We have conducted systematic structural modification, deconstruction, and reconstruction of the berberine core with the aim of lowering its cytotoxicity, investigating its pharmacophore, and ultimately, seeking novel synergistic agents to restore the effectiveness of fluconazole against fluconazole-resistant Candida albicans. A structure-activity relationship study of 95 analogues led us to identify the novel scaffold of N-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-2-(substituted phenyl)acetamides 7?a-l, which exhibited remarkable levels of in vitro synergistic antifungal activity. Compound 7?d (N-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-2-(2-fluorophenyl)acetamide) significantly decreased the MIC80 values of fluconazole from 128.0??g?mL(-1) to 0.5??g?mL(-1) against fluconazole-resistant C.?albicans and exhibited much lower levels of cytotoxicity than berberine toward human umbilical vein endothelial cells.
Proline-rich proteins (PRPs) are known to play important roles in sexual plant reproduction. Most of the known proteins in the family were found in styles or pollen and modulate pollen tube growth. Here, we identified a novel member of the gene family, NtProRP1, which is preferentially expressed in tobacco pollen grains, pollen tubes and zygotes. NtProRP1 could be secreted into the extracellular space including the cell wall, and the predicted N-terminal signal peptide is crucial for its secretion. In NtProRP1-RNAi plants, pollen germination and pollen tube growth were significantly slower and showed zigzag or swell morphology in vitro. Early embryogenesis also exhibited aberrant development, indicative of its critical role in both pollen tube growth and early embryogenesis. Further investigation revealed that NtProRP1 plays a crucial role in osmotic stress response during pollen tube growth and is likely regulated by Tsi, a stress-responsive gene, suggesting that the regulatory mechanism is also involved in the stress response during sexual plant reproduction. These data provide evidence that NtProRP1 functions as a downstream factor of Tsi1 in the stress response and converges the stress signal into the modulation of pollen tube growth and early embryogenesis.
Human Elongator complex, which plays a key role in transcript elongation in vitro assay, is incredibly similar in either components or function to its yeast counterpart. However, there are only a few studies focusing on its target gene characterization in vivo. We studied the effect of down-regulation of the human elongation protein 3 (hELP3) on the expression of HSP70 through antisense strategy. Transfecting antisense plasmid p1107 into HeLa cells highly suppressed hELP3 expression, and substantially reduced expression of HSP70 mRNA and protein. Furthermore, chromatin immunoprecipitation assay (ChIP Assay) revealed that hElp3 participates in the transcription elongation of HSPA1A in HeLa cells. Finally, complementation and ChIP Assay in yeast showed that hElp3 can not only complement the growth and slow activation of HSP70 (SSA3) gene transcription, but also directly regulates the transcription of SSA3. On the contrary, these functions are lost when the HAT domain is deleted from hElp3. These data suggest that hElp3 can regulate the transcription of HSP70 gene, and the HAT domain of hElp3 is essential for this function. These findings now provide novel insights and evidence of the functions of hELP3 in human cells.
Yeast Elongation protein 3 (yElp3), the catalytic subunit of the multi-subunit histone acetyltransferase elongator complex, is involved in histone acetylation and transcription, exocytosis and tRNA modification. To study the complex function of yElp3 in yeast, we amplified the yElp3 gene fragment encoding 73aa in the N-terminal from plasmid pYES2-yElp3, and then cloned it into pMXB10 to construct the recombinant plasmid pMXB10-yElp3-219. We expressed the fusion protein in E. coli BL21 (DE3), then purified it by chin affinity column, and finally obtained the soluble purified protein (8.0 kD), which was used to immune the rabbits for acquiring antiserum. ELISA and Western blotting indicated that the polyclonal antibody was of high titration and specificity. Chromatin immunoprecipitation (ChIP) assay with this antibody suggested that yhElp3 exerted the transcriptional regulatory function directly through its presence on the SSA3 gene; this might be the reason that it can rescue the delay activation of SSA3 in elp3delta cells.
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