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C3G (RapGEF1), a regulator of actin dynamics promotes survival and myogenic differentiation of mouse mesenchymal cells. - Biochimica et biophysica acta
RapGEF1 (C3G) is a ubiquitously expressed protein that is essential for mammalian embryonic development. We have shown earlier that C3G regulates cytoskeletal dynamics and is required for neuronal differentiation. To determine if C3G plays a wider role in differentiation of multiple tissue types, we examined its role in skeletal muscle differentiation using the model system of C2C12 cells in culture. C3G protein is highly expressed in mouse skeletal muscle and its transcript and protein levels increase as C2C12 cells are induced to differentiate. Increase in C3G was predominantly seen in the nuclei of myotubes. Ectopic expression of C3G promoted myotube formation when cells were cultured in growth as well as differentiation medium and, enhanced MHC levels were associated with C3G expression. C3G induced differentiation required its catalytic and protein interaction domains and was dependent on the function of cellular R-Ras. Knockdown of cellular C3G using small hairpin RNA reduced expression of muscle specific markers and Î²-catenin, resulting in impaired differentiation. Disabling C3G function also resulted in enhanced cell death suggesting that cellular C3G is required for cell survival. In cells grown in growth medium, over-expressed C3G increased Akt activity, and C3G knockdown reduced it. C3G expression also suppressed cyclin D1 levels, and induced p27 expression, molecules involved in regulating cell proliferation. Endogenous C3G localizes to focal adhesions in myotubes and C3G expressing cells show distinct stress fibers, elongation and parallel alignment. Expression of a dominant negative construct of C3G, disrupts actin cytoskeleton and formation of focal adhesions resulting in detachment of cells from the substratum and inhibition of differentiation. Our results provide evidence that C3G plays an important role in myogenic differentiation by coordinating cell cycle exit, actin dynamics and survival signaling.Copyright Â© 2015 Elsevier B.V. All rights reserved.
A Multivariate Computational Method to Analyze High-Content RNAi Screening Data. - Journal of biomolecular screening
High-content screening (HCS) using RNA interference (RNAi) in combination with automated microscopy is a powerful investigative tool to explore complex biological processes. However, despite the plethora of data generated from these screens, little progress has been made in analyzing HC data using multivariate methods that exploit the full richness of multidimensional data. We developed a novel multivariate method for HCS, multivariate robust analysis method (M-RAM), integrating image feature selection with ranking of perturbations for hit identification, and applied this method to an HC RNAi screen to discover novel components of the DNA damage response in an osteosarcoma cell line. M-RAM automatically selects the most informative phenotypic readouts and time points to facilitate the more efficient design of follow-up experiments and enhance biological understanding. Our method outperforms univariate hit identification and identifies relevant genes that these approaches would have missed. We found that statistical cell-to-cell variation in phenotypic responses is an important predictor of hits in RNAi-directed image-based screens. Genes that we identified as modulators of DNA damage signaling in U2OS cells include B-Raf, a cancer driver gene in multiple tumor types, whose role in DNA damage signaling we confirm experimentally, and multiple subunits of protein kinase A.Â© 2015 Society for Laboratory Automation and Screening.
Quantitative Measurements of HO2 and other products of n-butane oxidation (H2O2, H2O, CH2O, and C2H4) at elevated temperatures by direct coupling of a jet-stirred reactor with sampling nozzle and cavity ring-down spectroscopy (cw-CRDS). - Journal of the American Chemical Society
For the first time quantitative measurements of the hydroperoxyl radical (HO2) in a jet-stirred reactor were performed thanks to a new experimental setup involving fast sampling and near-infrared cavity ring-down spectroscopy at low pressure. The experiments were performed at atmospheric pressure and over a range of temperatures (550-900 K) with n-butane, the simplest hydrocarbon fuel exhibiting cool flame oxidation chemistry which represents a key process for the auto-ignition in internal combustion engines. The same technique was also used to measure H2O2, H2O, CH2O, and C2H4 under the same conditions. This new setup brings new scientific horizons for characterizing complex reactive systems at elevated temperatures. Measuring HO2 formation from hydrocarbon oxidation is extremely important in determining the propensity of a fuel to follow chain-termination pathways from R + O2 compared to chain branching (leading to OH), helping to constrain and better validate detailed chemical kinetics models.
The high prevalence of serine protease autotransporters of Enterobacteriaceae (SPATEs) in Escherichia coli causing neonatal septicemia. - European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology
Serine protease autotransporters of Enterobacteriaceae (SPATEs) are secreted proteins demonstrating diverse virulence functions. The distribution of SPATEs is studied among diarrheagenic and extraintestinal pathogenic Escherichia coli. However, the contribution of SPATEs to the virulence of neonatal septicemic Escherichia coli (NSEC) has not yet been elucidated. This study was undertaken to evaluate the prevalence and phylogenetic distribution of different subtypes of SPATEs among NSEC. The presence of virulence factors and subtypes of SPATEs among different E. coli isolates was determined by polymerase chain reaction (PCR). E. coli phylogrouping was done by triplex PCR. Clonality of the isolates was assessed by pulsed-field gel electrophoresis (PFGE). The presence of SPATEs was significantly higher among the septicemic isolates (89 %) than the fecal (7.5 %) and environmental isolates (2.5 %). Vat (vacuolating autotransporter toxin) and Sat (secreted autotransporter toxin) were found to be the two most predominant SPATEs. The incidence of SPATEs was high in septicemic isolates of phylogroups A and B1 (87 %), lacking other virulence factors. The high prevalence of SPATEs in the non-B2 phylogroups of septicemic isolates in comparison with fecal and environmental isolates indicates an association of SPATEs with NSEC. The NSEC isolates were found to be clonally distinct, suggesting that the high prevalence of SPATEs was not due to clonal relatedness of the isolates. This study is the first to show the association of SPATEs with NSEC. The presence of SPATEs in the septicemic/NSEC isolates may be considered as the most discriminatory trait studied here.
Reciprocal Negative Regulation between the Guanine Nucleotide Exchange Factor C3G and Î²-Catenin. - Genes & cancer
The guanine nucleotide exchange factor C3G (RAPGEF1) regulates proliferation, migration, and differentiation of cells and is essential for mammalian embryonic development. The molecular effectors of C3G dependent functions are poorly understood. Here we report that C3G functions as a negative regulator of Î²-catenin, a major player in pathways known to be deregulated in human cancers. In mammalian cells, C3G is present in a complex with cellular Î²-catenin. The proline rich Crk binding region of C3G and residues 90-525 of Î²-catenin are sufficient for the interaction. Knockdown of cellular C3G stimulated, and its overexpression repressed, Î²-catenin/TCF transcription activity. C3G acts by destabilizing Î²-catenin protein and inhibiting its nuclear accumulation. Nuclear extracts of C3G overexpressing cells showed reduced binding to TCF consensus oligos. C3G exerts its effects independent of its function as an exchange factor. It also inhibits stability and activity of an N-terminal deletion construct of Î²-catenin that is not subject to GSK3Î² dependent phosphorylation, suggesting that C3G exerts its effect independent of GSK3Î². Î²-catenin repression by C3G was not significantly altered in the presence of proteasome inhibitors, MG132 or lactacystin, suggesting that alternate mechanisms are engaged by C3G to cause Î²-catenin turnover. C3G expression represses Î²-catenin target gene expression, and stable clones of MCF-7 breast cancer cells expressing C3G showed reduced migration. Activation of cellular Î²-catenin or expression of constitutively active Î²-catenin resulted in reduced C3G expression, indicating that C3G gene expression is negatively regulated by Î²-catenin. Our results identify a novel property of C3G in functioning as a negative regulator of Î²-catenin signaling by promoting its degradation. In addition, we show that Î²-catenin inhibits C3G expression, forming a feedback loop.
Daxx is a key downstream component of receptor interacting protein kinase 3 mediating retinal ischemic cell death. - FEBS letters
Receptor-interacting protein 3 (RIP3) has been implicated in ischemic necrosis of retinal cells. An in silico analysis followed by experimental validation identified death associated protein (Daxx) as a novel substrate of RIP3. In vitro binding studies revealed that RIP3 binds to the serine/proline/threonine-rich domain (amino acid 625-740) of Daxx. Upon ischemic insult, RIP3 phosphorylated Daxx at Ser-668 in the retinal ganglion cells, triggering nuclear export of Daxx. Depletion of RIP3 significantly inhibited nuclear export of Daxx and attenuated cell death to a great extent. Collectively, the findings of this study demonstrate that phosphorylation of Daxx by RIP3 comprises an important part of ischemic necrosis in rat retinal ganglion cells.Copyright Â© 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Chemical modification of L-glutamine to alpha-amino glutarimide on autoclaving facilitates Agrobacterium infection of host and non-host plants: A new use of a known compound. - BMC chemical biology
Accidental autoclaving of L-glutamine was found to facilitate the Agrobacterium infection of a non host plant like tea in an earlier study. In the present communication, we elucidate the structural changes in L-glutamine due to autoclaving and also confirm the role of heat transformed L-glutamine in Agrobacterium mediated genetic transformation of host/non host plants.When autoclaved at 121Â°C and 15 psi for 20 or 40 min, L-glutamine was structurally modified into 5-oxo proline and 3-amino glutarimide (Î±-amino glutarimide), respectively. Of the two autoclaved products, only Î±-amino glutarimide facilitated Agrobacterium infection of a number of resistant to susceptible plants. However, the compound did not have any vir gene inducing property.We report a one pot autoclave process for the synthesis of 5-oxo proline and Î±-amino glutarimide from L-glutamine. Xenobiotic detoxifying property of Î±-amino glutarimide is also proposed.
Signalling to actin: role of C3G, a multitasking guanine-nucleotide-exchange factor. - Bioscience reports
C3G (Crk SH3-domain-binding guanine-nucleotide-releasing factor) is a ubiquitously expressed member of a class of molecules called GEFs (guanine-nucleotide-exchange factor) that activate small GTPases and is involved in pathways triggered by a variety of signals. It is essential for mammalian embryonic development and many cellular functions in adult tissues. C3G participates in regulating functions that require cytoskeletal remodelling such as adhesion, migration, maintenance of cell junctions, neurite growth and vesicle traffic. C3G is spatially and temporally regulated to act on Ras family GTPases Rap1, Rap2, R-Ras, TC21 and Rho family member TC10. Increased C3G protein levels are associated with differentiation of various cell types, indicating an important role for C3G in cellular differentiation. In signalling pathways, C3G serves functions dependent on catalytic activity as well as protein interaction and can therefore integrate signals necessary for the execution of more than one cellular function. This review summarizes our current knowledge of the biology of C3G with emphasis on its role as a transducer of signals to the actin cytoskeleton. Deregulated C3G may also contribute to pathogenesis of human disorders and therefore could be a potential therapeutic target.
Cytoskeletal remodeling by C3G to induce neurite-like extensions and inhibit motility in highly invasive breast carcinoma cells. - Biochimica et biophysica acta
Cytoskeletal remodeling is responsible for cell plasticity and facilitates differentiation, motility and adherence related functions. C3G (RAPGEF1), an exchange factor for Ras family of small GTPases, regulates cytoskeletal reorganization to induce filopodia in epithelial cells and neurite growth in neuroblastoma cells. Here we show that C3G overexpression induces neurite-like extensions (NLE) in MDA-MB-231 and BT549 breast carcinoma cells and not in a variety of other cancer cell lines examined. These processes were actin-rich with nodes, branches and microspikes. C3G associates with the cytoskeleton and its expression enabled stabilization of microtubules. NLE formation was dependent on Rap, Rac and Cdc42. C3G expression was associated with a decrease in cellular Î²-catenin levels specifically in MDA-MB-231 and BT549 cells. Î²-Catenin stabilization induced by GSK-3Î² inhibition, or coexpression of Î²-catenin, reduced C3G induced NLE formation. Time lapse analysis showed reduced motility of C3G expressing cells compared to GFP expressing cells. Our results suggest that C3G overexpression can induce phenotypic characteristics of neuronal cells in highly invasive breast cancer cells and inhibit their motility.Copyright Â© 2011 Elsevier B.V. All rights reserved.
Chemical kinetic study of the oxidation of a biodiesel-bioethanol surrogate fuel: methyl octanoate-ethanol mixtures. - The journal of physical chemistry. A
There is a growing interest for using bioethanol-biodiesel fuel blends in diesel engines but no kinetic data and model for their combustion were available. Therefore, the kinetics of oxidation of a biodiesel-bioethanol surrogate fuel (methyl octanoate-ethanol) was studied experimentally in a jet-stirred reactor at 10 atm and constant residence time, over the temperature range 560-1160 K, and for several equivalence ratios (0.5-2). Concentration profiles of reactants, stable intermediates, and final products were obtained by probe sampling followed by online FTIR, and off-line gas chromatography analyses. The oxidation of this fuel in these conditions was modeled using a detailed chemical kinetic reaction mechanism consisting of 4592 reversible reactions and 1087 species. The proposed kinetic reaction mechanism yielded a good representation of the kinetics of oxidation of this biodiesel-bioethanol surrogate under the JSR conditions. The modeling was used to delineate the reactions triggering the low-temperature oxidation of ethanol important for diesel engine applications.
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