RESUMO
Episomal expression of Leishmania histone H1 sense mRNAs in Leishmania major promastigotes was found previously to result in overexpression of this molecule and to reduce parasite infectivity in vitro. Herein, we evaluated the in vivo infectivity of these transfectants, in BALB/c mice, and showed that it is dramatically reduced. No lesions were observed in this group of mice and this was associated with an extremely low number of parasites both in the footpad and in the draining lymph nodes. Interestingly, the transfectants-reduced infectivity was associated with a delay in their cell-cycle progression and differentiation to axenic amastigotes, assessed in vitro. Therefore, the dramatic reduction in their infectivity may be attributed to the above-mentioned phenotypic modifications. As the metazoan linker histone H1(0) homologue is known to delay cell-cycle progression in mammalian cells we investigated whether its Leishmania counterpart, which possesses homology to its C-terminal region, when expressed in mammalian cells may also affect their cell-cycle progression. It was thus shown that Leishmania histone H1 expressed in COS7 and NIH 3T3 cells, delays cell-cycle progression in these cells too. The latter strengthens the phenotype observed in Leishmania and provides evidence that critical functions of histone H1 molecules are conserved throughout evolution.
Assuntos
Ciclo Celular/genética , Histonas/genética , Leishmania major/genética , Leishmania major/patogenicidade , Proteínas de Protozoários/genética , Ativação Transcricional , Animais , Células COS , Diferenciação Celular/genética , Chlorocebus aethiops , Leishmania donovani/citologia , Leishmania donovani/genética , Leishmania major/citologia , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Nocodazol/farmacologia , Virulência/genéticaRESUMO
BALB/c mice were given or not iron around the time of intradermal parasite inoculation, in their ears, of either 10(6) stationary-phase (designated "high-dose model") or 10(3)Leishmania major metacyclic promastigotes (designated "low-dose model"). Iron-loaded mice in the high-dose model displayed delayed and limited pathogenic processes, whereas in the low-dose model, the mice remained ear lesion-free over 12 months post-parasite inoculation. These phenotypes were coupled to an increased leukocyte oxidative burst displayed mainly by neutrophils: it was early and transient in the high-dose model, whereas it was sustained in the low-dose model. In the latter model, injection of an antioxidant (diphenyleneiodonium chloride) at week 2 post-L. major inoculation resulted in a significant decrease in oxidative burst and reversed the protective status. The increased and sustained oxidative burst displayed by the neutrophils, the sustained presence of IL-12 (p40/p70)-positive leukocytes in the ear dermis, the low number of inflammatory leukocytes in the ear dermis and their concomitant high number in the draining lymph node are three related features that likely contribute to the shaping of the protective status, the onset and dynamic maintenance of which are antioxidant sensitive.
Assuntos
Complexo Ferro-Dextran/farmacologia , Leishmania major/crescimento & desenvolvimento , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Leucócitos/imunologia , Explosão Respiratória/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Orelha/parasitologia , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Leishmaniose Cutânea/imunologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Linfonodos/imunologia , Linfonodos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Oniocompostos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/imunologia , Organismos Livres de Patógenos EspecíficosRESUMO
alpha(IIb)beta(3), a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma-membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif. However, elucidation of the ligand-binding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage. In this study, a detailed mapping of the extracellular region of the alpha(IIb) subunit is presented, using overlapping 20-peptides, in order to identify the binding sites of alpha(IIb) potentially involved in the platelet-aggregation event. Regions alpha(IIb) 313-332, alpha(IIb) 265-284 and alpha(IIb) 57-64 of alpha(IIb)beta(3) were identified as putative fibrinogen-binding domains because the corresponding peptides inhibited platelet aggregation and antagonized fibrinogen association, possibly by interacting with this ligand. The latter is further supported by the finding that the above peptides did not interfere with the binding of PAC-1 to the activated form of alpha(IIb)beta(3). Furthermore, alpha(IIb) 313-332 was found to bind to fibrinogen in a solid-phase binding assay. It should be emphasized that all the experiments in this study were carried out on activated platelets and consequently on the activated form of this integrin receptor. We hypothesize that RAD and RAE adhesive motifs, encompassed in alpha(IIb) 313-332, 265-284 and 57-64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets.