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Maja Tarailo-Graovac Jun Wang Jeffrey SC Chu Domena Tu David L Baillie Nansheng Chen 《BMC cell biology》2010,11(1):1-16
Background
The spindle assembly checkpoint (SAC) delays anaphase onset by inhibiting the activity of the anaphase promoting complex/cyclosome (APC/C) until all of the kinetochores have properly attached to the spindle. The importance of SAC genes for genome stability is well established; however, the roles these genes play, during postembryonic development of a multicellular organism, remain largely unexplored.Results
We have used GFP fusions of 5' upstream intergenic regulatory sequences to assay spatiotemporal expression patterns of eight conserved genes implicated in the spindle assembly checkpoint function in Caenorhabditis elegans. We have shown that regulatory sequences for all of the SAC genes drive ubiquitous GFP expression during early embryonic development. However, postembryonic spatial analysis revealed distinct, tissue-specific expression of SAC genes with striking co-expression in seam cells, as well as in the gut. Additionally, we show that the absence of MDF-2/Mad2 (one of the checkpoint genes) leads to aberrant number and alignment of seam cell nuclei, defects mainly attributed to abnormal postembryonic cell proliferation. Furthermore, we show that these defects are completely rescued by fzy-1(h1983)/CDC20, suggesting that regulation of the APC/CCDC20 by the SAC component MDF-2 is important for proper postembryonic cell proliferation.Conclusion
Our results indicate that SAC genes display different tissue-specific expression patterns during postembryonic development in C. elegans with significant co-expression in hypodermal seam cells and gut cells, suggesting that these genes have distinct as well as overlapping roles in postembryonic development that may or may not be related to their established roles in mitosis. Furthermore, we provide evidence, by monitoring seam cell lineage, that one of the checkpoint genes is required for proper postembryonic cell proliferation. Importantly, our research provides the first evidence that postembryonic cell division is more sensitive to SAC loss, in particular MDF-2 loss, than embryonic cell division. 相似文献38.
Differentiation of micronuclei (MN) caused by ionizing radiation from those caused by chemicals is a crucial step for managing treatment of individuals exposed to radiation. MN in binucleated lymphocytes in peripheral blood are widely used as biomarkers for estimating dose of radiation, but they are not specific for ionizing radiation. MN induced by ionizing radiation originate predominantly as a result of chromosome breaks (clastogenic action), whereas MN caused by chemical agents are derived from the loss of entire chromosomes (aneugenic action). C-banding highlights centromeres, which might make it possible to distinguish radiation induced MN, i.e., as a byproduct of acentric fragments, from those caused by the loss of entire chromosomes. To test the use of C-banding for identifying radiation induced MN, a blood sample from a healthy donor was irradiated with 3 Gy of Co-60 gamma rays and cultured. Cells were harvested and dropped onto slides, divided into a group stained directly with Giemsa and another processed for C banding, then stained with Giemsa. The frequency of MN in 500 binucleated cells was scored for each method. In preparations stained with Giemsa directly, the MN appeared as uniformly stained structures, whereas after C banding, some MN exhibited darker regions corresponding to centromeres that indicated that they were not derived from acentric fragments. The C-banding technique enables differentiation of MN from acentric chromosomal material. This distinction is useful for improving the specificity of the MN assay as a biomarker for ionizing radiation. 相似文献
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Background
While syndecan-2 is usually considered a mesenchymal transmembrane proteoglycan, it can be upregulated in some tumour cells, such as the malignant breast carcinoma cell line, MDA-MB231. Depletion of this syndecan by siRNA, but not other syndecans, has a marked effect on cell morphology, increasing spreading, microfilament bundle and focal adhesion formation, with reduced cell migration.Methods
A combination of siRNA transfection, immunofluorescence microscopy, phosphoprotein analysis and migration assays was used to determine how syndecan-2 may influence the cytoskeleton.Results
The altered adhesion upon syndecan-2 depletion was dependent on the RhoGTPases. p190ARhoGAP relocated to the margins of spreading cells, where it codistributed with syndecan-4 and active β1-integrin. This was accompanied by increased RhoGAP tyrosine phosphorylation, indicative of activity and RhoGTPase suppression. Consistent with this, GTP-RhoA was strongly present at the edges of control cells, but lost after syndecan-2 reduction by siRNA treatments. Further, RhoA, but not RhoC was shown to be essential for the anchored phenotype of these breast carcinoma cells that accompanied siRNA-mediated loss of syndecan-2.Conclusions
Syndecan-2 has a key role in promoting the invasive activity of these cells, in part by regulating the RhoGTPases.General significance
Syndecan-2, as a cell surface receptor is accessible for targeting to determine whether breast tumour progression is altered. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. 相似文献40.
The relative amount of high mannose structures within an N‐glycomic pool differs from one source to another, but quite often it predominates over the larger size complex type structures carrying biologically important glyco‐epitopes. An efficient method to separate these two classes of N‐glycans would significantly aid in detecting the lower abundant components by MS. Capitalizing on an initial observation that only high mannose type structures were recovered in the flow‐through fraction when peptide‐N‐glycosidase F digested peptides were passed through a C18 cartridge in 0.1% formic acid, we demonstrated here that native complex type N‐glycans can be retained by C18 cartridge and to be efficiently separated from both the smaller high mannose type structures, as well as de‐N‐glycosylated peptides by stepwise elution with increasing ACN concentration. The weak retention of the largely hydrophilic N‐glycans on C18 resin is dependent not only on size but also increased by the presence of α6‐fucosylation. This was shown by comparing the resulting N‐glycomic profiles of the washed and low‐ACN eluted fractions derived from both a human cancer cell line and an insect cell line. 相似文献