Real-time PCR for chloroquine sensitivity assay and for pfmdr1-pfcrt single nucleotide polymorphisms in Plasmodium falciparum

Plasmodium falciparum drug resistance is a major problem in malaria endemic areas. Molecular markers and in vitro tests have been developed to study and monitor drug resistance. However, none, used alone, can provide sufficient data concerning the level of drug resistance and to issue precise guidelines for drug use policies in endemic areas. We propose real-time PCR for the simultaneous detection of pfcrt and pfmdr1 genes mutations and to determine the half-maximal inhibitory response (IC(50)) of antimalarial drug. Using hybridization probes and SybrGreen technology on LightCycler instrument, point mutations of pfcrt and pfmdr1 genes have been successfully detected in 161 human blood samples and determination of IC values was applied to chloroquine-sensitive and chloroquine-resistant strains. Moreover, mixed infections caused by P. falciparum clones with wild-type or mutant alleles could be efficiency separated. The aim of this study was not to provide definitive data concerning the rate of mutations in an endemic area, but to describe a powerful method allowing the quantification of DNA for IC(50) determination and the detection of major pfmdr1 and pfcrt mutations.     

Identification of essential genes in the human fungal pathogen Aspergillus fumigatus by transposon mutagenesis

The opportunistic pathogen Aspergillus fumigatus is the most frequent cause of deadly airborne fungal infections in developed countries. In order to identify novel antifungal-drug targets, we investigated the genome of A. fumigatus for genes that are necessary for efficient fungal growth. An artificial A. fumigatus diploid strain with one copy of an engineered impala160 transposon from Fusarium oxysporum integrated into its genome was used to generate a library of diploid strains by random in vivo transposon mutagenesis. Among 2,386 heterozygous diploid strains screened by parasexual genetics, 1.2% had a copy of the transposable element integrated into a locus essential for A. fumigatus growth. Comparison of genomic sequences flanking impala160 in these mutants with that of the genome of A. fumigatus allowed the characterization of 20 previously uncharacterized A. fumigatus genes. Among these, homologues of genes essential for Saccharomyces cerevisiae growth have been identified, as well as genes that do not have homologues in other fungal species. These results confirm that heterologous transposition using the transposable element impala is a powerful tool for functional genomics in ascomycota, and they pave the way for defining the complete set of essential genes in A. fumigatus, the first step toward target-based development of new antifungal drugs.     

Rapamycin treatment results in GATA factor-independent hyperphosphorylation of the proline utilization pathway activator in Saccharomyces cerevisiae

Treatment of Saccharomyces cerevisiae cells with the immunosuppressive drug rapamycin results in a variety of cellular changes in response to perceived nutrient deprivation. Among other effects, rapamycin treatment results in the nuclear localization of the global nitrogen activators Gln3p and Nil1p/Gat1p, which leads to expression of nitrogen assimilation genes. The proline utilization (Put) pathway genes were shown to be among the genes induced by rapamycin. Having previously shown that the Put pathway activator Put3p is differentially phosphorylated in response to the quality of the nitrogen source, we examined the phosphorylation status of Put3p after rapamycin treatment. Treatment with rapamycin resulted in the hyperphosphorylation of Put3p, which was independent of Gln3p, Nil1p, and Ure2p. The relative contributions of global nitrogen (Gln3p and Nil1p) and pathway-specific (Put3p) activators to rapamycin-induced expression of the target gene PUT1 were also examined. We found that Nil1p and Put3p, but not Gln3p, play major roles in rapamycin-induced PUT1 expression. Our findings show that perceived nitrogen deprivation triggered by rapamycin treatment and steady-state growth in nitrogen-derepressing conditions are associated with hyperphosphorylation of Put3p and increased PUT1 expression. Rapamycin treatment and nitrogen derepression may share some, but not all, regulatory elements, since Gln3p and Nil1p do not participate identically in both processes and are not required for hyperphosphorylation. A complex relationship exists among the global and pathway-specific regulators, depending on the nature and quality of the nitrogen source.     

Developmentally regulated chromosome fragmentation linked to imprecise elimination of repeated sequences in paramecia

The chromosomes of ciliates are fragmented at reproducible sites during the development of the polyploid somatic macronucleus, but the mechanisms involved appear to be quite diverse in different species. In Paramecium aurelia, the process is imprecise and results in de novo telomere addition at locally heterogeneous positions. To search for possible determinants of chromosome fragmentation, we have studied an ~21-kb fragmentation region from the germ line genome of P. primaurelia. The mapping and sequencing of alternative macronuclear versions of the region show that two distinct multicopy elements, a minisatellite and a degenerate transposon copy, are eliminated by an imprecise mechanism leading either to chromosome fragmentation and the formation of new telomeres or to the rejoining of flanking sequences. Heterogeneous internal deletions occur between short direct repeats containing TA dinucleotides. The complex rearrangement patterns produced vary slightly among genetically identical cell lines, show non-Mendelian inheritance during sexual reproduction, and can be experimentally modified by transformation of the maternal macronucleus with homologous sequences. These results suggest that chromosome fragmentation in Paramecium is the consequence of imprecise DNA elimination events that are distinct from the precise excision of single-copy internal eliminated sequences and that target multicopy germ line sequences by homology-dependent epigenetic mechanisms.     

Dual benefit of reduced Cx43 on atherosclerosis in LDL receptor-deficient mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR(-/-)) mice were intercrossed with mice heterozygous for Cx43 (Cx43(+/-) mice). Male mice with normal (Cx43(+/+)LDLR(-/-)) or reduced (Cx43(+/-)LDLR(-/-)) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43(+/-)LDLR(-/-) mice compared with Cx43(+/+)LDLR(-/-) littermate controls. In addition, atheroma in Cx43(+/-)LDLR(-/-) mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

The early ontogeny of digestive and metabolic enzyme activities in two commercial strains of arctic charr (Salvelinus alpinus L.)

The extent to which growth performance is linked to digestive or energetic capacities in the early life stages of a salmonid species was investigated. We compared two strains of Arctic charr known to have different growth potentials during their early development (Fraser and Yukon gold). Trypsin, lipase, and amylase activities of whole alevins were measured at regular intervals from hatching through 65 days of development. To assess catabolic ability, we also measured five enzymes representing the following metabolic pathways: amino acid oxidation (amino aspartate transferase), fatty acid oxidation (beta-hydroxy acyl CoA-dehydrogenase), tricarboxylic acid cycle (citrate synthase), glycolysis (pyruvate kinase), and anaerobic glycolysis (lactate dehydrogenase). The measurement of these enzyme activities in individual fish allowed a clear evaluation of digestive capacity in relation to energetic demand. We also compared triploid and diploid individuals within the Yukon gold strain. For the whole experimental period, diploid Yukon gold fish exhibited the highest growth rate (1.08+/-0.18% length/day) followed by triploid Yukon gold fish (1.00+/-0.28% length/day) and finally Fraser strain fish (0.84+/-0.28% length/day). When differences in enzyme activities were observed, the Fraser strain showed higher enzyme activities at a given length than the Yukon gold strain (diploid and triploid). Higher growth performance appears to be linked to lower metabolic capacity. Our results suggest that fish may have to reach an important increase in the ratio of digestive to catabolic enzyme activities or a leveling off of metabolic enzyme activities before the onset of large increases in mass.     

MD studies of the DIS/DIS kissing complex solution and x-ray structures

As in all retroviruses, human immunodeficiency virus (HIV) genomic RNA is packaged into virions as a dimer. The two copies of the genome are noncovalently linked by their 5'-ends in the dimerization initiating site (DIS), which folds as a hairpin containing an apical autocomplementary sequence. In vitro, DIS is able to dimerize in two conformations: a kissing complex and an extended dimer. Both conformations have been resolved by NMR and x-ray diffraction. Here, we report molecular dynamics (MD) studies of the two available structures for the DIS/DIS kissing complex in aqueous solution and in the presence of sodium counterions. The two structures behave in two different manners. On one hand, the NMR structure displays a very stable behavior, and the simulated structure remains very close to the starting structure. On the other hand, the structure issued from crystallography displays a more dynamic behavior, in which residues A8 and A9 are seen in a new and surprising bulge-in conformation. The transition from the bulge-out to the bulge-in conformation is analyzed, and a new and simple dimerization process is proposed.     

Yeast communities associated with stingless bees

The yeast communities associated with the stingless bees Tetragonisca angustula, Melipona quadrifasciata and Frieseomelitta varia were studied. The bees T. angustula and F. varia showed a strong association with the yeast Starmerella meliponinorum. M. quadrifasciata more frequently carried a species related to Candida apicola, but also vectored low numbers of S. meliponinorum. Some of the yeasts isolated from adult bees were typical of species known to occur in flowers. Other yeast species found in adult bees were more typical of those found in the phylloplane. S. meliponinorum and the species in the C. apicola complex, also part of the Starmerella clade, may have a mutualistic relationship with the bees studied. Many yeasts in that group are often found in bees or substrates visited by bees, suggesting that a mutually beneficial interaction exists between them.     

Three-phase liquid-phase microextraction of weakly basic drugs from whole blood

The behaviour of weak basic analytes in liquid-phase microextraction (LPME) and the optimisation of parameters in whole blood are described. Benzodiazepines and non-benzodiazepine drugs were chosen as model substances. Liquid-phase microextraction based on disposable polypropylene hollow fibres was used in the three-phase extraction of five weak bases from whole blood. The sample work up with the liquid-phase microextraction technique can be impeded by low recovery due to incomplete trapping in the acceptor phase of weakly basic drugs and the complexity of the whole blood matrix. Different parameters related to this problem were experimentally studied. Additionally the stability of the analytes was examined because of low pH in the acceptor phase. The investigation resulted in optimised LPME conditions for the extraction of weak bases from whole blood. The parameters limiting the recovery were evaluated.