Structural study of DNA duplex containing an N-(2-deoxy-beta-D-erythro-pentofuranosyl) formamide frameshift by NMR and restrained molecular dynamics.

The presence of an N-(2-deoxy-beta-D-erythro-pentofuranosyl) formamide (F) residue, a ring fragmentation product of thymine, in a frameshift context in the sequence 5'-d-(AGGACCACG)*d(CGTGGFTCCT) has been studied by 1H and 31P nuclear magnetic resonance (NMR) and molecular dynamics. Two-dimensional NMR studies show that the formamide residue, whether the cis or trans isomer, is rotated out of the helix and that the bases on either side of the formamide residue in the sequence, G14 and T16, are stacked over each other in a way similar to normal B-DNA. The cis and trans isomers were observed in the ratio 3:2 in solution. Information extracted from 31P NMR data reveal a modification of the phosphodiester backbone conformation at the extrahelical site, which is also observed during the molecular dynamics simulations.

Solution structure as a function of pH of two central mismatches, C . T and C . C, in the 29 to 39 K-ras gene sequence, by nuclear magnetic resonance and molecular dynamics.

The DNA duplexes 5' d(GCCACCAGCTC) x d(GAGCTXGTGGC), where the base X is either cytosine or thymine, have been studied by one and two-dimensional nuclear magnetic resonance, energy minimization and molecular dynamics. The sequence studied corresponds to the region 29 to 39 of the K-ras gene and is a hot spot for mutations. The results show that both duplexes adopt a globally B-DNA-type structure. For the C x C mismatch, we observe a structural change as a function of pH with an apparent pK of 6.95. The neutral species has only one hydrogen bond between the two bases but shows two families of wobble structures where one base or the other is displaced in the major groove. The protonated species has two hydrogen bonds and two structures but of unequal populations. In both systems, the sugar puckers remain predominantly C2'-endo and no significant changes in the backbone structure are observed. The neutral C . T mismatch is stabilized by two hydrogen bonds but, surprisingly, it can also be protonated, although the apparent pK is much lower, 5.65. In this case, protonation does not result in an additional hydrogen bond but must be due to better base-stacking interactions for C+ x T. The NMR data show that the environment of the T imino proton is very similar for C x T and C+ x T, although the hydrogen bond acceptor would be expected to be a nitrogen atom in the former case and an oxygen atom in the latter. We propose that for both structures there is an intervening water molecule which in addition reduces backbone strain. We have also measured the fluctuations during molecular dynamics runs in these mismatches. All are greater than for Watson-Crick base-pairs and the C x C mismatch shows very pronounced mobility.

Helical parameters, fluctuations, alternative hydrogen bonding, and bending in oligonucleotides containing a mistmatched base-pair by NOESY distance restrained and distance free molecular dynamics.

We have analysed and compared the molecular structures and dynamics of the DNA duplex, that corresponds to the sequence 29 to 39 of the K-ras gene, where the central base-pair is the normal C.G base or a mismatch base-pair C.A, C.A+, A.G and A+.G. Molecular dynamics runs of 100 picoseconds without or with distance restraints derived from NOE measurements are analysed and compared in all cases. (1) The EMBO convention of helical parameters for nucleic acids is extended to account for the construction and the description of any DNA mismatched base-pair. (2) Both types of MD runs reproduce very well all NMR data, except the H8 H2 inter-residue distances where agreement is not as good. (3) Average parameter values and fluctuations are in good agreement with results derived from persistence length and torsion modulus measurements. (4) Our molecular dynamics suggest the presence, in certain cases, of three-centred hydrogen bonds, which should be viewed as an equilibrium between hydrogen-bonding alternatives. In the case of the C.A mismatch, we observe the correlation between transient DNA bending and possible hydrogen bonding between a base and its 5' neighbour on the opposite strand in the sequence CCA. (5) These molecular dynamics analyses and observations provide a coherent view for the results obtained from recent DNA crystal structures and for results derived from other techniques such as gel electrophoresis at C.A steps.

Solution structure of an oncogenic DNA duplex, the K-ras gene and the sequence containing a central C.A or A.G mismatch as a function of pH: nuclear magnetic resonance and molecular dynamics studies.

The DNA duplex 5' d(GCCACCAGCTC)-d(GAGCTGGTGGC) corresponds to the sequence 29 to 39 of the K-ras gene, which contains a hot spot for mutations. This has been studied by one and two-dimensional nuclear magnetic resonance, energy minimization and molecular dynamics. The results show that it adopts a globally B-DNA type structure. We have introduced, at the central base-pair, the mismatches C.A and A.G. The mismatch position is that of the first base of the Gly12 codon, the hot spot. For the C.A mismatch we observe a structural change as a function of pH with an apparent pKa of 7.2. At low pH, the mismatch pair adopts a structure close to a classic wobble conformation with the cytidine residue displaced into the major groove. It is stabilised by two hydrogen bonds in which the adenosine residue is protonated and the cytidine residue has a significant C3'-endo population. At high pH, the mispair structure is in equilibrium between wobble and reverse wobble conformations. Similar studies are reported on the A.G mismatch, which also undergoes a transition as a function of pH. 31P spectra have been recorded on all systems and as a function of pH. No evidence for BII phosphodiester backbone conformations was found. The NMR results are well corroborated by molecular dynamics calculations performed with or without distance constraints. The dynamics at the mismatch sites have been examined. Although the overall structures are close to B-DNA, helical parameters fluctuate differently at these sites. Different hydrogen bonding alternatives in dynamic equilibrium that can involve three-centred hydrogen bonds are observed.

Structure of oligonucleotides with either a strand break or a bulged nucleotide.

We report NMR and molecular modelling studies on a DNA duplex structure which is composed of three oligonucleotides and mimics a strand break. Although it retains a B form conformation our model suggests that it is kinked at the strand break. In the same sequence with an extra bulged adenosine at the centre for the major species this residue is stacked in the helix and a kink is observed in the model.

Structure of an oligonucleotide containing a N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide residue facing a guanine.

Formamide residue is a major oxidative DNA damage product from ionizing radiation on thymine residues in DNA. We report NMR and molecular modeling studies on a DNA duplex structure which contains guanine opposite formamide residue. Formamide residue exists as either the cis and trans isomer. For the trans and the cis isomers, we find that guanine and formamide are stacked inside the helix and are hydrogen bonded. The oligonucleotide adopts globally a B form structure for the two isomers. Conformational changes are observed between the two isomers.

Physicochemical characteristics of pentamidine-loaded polymethacrylate nanoparticles: implication in the intracellular drug release in Leishmania major infected mice.

This work describes the preparation, the physicochemical properties, the tolerance and the intracellular trafficking of pentamidine loaded nanoparticles. Pentamidine was bound to the polymer by ionic interaction. This interaction involved the carboxylic acid functions of methacrylic acid (10% of the polymer) and the amine groups of the drug. Pentamidine fixation and release were pH dependent. An acidic pH led to a decrease of fixation or a release. At pH 5, which is the pH value of lysosomes and parasitophorous vacuoles, the release reached up to 50%. At this pH value, pentamidine is ionized and therefore can not traverse the biological membranes. Unloaded nanoparticles and pentamidine-loaded nanoparticles were tested in vitro on U937 cells and no cytotoxicity was observed. In vivo, in Leishmania infected mice, no significant weight loss was found. Ultrastructural studies showed the different steps of drug loaded nanoparticles trafficking inside Leismania-infected Küpffer cells. The nanoparticle uptake by macrophagic cells led to the location of nanoparticles inside phagocytosis vacuoles which fused with primary lysosomes to form secondary lysosomes. Ultimate fusion of secondary lysosomes containing nanoparticles with parasitophorous vacuoles was also observed. Nanoparticles were identified close to amastigotes but internalization by the parasite was not observed.

[Ultrastructural observations on the blood stages of Garniidae (G. gonatodi G. uranoscodoni and Fallisia effusa) parasites of South American Lizards)]. / Observations ultrastructurales sur les formes sanguines des Garniidés (Garnia gonatodi, G. uranoscodoni et Fallisia effusa) parasites de lézards sud-américains.

The ultrastructural study of two species of Garnia (G. gonatodi and G. uranoscodoni) and of Fallisia effusa demonstrates that the Garniidae possess the fundamental structures of Haemosporidia. A vacuolar digestive system was not found and the absence of pigment granules is confirmed. A number of caracteristics (- centriolar plaque inside a nuclear pore; - highly developed cytosqueleton in the host cell of F. effusa; - advanced maturation stages of gamétocytes of F. effusa in the vertebrate host) allow comparisons between the Garniidae and Plasmodiidae, Leucocytozoidae and Haemoproteidae. The authors' conclusion is that Garnia and Fallisia, although clearly different from each other, have common ultrastructural characteristics which also separate them from the other Haemosporidian families.

Observations ultrastructurales sur les formes sanguines et la sporogonie de plasmodiums de lémuriens malgaches.

The ultrastructure of blood stages and oocysts of Plasmodium coulangesi and P. percygarnhami, both parasites of Madagascan lemurs, was studied. The main results are:

Description and ultrastructure of Leishmania zuckermani n. sp. amastigotes detected within the erythrocytes of the South African gecko Pachydactylus turneri Gray, 1864.

In erythrocytes recovered from blood of geckoes of the species Pachydactylus turneri collected in Gauteng Province, Republic of South Africa, leishmania zuckemani n. sp. were detected. Giemsa stained erythrocytes contained amastigotes, either single or numerous, in loose assemblies or in a compact rounded oggregates which may condense to become a round basophilic bodies with a central hollow. This new species of Leishmania differs from all previously described species in being almost exclusively parasitic in circulating erythrocytes. Three to seven amastigotes lodged all within one, or divided between several parasitophorous vacuoles were detected at the EM level. The amastigotes demonstrated essentially all the cytological components characteristic of leishmania species known to parasitize mammals. A point which emphasizes an already suggested close affiliation between mammalian and lizard Leishmania.

In vivo antileishmanial action of Ir-(COD)-pentamidine tetraphenylborate on Leishmania donovani and Leishmania major mouse models.

Ir-(COD)-pentamidine tetraphenylborate which has previously been studied on promastigote forms of Leishmania, was investigated for its antileishmanial properties compared with pentamidine used as reference compound. In vitro, the iridium complex had the same IC50 value on intracellular forms of Leishmania as pentamidine (15 microM). In vivo, the compound could not be injected intravenously due to the DMSO excipient so that the treatments were performed intraperitoneally or subcutaneously. On the L. donovani LV9/Balb/C mouse model, the iridium complex was not toxic after intraperitoneal treatment at 232 mg/kg/day x 5 or 147 mumoles/kg/day x 5, whereas all the mice died within five days when treated at the same dose with pentamidine isethionate. However, only 23% of parasite suppression was observed with the iridium complex. On a L. major MON 74/Balb/C mouse model, susceptible to intravenously administered pentamidine at 6.7 mumoles/kg/day x 5 (54% of parasite suppression), the iridium complex exhibited 32% of parasite suppression after a treatment at 76 mumoles/kg/day x 5 administered subcutaneously. This slight activity is of interest since pentamidine isethionate is not active under these conditions. Transmission electron microscopy of amastigotes from infected and treated mice show aggregation of ribosomal material, distension of the nuclear membrane and kDNA depolymerization. The mechanism of action therefore involves several targets: membranes, ribosomes and kDNA. According to our results, the Iridium complex is a suitable candidate to be encapsulated in drug carriers such as liposomes or nanoparticles.

Survival of rodent malaria merozoites in the lymphatic network: potential role in chronicity of the infection.

Experiments performed during the last few years, lead us to hypothesise the existence of latent asexual forms of murine Plasmodium. In the present report we examined the organs of infected animals and describe novel structures, which we call merophores, containing merozoites which have resisted lysis seen with other asexual stage parasites. We propose that these merozoites represent a latent form of the parasite. Merophores were also found in the lymphatic circulation, and were demonstrated by subinoculation to have retained their viability. Depending on the parasite species two types of merophores were observed. For P. yoelii nigeriensis merophore sacks, with the latent merozoites found inside vesicles, were usually observed. Merophore leucocytes, where latent merozoites dispersed in the cytoplasm of macrophages or neutrophils, were solely seen with P. vinckei petteri. Both structures were seen in P. chabaudi chabaudi infections. Merophores were found in lymph nodes of rodents after the asexual parasitaemia had apparently subsided. They were formed soon after schizogony, principally in the spleen, either by pitting or by macrophage phagocytosis. Merophore numbers appeared to be proportional to the number of maturing schizonts. We propose that merophore formation and their circulation in the lymphatics play an important role in the pattern of recrudescences and chronicity of rodent malaria infections. It is further suggested that the lymphatic network, a privileged pathway for many parasites, might play a similar role in human malaria infections.

Ultrastructural changes in parasites induced by nanoparticle-bound pentamidine in a Leishmania major/mouse model.

Drug targeting enhances drug efficacy. This principle was tested in the treatment of an experimental visceral leishmaniasis. Using transmission electron microscopy (TEM) we localized pentamidine-loaded polymethocrylate nanoparticles in the liver of mice infected with Leishmania major and compared the ultrastructural changes in the parasites of these mice when they were treated with bound versus free pentamidine. Between days 13 and 17 after infection, loaded nanoparticles treated group were injected i.v. with 3 doses of 0.17 mg/kg bound pentamidine loaded on 2 x 10(11) nanospheres; control groups received 2 x 10(11) unloaded nanospheres. Drug reference control groups received five doses of 200 mg/kg pentavalent antimony (Glucantime) or three doses of free pentamidine (0.17 mg/kg or 2.28 mg/kg). Mice treated with bound pentamidine displayed a 77% reduction in their parasite burden versus the untreated controls. Nanoparticles were located by TEM inside parasitized Küpffer cells, in the phagolysosomes without entering the Leishmania. The low dose of 0.17 mg/kg bound pentamidine damaged the Leishmania to the same extent as 2.28 mg/kg of free pentamidine (the usual dose in human chemotherapy). In the parasites inside the Küpffer cells, TEM showed a swollen mitochondrian with loss of cristae, destruction or fragmentation of the kinetoplast, loss of ribosomes and destruction of parasite structures except for the subpellicular microtubules. This study therefore shows that a dose of bound pentamidine 13 times smaller than the usual dose of free pentamidine has a similar effect on the parasite.

[Importance of drug carriers in the treatment of visceral leishmaniasis]. / Importance des médicaments vectorisés dans le traitement de la leishmaniose viscérale.

Visceral leishmaniasis is caused by hemoflagellate protozoa which are obligatory parasites of the mononuclear phagocyte system. Leishmaniasis causes high morbidity and mortality worldwide. The treatment of choice remains pentavalent antimonials, but high toxicity and failures have been reported. An alternative to conventional treatment is delivery anti-leishmania agents using colloidal carrier systems. Carriers improve drug activity against intracellular disease involving the mononuclear phagocyte system. The principle of drug delivery by carrier systems has been applied successfully for anticancer drugs. Recently complete remission of polyresistant visceral leishmaniasis was obtained by injection of liposomal amphotericin B. At present, no colloidal drug carrier for antimony derivatives is available, but pentamidine can be linked experimentally to methacrylate polymer nano-particles. Drug-loaded nanoparticles have been shown to be effective against amastigote leishmania both in vitro and in vivo. Another colloidal system of major interest for drug delivery, the liposome has already been loaded with amphotericin B and used for human therapy. The concept of particulate drug carriers opens the way for new chemotherapeutic approaches in the field of parasitology.

Site-specific insertion of nitroxide-spin labels into DNA probes by click chemistry for structural analyses by ELDOR spectroscopy.

A new approach is described for the insertion of nitroxide spin-labels at specific positions within DNA oligomers. The latter bioconjugaison strategy is based on a click chemistry 1,3-dipolar cycloaddition between a spin-labeling reagent, namely the 4-azido-TEMPO, and alkyne modified uridine-containing oligonucleotides. This highly efficient labeling method was applied for site-specific incorporation of two TEMPO units within a set of double-stranded DNA constructs. Then the determination of the inter-nitroxide distances was achieved by using a four-pulses DEER technique that successfully validates the new site-directed spin labeling strategy.

Ultrastructural changes following treatment with a microwave pulse in the oocyst of Eimeria magna Perard, 1925.

The ultrastructure of oocysts of Eimeria magna was studied before and after their exposure to unique pulse of microwaves (2.450 MHZ; 600 W) of different durations (10, 15 and 20 s). Following treatment, the progressive destruction of the three layers of the oocyst wall was observed, the innermost being destroyed first. Internal structures were also affected, resulting in swollen mitochondria, a loss of ribosomes and fragmentation of the rough endoplasmic reticulum; moreover, the wall-forming bodies were no longer identifiable. Further studies using microwave pulses on biological material should be carried out to improve our understanding of the consequences of such treatment and to investigate its utility in the control of transmissible pathogenic organisms.