A novel form of actin in Leishmania: molecular characterisation, subcellular localisation and association with subpellicular microtubules

To study the occurrence and subcellular distribution of actin in trypanosomatid parasites, we have cloned and overexpressed Leishmania donovani actin gene in bacteria, purified the protein, and employed the affinity purified rabbit polyclonal anti-recombinant actin antibodies as a probe to study the organisation and subcellular distribution of actin in Leishmania cells. The Leishmania actin did not cross react with antimammalian actin antibodies but was readily recognized by the anti-Leishmania actin antibodies in both the promastigote and amastigote forms of the parasite. About 106 copies per cell of this protein (Mr 42.05 kDa) were present in the Leishmania promastigote. Unlike other eukaryotic actins, the oligomeric forms of Leishmania actin were not stained by phalloidin nor were dissociated by actin filament-disrupting agents, like Latrunculin B and Cytochalasin D. Analysis of the primary structure of this protein revealed that these unusual characteristics may be related to the presence of highly diverged amino acids in the DNase I-binding loop (amino acids 40-50) and the hydrophobic plug (amino acids 262-272) regions of Leishmania actin. The subcellular distribution of actin was studied in the Leishmania promastigotes by employing immunoelectron and immunofluorescence microscopies. This protein was present not only in the flagella, flagellar pocket, nucleus and the kinetoplast but it was also localized on the nuclear, vacuolar and cytoplasmic face of the plasma membranes. Further, the plasma membrane-associated actin was colocalised with subpellicular microtubules, while most of the actin present in the kinetoplast colocalised with the k-DNA network. These results clearly indicate that Leishmania contains a novel form of actin which may structurally and functionally differ from other eukaryotic actins. The functional significance of these observations is discussed

ADF/cofilin-driven actin dynamics in early events of Leishmania cell division

ADF/cofilin is an actin-dynamics-regulating protein that is required for several actin-based cellular processes such as cell motility and cytokinesis. A homologue of this protein has recently been identified in the protozoan parasite Leishmania, which has been shown to be essentially required in flagellum assembly and cell motility. However, the role of this protein in cytokinesis remains largely unknown. We show here that deletion of the gene encoding ADF/cofilin in these organisms results in several aberrations in the process of cell division. These aberrations include delay in basal body and kinetoplast separation, cleavage furrow progression and flagellar pocket division. In addition to these changes, the intracellular trafficking and actin dynamics are also adversely affected. All these abnormalities are, however, reversed by episomal complementation. Together, these results indicate that actin dynamics regulates early events in Leishmania cell division

Trafficking activity of myosin XXI is required in assembly of Leishmania flagellum

Actin-based myosin motors have a pivotal role in intracellular trafficking in eukaryotic cells. The parasitic protozoan organism Leishmania expresses a novel class of myosin, myosin XXI (Myo21), which is preferentially localized at the proximal region of the flagellum. However, its function in this organism remains largely unknown. Here, we show that Myo21 interacts with actin, and its expression is dependent of the growth stage. We further reveal that depletion of Myo21 levels results in impairment of the flagellar assembly and intracellular trafficking. These defects are, however, reversed by episomal complementation. Additionally, it is shown that deletion of the Myo21 gene leads to generation of ploidy, suggesting an essential role of Myo21 in survival of Leishmania cells. Together, these results indicate that actin-dependent trafficking activity of Myo21 is essentially required during assembly of the Leishmania flagellum

A novel homologue of coronin colocalizes with actin in filament-like structures in Leishmania

The presence of actin in Leishmania has recently been demonstrated, but the functional form of this protein (filamentous actin) has not yet been identified. We report here that the putative coronin homologue identified in the Leishmania genome is invariably associated with the filament-like structures of actin in Leishmania promastigotes. The occurrence of filamentous structures is significantly increased upon overexpression of Leishmania coronin as its GFP fusion product in Leishmania cells. However, expression of Leishmania actin or coronin alone in mammalian cells does not result in formation of any filament-like structures of Leishmania actin or association of Leishmania coronin with mammalian filamentous actin, but coexpression of both the proteins in these cells leads to formation of filamentous structures containing Leishmania actin and coronin. The high specificity of Leishmania coronin for Leishmania actin could be attributed to its unique structure as it differs from other coronins not only in the unique region but also in the actin-binding site and leucine zipper motif. These results taken together indicate that Leishmania contains a novel form of coronin which colocalizes with actin in filament-like structures in these cells

Ultrastructural immunogold localization of nitric oxide synthase isoforms in rat and human eosinophils

The involvement of nitric oxide (NO) as both pro and anti-inflammatory agent in allergic, airway inflammatory, and asthmatic diseases and the active participation of eosinophils in such ailments have been previously suggested. NO modulates eosinophil number, migration and their survival. The microenvironment of NO synthase (NOS) in subcellular organelles determines its rate and efficiency of catalysis, which in turn influences NO generation at distinct intracellular locales. The present study was undertaken to assess the intracellular distribution of NOS isoforms by transmission electron microscopy followed by morphometric analysis in human and rat eosinophils. Rat eosinophils were explored in parallel, and since they are widely used as model systems to mimic human diseases, a comparative study on NOS localization patterns might provide useful information in deciphering NO role in diverse aspects of eosinophil-related inflammatory ailments. The results demonstrated predominance of neuronal NOS (nNOS) in the eosinophilic granules and even distribution of inducible NOS (iNOS) and nNOS in the cytoplasm and nucleus of human eosinophils. In rat eosinophils, however, iNOS was mainly localized in the eosinophilic granules and nucleus, while nNOS was distributed evenly in cytoplasm and nucleus. Distribution of endothelial NOS (eNOS) in eosinophils was scanty. Differences in NOS isoforms and their localization in human and rat cells might have implications in differential mode of catalysis and functional contribution to eosinophil physiology and pathology, warranting detailed investigations. The present study highlights species-specific differences in the relative abundance and distribution pattern of NOS isoforms in rat and human eosinophils, which should be considered cautiously in interpreting the rat data to humans

Neutrophil extracellular trap formation by nitric oxide donors: involvement of nitric oxide synthase and myeloperoxidase derived free radicals

Presence of neutrophil extracellular traps (NET) at the site of inflammation and infection is demonstrated in various pathological conditions. Neutrophils migration and augmentation in NO availability are mostly associated with inflammation and pathogen infections. So far role of NO has not been explored in NET formation. Present study reports for the first time that incubation of human neutrophils with NO donors, sodium nitroprusside (SNP) or S-nitroso-N-acetyl-penicillamine (SNAP) led to the NET release and free radical generation, which was blocked in the presence of free radical scavengers (ebselen, NAC) or pretreatment of PMNs with inhibitors of NOS reductase domain (7-NI, DPI, imidazole) and myeloperoxidase (ABAH), while NOS oxygenase domain inhibitors had no effect. We thus propose that NO mediated NET release was dependent on the NO synthase (NOS) uncoupling and MPO derived free radicals, but independent of NADPH-oxidase. Moreover, NO dependent free radical generation, NET release and phosphorylation of MEK/ERK, p38MAPK was blocked by inhibitors of MEK/ERK (U0126), p38MAPK (SB202190 ), PKC (Rottlerin) suggesting for the first time role of ERK and p38MAPK in NET formation. The results of the present study seem to have important bearing on the role of NO in inflammatory pathological conditions

δ-Opioid receptor antagonist inhibits immunomodulation by met-enkephalin analogs

The methionine-enkephalin (Met-enkephalin, Tyr-Gly- Gly-Phe-Met) analogs Tyr-D-Ala-Gly-MePhe-Met NHC3H7- iso (1) and Tyr-D-Ala-Gly-MePhe-Gly-NHC3H7-iso (2) have been shown to enhance human T cell proliferation in in vitro treatment. Their immunomodulatory activities were completely blocked by naloxone, an opioid antagonist. Now we demonstrate that a selective δ -opioid receptor antagonist, ICI-174,864, completely blocks enhancement of T cell proliferation by analogs (1) and (2). The T cell-stimulatory effect was only partially inhibited by the μ -receptor-selective antagonist, β -funaltrexamine hydrochloride. The k -opioid receptor antagonist, nor-binaltorphimine dihydrochloride, showed no effect on T cell-proliferation stimulated by analogs (1) and (2). These observations suggest that analogs (1) and (2) of Met-enkephalin stimulate T cell proliferation predominantly via δ -opioid receptor present on T cells

Molecular and biochemical characterization of nitric oxide synthase isoforms and their intracellular distribution in human peripheral blood mononuclear cells

Nitric oxide synthase (NOS) expression and catalytic status in human peripheral blood mononuclear cells (PBMCs) is debatable, while its sub-cellular distribution remains unascertained. The present study characterizes NOS transcripts by real time PCR, NOS protein by immunoprecipitation (IP)/Western blot (WB), nitric oxide (NO) generation by DAF-2DA and NOS sub-cellular distribution by immunogold electron microscopy in resting PBMCs, monocytes and lymphocytes obtained from healthy donors. We observed constitutive expression of full length NOS isoforms (nNOS, iNOS and eNOS) in PBMCs: with the highest expression of iNOS in comparison to nNOS and eNOS. Isolated monocytes expressed more eNOS transcript and protein as compared to nNOS and iNOS. Lymphocytes however had more iNOS transcripts and protein than nNOS and eNOS. NOS was catalytically active in PBMCs, monocytes as well as in lymphocytes as evident by NO generation in the presence of substrate and cofactors, which was significantly reduced in the presence of NOS inhibitor. Immunogold electron microscopy and morphometric analysis revealed the distinct pattern of NOS distribution in monocytes and lymphocytes and also exhibited differences in the nuclear–cytoplasmic ratio. nNOS localization was much more in the cytosol than in the nucleus among both monocytes and lymphocytes. Interestingly, iNOS distribution was comparable in both cytosol and nucleus among monocytes, but in lymphocytes iNOS was predominantly localized to the cytosol. The present study exhibits constitutive presence of all the NOS isoforms in PBMCs and reports the distinct pattern of NOS distribution among monocytes and lymphocytes

Nitric oxide dependent increase in free radical generation mediates release of extracellular traps from human neutrophils

High availability of NO at the inflammatory/infection site is noticed often with oxidative stress and neutrophil extracellular traps (NETs) contents, but role of NO remains unexplored in NETs formation. In the present study incubation of adhered human neutrophils with DETA-NONOate led to NETs release in a time and concentration dependent manner, as assessed by confocal microscopy and by measuring extracellular DNA and NET-bound elastase, which was blocked by N-acetyl cysteine, suggesting role of free radicals. A time and concentration dependent augmentation in free radical formation by NO donors was measured by using DCF-DA, NBT, and by p47 phox migration to the neutrophils membrane. NO mediated formation of NETs and free radicals was significantly attenuated by pretreatment of neutrophils with diphenyleneiodonium, a dual inhibitor of NADPH-oxidase/NO synthase (NOS), 4-aminobenzoic acid hydrazide, a myeloperoxidase inhibitor and 7-nitroindazole, a NOS inhibitor, suggesting enzymatic free radical generation. We thus provide first experimental evidence that NO by augmenting free radical formation in human neutrophils mediates NETs release