Phenotypic and genotypic characteristics of recently adapted isolates of Plasmodium falciparum from Thailand.
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The drug sensitivity characteristics and Plasmodium falciparum pfmdr1 status of five isolates of P. falciparum recently isolated from patients presenting for treatment from the Thailand/Myanmar border have been investigated. The aim of the study was to avoid the criticisms of some earlier studies by focusing on newly collected isolates from a specific geographic location. Three of the isolates studied exhibited clear resistance to chloroquine similar to that observed in the K1 Thai standard isolate obtained in the 1970s, and the other two isolates were of intermediate sensitivity to chloroquine with concentrations of drug that inhibit parasite growth by 50% of 50 and 43 nmol. The sensitivity of all isolates was enhanced by verapamil but we found no clear association between chloroquine sensitivity and gene copy number or intra-allelic variation of pfmdr1. In contrast, clear cross-resistance was seen between mefloquine and halofantrine, with the most sensitive isolates carrying the K1 mutation in pfmdr1. (+info)
Polycyclic aromatic hydrocarbon metabolism by white rot fungi and oxidation by Coriolopsis gallica UAMH 8260 laccase.
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We studied the metabolism of polycyclic aromatic hydrocarbons (PAHs) by using white rot fungi previously identified as organisms that metabolize polychlorinated biphenyls. Bran flakes medium, which has been shown to support production of high levels of laccase and manganese peroxidase, was used as the growth medium. Ten fungi grown for 5 days in this medium in the presence of anthracene, pyrene, or phenanthrene, each at a concentration of 5 microg/ml could metabolize these PAHs. We studied the oxidation of 10 PAHs by using laccase purified from Coriolopsis gallica. The reaction mixtures contained 20 microM PAH, 15% acetonitrile in 60 mM phosphate buffer (pH 6), 1 mM 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), and 5 U of laccase. Laccase exhibited 91% of its maximum activity in the absence of acetonitrile. The following seven PAHs were oxidized by laccase: benzo[a]pyrene, 9-methylanthracene, 2-methylanthracene, anthracene, biphenylene, acenaphthene, and phenanthrene. There was no clear relationship between the ionization potential of the substrate and the first-order rate constant (k) for substrate loss in vitro in the presence of ABTS. The effects of mediating substrates were examined further by using anthracene as the substrate. Hydroxybenzotriazole (HBT) (1 mM) supported approximately one-half the anthracene oxidation rate (k = 2.4 h(-1)) that ABTS (1 mM) supported (k = 5.2 h(-1)), but 1 mM HBT plus 1 mM ABTS increased the oxidation rate ninefold compared with the oxidation rate in the presence of ABTS, to 45 h(-1). Laccase purified from Pleurotus ostreatus had an activity similar to that of C. gallica laccase with HBT alone, with ABTS alone, and with 1 mM HBT plus 1 mM ABTS. Mass spectra of products obtained from oxidation of anthracene and acenaphthene revealed that the dione derivatives of these compounds were present. (+info)
Novel nitrated derivatives of 5,8-diazabenzo[c]phenanthrene and 9,14-diazadibenz[a,e]acephenanthrylene: new classes of potent mutagenic compounds.
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We report the synthesis of 4-nitro-5,8-diazabenzo[c]phenanthrene (4-NDBP) and 11-nitro-9,14-diazadibenz[a,e]acephenanthrylene (11-NDDA) and the remarkable mutagenic activity of the latter. These two compounds and their non-nitrated parents, 5, 8-diazabenzo[c]phenanthrene (DBP) and 9,14-diazadibenz[a, e]acephenanthrylene (DDA), were screened in Ames plate incorporation assays against Escherichia coli WP2uvrA and Salmonella typhimurium TA98 both in the presence and absence of S9 liver fraction from Aroclor 1254-induced rats. None of the four compounds were cytotoxic up to the limits of their solubility and none showed mutagenic activity in E.coli WP2uvrA, which suggested that any such activity they may have had was not mediated via a base substitution mechanism. DBP and DDA also displayed a lack of activity in TA98 up to their precipitating doses (560 and 33.5 microg/plate, respectively). The two nitrated compounds, however, were genotoxic. 4-NDBP was active at a dose of 500 ng/plate, in the absence of S9, producing 80.0 +/- 28.0 prototrophic organisms (equivalent to 44 revertants/nmol) and at 0.5 ng/plate, in the presence of S9, giving 147 +/- 6.6 revertants (equivalent to 81 000/nmol) and allowed the description of this tetracycle as a potent mutagen. Much more striking was the activity of 11-NDDA: in the absence of S9 a dose of 8.0 ng produced 2000 revertants/nmol and, remarkably, in the presence of S9 80 pg produced the equivalent of 643 000 revertants/nmol. This makes the hexacyclic 11-NDDA the most potent mutagen to date, in the Ames procedures described here. (+info)
Molecular epidemiology of malaria in Yaounde, Cameroon V. analysis of the omega repetitive region of the plasmodium falciparum CG2 gene and chloroquine resistance.
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A novel Plasmodium falciparum gene, denoted cg2 gene, has been recently discovered, and a distinct genotype, characterized by 12 point mutations and 3 size polymorphisms, has been shown to be associated with chloroquine resistance in laboratory-adapted parasite strains. One of the polymorphic regions, denoted the omega region, consists of 16 tandem repeat units in chloroquine-resistant strains, while the chloroquine-sensitive strains have either < or = 15 or > or = 17 repeat units. In this study, the in vivo and in vitro responses were compared with the number of repeat units in the omega region of the cg2 gene for 75 Cameroonian isolates determined either by DNA sequencing or agarose gel electrophoresis. The 16-repeat units that characterize the resistant strains were found in 10 chloroquine-sensitive isolates (50% inhibitory concentration [IC50] < 100 nM) and 30 chloroquine-resistant isolates (IC50 > or = 100 nM). Thirty-five isolates (28 chloroquine-sensitive isolates and 7 chloroquine-resistant isolates) displayed < or = 15 or > or = 17 repeat units. Of the 18 patients responding with treatment failure, 15 were infected with parasites carrying 16 repeat units. Twenty-eight patients (11 with isolates carrying 16 repeat units and 17 with isolates carrying < or = 15 or > or = 17 repeat units) showed an adequate clinical response. The sensitivity, specificity, and predictive value were 81% (83%), 74% (61%), and 75% (58%), respectively compared with in vitro (or in vivo) responses. Neither the level of IC50 nor the key P. falciparum multidrug resistance gene 1 (pfmdr 1) allele at position 86 was associated with the number of omega repeat units. Although in vitro and in vivo resistance to chloroquine was statistically associated with the presence of 16 repeat units in the omega region (P < 0.05), the number of omega repeat units did not adequately discriminate patients infected with chloroquine-resistant parasites from those infected with chloroquine-sensitive parasites. Further studies on the cg2 gene are needed to determine whether cg2 gene is a reliable genetic marker for chloroquine resistance. (+info)
The effect of W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride] on Ca(2+) signaling in Madin-Darby canine kidney cells was investigated. W-7 (0.1-1 mM) induced a [Ca(2+)](i) increase, which comprised an initial increase and a plateau. Ca(2+) removal inhibited the Ca(2+) signals by 80%, suggesting that W-7 activated external Ca(2+) influx and internal Ca(2+) release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (2 microM) and the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (1 microM) abolished the internal Ca(2+) release induced by 0.5 mM W-7; conversely, pretreatment with W-7 prevented thapsigargin and carbonylcyanide m-chlorophenylhydrazone from releasing internal Ca(2+). W-7 (0.2 mM) induced Mn(2+) quench of fura-2 fluorescence, which was inhibited by La(3+) (0.1 mM) by 80%. La(3+) (0.1 mM) partly inhibited 0.2 mM W-7-induced [Ca(2+)](i) increase. Addition of 5 mM Ca(2+) induced a significant [Ca(2+)](i) increase after pretreating with 0.2 to 1 mM W-7 in Ca(2+)-free medium for 5 min, suggesting that W-7 induced capacitative Ca(2+) entry. W-7 (0.5 mM) potentiated the capacitative Ca(2+) entry induced by 1 microM thapsigargin by 15%. Pretreatment with aristolochic acid (40 microM) to inhibit phospholipase A(2) reduced 0.5 mM W-7-induced internal Ca(2+) release and external Ca(2+) influx by 25 and 80%, respectively. Inhibition of phospholipase C with U73122 (2 microM) or inhibition of phospholipase D with propranolol (0.1 mM) had no effect on the internal Ca(2+) release induced by 0.5 mM W-7. It remains unclear whether W-7 induced [Ca(2+)](i) increases via inhibition of calmodulin. Three other calmodulin inhibitors (phenoxybenzamine, trifluoperazine, and fluphenazine-N-chloroethane) did not alter resting [Ca(2+)](i). (+info)
Use of sublimation to prepare solid microbial media with water-insoluble substrates.
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A method was developed to deposit a visible layer of water-insoluble compounds via sublimation onto the surface of solid media. The compound is sublimed from a heated aluminum dish containing the compound onto the surface of an inverted, ice-cooled, inoculated agar petri dish. The method results in the deposition of a thin, even layer on the agar surface without the use of solvent. After incubation, clearing zones around colonies indicate the presence of compound-degrading microorganisms. (+info)
Tanshinone IIA, an ingredient of Salvia miltiorrhiza BUNGE, induces apoptosis in human leukemia cell lines through the activation of caspase-3.
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Tanshinone II-A is a derivative of phenanthrene-quinone isolated from Salvia miltiorrhiza BUNGE, a traditional herbal medicine that is known to induce antiinflammatory, anti-oxidative and cytotoxic activity. We have examined cellular effects of Tanshione II-A on HL60 human promyelocytic leukemic cells and K562 human erythroleukemic cells. Tanshione II-A induced a dose- and time-dependent DNA fragmentation into the multiples of 180 bp and specific proteolytic cleavage of poly(ADP-ribose) polymerase in both cell lines. PI-staining and flow cytometry analysis of K562 cells following Tanshione II-A treatment showed an increase of the cells possessing hypodiploid DNA indicative of apoptotic state of cells. Caspase-3 activity was significantly increased during Tanshinone II-A treatment of both HL60 and K562 cells, whereas caspase-1 activity was not changed. These results suggest that Tanshione II-A induced HL60 and K562 cellular apoptosis that may be associated with the selective members of caspase family. (+info)
Using polymerase arrest to detect DNA binding specificity of aristolochic acid in the mouse H-ras gene.
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The distribution of DNA adducts formed by the two main components, aristolochic acid I (AAI) and aristolochic acid II (AAII), of the carcinogenic plant extract aristolochic acid (AA) was examined in a plasmid containing exon 2 of the mouse c-H-ras gene by a polymerase arrest assay. AAI and AAII were reacted with plasmid DNA by reductive activation and the resulting DNA adducts were identified as the previously characterized adenine adducts (dA-AAI and dA-AAII) and guanine adducts (dG-AAI and dG-AAII) by the (32)P-post-labeling method. In addition, a structurally unknown adduct was detected in AAII-modified DNA and shown to be derived from reaction with cytosine (dC-AAII). Sites at which DNA polymerase progress along the template was blocked were assumed to be at the nucleotide 3' to the adduct. Polymerase arrest spectra showed a preference for reaction with purine bases in the mouse H-ras gene for both activated compounds, consistent with previous results that purine adducts are the principal reaction products of AAI and AAII with DNA. Despite the structural similarities among AAI-DNA and AAII-DNA adducts, however, the polymerase arrest spectra produced by the AAs were different. According to the (32)P-post-labeling analyses reductively activated AAI showed a strong preference for reacting with guanine residues in plasmid DNA, however, the polymerase arrest assay revealed arrest sites preferentially at adenine residues. In contrast, activated AAII reacted preferentially with adenine rather than guanine residues and to a lesser extent with cytosine but DNA polymerase was arrested at guanine as well as adenine and cytosine residues with nearly the same average relative intensity. Thus, the polymerase arrest spectra obtained with the AA-adducted ras sequence do not reflect the DNA adduct distribution in plasmid DNA as determined by (32)P-post-labeling. Arrest sites of DNA polymerase associated with cytosine residues confirmed the presence of a cytosine adduct in DNA modified by AAII. For both compounds adduct distribution was not random; instead, regions with adduct hot spots and cold spots were observed. Results from nearest neighbor binding analysis indicated that flanking pyrimidines displayed the greatest effect on polymerase arrest and therefore on DNA binding by AA. (+info)