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M Ouellette

Publications and source records attributed to M Ouellette.

At least 73 records · Page 4Linked to original sources
PubMed1995

New mechanisms of drug resistance in parasitic protozoa.

The main line of defense now available against parasitic protozoa--which are responsible for major diseases of humans and domestic animals--is chemotherapy. This defense is being eroded by drug resistance and, with few new drugs in the pipeline, prevention and circumvention of resistance are medical and veterinary priorities. Although studies of resistance mechanisms in parasites have lagged behind similar studies in bacteria and cancer cells, the tools to tackle this problem are rapidly improving. Transformation with exogenous DNA is now possible with all major parasitic protozoa of humans. Hence, putative resistance genes can be tested in sensitive protozoa, allowing an unambiguous reconstruction of resistance mechanisms. Gene cloning, the polymerase chain reaction, and monoclonal antibodies against resistance-related proteins have made it possible to analyze potential resistance mechanisms in the few parasites that can be obtained from infected people. Hence, the prospect of applying new knowledge about resistance mechanisms to parasites in patients is good, even though today virtually all knowledge pertains to parasites selected for resistance in the laboratory. Resistance mechanisms highlighted in this review include: 1. Decrease of drug uptake because of the loss of a transporter required for uptake. This decrease contributes to resistance to arsenicals and diamidines in African trypanosomes. 2. The export of drugs from the parasite by P-glycoproteins and other traffic ATPases. This export could potentially be an important mechanism of resistance, as these proteins are richly represented in the few protozoa analyzed. There are indications that such transmembrane transporters can be involved in resistance to emetine in Entamoeba spp., to mefloquine in Plasmodium spp., and to antimonials in Leishmania spp. 3. The possible involvement of the P-glycoprotein encoded by the Plasmodium falciparum pfmdr1 gene in chloroquine resistance. We present the available data that lead to the conclusion that overproduction of the wild-type version of this protein results in chloroquine hypersensitivity rather than resistance. 4. The involvement of the PgpA P-glycoprotein of Leishmania spp. in low-level resistance to arsenite and antimonials. We raise the possibility that this protein transports glutathione conjugates of arsenite and antimonials rather than the compounds themselves. 5. Loss of drug activation as the main mechanism of metronidazole resistance in Trichomonas and Giardia spp. Recent evidence indicates that a decrease of the proximal cellular electron donor for metronidazole activation, ferredoxin, is the main cause of resistance in Trichomonas. 6. Resistance arising through alteration of drug targets. The amino acid substitutions in the dihydrofolate reductase-thymidylate synthase of Plasmodium spp. are good examples of this mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

P-glycoprotein overexpression in methotrexate-resistant Leishmania tropica.

A methotrexate (MTX)-resistant Leishmania tropica line develops a stable drug-resistant phenotype in which the resistance mechanism is associated with a significant reduction in MTX accumulation. After a 2 hr exposure to [3H]MTX, a L. tropica line resistant to 1000 microM of MTX did not accumulate more than 3% of the amount of drug incorporated by wild-type cells. The same resistant cell line was found to be cross-resistant to several unrelated drugs. The monoclonal antibody C219, directed against the cytoplasmic domain of mammalian P-glycoproteins, recognized a putative P-glycoprotein of 240 kDa overexpressed in the resistant line. Also, this resistant line showed the overexpression of the putative homolog of the ltpgpE gene, as determined by northern blot analysis using gene-specific probes for the P-glycoprotein genes of Leishmania tarentolae. This overexpression was not correlated with a proportional increase in the copy number of the gene, but Southern blot analysis suggested that the ltpgpE homolog was overexpressed as a consequence of gene rearrangement. This would be considered as an epiphenomenon that probably does not arise from the same MTX-resistant mechanism.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Contribution of the Leishmania P-glycoprotein-related gene ltpgpA to oxyanion resistance.

Oxyanions in the form of pentavalent antimony compounds are currently the drug of choice for treating leishmaniasis. Leishmania mutants resistant to high concentrations of the oxyanion arsenite were obtained in a stepwise selection procedure. Amplification of the H locus P-glycoprotein-related gene ltpgpA, as part of extrachromosomal circles, is a frequent event in arsenite-resistant cells, but was observed only in cells resistant to high concentrations of the metalloid salts. Revertants grown in the absence of the drug lost their ltpgpA-containing amplicon and part of their resistance. The results of previous transfection experiments in Leishmania had suggested that ltpgpA is only involved in low level resistance to arsenite and antimonite. The results of this study using transfection of ltpgpA alleles isolated from arsenite-resistant mutants or of whole circular amplicons containing ltpgpA demonstrate clearly that this P-glycoprotein-related gene is involved in low level resistance to oxyanions, including the pentavalent antimony-containing drug Pentostam. By site-directed mutagenesis, the LtpgpA protein was shown to require an intact nucleotide-binding site to confer arsenite resistance. Under the experimental conditions used, decreased accumulation of the 73AsO2- drug was not observed in the ltpgpA transfectants. One possibility is that LtpgpA-mediated arsenite resistance could result from sequestration of the toxic anion in an intracellular compartment. The results indicate that despite the fact that ltpgpA amplification is a frequent event in oxyanion-resistant mutants, it contributes only slightly to the overall resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Changes in folate and pterin metabolism after disruption of the Leishmania H locus short chain dehydrogenase gene.

The short chain dehydrogenase gene ltdh, which is derived from the H locus of Leishmania, confers high level resistance to the dihydrofolate reductase inhibitor methotrexate via a novel mechanism. Resistance is correlated with LTDH overproduction. High level resistance of ltdh transfectants was observed even in poor media where reduced folates are essential for the synthesis of thymidylate precursors. The ltdh transfectants were also capable of growing for several passages, at slightly reduced rates, in unsupplemented folate-deficient medium (fdDME-L), unlike the wild-type cells that could grow only in fdDME-L if supplemented with various folates or pterins. An homozygous ltdh mutant was obtained by gene targeting. This mutant became hypersensitive to methotrexate, but unlike wild-type cells, methotrexate toxicity could not be circumvented by the addition of thymidine. Although the homozygous mutant was capable of growing in fdDME-L supplemented with folate derivatives, it lost its ability to thrive in fdDME-L supplemented with pterins. Our results support the model in which LTDH is a key enzyme responsible for the conversion of a pterin derivative into an essential cofactor. This essential cofactor can also be converted into reduced folates at sufficient levels for growth when LTDH is overproduced, rendering dihydrofolate reductase dispensable. The novelty and uniqueness of LTDH opens the possibility of developing parasite-specific inhibitors.

Animals↗
PubMed1994

The P-glycoprotein-related gene family in Leishmania.

P-glycoprotein gene amplification has been described in several drug-resistant parasitic protozoa. The first P-glycoprotein related gene described in Leishmania was ltpgpA, a gene frequently amplified in arsenite resistant Leishmania. Hybridization experiments indicated that ltpgpA was part of a gene family. In addition to ltpgpA, four novel genes were cloned that are present in two loci: ltpgpB and ltpgpC tandemly linked to ltpgpA on a 800-kb chromosome; and ltpgpD and ltpgpE closely linked on a chromosome ranging from 950 kb to 1400 kb, depending on the Leishmania species. Another P-glycoprotein gene, homologous to the more recently described ldmdr1, was linked to ltpgpD and ltpgpE. Nucleotide sequencing of ltpgpB and ltpgpE revealed that the Leishmania P-glycoprotein-related genes have diverged considerably from the main branch of P-glycoproteins and are more homologous to the recently described multidrug resistance-associated protein found in multidrug-resistant human lung cancer cell lines. Cross-resistance studies and gene transfection experiments indicated that under the conditions tested only ltpgpA and ldmdr1 are involved in resistance to arsenite and antimonials or hydrophobic drugs such as vinblastine respectively.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

High level arsenite resistance in Leishmania tarentolae is mediated by an active extrusion system.

Leishmania tarentolae cells selected for resistance to the oxyanions pentavalent or trivalent antimonials or to trivalent arsenicals exhibited cross-resistance to the other oxyanions. The basis for resistance in these mutants was studied by transport experiments using radioactive arsenite. All mutants exhibiting high level resistance to arsenite showed a marked decrease in the steady-state accumulation of arsenite. Decreased accumulation was also observed in antimonials-resistant mutants cross-resistant to various concentrations of arsenite. Cells depleted of endogenous energy reserves with metabolic inhibitors were loaded with radioactive arsenite; following addition of glucose, rapid efflux of arsenite was observed from arsenite mutant cells. Mutants resistant to high levels of arsenicals exhibited amplification of the P-glycoprotein related gene ltpgpA or of a linear amplicon of unknown function. However, the efflux-mediated arsenite resistance did not correlate with the amplification of the ltpgpA gene or with the presence of the linear amplicon. The calcium channel blocker verapamil and arsenite act in synergy in cells exhibiting the efflux system. Overall the oxyanion efflux system in Leishmania shares several properties with other resistance efflux systems mediated by transporters.

Animals↗
PubMed1994

Autonomous replication of bacterial DNA plasmid oligomers in Leishmania.

Extrachromosomal amplicons are frequently observed in drug-resistant Leishmania. A dominant selectable marker, the neomycin phosphotransferase gene, was introduced by gene targeting in a circular amplicon derived from the H locus of Leishmania in a mutant cell. This recombinant amplicon was isolated and transfected in a wild-type cell. The amplicon was kept in the wild-type cells, provided the selective pressure was maintained, suggesting that it was capable of autonomous replication. Novel Leishmania expression vectors suited for stable transfections were made to isolate, by a high transformation assay, the putative origin of replication in the amplicons. However, these plasmids, which did not contain a single Leishmania nucleotide, were found as extrachromosomal circular oligomers in Leishmania transfectants. Their relative stability, in addition to changes in their methylation pattern, indicated that these plasmids were most likely replicating. No specific sequences seem to be required for replication (and expression) in Leishmania, therefore precluding the isolation of origins of replication by genetic transformation.

Animals↗
PubMed1994

Microbial multidrug-resistance ABC transporters.

Multidrug resistance in tumor cells is often caused by the increased efflux of a wide variety of drugs, mediated by P glycoprotein, a member of the superfamily of ATP-binding cassette (ABC) transporters. The genes encoding members of this superfamily have also been isolated from drug-resistant microorganisms, and the role of microbial ABC transporters in drug resistance is being investigated.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Molecular cloning and characterization of the hamster preproenkephalin A cDNA.

The cDNA for hamster preproenkephalin A (ENK) was cloned from an adrenal gland cDNA library constructed in the lambda ZapII vector. A nearly full-length cDNA was obtained and its 5' end region was completed using the technique of rapid amplification of cDNA ends (RACE). The coding and 3' untranslated regions of the hamster ENK cDNA share a high sequence identity with the rat, human, and bovine cDNAs, whereas the sequence identity is lower for the 5' untranslated region. Southern blot analysis of genomic DNA digests showed that a single copy of the ENK gene is present in the hamster haploid genome. Northern blot analysis of poly(A)+RNA from various hamster tissues indicated the following rank order for ENK messenger RNA abundance: adrenal glands > right atrium > brain > left atrium > right ventricle > ventricular septum > left ventricle, whereas primer extension analysis showed a single, identical transcriptional initiation site for the ENK mRNA in all these tissues. The sequence of the 5' untranslated region of the heart ENK cDNA was found to be identical to that from adrenal glands. This rules out the possibility that structural divergences in the 5' untranslated region of the heart ENK mRNA could decrease its translation efficiency and contribute to the very low level of enkephalin-containing peptides in the heart, compared to the adrenal glands.

Adrenal Glands↗
PubMed1993

Frequent amplification of a short chain dehydrogenase gene as part of circular and linear amplicons in methotrexate resistant Leishmania.

The H locus of Leishmania codes for a short chain dehydrogenase gene (ltdh) that is involved in antifolate resistance. Leishmania tarentolae cells, selected in a step by step fashion for resistance to the antifolate methotrexate (MTX), frequently amplified ltdh in response to drug selection. Both circular and linear extrachromosomal amplicons were generated de novo from the chromosomal H locus and several contained inverted duplications. At least four different rearrangement points were used during the formation of amplicons, with one of them used preferentially. All mutants highly resistant to MTX, whether or not they have the H locus amplified, showed a decreased steady-state accumulation of MTX. Nevertheless, two types of transport mutants were clearly discernable. In the first type, accumulation was reduced four to five-fold, whereas in the second class of mutants, accumulation was reduced more than 50-fold. The ltdh gene was amplified in all the mutants with the transport mutation of the first type, but not in all the mutants with a more pronounced decrease in the steady-state accumulation of MTX. Both types of transport mutation, leading to the reduction in MTX accumulation, arose early during the selection process and were stable even when cells were grown in absence of the drug for prolonged period.

Animals↗
PubMed1993

Homologous recombination between direct repeat sequences yields P-glycoprotein containing amplicons in arsenite resistant Leishmania.

The protozoan parasite Leishmania often responds to drug pressure by amplifying part of its genome. At least two loci derived from the same 800 kb chromosome were amplified either as extrachromosomal circles or linear fragments after sodium arsenite selection. A 50 kb linear amplicon was detected in six independent arsenite mutants and revertants grown in absence of arsenite rapidly lost the amplicon and part of their resistance. The circular extrachromosomal amplicons, all derived from the H locus of Leishmania, were characterized more extensively. In all cases, direct repeated sequences appeared to be involved in the formation of circular amplicons. Most amplicons were generated after homologous recombination between two linked P-glycoprotein genes. This recombination event was, in two cases, associated with the loss of one allele of the chromosomal copy. A novel rearrangement point was found in a mutant where the amplicon was created by recombination between two 541 bp direct repeats surrounding the P-glycoprotein gene present at the H locus. It is also at one of these repeats that an H circle with large inverted duplications was formed. We propose that the presence of repeated sequences in the H locus facilitates the amplification of the drug resistance genes concentrated in this locus.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Mechanisms of drug resistance in Leishmania.

The emergence of drug resistance in protozoan parasites is a major obstacle to their control. Since vaccines are not yet in sight for several of these parasites, there is on urgent need to develop new and better drugs. These antimicrobial agents will possibly be more expensive, and will therefore impose on additional burden in health-care costs and in the planning of public health policies of the developing countries. A better understanding of drug resistance, to try to circumvent or overcome it, and the search for new specific cellular targets of parasites are warranted. The development, in vitro, of drug-resistant parasite cell lines has been instrumental in our understanding of the mechanisms of drug resistance in parasitic protozoans. Marc Ouellette and Barbara Popodopoulou here present on overview of the recent progress on the elucidation of mechanisms of drug resistance in the protozoan parasite Leishmania, selected under laboratory conditions.

Journal Article↗
PubMed1992

A novel antifolate resistance gene on the amplified H circle of Leishmania.

In several Leishmania spp., resistance to methotrexate and other drugs is often associated with amplification of the chromosomal H region in the form of extrachromosomal H circles. We report here that the H circle of Leishmania tarentolae contains an 867 bp open reading frame, ltdh, which mediates high levels of resistance to methotrexate and other antifolates, after transfection. The predicted amino acid sequence of the ltdh gene product has significant similarities to a family of short-chain dehydrogenases, enzymes that are involved in several oxido-reduction reactions in a wide range of organisms. To resist antifolates, Leishmania amplifies the ltdh gene as part of the H circle. We propose that LTDH might be involved in an alternative pathway for the synthesis of reduced folates and that ltdh overproduction represents a novel mechanism for resistance to antifolates. Our results support the hypothesis that the H region of the Leishmania genome contains several drug resistance genes and that preferential amplification of this region has evolved as a defense mechanism against cytotoxic drugs.

Amino Acid Sequence↗
PubMed1991

Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania.

The H circle of Leishmania species contains a 30 kb inverted duplication separated by two unique DNA segments, a and b. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We show here that the chromosomal segments a and b are flanked by inverted repeats (198 and 1241 bp) and we discuss how these repeats could lead to formation of H circles from chromosomal DNA. Selection of Leishmania tarentolae for methotrexate resistance indeed resulted in the de novo formation of circles with long inverted duplication, but two mutants selected for arsenite resistance contained new H region plasmids without such duplications. One of these plasmids appears due to a homologous recombination between two P-glycoprotein genes with a high degree of sequence homology. Our results show how the same DNA region in Leishmania may be amplified to give plasmids with or without long inverted duplications and apparently by different mechanisms.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Circadian regulation of the biosynthesis of cardiac Met-enkephalin and precursors in normotensive and spontaneously hypertensive rats.

Preproenkephalin A mRNA (ppEnk mRNA) and immunoreactive Met-enkephalin (ir-Met-Enk) were measured in the heart of 4, 8 and 16 week-old normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. WKY rats displayed a small decrease in their cardiac concentration of free (1.3 to 1.0 pmol/g) and cryptic (enzyme processed: 5.3 to 3.7 pmol/g) ir-Met-Enk with aging while the abundance of ppEnk mRNA increased by 3.2 fold between 4 and 16 week-old animals. Similar decreases in free (1.5 to 1.0 pmol/g) and cryptic (5.6 to 4.2 pmol/g) ir-Met-Enk levels were observed in SHR with aging but the rise in the level of ppEnk mRNA was much more pronounced reaching at 16 week-old levels of 7.3 times higher than at 4 week-old and 4.3 times higher than in age-matched WKY. The lack of correlation between the concentration of free and cryptic ir-Met-Enk and the abundance of ppEnk mRNA led us to measure the level of peptides in the heart of 16 week-old animals sacrificed at 4 hr intervals over a 24 hr period. SHR rats displayed circadian variations in their heart content of free and cryptic ir-Met-Enk and increased levels (1.6 fold) of cryptic peptide as compared with WKY at the beginnings of light (6 hr) and dark (18 and 22 hr) periods, suggesting the occurrence of cyclic and transitory upregulation of cDNA transcription and/or derepression of mRNA translation.

Animals↗
PubMed1991

Expression of the preproenkephalin A gene in the heart of cardiomyopathic hamsters.

The expression of the preproenkephalin A gene (Enk gene), which codes for the precursor of enkephalins, was investigated in the heart of hamsters with a hereditary cardiomyopathy at four different stages of the disease: the prenecrotic stage (30 days), the necrotic stage (60 days), the hypertrophic stage (120 days), and the final stage (200 days). In control atria and ventricles, the relative abundance of the Enk mRNA, as assessed by Northern blot analysis, did not change upon ageing. In the ventricles of cardiomyopathic hamsters, however, it increased about two- to three-fold at the necrotic stage, but was unaltered at the other time points studied; whereas in the atria, it progressively decreased to reach about half that of control hamsters at the final stage. Enkephalin levels, as measured by radioimmunoassay, decreased at 60 days in both the atria and ventricles of control hamsters, and also in the atria of cardiomyopathic hamsters, and remained stable thereafter, corresponding to one-third to one-half of those at 30 days. However, in the ventricles of cardiomyopathic hamsters, the peptide level decreased only slightly, the consequence being that at 60, 120, and 200 days, it was about two- to three-fold that of control hamsters. The lack of correlation between peptide levels and the relative abundance of the Enk mRNA suggests that translational and (or) post-translational mechanisms are important in the control of the expression of the Enk gene in the heart of hamsters.

Aging↗
PubMed1991

Molecular biology of myogenic regulatory factors.

A family of proteins has recently been identified, each member of which has the capacity to initiate muscle differentiation in many non-muscle cell types. These factors, which include MyoD1, myogenin, myf-5 and MRF4, share homologies with each other and belong to a superfamily of Myc-related proteins. Expression of these regulatory proteins results in auto-activation and cross-activation of other members of the family and in the transcriptional activation of the markers of terminal differentiation. Sequence analysis has shown a conserved basic domain in each protein that is required for binding to specific DNA sequences of the E-box type and for myogenic activation. A conserved helix-loop-helix (HLH) domain allows homo- and heterodimerization of these muscle-specific proteins with each other and with ubiquitously expressed proteins such as the E2A gene products (E12/E47). This review describes the discovery and characterization of these muscle regulatory proteins and their actions in the context of proposed models for the determination and differentiation of muscle tissue.

Amino Acid Sequence↗