The Saccharomyces cerevisiae ubiquitin ligase SCF Met30 is essential for cell cycle progression. To identify and characterize SCF Met30 -dependent cell cycle steps, we used temperature-sensitive met30 mutants in cell cycle synchrony experiments. These experiments revealed a requirement for Met30 during both G 1 /S transition and M phase, while progression through S phase was unaffected by loss of Met30 function. Expression of the G 1 -specific transcripts CLN1, CLN2, and CLB5 was very low in met30 mutants, whereas expression of CLN3 was unaffected. However, overexpression of Cln2 could not overcome the G 1 arrest. Interestingly, overexpression of Clb5 could induce DNA replication in met30 mutants, albeit very inefficiently. Increased levels of Clb5 could not, however, suppress the cell proliferation defect of met30 mutants. Consistent with the DNA replication defects, chromatin immunoprecipitation experiments revealed significantly lower levels of the replication factors Mcm4, Mcm7, and Cdc45 at replication origins in met30 mutants than in wild-type cells. These data suggest that Met30 regulates several aspects of the cell cycle, including G 1 -specific transcription, initiation of DNA replication, and progression through M phase.
The aim of this paper was to evaluate the skin irritant potential of five plant extracts for cosmetic use by in vitro and in vivo methods and to analyze the correlation between these different methods. Cell viability in fibroblasts and in the reconstructed human skin model was measured using the MTT assay to test cytotoxicity, and ELISA was used to detect IL-8 release. Finally, human skin patch tests were performed to validate the results obtained using the in vitro methods. The human patch results were compared with IL-8 release results in fibroblasts and the MTT assay findings in Episkin TM . The MTT assay in fibroblasts showed that the five plant extracts exhibited low cytotoxicity. The human patch results showed that four of the five plant extracts showed weak irritant potential. The MTT assay results in Episkin TM were best correlated with the human patch test results with a Spearman's correlation coefficient of 0.6156. The Spearman's correlation coefficient between the human patch test and IL-8 release results was 0.4617. The MTT assay in Episkin TM was more sensitive than the IL-8 release assay in fibroblasts to evaluate skin irritant potential. Our findings indicate that the five plant extracts are safe for cosmetic use.
Sodium dodecyl sulfate (SDS) is widely used as an irritant. Inflammatory and immune related cytokines/ chemokines are released by keratinocytes following SDS irritation. However, a specific effect of SDS on keratinocytes and the mechanism of skin irritation caused by SDS have not been investigated. To explore the irritant mechanism of SDS on keratinocytes, a gene microarray was used to detect the changes in gene expression after treating keratinocytes with SDS for different amounts of time. After 0.5 h and 1 h SDS exposure, there were changes mainly in genes in the rheumatoid arthritis pathway (CCL5, IL-6, FOS, CSF1, and HLA-DPB1) and TNF signaling pathway (TNF, CSF2, CXCL3, TNFAIP3, PTGS2, CXCL8, CCL20, and PIK3CA) to cause a pro-inflammatory reaction as well as autoimmune activation. After treating with SDS for 2 h and 4 h, there were changes in the expression of genes (LIF, PIM1, MAP3K8, CCNA1, RUNX1, and CYP1) that are related to cell apoptosis and cancerization. This was related to changes in transcriptional activity in the cancer and tryptophan metabolism pathway. Overall, SDS irritation caused different changes in gene expression over time, which altered the state of keratinocytes affecting processes of inflammation, autoimmune response, cell apoptosis, and cancerization. These results provide insight into the irritation process of SDS and provide reference for the future evaluation of skin irritation.
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