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.

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.

Ultrastructure of developmental stages of Hemolivia stellata (Apicomplexa: Haemogregarinidae) in the cane toad Bufo marinus and in its vector tick Amblyomma rotondatum.

The fine structure of the hemogregarine Hemolivia stellata Petit, Landau, Baccam and Lainson, 1990 developmental stages in the cane toad Bufo marinus L. and the vector tick Amblyomma rotondatum Koch, 1844 are described. In the liver of the toad, merozoites bound by a pellicle were located free and in the cytoplasm, and young and encased mature polynucleated meronts were located in a parasitophorous vacuole (PV). Premature gametocytes in the erythrocytes were bound by a bilayered membranous wall and the mature gametocytes were encased in a bivalved capsule, suture sites occurring at both gametocyte extremities. In the tick gut cells, oocysts located within a PV formed oblong, pellicle-bound sporokinetes, with a small apical complex, a few short rhoptries, and a fragmented crystalloid body. Liberated sporokinetes re-entered gut cells to proceed with their differentiation into sporocysts within a PV with elaborate rims which suggested engagement in active metabolite cross-transport. With maturity, the sporocyst wall gradually transformed into a hard capsule. Differences in fine structural development between species of Hemolivia and the insect-transmitted Hepatozoon are conspicuous. The fine structure and course of development of H. stellata are very similar to those of the previously described Hemolivia mariae; their sporokinetes differ, however, in having conspicuous rhoptries rather than spherical-granular anlagen bodies, and fragmented rather than continuous crystalline bodies.

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.

Multiple structures for the 2-aminopurine-cytosine mispair.

The base pair formed between 2-aminopurine (2AP) and cytosine (C) is an intermediate in transition mutations generated by 2AP. To date, several structures have been proposed for the 2AP-C mispair, including those involving a rare tautomer, a protonated base pair, and a neutral wobble structure. In this paper, we describe a series of UV, fluorescence, and NMR studies which demonstrate that an equilibrium exists between the neutral wobble and the protonated Watson-Crick structures. The apparent pK value for the transition between the structures is 5.9-6.0. Formation of a Watson-Crick base pair is accomplished predominantly by protonation of the 2AP residue as indicated by UV spectral changes, fluorescence quenching, and changes in proton chemical shifts. Rapid transfer of the shared proton between the 2AP and cytosine residues is indicated by the rapid exchange of the cytosine amino protons of the protonated Watson-Crick configuration. The relative contribution of the neutral wobble and protonated Watson-Crick configurations to 2AP-induced transition mutations is discussed.

Solution structure by NMR and molecular dynamics of a duplex containing a guanine opposite a N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide lesion.

One- and two-dimensional NMR spectroscopy has been used combined with molecular dynamics to determine the fine structure of the DNA duplex 5'-d(AGGAGCCACG).d(CGTGGFTCCT) where F is the N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide residue which is a ring fragmentation product of thymine. The formamide deoxyribose exists as two isomers with respect to the orientation about the peptide bond. The two isomers (trans and cis) are observed in a ratio 3:2 in solution. For both species, the oligonucleotide adopts a globally B form structure although conformational changes are observed around the mismatch site. The formamide residue, whatever the isomer, is intrahelical and can pair with the guanine on the opposite strand with one hydrogen bond. For the cis isomer, the residue adopts a syn orientation and is able to form a second hydrogen bond with the guanine on the 5' side on the same strand. Off-resonance ROESY experiments have been used to investigate the chemical exchange observed at low temperature of the duplex. Conformational exchange has only been found for the oligonucleotide with the formamide residue in the trans conformation.

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.

Solution structures of a duplex containing an adenine opposite a gap (absence of one nucleotide). An NMR study and molecular dynamic simulations with explicit water molecules.

We investigated the behaviour of a 15mer DNA duplex, [5'd(CAGAGTCACTGGCTC)3']. [5'd(GAGCCAG)3' + 5'd(GACTCTG)3'] which contained an adenine opposite the gap. Analysis of the NMR data showed the existence of one major species, which was in equilibrium with two minor species. Their relative concentrations varied as a function of pH with a pKa of approximately 4.5. For the major species, the duplex was globally in B conformation with the central adenine stacked in the helix. The two G.C base pairs adjacent to the central adenine were well formed and a gap was present in front of this adenine. For the minor species, major structural perturbations occurred in the centre of the duplex. At neutral pH, the central adenine was involved in a G.A mismatch with G23 adjacent to the gap. Cytosine C7 was then extrahelical and no gap was observed. Under these conditions, the major neutral species corresponded to 70% of the total and the minor species to 30%. At acidic pH, the central adenine of the minor species was protonated and was involved in a G(syn).A+(anti) mismatch. The difference is that C9 is now extrahelical and G22 is implicated in the mispair. Three-dimensional models were built to initiate molecular dynamic simulations, which were in good agreement with the NMR data. Their structural stability in terms of hydrogen bonding and their flexibility are discussed and the biological significance for the interaction with DNA polymerase is evoked.

Final stage of maturation of the erythrocytic schizonts of rodent Plasmodium in the lungs.

Schizonts of all rodent Plasmodium studied (Plasmodium yoelii, P. chabaudi, P. vinckei) show a characteristic morphology when they are completely mature: rounded or slightly elongate merozoites, completely detached from the pigment mass. At this stage, they are localized principally in the spleen and the lungs but, in impression smears of these organs they show two different aspects. In the spleen, schizonts are either inside the host erythrocyte or extraglobular but still close to a pigment mass; free merozoites are rare. In the lungs, on the contrary, merozoites are often free and dispersed; electron microscopy showed them to lie against the endothelium. Work by physiologists has shown the blood circulation in the alveoli to be much slowed down. Free merozoites, lined against the endothelium of relatively rigid capillaries, are in the best possible conditions to make contact with the intact red blood cells. Lungs appear to be the privileged site for the invasion of erythrocytes by the merozoites.

Selective and reversible effects of vinca alkaloids on Trypanosoma cruzi epimastigote forms: blockage of cytokinesis without inhibition of the organelle duplication.

Vinca alkaloids, vincristine and vinblastine, produce differential effects on the cell division of Trypanosoma cruzi epimastigote forms depending on drug concentrations. These effects are related to different microtubule-based mechanisms. For 15 microM vinblastine and 50 microM vincristine, the drugs inhibit both nuclear division and cytokinesis, and affect cell shape. At 3 microM vinblastine and 10 microM vincristine, however, cytokinesis is inhibited without major effect on the progression of the cell cycle; this yields giant cells having multiple nuclei, kinetoplasts and flagella. Cultures maintained over 1 week with daily drug replacement produced cells with more than 16 nuclei and 24 kinetoplasts, indicating that an equivalent of a fifth cell cycle was initiated. The ultrastructure of the multinucleate cells showed a basic organization closely similar to that of trypanosomes. Cytokinesis inhibition by vinca alkaloids seems to result from modulations of interactions between microtubules and associated proteins, rather than from an inhibition of microtubule dynamics as is usually proposed for vinca alkaloids. Cytokinesis inhibition is reversible: after removing the drug, epimastigotes emerge from the multinucleate cells. The emerging process follows a precise axis and polarity which are determined by the position of the flagellum/kinetoplast complex. This region could play an essential role in cell morphogenesis since zoids (cells without a nucleus) are frequently observed.

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.

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.

Conformations of nicked and gapped DNA structures by NMR and molecular dynamic simulations in water.

We have analyzed and compared the molecular structures and dynamics of DNA duplexes containing a nick or a gap of one nucleotide where the base in front of the gap is a guanine. The continuous strand has the sequence 5'(CAGAGTCXCTGGCTC) where the residue X is absent for the nick, 14-mer, and where it is a G residue for the gap. Duplexes were formed with the two corresponding 7-mers. Neither of these is phosphorylated adjacent at the nick site, but it is a good model for a single strand break. For the nick structure, the quantitative NMR data show that the global conformation is very close to canonical B-form DNA, but it displays enhanced local flexibility. For the gap structure, we observe only one species in which the extra G is well stacked into the helix. The two half-helices around this residue also show a B-form conformation. As with the nick duplex, the adjacent G imino protons show enhanced exchange with solvent. The gap does not close completely. Using distance constraints, MD calculations show that the nick conformation is very close to a duplex with no lesion but is indeed more flexible in the central part. The gapped structure shows two families of conformations. One is close to B-DNA, the other is significantly kinked at the gap which reduces the size of the cavity. We observe a spine of hydration within the cavities, similar, but of different geometry in the two cases.

Host cell and malarial targets for docetaxel (Taxotere) during the erythrocytic development of Plasmodium falciparum.

The microtubular stabilizing agent docetaxel (Taxotere) is known to inhibit the intraerythrocytic development of Plasmodium falciparum. To investigate the mechanism(s) of inhibition, we analyzed the structural organization of the mitotic spindle by immunofluorescence and electron microscopy. When 30 microM docetaxel was applied for five hours on ring forms, alterations in the mitotic spindles leading to abnormal nuclear divisions were observed. At the trophozoite- and schizont-stage, docetaxel pulses prevent mitosis by stabilizing microtubular structures associated with the mitotic apparatus, giving abnormal spindles. However, this inhibition did not interfere with parasite DNA synthesis indicating the absence of a checkpoint that couples exit from mitosis with proper spindle assembly as observed in higher eukaryotic cells. In parallel, intraerythrocytic concentration of docetaxel was measured in parasitized erythrocytes, after incubation of cells with 3H-docetaxel for five hours. It was found to be 14-fold increased at the ring-stage of infected erythrocytes compared to normal ones, 170-fold increased at the trophozoite-stage and 1,500-fold increased at the schizont-stage. Our data show that, even though the overall intracellular concentration of docetaxel is low in docetaxel-pulsed rings, the agent might be sufficient to disturb the spindle organization. However, the existence of targets for docetaxel other than mitotic spindle microtubules, i.e. erythrocyte membrane components could interfere with mitotic spindle formation.

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.

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.

The chemotherapy of rodent malaria. LIII. 'Fenozan B07' (Fenozan-50F), a difluorinated 3,3'-spirocyclopentane 1,2,4-trioxane: comparison with some compounds of the artemisinin series.

Fenozan B07, a difluorinated 3,3'-spirocyclopentane, 1,2,4-trioxane, is a novel, second-generation antimalarial endoperoxide which is a potent blood schizontocide against strains of rodent malaria that are highly resistant to a wide spectrum of classical antimalarials. Like compounds of the artemisinin series, its action is limited to the intra-erythrocytic stages, both asexual and sexual, and it is devoid of causal prophylactic activity. Both Fenozan B07 and the artemisinins are potent gametocytocides. In contrast to arteether, in a model using synchronous infection with Plasmodium vinckei petteri, Fenozan B07 inhibits the development of all asexual stages except preschizonts, as well as gametocytes. The activity of the artemisinin series in rodent-malaria models is limited to the rings and young trophozoites. The combined effect of Fenozan B07 with artesunate against P. v. petteri was only additive. A slight degree of potentiation was found in mice infected with asynchronous, drug-sensitive P. berghei but the combination was only additive against CQ-resistant P. yoelli ssp. NS. On the other hand, a significant degree of synergism was observed when mice infected with the artemisinin-resistant ART line of P. yoelii ssp. NS received combinations of Fenozan B07 with artemisinin. The conclusion is drawn from these and other data that there are significant differences between the blood schizontocidal actions of Fenozan B07 and the artemisinins. The basis of these differences remains to be determined.

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.