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1.
Mandy L. Roberts-Crowley Tora Mitra-Ganguli Liwang Liu Ann R. Rittenhouse 《Cell calcium》2009,45(6):589-601
Great skepticism has surrounded the question of whether modulation of voltage-gated Ca2+ channels (VGCCs) by the polyunsaturated free fatty acid arachidonic acid (AA) has any physiological basis. Here we synthesize findings from studies of both native and recombinant channels where micromolar concentrations of AA consistently inhibit both native and recombinant activity by stabilizing VGCCs in one or more closed states. Structural requirements for these inhibitory actions include a chain length of at least 18 carbons and multiple double bonds located near the fatty acid's carboxy terminus. Acting at a second site, AA increases the rate of VGCC activation kinetics, and in CaV2.2 channels, increases current amplitude. We present evidence that phosphatidylinositol 4,5-bisphosphate (PIP2), a palmitoylated accessory subunit (β2a) of VGCCs and AA appear to have overlapping sites of action giving rise to complex channel behavior. Their actions converge in a physiologically relevant manner during muscarinic modulation of VGCCs. We speculate that M1 muscarinic receptors may stimulate multiple lipases to break down the PIP2 associated with VGCCs and leave PIP2's freed fatty acid tails bound to the channels to confer modulation. This unexpectedly simple scheme gives rise to unanticipated predictions and redirects thinking about lipid regulation of VGCCs. 相似文献
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Alan Tin Lun Lam Valerie Ho Svetlan Vassilev Shaul Reuveny Steve Kah Weng Oh 《Cell proliferation》2022,55(8)
ObjectivesInduced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges.Materials and MethodsFive sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures.ResultsImprovement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50‐fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (β‐catenin, E‐cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ‐layers, cardiomyocytes and haematopoietic stem cells were further demonstrated.ConclusionsOur method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.We have developed an allied protocol for reprogramming, selecting, expanding and differentiating human pluripotent stem cells on Microcarriers (designated as RepMC). This method allows faster reprogramming, selecting 30‐50‐fold more candidates for characterization and also allows us to find high quality candidates that differentiate to cardiomyocytes and blood lineages. Mechanistically, this method appears to accelerate the induction, maturation and stabilization phases of reprogramming. Our findings help simplify the process of deriving and expanding iPSCs for therapeutic applications, offering a robust and scalable suspension platform for large‐scale generation of clinical grade iPSCs. 相似文献
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BACKGROUND: Wee1 kinases delay entry into mitosis by phosphorylating and inactivating cyclin-dependent kinase 1 (Cdk1). Loss of this activity in many systems, including Drosophila, leads to premature mitotic entry. RESULTS: We report here that Drosophila Wee1 (dwee1) mutant embryos show mitotic-spindle defects that include ectopic foci of microtubule organization, formation of multipolar spindles from adjacent centrosome pairs, and promiscuous interactions between neighboring spindles. Furthermore, centrosomes are displaced from the embryo cortex in dwee1 mutants. These defects are not observed to the same extent in embryos in which nuclei also enter mitosis prematurely as a result of a lack of checkpoint control or in embryos with elevated Cdk1 activity. dWee1 physically interacts with members of the gamma-tubulin ring complex (gammaTuRC), and gamma-tubulin is phosphorylated in a dwee1-dependent manner in embryo extracts. CONCLUSIONS: Some of the abnormalities in dwee1 mutant embryos cannot be explained by premature entry into mitosis or bulk elevation of Cdk1 activity. Instead, dWee1 is also required for phosphorylation of gamma-tubulin, centrosome positioning, and mitotic-spindle integrity. We propose a model to account for these requirements. 相似文献
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Joe Carver Domingos Ng Michelle Zhou Peggy Ko Dejin Zhan Mandy Yim David Shaw Brad Snedecor Michael W. Laird Steven Lang Amy Shen Zhilan Hu 《Biotechnology progress》2020,36(4):e2967
Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system. 相似文献
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We show that the MutY protein competes with the MutS-dependent mismatch repair system to process at least some A. C mispairs in vivo, converting them to G. C pairs. In the presence of an increased dCTP pool resulting from the loss of nucleotide diphosphate kinase, the frequency of A. T-->G. C transitions at a hot spot in the rpoB gene is 30-fold lower in a MutY-deficient derivative than in the wild type. 相似文献
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Timo Frensing Antje Pflugmacher Mandy Bachmann Britta Peschel Udo Reichl 《Applied microbiology and biotechnology》2014,98(21):8999-9008
During the replication of influenza viruses, defective interfering particles (DIPs) can be generated. These are noninfectious deletion mutants that require coinfection with a wild-type virus but interfere with its helper virus replication. Consequently, coinfected cells mainly produce DIPs. Little is known about how such noninfectious virus particles affect the virus yield of cell culture-based influenza vaccine production. We compared infections of Madin-Darby canine kidney cells with two seed virus preparations of the influenza virus strain A/Puerto Rico/8/34 that contain different amounts of DIPs. A combination of conventional RT-PCR, RT-qPCR, and flow cytometry revealed that DI genomes indeed strongly accumulate in coinfected cells and impede the viral RNA synthesis. Additionally, cells infected at the higher DIP concentration showed a stronger antiviral response characterized by increased interferon-β expression and apoptosis induction. Furthermore, in the presence of DIPs, a significant fraction of cells did not show any productive accumulation of viral proteins at all. Together, these effects of DIPs significantly reduce the virus yield. Therefore, the accumulation of DIPs should be avoided during influenza vaccine production which can be achieved by quality controls of working seed viruses based on conventional RT-PCR. The strategy for the depletion of DIPs presented here can help to make cell culture-based vaccine production more reliable and robust. 相似文献