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171.
Suppression of high-fidelity double-strand break repair in mammalian chromosomes by pifithrin-alpha,a chemical inhibitor of p53 总被引:1,自引:0,他引:1
We investigated the effect of pifithrin-alpha (PFTalpha), a chemical inhibitor of p53, on DNA double-strand break (DSB) repair in mammalian chromosomes. Thymidine kinase-deficient mouse fibroblasts were stably transfected with DNA substrates containing one or two recognition sites for yeast endonuclease I-SceI embedded within a herpes simplex virus thymidine kinase gene. Genomic DSBs were induced by introducing an I-SceI expression plasmid into cells in the presence or absence of 20 microM PFTalpha. From cells containing the DNA substrate with a single I-SceI site we recovered low-fidelity nonhomologous end-joining (NHEJ) events in which one or more nucleotides were deleted or inserted at the DSB. From cells containing the substrate with two I-SceI sites we recovered high-fidelity DNA end-joining (precise ligation (PL)) events. We found that treatment of cells with PFTalpha caused a 5-10-fold decrease in recovery of PL but decreased recovery of NHEJ by less than two-fold. Deletion sizes associated with NHEJ were unaffected by treatment with PFTalpha. Our work suggests the possibility that p53 facilitates high-fidelity DSB repair while playing little or no role in mutagenic NHEJ. 相似文献
172.
Macchi P Hemraj I Goetze B Grunewald B Mallardo M Kiebler MA 《Molecular biology of the cell》2003,14(4):1570-1582
An inducible fluorescent system based on GFP is presented that allows for the uncoupling of dendritic mRNA transport from subsequent protein synthesis at the single cell level. The iron-responsive element (IRE) derived from ferritin mRNA in the 5'-UTR of the GFP reporter mRNA renders translation of its mRNA dependent on iron. The addition of the full-length 3'-UTR of the Ca(2+)/calmodulin-dependent protein kinase II alpha (CaMKIIalpha) after the stop codon of the GFP reading frame targets the reporter mRNA to dendrites of transfected fully polarized hippocampal neurons. As we show by time-lapse videomicroscopy, iron specifically turns on GFP reporter protein synthesis in a single transfected hippocampal neuron. We investigate whether GFP expression is affected--in addition to iron--by synaptic activity. Interestingly, synaptic activity has a clear stimulatory effect. Most importantly, however, this activity-dependent protein synthesis is critically dependent on the presence of the full-length 3'-UTR of CaMKIIalpha confirming that this sequence contains translational activation signals. The IRE-based system represents a new convenient tool to study local protein synthesis in mammalian cells where mRNA localization to a specific intracellular compartment occurs. 相似文献
173.
174.
L1210 leukemic cells injected in vivo are eliminated from the blood and disintegrated in organs such as the lungs and liver. We present a compartmental model which reproduces one type of in vivo experiment, based on the so-called perfusion curves. Although the data are not complete and some are only approximated, modeling gives a consistent picture of the process. 相似文献
175.
176.
177.
Erasmus Schneider Barbara Müller Richard Schindler 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1983,741(1):77-85
Two heat-sensitive (arrested in G1 at 39.5°C) and two cold-sensitive (arrested in G1 at 33°C) clonal cell-cycle mutants that had been isolated from the same clone (K 21), of the murine mastocytoma P-815 cell line, were tested for thymidine kinase (EC 2.7.1.21) activity. After shift of mutant cells to the nonpermissive temperature, thymidine kinase activity decreased, and minimal levels (i.e., less than 3% of those observed for ‘wild-type’ K 21 cells at the respective temperature) were attained within 16 h in heat-sensitive and after 3–4 days in cold-sensitive mutants, which is in good agreement with kinetics of accumulation of heat-sensitive and cold-sensitive cells in G1 phase. After return of arrested mutant cells to the permissive temperature, thymidine kinase of heat-sensitive cells increased rapidly and in parallel with entry of cells into the S phase. In cultures of cold-sensitive cells, however, initiation of DNA synthesis preceded the increase of thymidine kinase activity by approx. one cell-cycle time. Thymidine kinase activities in revertants of the heat-sensitive and cold-sensitive mutants were similar to those of ‘wild-type’ cells. In ‘wild-type’ K 21 cells incubated at 39.5°C, thymidine kinase activity was approx. 30% of that at 33°C. This difference is attributable, at least in part, to a higher rate of inactivation of the enzyme at 39.5°C, as determined in cultures incubated with cycloheximide. The rapid increase of thymidine kinase activity that occurred after shift of K 21 cells and of arrested heat-sensitive mutant cells from 39.5°C to 33°C was inhibited by actinomycin D and cycloheximide. 相似文献
178.
pp60
c-src
kinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443–23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependant activation of pp60
c-src
but failed to increase hormone independent (basal) pp60
c-src
activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60
c-src
was not detected in response to PDGF or in PTPase+cells. PDGF increased the intrinsic tyrosine kinase activity of pp60
c-src
in both control and PTPase+cells but the effect was smaller in PTPase+cells. In anin vitro assay, hormone-stimulate pp60
c-src
autophosphorylation from PTPase+ cells was decreased 64±22%, and substrate phosphorylation by pp60
c-src
was reduced 54±16% compared to controls. Hormone-independent pp60
c-src
kinase activity was unchanged by expression of the PTPase. pp60
c-src
was, however, anin vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr527) and kinase domain (Tyr416) residues. In addition,in vitro dephosphorylation by CD45 increased pp60
c-src
activity. These findings suggest that the PDGF receptor was anin vivo substrate of CD45 but pp60
c-src
was not. The lack of activation of pp60
c-src
in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions.Abbreviations PTPase
phosphotyrosine phosphatase
- PDGF
platelet-derived growth factor
- PMSF
phenylmethylsulfonyl fluoride
- LCA, CD45
leukocyte common antigen
- PBS
phosphate buffered saline
- SDS
sodium dodecyl sulfate
- PAGE
polyacrylamide gel electrophoresis
- DTT
dithiothreitol
- Na3VO4
sodium orthovanadate
- PV
pervanadate
- -ME
-mercaptoethanol 相似文献
179.
Edda Siegl Barbara Nebe Heidrun Blunk Joachim Rychly 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》1998,119(4):2047
Flow cytometric analysis of the forward/side light scatter (FSC/SSC) of density gradient-separated head kidney cells of the rainbow trout revealed three distinctly separated populations, which we defined as population 1, 2 and 3. In spleen cells, populations 1 and 2 were also found, whereas population 3 was not detected. Further characterization regarding the surface Ig (sIg) revealed that population 2 of the head kidney and spleen contained 37.4 and 34.4% sIg+-cells, respectively. Incubation of the head kidney and spleen cells with different concentrations of concanavalin A (ConA), phytohemagglutinin (PHA) and [PWM] induced a pronounced intracellular calcium increase only in cells of population 2. This reaction was concentration dependent and caused by a release of intracellular Ca2+-stores. FMLP, a chemotactic peptide, had no effect on intracellular calcium response in all three populations. Similarly, the stimulation with PMA had no effect. This indicates that population 2 of the head kidney as well as the spleen is characterized by a high forward and low side light scatter and contains both subpopulation of lymphocytes, B- and T-cells. We demonstrated that the analysis of intracellular calcium increase due to mitogens is a suitable approach to identify lymphocytes in fish and enables further functional studies in these cells. 相似文献
180.
Barbara C. Banz Jia Wu Michael J. Crowley Marc N. Potenza Linda C. Mayes 《The Yale journal of biology and medicine》2016,89(2):143-151
Adolescence and prenatal cocaine exposure can impact risk-taking. In this study, we evaluated risk-taking and gender-related differences in adolescents with prenatal cocaine exposure in terms of electrophysiological correlates of inhibitory control and sustained attention. No differences related to gender were found within measures of risk-taking, or electrophysiological response relating to risk-taking. Greater responses during inhibition versus attention trials support previous studies, with boys showing the largest responses. Gender-related differences were found when comparing the trials before and after frustration was induced, with greater initial attention indices for girls in both trial types and greater sustained attention for both genders during inhibition trials and for boys during attention trials. These data suggest neural correlates of response inhibition show important gender-related differences in this population. Considering these relationships allows us to further understand underlying processes among adolescents who, as a group, tend to be more inclined toward greater risk behaviors. 相似文献