Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment.

The suppression levels induced by gentamicin on premature stop codons, caused by primary nonsense mutations found in muscular dystrophy patients, were assessed using a very sensitive dual reporter gene assay. Results show that: (i) the effect of gentamicin on readthrough is similar in cultured cells and in vivo in murine skeletal muscle; (ii) a wide variability of readthrough efficiency is obtained, depending on the mutation tested; (iii) due to the complexity of readthrough regulation, efficiency cannot be predicted by the nucleotide context of the stop codon; (iv) only a minority of premature stop codons found in patients show a significant level of readthrough, and would thus be amenable to this pharmacological treatment, given our present understanding of the problem. These results probably provide an explanation for the relative failure of clinical trials reported to date using gentamicin to treat diseases due to premature stop codons, and emphasize that preliminary assays in cell culture provide valuable information concerning the potential efficiency of pharmacological treatments.

Impact of the six nucleotides downstream of the stop codon on translation termination.

The efficiency of translation termination is influenced by local contexts surrounding stop codons. In Saccharomyces cerevisiae, upstream and downstream sequences act synergistically to influence the translation termination efficiency. By analysing derivatives of a leaky stop codon context, we initially demonstrated that at least six nucleotides after the stop codon are a key determinant of readthrough efficiency in S. cerevisiae. We then developed a combinatorial-based strategy to identify poor 3' termination contexts. By screening a degenerate oligonucleotide library, we identified a consensus sequence -CA(A/G)N(U/C/G)A-, which promotes >5% readthrough efficiency when located downstream of a UAG stop codon. Potential base pairing between this stimulatory motif and regions close to helix 18 and 44 of the 18S rRNA provides a model for the effect of the 3' stop codon context on translation termination.

Translational errors as an early event in prion conversion.

A prion is an infectious, altered form of a cellular protein which can self-propagate and affect normal phenotype. Prion conversion has been observed for mammalian and yeast proteins but molecular mechanisms that trigger this process remain unclear. Up to now, only post-translational models have been explored. In this work, we tested the hypothesis that co-translational events may be implicated in the conformation changes of the Ure2p protein of Saccharomyces cerevisiae. This protein can adopt a prion conformation leading to an [URE3] phenotype which can be easily assessed and quantified. We analyzed the effect of two antibiotics, known to affect translation, on [URE3] conversion frequency. For cells treated with G418 we observed a parallel increase of translational errors rate and frequency of [URE3] conversion. By contrast, cycloheximide which was not found to affect translational fidelity, has no influence on the induction of [URE3] phenotype. These results raise the possibility that the mechanism of prion conversion might not only involve alternative structures of strictly identical molecules but also aberrant proteins resulting from translational errors.

Nonsense-mediated decay mutants do not affect programmed -1 frameshifting.

Sequences in certain mRNAs program the ribosome to undergo a noncanonical translation event, translational frameshifting, translational hopping, or termination readthrough. These sequences are termed recoding sites, because they cause the ribosome to change temporarily its coding rules. Cis and trans-acting factors sensitively modulate the efficiency of recoding events. In an attempt to quantitate the effect of these factors we have developed a dual-reporter vector using the lacZ and luc genes to directly measure recoding efficiency. We were able to confirm the effect of several factors that modulate frameshift or readthrough efficiency at a variety of sites. Surprisingly, we were not able to confirm that the complex of factors termed the surveillance complex regulates translational frameshifting. This complex regulates degradation of nonsense codon-containing mRNAs and we confirm that it also affects the efficiency of nonsense suppression. Our data suggest that the surveillance complex is not a general regulator of translational accuracy, but that its role is closely tied to the translational termination and initiation processes.

Competitive reverse transcription-polymerase chain reaction for quantifying murine AIDS virus.

The causative agent of murine AIDS (MAIDS) is the defective murine leukemia virus BM5d, that requires the replication-competent ecotropic MuLV (BM5e) helper virus. We developed a competitive quantitative PCR method including specific internal standards to quantify the expression of BM5d in the spleen of infected mice and to characterize BM5d expression kinetics following experimental infection. Specimen RNA was reverse-transcribed and co-amplified with a competitive template containing a gag sequence specific for BM5d that can be discriminated from that corresponding to wild-type cDNA by the presence of a unique restriction site, Bg/II. PCR products were quantified by means of densitometric analysis after ethidium bromide staining of gels. To standardise the RNA extraction and reverse transcription steps, the amount of defective-virus mRNA was compared to a constant copy number of murine beta actin mRNA. LP-BM5 production was measured in the spleen of infected mice. Defective gag mRNA production was compared to that of the ecotropic virus. The mRNA level of the defective virus and the titre of replicative virus increased with the duration of infection, and the amount of defective virus mRNA correlated with the titre of replicating virus.

Molecular characterisation of the ADP/ATP-transporter cDNA from the human malaria parasite Plasmodium falciparum.

We have isolated a cDNA sequence encoding the ADP/ATP transporter in Plasmodium falciparum. The sequence analysis revealed an open reading frame encoding 301 amino acids and showed significant similarities to known eukaryotic translocases such as that of Chlorella (up to 67.2% identity) and the human transporter (61.2%). RNA blot analysis showed the presence of mRNA encoding for a 33.7-kDa ADP/ATP transporter. During the cell cycle of the parasite the expression levels of the transcripts fluctuate. The mitochondrial ADP/ATP transporter could play a role in energy metabolism of P. falciparum and makes this transporter an excellent target for chemotherapy.

An alternative to DNA extraction for the diagnosis of Pneumocystis carinii pneumonia by polymerase chain reaction using a new oligonucleotide probe.

We have used new specific primers and probe in a polymerase chain reaction (PCR) followed by Southern blot assays to detect Pneumocystis carinii in human bronchoalveolar lavage samples obtained from HIV-infected patients with pulmonary symptoms. To facilitate the procedure we developed a filtration technique without DNA extraction yielding a high sensitivity (18/18 positive results). The high specificity of the technique was shown by testing immunosuppressed patients without P. carinii pneumonia.

Susceptibility of Plasmodium falciparum strains to mefloquine in an urban area in Senegal.

A total of 47 nonimmune febrile patients from Pikine, Senegal, with greater than 1,000 Plasmodium falciparum asexual forms per microliter whole blood were given 12.5 mg per kg body weight of mefloquine in a single oral dose and were followed up daily until day 7 and also on day 14 of the study. Seven of the patients who vomited, four who had 4-aminoquinolines in their blood, and five dropouts were excluded. Fever and parasitaemia were suppressed within four days until day fourteen in 29 of the 31 remaining patients, including 10 with P. falciparum strains that had a low sensitivity to mefloquine. Two failures were due to poor absorption of mefloquine. The presence of P. falciparum strains with low in vitro susceptibility to mefloquine did not affect, within 14 days, the clinical and parasitological efficacy of a single oral dose mefloquine regimen in patients who had received no previous antimalarial treatment and who did not have partial immune protection.

Susceptibility of Plasmodium falciparum strains to mefloquine in an urban area in Senegal

A total of 47 nonimmune febrile patients from Pikine, Senegal, with greater than 1,000 Plasmodium falciparum asexual forms per microliter whole blood were given 12.5 mg per kg body weight of mefloquine in a single oral dose and were followed up daily until day 7 and also on day 14 of the study. Seven of the patients who vomited, four who had 4-aminoquinolines in their blood, and five dropouts were excluded. Fever and parasitaemia were suppressed within four days until day fourteen in 29 of the 31 remaining patients, including 10 with P. falciparum strains that had a low sensitivity to mefloquine. Two failures were due to poor absorption of mefloquine. The presence of P. falciparum strains with low in vitro susceptibility to mefloquine did not affect, within 14 days, the clinical and parasitological efficacy of a single oral dose mefloquine regimen in patients who had received no previous antimalarial treatment and who did not have partial immune protection

[Sensitivity of Plasmodium falciparum to quinolines and therapeutic strategies: comparison of the situation in Africa and Madagascar between 1983 and 1986]. / Sensibilité de Plasmodium falciparum aux quinoléines et stratégies thérapeutiques: comparaison de la situation en Afrique et à Madagascar entre 1983 et 1986.

One thousand and twenty six P. falciparum strains isolated from cases imported in France and field surveys in four regions of Africa and Madagascar were studied in vitro against chloroquine, monodesethylamodiaquine, quinine and mefloquine, 917 in vivo tests were performed during field studies with chloroquine (10 and 25 mg/kg) and amodiaquine (10, 25, 35 mg/kg). In Madagascar, the chemoresistance remained low and stable during the study period, concerning mostly chloroquine (11% in vitro and in vivo) without obvious geographical variation. 25 mg/kg chloroquine or amodiaquine were satisfactory as respectively first and second line therapeutic regimen. In Central Africa, chemoresistance emerged with an epidemic profile and increased dramatically in disseminated urban focus. High level and prevalence of chloroquine resistance and multiresistance were observed few months after the index cases in these foci. In South West Cameroon, amodiaquine remained efficient as curative treatment but only at a dose of 35 mg/kg/5 days. Decrease of in vitro sensitivity and in vivo efficacy of quinine is a matter of concern. Given the heterogeneous and evolutive situation of drug resistance, the need for epidemiological surveillance and monitoring of P. falciparum drug sensitivity in Africa is obvious to adjust therapeutic regimen.

Chloroquine uptake by Plasmodium falciparum-infected human erythrocytes during in vitro culture and its relationship to chloroquine resistance.

Chloroquine uptake by Plasmodium falciparum-infected human erythrocytes (RBC) was studied in vitro before and during culture by measuring the chloroquine gradient between the cells and medium (C/M) by high-pressure liquid chromatography. The C/M values were 5.9 +/- 2.7 (n = 23) for uninfected RBC, 13 to 34 for six chloroquine-susceptible isolates (concentration required to inhibit 50% of parasite growth, less than 100 nmol/liter) in partially infected RBC (parasitemia from 0.3 to 5%) (n = 28), and 8.4 to 4.9 for four chloroquine-resistant isolates (concentration required to inhibit 50% of parasite growth, 320 to 1,500 nmol/liter) in partially infected RBC (parasitemia from 0.4 to 5%) (n = 26). Two isolates were studied before and after adaptation to continuous culture. C/M was found to decrease (34.2 to 2.1 and 19.3 to 4.9), whereas the concentration required to inhibit 50% of parasite growth increased (35 to 1,400 and 54 to 1,500 nmol/liter), thus indicating the acquisition of chloroquine resistance. These results demonstrate that chloroquine uptake decreased in RBC in which the infective strain, initially susceptible, became resistant in culture and imply that the drug is bound to ferriprotoporphyrin IX to a lesser extent or that a parasite protein competes with ferriprotoporphyrin IX to a greater extent. We suggest that genotypic modifications in the mechanism of chloroquine uptake might occur in the parasite.