The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport. Here we report an experimental structure of drug-bound EmrE in phospholipid bilayers, determined using 19F and 1H solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F4-TPP+). The drug-binding site, constrained by 214 protein-substrate distances, is dominated by aromatic residues such as W63 and Y60, but is sufficiently spacious for the tetrahedral drug to reorient at physiological temperature. F4-TPP+ lies closer to the proton-binding residue E14 in subunit A than in subunit B, explaining the asymmetric protonation of the protein. The structure gives insight into the molecular mechanism of multidrug recognition by EmrE and establishes the basis for future design of substrate inhibitors to combat antibiotic resistance.

The antioxidant activities of the methanolic extracts of 9 Salvia species and 15 other Lamiaceae plants growing in Iran were evaluated using ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assays. FRAP values ranged form 8.5 to 79.0 μM quercetin equivalents/g dry weight, and IC 50 values in the DPPH assay from 115.7 to 1350.2 μg dry weight/mL. Salvia species showed the highest antioxidant activities. S. santolinifolia, S. eremophila and S. palestina, which have not been studied before, were the most active plants. These were more active than the previously studied species from this family, such as S. multicaulis and Marrubium vulgare. S. hydrangea and Gontscharovia popovii also showed high antioxidant activities. FRAP and DPPH assay results showed good correlations with the total phenolic contents of the plants, measured by the Folin-Ciocalteau assay (r 2 = 0.925 and 0.799, respectively, p < 0.0001). In conclusion, our study shows that some Lamiaceae plants growing in Iran represent good potential sources of natural antioxidants useful for either prevention or treatment of oxidative stress-related diseases.

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Bioassay-guided fractionation of Haplophyllum canaliculatum Boiss. (Rutaceae) extract resulted in isolation of five quinoline alkaloids: 7-isopentenyloxy-gamma-fagarine, atanine, skimmianine, flindersine and perfamine. This is the first isolation of these compounds from this endemic species. The antitumor activity of these five isolates was evaluated against RAJI, Jurkat, KG-1a, HEP-2, MCF-7, HL-60 and HL-60/MX1 tumor cell lines. The highest cytotoxic effect was observed on acute lymphoblastic leukemia cell lines. 7-Isopentenyloxy-gamma-fagarine, atanine, skimmianine and flindersine exhibited very high cytotoxicity against the RAJI cell line with IC(50) values of 1.5, 14.5, 15.6 and 14.9 microg/mL, respectively and 7-isopentenyloxy-gamma-fagarine, atanine and skimmianine exhibited very high cytotoxicity against the Jurkat cell line with IC(50) values of 3.6, 9.3 and 11.5 microg/mL, respectively. 7-Isopentenyloxy-gamma-fagarine was also highly cytotoxic against the MCF-7 cell line (IC(50) = 15.5 microg/mL), while atanine, skimmianine, flindersine and perfamine showed moderate to low activity against these cells. All alkaloids had moderate to low cytotoxicity against KG-1a and HEP-2. Investigation of the toxic potential of the alkaloids on HL-60 and HL-60/MX1 showed a significantly higher effect against HL-60/MX1, a multidrug-resistant cell line, compared with the control etoposide (p < 0.05). In all cytotoxicity experiments, peripheral blood mononuclear cells (PBMC) were used as a control for normal hematopoietic cells. Flow cytometry analysis of the compounds resulted in the arrest of cell cycle progression at the sub-G1 phase of the RAJI and Jurkat cell lines in a dose-dependent manner. According to computational analyses, the similar cytotoxic trend in the cell lines could be indicative of the fact that these compounds may act through parallel mechanisms.

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