No abstract

We used comparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry methodologies to highlight and identify proteins that are differentially expressed in the intracellular stage of the parasite Leishmania donovani infantum, a causative agent of visceral leishmaniasis. During its digenetic life cycle, Leishmania alternates between the alimentary tract of the sandfly vector as an extracellular promastigote and the acidic phagolysosomes of macrophage cells as an intracellular amastigote. Proteins differentially expressed in the intracellular form of the parasite are thought to be important for intracellular survival and pathogenesis. We used narrow pH range strips for isoelectric focusing to resolve soluble proteins of both developmental stages of L. infantum. More than 62 proteins differentially expressed in amastigotes were detected among approximately 2000 protein spots resolved by 2-DE. A quadrupole time-of-flight analysis of few selected protein spots, specifically expressed in the amastigote stage, permitted the identification of two proteins, part of the energetic metabolism pathways, the isocitrate dehydrogenase and the glycolytic enzyme triosephosphate isomerase. The kinetic parameters of these two enzymes were measured in both developmental stages of the parasite and their activity was indeed found to be higher in amastigotes. These findings bring a new insight in our understanding of metabolic and energy requirements of the intracellular form of Leishmania. Comparative analysis of the proteome of both developmental stages of the protozoan parasite Leishmania should permit the identification of protein candidates for the development of vaccines and new drugs.

Lipid rafts are plasma membrane microdomains that are highly enriched in signaling molecules and that act as signal transduction platforms for many immune receptors. The involvement of these microdomains in HLA-DR-induced signaling is less well defined. We examined the constitutive presence of HLA-DR molecules in lipid rafts, their possible recruitment into these microdomains, and the role of these microdomains in HLA-DR-induced responses. We detected significant amounts of HLA-DR molecules in the lipid rafts of EBV ؉ and EBV ؊ B cell lines, monocytic cell lines, transfected HeLa cells, tonsillar B cells, and human monocytes. Localization of HLA-DR in these microdomains was unaffected by the deletion of the cytoplasmic domain of both the ␣ and ␤ chains. Ligation of HLA-DR with a bivalent, but not a monovalent, ligand resulted in rapid tyrosine phosphorylation of many substrates, especially Lyn, and activation of ERK1/2 MAP kinase. However, the treatment failed to induce further recruitment of HLA-DR molecules into lipid rafts. The HLA-DR-induced signaling events were accompanied by the induction of cellcell adhesion that could be inhibited by PTK and Lyn but not ERK1/2 inhibitors. Disruption of lipid rafts by methyl-␤-cyclodextrin (M␤CD) resulted in the loss of membrane raft association with HLA-DR molecules, inhibition of HLA-DR-mediated protein tyrosine phosphorylation and cell-cell adhesion. M␤CD did not affect the activation of ERK1/2, which was absent from lipid rafts. These results indicate that although all the HLA-DRinduced events studied are dependent on HLA-DR dimerization, some require the presence of HLA-DR molecules in lipid rafts, whereas others do not. Although major histocompatibility complex (MHC)1 class II molecules do not possess any known signaling motifs in their cytoplasmic and transmembrane domains, they act as signal transducers in addition to playing a critical role in antigen presentation and autoimmune disease susceptibility and severity (1, 2). Corley et al. (3) reported that the recognition of MHC class II-peptide complexes by specific T cell receptors (TCR) leads to the activation of both T lymphocytes and antigenpresenting cells (APCs), suggesting that MHC class II molecules can act as signal transducers. To confirm this possibility, anti-MHC class II antibodies (Abs) and superantigens (SAgs), which act as natural MHC class II ligands, were used to mimic MHC class II-peptide complex recognition by specific TCRs. This ligation led to various cellular events such as homotypic and heterotypic cell-cell adhesion (4), B cell proliferation and differentiation (5), cytokine production, and expression of costimulatory molecules (6) and, under certain conditions, cell death (7). Like other ligand-receptor interactions, some of these events are dependent on MHC class II ligand dimerization (8, 9), generate cAMP and intracellular calcium flux (1), and are mediated by signaling pathways and secondary messengers including protein kinase C (PKC), protein tyrosine kinase (PTK), cyclooxygenase 2,...

No abstract

No abstract