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221.
Matthias Weiand Paula Ballmaier Christoph Niemietz Hartmut Schmidt Andree Zibert 《Biochemistry and Biophysics Reports》2022
Human primary cells, including urine-derived cells (UCs), are an excellent source for generation of pluripotent stem cells (iPSCs) to model disease. However, replicative senescence starts early and shortens the time window for generation of iPSCs. We addressed the question whether combinations of transgenes allows efficient immortalization of UCs, iPSC generation, and differentiation into hepatocyte-like cells (HLCs). Retroviral transfer of three gene cassettes HPVE6E7 (H), hTERT/p53DD (T), cyclinD1/CDK4R24C (C) encoding five genes was established in primary UCs. Long-term cell proliferation was observed in cells carrying transgenes H, HT, HC, and HCT, whereas cells carrying transgenes C, T and CT showed early senescence similar to UCs. iPSCs could be exclusively generated from immortalized UCs transduced with transgenes HCT and HC. iPSC colonies appeared however later and in smaller number as compared to UCs. Using an established hepatic differentiation protocol, HLCs were obtained with high efficacy. Of note, a high expression of individual transgenes was observed in immortalized UCs, which was down-regulated after reprogramming in four out of five genes. One transgene was re-expressed in HLCs as compared to iPSCs. Our data suggest that individual transgene combinations result in advanced growth rates of immortalized cells and do not prevent iPSC formation and HLC differentiation. Retroviral transgene expression is mostly silenced in iPSCs but can be rarely re-expressed after hepatic differentiation. An extended time window for iPSC establishment can be proposed that allows straightforward functional analyses of differentiated cells. 相似文献
222.
Rosentreter A Hofmann A Xavier CP Stumpf M Noegel AA Clemen CS 《Experimental cell research》2007,313(5):878-895
The actin interaction of coronin 3 has been mainly documented by in vitro experiments. Here, we discuss coronin 3 properties in the light of new structural information and focus on assays that reflect in vivo roles of coronin 3 and its impact on F-actin-associated functions. Using GFP-tagged coronin 3 fusion proteins and RNAi silencing we show that coronin 3 has roles in wound healing, protrusion formation, cell proliferation, cytokinesis, endocytosis, axonal growth, and secretion. During formation of cell protrusions actin accumulation precedes the focal enrichment of coronin 3 suggesting a role for coronin 3 in events that follow the initial F-actin assembly. Moreover, we show that coronin 3 similar to other coronins interacts with the Arp2/3-complex and cofilin indicating that this family in general is involved in regulating Arp2/3-mediated events. 相似文献
223.
Cholesterol depletion alters detergent-specific solubility profiles of selected tight junction proteins and the phosphorylation of occludin 总被引:3,自引:0,他引:3
Lynch RD Francis SA McCarthy KM Casas E Thiele C Schneeberger EE 《Experimental cell research》2007,313(12):2597-2610
Differential centrifugation of Triton X-100 or CHAPS lysates from control and cholesterol (CH)-depleted MDCK II cells, segregated integral tight junction (TJ) proteins associated with detergent-resistant membranes (DRMs) into two groups. Group A proteins (occludin, claudin-2 and -3) were detected in large, intermediate and small aggregates in both detergents, whereas group B proteins (claudin-1, -4 and -7) were observed in small aggregates in TX-100 and in intermediate and small aggregates in CHAPS. Depletion of CH altered the distribution of group A and B proteins among the three size categories in a detergent-specific manner. In lysates produced with octyl glucoside, a detergent that selectively extracts proteins from DRMs, group A proteins were undetectable in large aggregates and CH depletion did not alter the distribution of either group A or B proteins in intermediate or small aggregates. Neither occludin (group A) nor claudin-1 (group B) was in intimate enough contact with CH to be cross-linked to [(3)H]-photo-cholesterol. However, antibodies to either TJ protein co-immunoprecipitated caveolin-1, a CH-binding protein. Unlike claudins, occludin's presence in TJs and DRMs did not require palmitoylation. Equilibrium density centrifugation on discontinuous OptiPrep gradients revealed detergent-related differences in the densities of TJ-bearing DRMs. There was little or no change in those densities after CH depletion. Removing CH from the plasma membrane increased tyrosine and threonine phosphorylation of occludin, and transepithelial electrical resistance (TER) within 30 min. After 2 h of CH efflux, phospho-occludin levels and TER fell below control values. We conclude that the association of integral TJ proteins with DRMS, pelleted at low speeds, is partially CH-dependent. However, the buoyant density of TJ-associated DRMs is a function of the detergent used and is insensitive to decreases in CH. 相似文献
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225.
Genetic load in sexual and asexual diploids: segregation, dominance and genetic drift 总被引:1,自引:0,他引:1
In diploid organisms, sexual reproduction rearranges allelic combinations between loci (recombination) as well as within loci (segregation). Several studies have analyzed the effect of segregation on the genetic load due to recurrent deleterious mutations, but considered infinite populations, thus neglecting the effects of genetic drift. Here, we use single-locus models to explore the combined effects of segregation, selection, and drift. We find that, for partly recessive deleterious alleles, segregation affects both the deterministic component of the change in allele frequencies and the stochastic component due to drift. As a result, we find that the mutation load may be far greater in asexuals than in sexuals in finite and/or subdivided populations. In finite populations, this effect arises primarily because, in the absence of segregation, heterozygotes may reach high frequencies due to drift, while homozygotes are still efficiently selected against; this is not possible with segregation, as matings between heterozygotes constantly produce new homozygotes. If deleterious alleles are partly, but not fully recessive, this causes an excess load in asexuals at intermediate population sizes. In subdivided populations without extinction, drift mostly occurs locally, which reduces the efficiency of selection in both sexuals and asexuals, but does not lead to global fixation. Yet, local drift is stronger in asexuals than in sexuals, leading to a higher mutation load in asexuals. In metapopulations with turnover, global drift becomes again important, leading to similar results as in finite, unstructured populations. Overall, the mutation load that arises through the absence of segregation in asexuals may greatly exceed previous predictions that ignored genetic drift. 相似文献
226.
Mazanek M Mituloviae G Herzog F Stingl C Hutchins JR Peters JM Mechtler K 《Nature protocols》2007,2(5):1059-1069
We have developed a new offline chromatographic approach for the selective enrichment of phosphorylated peptides that is directly compatible with subsequent analysis by online nano electrospray ionization tandem mass spectrometry. In this technique, a titanium dioxide (TiO2)-packed pipette tip is used as a phosphopeptide trap that acts as an offline first-dimension separation step in a two-dimensional chromatography system. This is followed by online nano reversed-phase high-performance liquid chromatography. Here, we present suitable methods for enrichment, optimized separately for each step: sample loading, washing and elution from the TiO2-filled tips. To increase the trapping selectivity of the TiO2 column, we used the sodium salt of 1-octanesulfonic acid combined with 2,5-dihydroxybenzoic acid as ion-pairing agents and displacers for acidic peptides. These agents also improve the binding of phosphorylated peptides and block the binding of non-phosphorylated ones. This enrichment procedure takes 30 min, followed by a 100-min HPLC program, including washing and an elution gradient. 相似文献
227.
Background
One central goal of computational systems biology is the mathematical modelling of complex metabolic reaction networks. The first and most time-consuming step in the development of such models consists in the stoichiometric reconstruction of the network, i. e. compilation of all metabolites, reactions and transport processes relevant to the considered network and their assignment to the various cellular compartments. Therefore an information system is required to collect and manage data from different databases and scientific literature in order to generate a metabolic network of biochemical reactions that can be subjected to further computational analyses. 相似文献228.
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