Caspar-like gene depletion reduces Leishmania infection in sand fly host Lutzomyia longipalpis

Female phlebotomine sand flies Lutzomyia longipalpis naturally harbor populations of the medically important Leishmania infantum (syn. Leishmania chagasi) parasite in the gut, but the extent to which the parasite interacts with the immune system of the insect vector is unknown. To investigate the sand fly immune response and its interaction with the Leishmania parasite, we identified a homologue for caspar, a negative regulator of immune deficiency signaling pathway. We found that feeding antibiotics to adult female L. longipalpis resulted in an up-regulation of caspar expression relative to controls. caspar was differentially expressed when females were fed on gram-negative and gram-positive bacterial species. caspar expression was significantly down-regulated in females between 3 and 6 days after a blood feed containing Leishmania mexicana amastigotes. RNA interference was used to deplete caspar expression in female L. longipalpis, which were subsequently fed with Leishmania in a blood meal. Sand fly gut populations of both L. mexicana and L. infantum were significantly reduced in caspar-depleted females. The prevalence of L. infantum infection in the females fell from 85 to 45%. Our results provide the first insight into the operation of immune homeostasis in phlebotomine sand flies during the growth of bacterial and Leishmania populations in the digestive tract. We have demonstrated that the activation of the sand fly immune system, via depletion of a single gene, can lead to the abortion of Leishmania development and the disruption of transmission by the phlebotomine sand fly.     

Platelet immune complex interaction in pathogenesis of Kawasaki disease and childhood polyarteritis.

The role of platelets in the pathogenesis of vasculitis and the formation of coronary artery aneurysms was studied in 19 children with Kawasaki disease and five with polyarteritis. All patients with Kawasaki disease developed thrombocytosis in the third week of illness. The peak platelet count was significantly correlated (p less than 0.005) with the subsequent development of coronary artery aneurysms. The rise in platelet count was associated with the appearance in the circulation of a factor that induced aggregation and serotonin release in normal platelets. This factor was shown to be of high molecular weight, and its activity was lost at low pH--features suggestive of an immune complex. Immune complexes, detected by precipitation with polyethylene glycol, also appeared in the circulation as the platelet count increased. These complexes induced platelet aggregation, and there was a significant correlation (p less than 0.001) between the concentrations of IgG and IgA in the polyethylene glycol precipitated material and the platelet aggregating activity. Similar platelet aggregating activity was also detected in patients with polyarteritis but followed a different time course, persisting in the circulation for several months in association with continued disease activity. These findings imply that different mechanisms have a role in distinct phases of Kawasaki disease. The initial feverish phase (probably infective) is probably followed by an immune complex vasculitis that occurs when antibodies to the initiating agent appear in the circulation. The immune complexes aggregate platelets and induce release of serotonin. Platelet derived vasoactive mediators may increase vascular permeability and facilitate further deposition of complexes in the tissues.     

Lead dynamics in the forest floor and mineral soil in south-central Ontario

Recent studies have suggested that the residence time of Pb in the forest floor may not be as long as previously thought, and there is concern that the large pulse of atmospheric Pb deposited in the 1960s and early 1970s may move rapidly through mineral soils and eventually contaminate groundwater. In order to assess Pb mobility at a woodland (JMOEC) in south-central Ontario, a stable Pb isotope tracer ²⁰⁷Pb (8?mg?m^?2) was added to the forest floor in white pine (Pinus strobus) and sugar maple (Acer saccharum) stands, respectively, and monitored over a 2-year period. Excess ²⁰⁷Pb was rapidly lost from the forest floor. Applying first-order rate coefficients (k) of 0.57 (maple) and 0.32 (pine) obtained from the tracer study, and estimates of Pb deposition in the region, current predicted Pb concentrations in the forest floor are 1.5–3.1 and 2.1–5.8?mg?kg^?1 in the maple and pine plots, respectively. These values compare favorably with measured concentrations (corrected for mineral soil contamination) of 3.1–4.3?mg?kg^?1 in the maple stand and 2.6–3.6?mg?kg^?1 in the pine stand. The response time (1/k) of Pb in the forest floor at the sugar maple and white pine plots was estimated to be 1.8 and 3.1 years, respectively. The rapid loss of Pb from the forest floor at the JMOEC is much greater than previously reported, and is probably due to the rapid rate of litter turnover that is characteristic of forests with mull-type forest floors. In a survey of 23 forested sites that border the Precambrian Shield in south-central Ontario, Pb concentrations in the forest floor increased exponentially with decreasing soil pH. Lead concentrations in the forest floor at the most acidic survey sites, which exhibited mor-type forest floors, were approximately 10 times higher (～80?mg?kg^?1) than at the JMOEC, and pollution Pb burdens were up to 25 times greater. Despite the rapid loss of Pb from the forest floor at the JMOEC, the highest pollution Pb concentrations were found in the upper (0–1?cm) mineral soil horizon. Lead concentrations in the upper 30?cm of mineral soil were strongly correlated with organic matter content, indicating that pollution Pb does not move as a pulse down the soil profile, but instead is linked with organic matter distribution, indicating groundwater contamination is unlikely.     

Novel isosorbide di-ester compounds as inhibitors of acetylcholinesterase

We report herein that a variety of isosorbide di-esters, previously reported to be novel substrates for butyrylcholinesterase (BuChE, EC 3.1.1.8), are in fact inhibitors of the homologous enzyme acetylcholinesterase (AChE), with IC(50) values in the micromolar range. In vitro studies show that they are mixed inhibitors of the enzyme, and thus the ternary enzyme-inhibitor-substrate complex can form in acetylcholinesterase. This is rationalised by molecular modelling which shows that the compounds bind in the mid-gorge area. In this position, simultaneous substrate binding might be possible, but the hydrolysis of this substrate is prevented. The di-esters dock within the butyrylcholinesterase gorge in a very different manner, with the ester sidechain at the 5-position occupying the acyl pocket at residues Leu286 and Val288, and the 2-ester binding to Trp82. The carbonyl group of the 2-ester is susceptible to nucleophilic attack by Ser198 of the catalytic triad. The larger residues of the acyl pocket in acetylcholinesterase prevent binding in this manner. The results complement each other and explain the differing behaviours of the esters in the cholinesterase enzymes. These findings may prove very significant for future work.     

N-methyl-N'-nitro-N-nitrosoguanidine activates multiple cell death mechanisms in human fibroblasts

Response to genotoxic stress may trigger the activation of distinct mechanisms that serve to promote cell death, including apoptosis and necrosis. In this study we examined the response of human fibroblasts, either proficient or deficient for the damage-activated protein kinase ataxia telangiectasia-mutated (ATM), to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Analysis of both long- and short-term viability shows that both ATM-proficient YZ-5 and ATM-deficient EBS-7 fibroblasts display a cytotoxic response to MNNG. Consistent with activation of apoptosis in response to MNNG, we observed increased caspase-3 cleavage and activity, appearance of fragmented nuclei, and increased staining with annexin V in both ATM-proficient and -deficient fibroblasts. Flow cytometry demonstrated that these cell lines also display a nonapoptotic cell death in response to MNNG. This form of cell death is associated with activation of poly-ADP ribose polymerase (PARP), and analysis of PARP activity indicated increased protein poly(ADP-ribosylation) in YZ-5 when compared to EBS-7. This PARP activity was accompanied by apoptosis-inducing factor release and translocation from the mitochondria to the nucleus. Finally, the PARP inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ) or the caspase-3 inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone dramatically diminished the cytotoxic response to MNNG, reinforcing the roles for apoptotic and nonapoptotic cell death in human fibroblasts treated with MNNG. From these findings, we conclude that MNNG induces a heterogeneous death response in human fibroblasts.