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Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. The enzyme which converts the first intermediate in the pathway, 2,4,5-trichlorophenol, to 5-chlorohydroquinone has been purified and consists of two subunits of 58 and 22 kDa, encoded by the tftC and tftD genes (48). A degenerate primer was designed from the N terminus of the 58-kDa polypeptide and used to isolate a clone containing the tftC and tftD genes from a genomic library of AC1100. The derived amino acid sequences of tftC and tftD show significant homology to the two-component monooxygenases HadA of Burkholderia pickettii, HpaBC of Escherichia coli, and HpaAH of Klebsiella pneumonia. Expression of the tftC and tftD genes appeared to be induced when they were grown in the presence of 2,4,5-T, as shown by RNA slot blot and primer extension analyses. Three sets of cloned tft genes were used as probes to explore the genomic organization of the pathway. Pulsed-field gel electrophoresis analyses of whole chromosomes of B. cepacia AC1100 demonstrated that the genome is comprised of five replicons of 4.0, 2.7, 0.53, 0.34, and 0.15 Mbp, designated I to V, respectively. The tft genes are located on the smaller replicons: the tftAB cluster is on replicon IV, tftEFGH is on replicon III, and copies of the tftC and the tftCD operons are found on both replicons III and IV. When cells were grown in the absence of 2,4,5-T, the genes were lost at high frequency by chromosomal deletions and rearrangements to produce 2,4,5-T-negative mutants. In one mutant, the tftA and tftB genes translocated from one replicon to another, with the concomitant loss of tftEFGH and one copy of tftCD. PMID:9603818
We have investigated the cytotoxic assay of Fe-aminoclay (FeAC) nanoparticles (NPs) and simultaneous imaging in HeLa cells by photoluminescent carbon nanodots (CD) conjugation. Non-cytotoxic, photostable, and CD NPs are conjugated with cationic FeAC NPs where CD NPs play a role in bio-imaging and FeAC NPs act as a substrate for CD conjugation and help to uptake of NPs into cancer cells due to positively charged surface of FeAC NPs in physiological media. As increase of CD-FeAC NPs loading in HeLa cell in vitro, it showed slight cytotoxicity at 1000 μg/mL but no cytotoxicity for normal cells up to concentration of 1000 μg/mL confirmed by two 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR) assays, with further observations by 4',6-diamidino-2-phenylindole (DAPI) stained confocal microscopy images, possessing that CD-FeAC NPs can be used as potential drug delivery platforms in cancer cells with simultaneous imaging. Graphical abstract CD conjugation with organo-building blocks of delaminated FeAC NPs.
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