Use of resazurin to detect mefloquine as an efflux-pump inhibitor in Pseudomonas aeruginosa and Escherichia coli

Multi-drug-resistant bacteria can cause serious infections that are extremely difficult to treat. Bacterial efflux pumps are known to contribute to multi-drug resistance and, thus, constitute a promising target for novel antibacterial agents. Resazurin is widely used to monitor bacterial growth because resazurin is reduced to the fluorescent resorufin by live cells. We have shown by flow cytometric analysis and by accumulation studies with wild type and efflux deficient strains that resazurin is a substrate of efflux pumps in Escherichia coli and Pseudomonas aeruginosa. Our investigations showed that the conversion rate of resazurin to resorufin is affected by efflux pumps. This finding was used to design an assay useful to detect efflux pump activity and to find potential efflux-pump inhibitors in a microtiter plate format. Mefloquine was detected as efflux-pump inhibitor when a panel of selected chemical compounds was tested for assay validation purposes.     

Role of platelet factors and serum complement in growth of fibroblasts with high-affinity C1q complement receptors

Summary Cultures of human diploid fibroblasts are heterogeneous in that a subpopulation interacts via high-affinity receptors with the globular head regions of the C1q complement protein. Growth and synthetic properties of these cells are characteristic of cells residing in healing wounds and inflammatory lesions. At these sites, fibroblasts are exposed to regulatory molecules such as complement components and factors released from blood platelets. We assessed the effects of native complement proteins and platelet-derived factors on proportions and phenotypic stability of high-affinity and low-affinity receptor cells generated from explants of adult and embryonic connective tissue, using radioligand binding assays and immunofluorescence analysis by flow cytometry. Fibroblasts expressing high-affinity C1q receptors could be generated from explants only when factors from platelets were present in the medium; native complement proteins were not essential. High-affinity receptor cells could be generated only from tissue; they could not be generated by incubating cultures of the low-affinity receptor phenotype in medium containing platelet-derived factors. High-affinity receptor cells, once established from explants in the presence of platelet-derived factors, persisted through many replications in the absence of platelets. We obtained the same fibroblast phenotypes from embryonic skin as from adult gingiva, but the proportion of high-affinity receptor cells from skin was much greater. We conclude that factors derived from platelets are essential for generating cultures containing fibroblasts expressing high-affinity C1q receptors, but not for their maintenance. High-affinity receptor cells may comprise a rapidly dividing subpopulation giving rise only to like progeny or to other, more differentiated cells. Supported by grants DE03301 and DE02600 from the National Institutes of Health, Bethesda, MD.     

Hypoxoside production in tissue cultures of Hypoxis rooperi

Three culture types of Hypoxis rooperi T. Moore were examined to determine whether hypoxoside was present. Of these cultures, only root-type cultures were found to contain hypoxoside. Quantification of this compound within these tissues using HPLC, indicated that malformed root (MR) cultures contained the highest levels of hypoxoside. In MR cultures initiated from corm explants, the hypoxoside content was found to fluctuate. Contrary to most reports, neither an increase in sucrose concentration in the basal medium (BM) nor light, stimulated hypoxoside synthesis within this tissue. Alternatively a lowering of the levels of inorganic nitrogen in the BM and the incubation of cultures in continuous darkness, enhanced hypoxoside production.     

Identification of two catalases in Azotobacter vinelandii: a KatG homologue and a novel bacterial cytochrome c catalase, CCCAv

Azotobacter vinelandii produces two detectable catalases during growth on minimal medium. The heat-labile catalase expressed during exponential growth phase was identified as a KatG homologue by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a mixed protein sample. The second catalase was heat resistant and had substantial residual activity after treatment at 90°C. This enzyme was purified by anion-exchange and size exclusion chromatography and was found to exhibit strong absorption at 407 nm, which is often indicative of associated heme moieties. The purified protein was fragmented by proteinase K and identified by LC-MS/MS. Some identity was shared with the MauG/bacterial cytochrome c peroxidase (BCCP) protein family, but the enzyme exhibited a strong catalase activity never before observed in this family. Because two putative c-type heme sites (CXXCH) were predicted in the peptide sequence and were demonstrated experimentally, the enzyme was designated a cytochrome c catalase (CCC_Av). However, the local organization of the CCC_Av heme motifs differed significantly from that of the BCCPs as the sites were confined to the C-terminal half of the catalase. A possible Ca²⁺ binding motif, previously described in the BCCPs, is also present in the CCC_Av peptide sequence. Some instability in the presence of EGTA was observed. Expression of the catalase was abolished in cccA mutants, resulting in a nearly 8,700-fold reduction in peroxide resistance in stationary phase.