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AbstractWe studied the uptake and intracellular distribution of an FITC labelled phosphodiester oligodeoxynucleotide (ODN) vectorized by a dendrimeric structure in cell culture.

Long-lasting relapsing or lingering rheumatic musculoskeletal pain (RMSP) is the hallmark of Chikungunya virus (CHIKV) rheumatism (CHIK-R). Little is known on their prognostic factors. The aim of this prognostic study was to search the determinants of lingering or relapsing RMSP indicative of CHIK-R.

Background: Data about anti-malarial drugs prescription practices in Europe and the safety of imported malaria treatments are scanty. In 1999, a French consensus development conference published guidelines for the prevention and treatment of imported P. falciparum malaria. The impact of these guidelines has not been evaluated. Aim: To investigate the impact of these guidelines on the prescription of anti-malarials, and to evaluate the incidence of acute drug events (ADEs) leading to discontinuation of treatment. Design: Cross-sectional survey. Methods: Members of the medical staff in 14 French infectious and tropical disease wards completed a standardized form for each patient treated for imported malaria in 2001. A propensity score matching technique was used to estimate the risk of ADEs leading to discontinuation of the regimen. Results: In the 474 patients studied, quinine was the first-line anti-malarial most often prescribed. Only 3% of patients received halofantrine. Mefloquine was associated with a RR of 4.9 (95%CI 3.2–7.4, p<0.00001) risk of discontinuation of treatment due to ADEs. Discussion: The very limited use of halofantrine indicates that the main practice recommendations of the guidelines have been taken into account. Mefloquine was associated with a substantial risk of discontinuing the treatment because of ADEs. This is a serious limitation for the use of mefloquine in the treatment of out-patients with imported malaria.

For Abstract see ChemInform Abstract in Full Text.

For Abstract see ChemInform Abstract in Full Text.

Both vitamin A deficiency and trachoma are important causes of preventable ocular diseases. We examined 10 559 children in Mali for clinical signs of trachoma and of xerophthalmia. In five regions, the mean prevalence of trachoma was 39·3% (95% CI: 37·4–41·2). Clinical xerophthalmia was present among 1·89 % of children (1·59–2·25). The diagnosis of xerophthalmia appeared to be associated with active trachoma (OR=204 [1·52–2·74]). This association remained after controlling for socio-economic status.

In our previous works we have shown that the oligonucleotides 5′-GGGGAGGGGGAGG-3′ and 5′-GGAGGGGGAGGGG-3′ give very stable and specific triplexes with their target double stranded DNAs [Svinarchuk, F., Bertrand, J.-R. and Malvy, C. (1994) Nucleic Acids Res., 22, 3742-3747; Svinarchuk, F., Paoletti, J. and Malvy, C. (1995) J. Biol Chem., 270, 14 068-14 071]. The target for the invariable part of these oligonucleotldes, 5′-GGAGGGGGAGG-3′, is found in a highly conserved 20 bp long purine/pyrimidine tract of the vpx gene of the SIV and HIV-2 viruses and could be a target for oligonucleotide directed antivirus therapy. Here we report on the ability of four purine oligonucieotides with different lengths (11-, 14-, 17- and 20-mer) to form triplexes with the purine/pyrimidine stretch of the vpx gene. Triplex formation was tested by Joint dimethyl sulfate (DMS) footprint, gel-retardation assay, circular dichrolsm (CD) and UV-melting studies. Dimethyl sulfate footprint studies revealed the antiparallel orientation of the third strand to the purine strand of the Watson-Crick duplex. However, the protection of the guanines at the ends of the target sequence decreased as the length of the third strand oligonucleotide increased. Melting temperature studies provided profiles with only one transition for all of the tripiexes. The melting tenpe t werei as for the targeted duplex in the case of the 11- and 14-mer third strands while for the 17- and 20-mer third strands the melting temperature of the triplexes were correspondingly 4 and 8°C higher than for the duplex. Heating and cooling melting curves were reversible for all of the tested triplexes except one with the 20-mer third strand oligonucleo tide. Circular dichroism spectra showed the ability of the target DNA to adopt an A-like DNA conformation.Upon triplex formation the A-DNA form becomes even more pronounced. This effect depends on the length of the thlrd strand oligonucleotide: the CD spectrum shows a ‘classical’ A-DNA shape wtth the 20-mer. This is not observed wtth the purin/pyrimidine stretch of the HN-1 DNA which keeps an-like spectrum even after triplex formation. We suggest, that an A-like duplex DNA is required for the formation of a stable DNA purlne(purine-pyrimidine) triplex.

α and ß-anomeric d(G2T12G2) oligodeoxyribonucleotides were compared for their hybridization to rA12 : the observed melting temperatures are 27°C for ß-oligodeoxyribonucleotide/RNA hybrid and 53°C for α-oligodeoxyribonucleotide/RNA. α-oligonucleotides with the four bases, complementary to natural mRNAs, were synthesized for the first time, labeled at their 5′-end with [32P] and used as probes in Northern blot experiments. In spite of these higher affinities for their target RNA's, they were unable to block translation of natural or synthetic mRNA's in rabbit reticulocyte lysate. We have studied the RNase H activity on model rA12:α- or ß-d(G2T12G2) hybrids or on mRNA:α- or ß-oligonucleotides hybrids. Specific hybridization protects RNA strech when using α-oligonucleotides but not ß-oligonucleotides. Thus, our results show the inability of RNase H to degrade RNA in α-oligodeoxyribonucleotides:RNA duplexes.

A homopurine-homopyrimidine region of murine c pim-1 proto-oncogene was chosen as a target for triplehelix-forming oligonucleotide. Oligonucleotide 5′-GGGGAGGGGGAGG-3′ was shown to bind to its target sequence in the presence of 50 mM Na$ or K$, 10 mM MgCI2 and 20 mM Tris-acetate, pH 7.5. This oligonucleotide is bound in an antiparallel orientation with respect to the homopurine sequence. As was shown by co-migration assay the triplex is stable up to 65°C. At 37°C it was practically irreversible: after 24 hours of co-migration assay there was no traces of triplex dissociation. The rate of triplex formation was highly accelerated with increase of temperature and Mg2$ concentration. This rate was higher for superhelical DNA when compared to the linear and circular ones and the preference was dependent from temperature and Mg2$ concentration. The precision of this interaction is extremely high: sequences in c-pim-1 promoter region with only one substitution when compared to the target gave negligible triplex formation under investigated conditions. These data suppose that natural triplex structures could play an important role in eukaryotic gene regulation and/or chromatin structure formation.

Serum antioxidant vitamins A (retinol) and E (α-tocopherol), β-carotene, zinc and selenium for 418 children with newly diagnosed malignancy were compared with those of 632 cancer-free controls. Incident cancer cases and controls were 1–16 years old and recruited in 1986–1989. Age- and sex-adjusted serum concentrations of retinol, β-carotene and α-tocopherol were significantly inversely associated with cancer. In similar models, the odds ratio (OR) comparing the highest with the lowest quintile was 2.06 (95% confidence interval [CI] 1.40–3.02) for retinol, 3.87 (95% CI:2.54–5.90) for β-carotene, 2.15 (95% CI:1.48–3.10) for α-tocopherol, 1.29 (95% CI:0.75–2.23) for selenium, and 1.94 (95% CI:1.17–2.23) for zinc. The cancer sites that were associated with serum β-carotene were, in general, leukaemia, lymphoma, central nervous system, bone and renal tumours. Moreover, leukaemia was associated with low mean serum levels of retinol, selenium and zinc. Subjects with lymphoma, bone and renal tumours also had lower mean retinol and α-tocopherol levels than controls. Brain tumour patients had low vitamin E levels. Low serum values of antioxidant vitamins were associated with childhood neoplasm occurrence. Some site-specific effect was reported. Low peripheral nutrient levels are not considered as cancer promoters but rather as an impairment of the body's defence mechanism occurring during the cancer-related metabolic and nutritional disturbances and inflammation processes.

Age- and sex-specific reference intervals of data derived from a healthy paediatric population are presented for retinol, β-carotene, α-tocopherol, cholesterol and related proteins in serum. Age was an important covariate for the micronutrient concentration values. Retinol was highly correlated with a-tocopherol and cholesterol. Strong correla tion was found with both β-carotene and cholesterol. Serum levels were considered for their appropriateness as indicators of micronutrient status.

In a previous work we showed that a short triple helix-forming oligonucleotide (TFO) targeted to the murine c-pim-1 proto-oncogene promoter gives a very stable triple helix under physiological conditions in vitro. Moreover, this triplex was stable inside cells when preformed in vitro. However, we failed to detect triplex formation for this sequence inside cells in DMS footprinting studies. In the present work, in order to determine whether our previous in vivo results are limited to this particular short triplex or can be generalized to other purine·(purine/pyrimidine) triplexes, we have tested three other DNA targets already described in the literature. All these purine·(purine/pyrimidine) triplexes are specific and stable at high temperature in vitro. In vivo studies have shown that the preformed triplexes are stable inside cells for at least 3 days. This clearly demonstrates that intracellular conditions are favourable for the existence of purine·(purine/pyrimidine) triplexes. The triplexes can also be formed in nuclei. However, for all the sequences tested, we were unable to detect any triple helix formation in vivo in intact cells by DMS footprinting. Our results show that neither (i) chromatinization of the DNA target, (ii) intracellular K+ concentration nor (iii) cytoplasmic versus nuclear separation of the TFO and DNA target are responsible for the intracellular arrest of triplex formation. We suggest the existence of a cellular mechanism, based on a compartmentalization of TFOs and/or TFO trapping, which separates oligonucleotides from the DNA target. Further work is needed to find oligonucleotide derivatives and means for their delivery to overcome the problem of triplex formation inside cells.

G,A-containing purine oligonucleotides of various lengths form extremely stable and specific triplexes with the purine-pyrimidine stretch of the vpx gene [Svinarchuk,F., Monnot,M., Merle,A., Malvy,C. and Fermandjian,S. (1995) Nucleic Acids Res., 22, 3742–3747]. The potential application of triple-helix-forming oligonucleotides (TFO) in gene-targeted therapy has prompted us to study triplex formation mimicking potassium concentrations and temperatures in cells. Triplex formation was tested by dimethyl sulphate (DMS) footprinting, gel-retardation, UV melting studies and electron microscopy. In the presence of 10 mM MgCl2, KCl concentrations up to 150 mM significantly lowered both efficiency (triplex: initial duplex) and rate constants of triplex formation. The KCl effect was more pronounced for 11mer and 20mer TFOs than for 14mer TFO. Since the dissociation half-life for the 11mer TFO decreases from 420 min in the absence of monovalent cations to 40 min in the presence of 150 mM KCl, we suggest that the negative effect could be explained by a decrease in triplex stability. In contrast, for the 20mer TFO no dissociation of the triplex was observed during 24 h of incubation either in the absence of monovalent cations or in the presence of 150 mM KCl. We suppose that in the case of the 20mer TFO the negative effect of KCl on triplex formation is probably due to the self-association of the oligonucleotide in competitive structures such as parallel duplexes and/or tetraplexes. This negative effect may be overcome by the prior formation of a short duplex either on the 3′- or 5′-end of the 20mer TFO. We refer to these partial duplexes as ‘zipper’ TFOs. It was demonstrated that a ‘zipper’ TFO can form a triplex over the full length of the target, thus unzipping the short complementary strand. The minimal single-stranded part of the ‘zipper’ oligonucleotide which is sufficient to initiate triplex formation can be as short as three nucleotides at the 3′-end and six nucleotides at the 5′-end. We suggest that this type of structure may prove useful for in vivo applications.

The potential use of α-β-anomeric duplex oligonucleotides to inhibit transcription factor activity by the decoy approach is investigated in this report. Indeed, several α β-anomeric heteroduplexes display a sequence-specific interaction with the p50 subunit of the transcription factor NFxB. Used in a decoy approach, these duplexes interact strongly enough with this transcription factor to modulate the expression of a reporter gene, under the control of NFxB. However, all the a-jS-anomeric heteroduplexes do not interact with the p50 subunit; the sequence of the chirally natural β-anomeric strand may explain the different recognition properties of the protein. The analysis of the appropriate β-anomeric sequences is consistent with a preferential interaction of the p50 subunit with one strand of double-stranded DNA.

Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a hairpin-TFO able to recruit transcription factors to a target DNA. The designed oligonucleotide contains a triplex-forming sequence, linked through a nucleotide loop to a double-stranded hairpin including the SRE enhancer of the c-fos gene promoter. We show here that this oligonucleotide can specifically recognise its DNA target at physiological salt and pH conditions. The stability of the triplex formed under these conditions is very high: >90% of the triplex remains intact after 24 h of incubation. Bound to the double-stranded target DNA, the oligonucleotide retains its ability to interact specifically with transcription factors, recruiting them to the proximity of the target DNA. Our results suggest that this type of oligonucleotide may prove useful in the design of new tools for artificial modulation of gene expression.

This paper describes for the first time the synthesis of α-oligonucleotides containing the four usual bases. Two unnatural hexadeoxyribonucleotidesi α-[d(CPAPTPGPCPG)] and α-[d(CPGPCPHPTPG)], consisting only of a-anomeric nucleotide units, were obtained by an improved phosphotriester method, in solution. Starting material was the four base-protected α-deoxyribo-nucleosides 3-d. Pyrimidine a-deoxynucleosides 3a and 3b were prepared by self-anomerization reactions followed by selective deprotection of sugar hydroxyles, while the two purine α-deoxynucleosides 3c and 3d were prepared by glycosylation reactions. In the case of guanine α-nucleoside derivative a supplementary base-protecting group: N, N-diphenylcarbamoyl was introduced on O6-position in order to avoid side-reactions during oligonucleotide assembling. The hexadeorynucleotide α-[d(CPAPTPGPCPG)] was tested as substrate of selected endo- and exonucleases. In conditions where the natural corresponding β-hexamer was completely degradated by nuclease s1 and calf spleen phosphodiesterase, the α-oligonucleotide remained almost intact.

In our previous work we have shown that the oligonucleotide 5′-GGGGAGGGGGAGG-3′ gives a very stable and specific triplex with the promoter of the murine c-pim-1 proto-oncogene in vitro [Svinarchuk,F., Bertrand,J.-R. and Malvy,C. (1994) Nucleic Acids Res., 22, 3742–3747]. In the present work, we have tested triplex formation with some derivatives of this oligonucleotide which are designed to be degradation-resistant inside the cells, and we show that phosphorothioate and the oligonucleotide with a 3′ terminal amino group are still able to form triplexes. Moreover these oligonucleotides, like the 13mer oligonucleotide of similar composition [Svinarchuk,F., Paoletti,J., and Malvy,C. (1995) J. Biol. Chem., 270, 14068–14071], are able to stabilize the targeted duplex. In vivo DMS footprint analysis after electroporation of the pre-formed triplex into the cell have shown the presence of the triple helix inside the cells. This triplex structure partially blocks c-pim-1 promoter activity as shown by transient assay with a c-pim-1 promoter-luciferase gene construct. To our knowledge these data are the first direct evidence that conditions inside cells are favorable for triplex stability with non-modified oligonucleotides. However we were unable to show triplex formation inside living cells using various methods of oligonucleotide delivery. We suppose that this may be due to the oligonucleotide being sequestered by cellular processes or proteins. Further work is needed to find oligonucleotide derivatives and ways of their delivery to overcome the problem of triplex formation inside the cells.

A 13 mers abasic oligonucleotide was synthetized. It was therefore possible to compare thermal stability and reactivity of duplex oligonucleotides either with an apurinic/apyrimidinic site or without any lesion. An important decrease in the melting temperature appeared for duplexes with an abasic site. The chemical reaction of these modified oligonucleotides with the intercalating agent 9-aminoellipticine was studied by gel electrophoresis and by fluorescence. The formation of a Schiff base between 9-aminoellipticine and abasic sites was rapid and complete with duplexes at 11°C. Schiff base related fluorescence and β-elimination cleavage were more important with the apyrimidinic sites than with the apurinic ones. When compared to previous results obtained with the model d(TprpT) some unexpected behaviours appeared with longer and duplex oligonucleotides. For instance only partial β-elimination cleavage was observed. It is likely that stacking parameters in the double helix play a great role in the studied reaction.