently normal and xeroderma pigmentosum lymphoblastoid cell lines with various doses of benzo(a)pyrene diol epoxide (BPDE) and harvested cells at different time points. A polyclonal antiserum against BPDE-DNA was used to quantitate levels of adducts by immunoslot-blot and immunohistochemistry. Selected conditions included treatment with 10 ,M BPDE, a 4-hr culture in mutagen-free medium, and immunohistochemical measurement of BPDE-DNA adducts. The method was then applied in a pilot study to 50 lymphoblastoid lines from sisters discordant for breast cancer. There was no significant difference between cases and controls in the level of BPDE-DNA adducts in lymphoblasts harvested immediately after BPDE treatment. However, after a 4-hr culture in mutagen-free medium, the level of adducts was significantly higher (P = 0.006) among cases than in controls. There was a two-fold increase in mean adduct removal in lines from nonaffected as compared to affected sisters (44% and 22% decrease, respectively). DNA repair capacity was predictive of case status (P = 0.04) in logistic regression analysis. This method, which can be easily applied to large numbers of samples, should be useful in studies to investigate the role of DNA repair in cancer risk. Environ. Mol. Mutagen. 40:93,100, 2002. © 2002 Wiley-Liss, Inc. [source] Echistatin inhibits pp125FAK autophosphorylation, paxillin phosphorylation and pp125FAK,paxillin interaction in fibronectin-adherent melanoma cellsFEBS JOURNAL, Issue 16 2000Rossella Della Morte Echistatin, a snake-venom RGD-containing protein, was previously shown to disrupt cell-matrix adhesion by a mechanism that involves the reduction of pp125FAK tyrosine phosphorylation levels. The aim of this study was to establish the sequence of events downstream pp125FAK dephosphorylation that could be responsible for echistatin-induced disassembly of actin cytoskeleton and focal adhesions in fibronectin-adherent B16-BL6 melanoma cells. The results obtained show that echistatin induces a decrease of both autophosphorylation and kinase activity of pp125FAK. One hour of cell exposure to echistatin caused a 39% decrease of pp125FAK Tyr397 phosphorylation and a 31% reduction of pp125FAK autophosphorylation activity as measured by immune-complex kinase assay. Furthermore, 1 h of cell treatment by echistatin produced a 63% decrease of paxillin phosphorylation, as well as a reduction in the amount of paxillin bound to pp125FAK. Immunofluorescence analysis of echistatin treated cells showed the concomitant disappearance of both paxillin and pp125FAK from focal adhesions. The reduction of paxillin phosphorylation may represent a critical step in the pathway by which disintegrins exert their biological activity, including the inhibition of experimental metastasis in vivo. [source] Apoptosis induction by interleukin-2-activated cytotoxic lymphocytes in a squamous cell carcinoma cell line and Daudi cells , involvement of reactive oxygen species-dependent cytochrome c and reactive oxygen species-independent apoptosis-inducing factorsIMMUNOLOGY, Issue 2 2003Tetsuya Yamamoto Summary Investigation of the induction of apoptosis by cytotoxic lymphocytes has mainly focused on the signalling associated with Fas and its adaptor proteins. The signal pathway via mitochondria, however, has not been sufficiently elucidated in cytotoxic lymphocyte-induced apoptosis. We examined the release of mitochondrial proapoptotic factors by lymphokine-activated killer (LAK) cells in two cell lines. LAK cell-induced DNA fragmentation of the target cells was suppressed to approximately 50% of control levels by the addition of neutralizing monoclonal antibody to Fas and a granzyme B inhibitor. When intracellular reactive oxygen species (ROS) were scavenged, the LAK cell-induced DNA fragmentation was decreased to approximately 60% of the non-treated cell level. Co-cultivation of Daudi cells with LAK cells increased cytosolic and mitochondrial ROS levels. Activation of procaspase-3 and apoptosis by treatment of oral squamous cell carcinoma cells (OSC) with LAK cells was partially inhibited by pretreatment of OSC cells with ROS scavengers and mitochondrial complex inhibitors. Furthermore, cytochrome c and apoptosis-inducing factor (AIF) were released from mitochondria by OSC cell treatment with supernatants of LAK cells. The supernatant-induced cytochrome c release was suppressed by mitochondrial complex inhibitors, but the inhibitors did not inhibit the release of AIF. These results indicate that LAK cells induce target cell apoptosis via not only the Fas/Fas ligand system and granzyme B, but also ROS-dependent cytochrome c and ROS-independent AIF release. [source] RANKL Treatment Releases the Negative Regulation of the Poly(ADP-Ribose) Polymerase-1 on Tcirg1 Gene Expression During Osteoclastogenesis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2006Guillaume E Beranger Abstract The Tcirg1 gene encodes the osteoclast-specific a3 isoform of the V-ATPase a subunit. Using the mouse osteoclastic model RAW264.7 cells, we studied Tcirg1 gene expression, and we identified PARP-1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis. Introduction: The TCIRG1 gene encodes the a3 isoform of the V-ATPase a subunit, and mutations at this locus account for ,60% of infantile malignant osteopetrosis cases. Using RAW264.7 cells as an osteoclastic differentiation model, we undertook a transcriptional study of the mouse Tcirg1 gene focused on the 4-kb region upstream of the transcription starting point. Materials and Methods: The promoter activity of serial-deletion fragments of the Tcirg1 gene promoter was monitored throughout the RAW264.7 cell differentiation process. We next performed EMSA, UV cross-linking, affinity purification, mass spectrometry analysis, gel supershift, and siRNA transfection experiments to identify the factor(s) interacting with the promoter. Results: The ,3946/+113 region of the mouse Tcirg1 gene displayed a high basal promoter activity, which was enhanced by RANKL treatment of RAW264.7 cells. Constructs deleted up to ,1589 retained this response to RANKL. A deletion up to ,1402 induced a 3-fold enhancement of the basal activity, whereas RANKL response was not affected. EMSA experiments led us to identify within the ,1589/,1402 region, a 10-nucleotide sequence, which bound a nuclear protein present in nondifferentiated RAW264.7 cells. This interaction was lost using nuclear extracts derived from RANKL-treated cells. Affinity purification followed by mass spectrometry analysis and gel supershift assay allowed the identification of poly(ADP-ribose) polymerase-1 (PARP-1) as this transcriptional repressor, whereas Western blot experiments revealed the cleavage of the DNA-binding domain of PARP-1 on RANKL treatment. Finally, both PARP-1 depletion after siRNA transfection and RAW264.7 cell treatment by an inhibitor of PARP-1 activity induced an increase of a3 mRNA expression. Conclusions: We provide evidence that the basal transcription activity of the Tcirg1 gene is negatively regulated by the binding of PARP-1 protein to its promoter region in mouse pre-osteoclast. On RANKL treatment, PARP-1 protein is cleaved and loses its repression effect, allowing an increase of Tcirg1 gene expression that is critical for osteoclast function. [source] Butterfat fatty acids differentially regulate growth and differentiation in Jurkat T-cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2005Paolo Bergamo Abstract Synthetic Conjugated Linoleic Acid mixture (CLA; c9,t11; t10,c12-18:2) has been previously shown to inhibit growth, and enhance apoptosis and IL-2 mRNA synthesis in human lymphoblastic Jurkat T-cells. In this study, two different butterfat types were evaluated and compared for their effects on Jurkat cell viability, oxidative stress, pro-apoptotic activity, and cytokine synthesis: the conventionally produced butterfat (CBF), and organic butterfat (OBF) containing significantly higher amounts of c9,t11 (Rumenic Acid, RA), trans-vaccenic acid (VA; t11-18:1), ,-linolenic acid (ALA), and lower levels of linoleic acid (LA). Results from cell treatment with both butterfat mixtures showed comparable oxidative stress (superoxide production, intracellular GSH depletion,and lipid peroxides yield), NADPH oxidase activation, cytotoxicity (LDH release), and IL-2 transcript level, whereas the effects of enhanced growth-inhibitory and pro-apoptotic activities were associated with OBF treatment. To then investigate each butterfat-induced effect caused by RA, VA, LA, and ALA, cells were exposed to synthetic FA concentrations similar to those from the different butterfats. Higher oxidative stress (superoxide production, intracellular GSH depletion) was induced by ,-linolenic (ALA) and linoleic (LA) incubation (P,<,0.01) and superoxide production was suppressed by specific PKC, inhibitor (Gö 6976) and linked to increased toxicity and IL-2 synthesis inhibition. By contrast, cell treatment with RA increased apoptosis and IL-2 synthesis. These results suggest that a supply of ALA and LA is responsible for BF-induced oxidative stress via PKC,-NADPH oxidase pathway, and that enhanced antiproliferative effects in OBF treated cells is essentially determined by RA-induced pro-apoptotic activity. © 2005 Wiley-Liss, Inc. [source] Screening for GALC to make neonatal diagnosis and initial neonatal stem cell treatment with umbilical cord bloodPEDIATRIC TRANSPLANTATION, Issue 2 2003Patricia A. Galvin-Parton MD No abstract is available for this article. [source] Global Model of He/O2 and Ar/O2 Atmospheric Pressure Glow DischargesPLASMA PROCESSES AND POLYMERS, Issue 6 2008Ganyoung Park Abstract Atmospheric pressure glow discharges (APGDs) have widespread applications, including sterilization, cancer cell treatment, deposition, and surface modification due to their rather simple configurations, thanks to no need for vacuum system and their great capability to generate reactive species such as radical oxygen species. Helium and argon are widely used as feeding gases, achieving stable operations for wide ranges of parameters in atmospheric pressure, and oxygen is added into these gases to generate more reactive oxygen species (ROS), which play a significant role in sterilization. As the measurements of species densities and electron temperature in APGDs are difficult, we have developed the zero-dimensional global model of He/O2 and Ar/O2 APGDs to calculate the densities of several kinds of species and electron temperature. It was shown that even though small fraction of oxygen less than 1% was added to helium or argon, electrons dissipated most of their energy through collisions with oxygen molecules rather than helium or argon atoms. The densities of electron, atomic oxygen, and ozone of Ar/O2 were higher than those of He/O2; however, the electron temperature of He/O2 was higher than that of Ar/O2. When the pulsed power is applied, the time-averaged electron temperature for the shorter pulse period and the larger duty ratio increased, and the electron density decreased as the duty ratio increased. [source] Enzymatic production of ,- D -glucose-1-phosphate from trehaloseBIOTECHNOLOGY JOURNAL, Issue 9 2010Jef Van der Borght Abstract ,- D -Glucose-1-phosphate (,Glc1P) is an efficient glucosyl donor for both enzymatic and chemical glycosylation reactions but is currently very costly and not available in large amounts. This article provides an efficient production method of ,Glc1P from trehalose and phosphate using the thermostable trehalose phosphorylase from Thermoanaerobacter brockii. At the process temperature of 60°C, Escherichia coli expression host cells are lysed and cell treatment prior to the reaction is, therefore, not required. In this way, the theoretical maximum yield of 26% could be easily achieved. Two different purification strategies have been compared, anion exchange chromatography or carbohydrate removal by treatment with trehalase and yeast, followed by chemical phosphate precipitation. In a next step, ,Glc1P was precipitated with ethanol but this did not induce crystallization, in contrast to what is observed with other glycosylphosphates. After conversion of the product to its cyclohexylammonium salt, however, crystals could be readily obtained. Although both purification methods were quantitative (>99% recovery), a large amount of product (50%) was lost during crystallization. Nevertheless, a production process for crystalline ,Glc1P is now available from the cheap substrates trehalose and inorganic phosphate. [source] | |||||||||||||