Close association of invading Plasmodium berghei and beta integrin in the Anopheles gambiae midgut

We have used confocal microscopy and an antibody against Anopheles gambiae beta integrin to study this protein's distribution in the mosquito midgut and its relationship to invading Plasmodium berghei parasites. An extensive reorganization of integrin is seen to take place in the midgut epithelial cells following the uptake of either non-infected or parasite-infected blood meal, probably reflecting the reshaping of the gut due to the presence of the food bolus and the peritrophic membrane that surrounds it. Furthermore, malaria parasites are coated with beta integrin immediately upon entry into the epithelium, independent of whether they develop intra- or extracellularly. Although this coat is shed a few days after the invasion, beta integrin remains concentrated in the cells surrounding the maturing oocyst for several days. Finally, the antibody detects a structural change in the midgut epithelial cells in the immediate vicinity of the invading ookinete, which is consistent with Plasmodium-induced apoptosis followed by wound healing. This intimate association suggests a specific role of beta integrin in the invasion process.     

Plasmodium berghei ookinetes bind to Anopheles gambiae and Drosophila melanogaster annexins

Using a proteomic approach we identified polypeptides from Anopheles gambiae and Drosophila melanogaster protein extracts that selectively bind purified Plasmodium berghei ookinetes in vitro; these were two and three distinct polypeptides, respectively, with an apparent molecular weight of about 36 kDa. Combining two-dimensional electrophoresis and MALDI-TOF (matrix-associated laser desorption ionization time of flight) mass spectrometry we determined that the polypeptides correspond to isomorphs of the annexin B11 protein of the fruit fly. When protein extracts derived from A. gambiae and D. melanogaster tissue culture cells were further fractionated, the binding activity matching the annexin protein could be localized in the fraction derived from cell membranes in both diptera. Antibody staining showed that annexin also binds to ookinetes during the invasion of the mosquito midgut. Finally, inclusion of antiannexin antisera in a mosquito blood meal impaired parasite development, suggesting a facilitating role for annexins in the infection of the mosquito by Plasmodium.     

An inhibitor of glucosylceramide synthase inhibits the human enzyme, but not enzymes from other organisms

Specific inhibitors of glucosylceramide biosynthesis are used as drugs for the treatment of some human diseases correlated to glycosphingolipid metabolism. The target of the presently available inhibitors is the human glucosylceramide synthase (GCS), but effects on enzymes from other organisms have not been studied. We expressed cDNAs encoding GCS enzymes from lower animals, plants, fungi, and bacteria in the yeast P. pastoris. In vitro GCS assays with the GCS inhibitor D-threo-1-(3',4'-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol showed that this inhibitor did not affect non-human GCS enzymes.     

LmxMPK4, a mitogen-activated protein (MAP) kinase homologue essential for promastigotes and amastigotes of <Emphasis Type="Italic">Leishmania mexicana</Emphasis>

Background  

Leishmania parasites undergo profound morphological and biochemical changes while passing through their life cycle. Protein kinases have been shown to be involved in the differentiation from the extracellular flagellated promastigotes to the intracellular "non-flagellated" amastigotes and vice versa. Moreover, these enzymes are likely involved in the regulation of the proliferation of the different life stages.     

Characterization of the S-denitrosation activity of protein disulfide isomerase

S-nitrosoglutathione (GSNO) denitrosation activity of recombinant human protein disulfide isomerase (PDI) has been kinetically characterized by monitoring the loss of the S-NO absorbance, using a NO electrode, and with the aid of the fluorogenic NOx probe 2,3-diaminonaphthalene. The initial rates of denitrosation as a function of [GSNO] displayed hyperbolic behavior irrespective of the method used to monitor denitrosation. The Km values estimated for GSNO were 65 +/- 5 microm and 40 +/- 10 microm for the loss in the S-NO bond and NO production (NO electrode or 2,3-diaminonaphthalene), respectively. Hemoglobin assay provided additional evidence that the final product of PDI-dependent GSNO denitrosation was NO*. A catalytic mechanism, involving a nitroxyl disulfide intermediate stabilized by imidazole (His160 a-domain or His589 a'-domain), which after undergoing a one-electron oxidation decomposes to yield NO plus dithiyl radical, has been proposed. Evidence for the formation of thiyl/dithiyl radicals during PDI-catalyzed denitrosation was obtained with 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl. Evidence has also been obtained showing that in a NO- and O2-rich environment, PDI can form N2O3 in its hydrophobic domains. This "NO-charged PDI" can perform intra- and intermolecular S-nitrosation reactions similar to that proposed for serum albumin. Interestingly, reduced PDI was able to denitrosate S-nitrosated PDI (PDI-SNO) resulting in the release of NO. PDI-SNO, once formed, is stable at room temperature in the absence of reducing agent over the period of 2 h. It has been established that PDI is continuously secreted from cells that are net producers of NO-like endothelial cells. The present demonstration that PDI can be S-nitrosated and that PDI-SNO can be denitrosated by PDI suggests that this enzyme could be intimately involved in the transport of intracellular NO equivalents to the cell surface as well as the previous demonstration of PDI in the transfer of S-nitrosothiol-bound NO to the cytosol.     

Mitochondrial dysfunction and oxidative damage in parkin-deficient mice

Loss-of-function mutations in parkin are the predominant cause of familial Parkinson's disease. We previously reported that parkin-/- mice exhibit nigrostriatal deficits in the absence of nigral degeneration. Parkin has been shown to function as an E3 ubiquitin ligase. Loss of parkin function, therefore, has been hypothesized to cause nigral degeneration via an aberrant accumulation of its substrates. Here we employed a proteomic approach to determine whether loss of parkin function results in alterations in abundance and/or modification of proteins in the ventral midbrain of parkin-/- mice. Two-dimensional gel electrophoresis followed by mass spectrometry revealed decreased abundance of a number of proteins involved in mitochondrial function or oxidative stress. Consistent with reductions in several subunits of complexes I and IV, functional assays showed reductions in respiratory capacity of striatal mitochondria isolated from parkin-/- mice. Electron microscopic analysis revealed no gross morphological abnormalities in striatal mitochondria of parkin-/- mice. In addition, parkin-/- mice showed a delayed rate of weight gain, suggesting broader metabolic abnormalities. Accompanying these deficits in mitochondrial function, parkin-/- mice also exhibited decreased levels of proteins involved in protection from oxidative stress. Consistent with these findings, parkin-/- mice showed decreased serum antioxidant capacity and increased protein and lipid peroxidation. The combination of proteomic, genetic, and physiological analyses reveal an essential role for parkin in the regulation of mitochondrial function and provide the first direct evidence of mitochondrial dysfunction and oxidative damage in the absence of nigral degeneration in a genetic mouse model of Parkinson's disease.     

Caspase-12 cleavage and increased oxidative stress during motoneuron degeneration in transgenic mouse model of ALS

Glucose-dependent insulinotropic polypeptide is an incretin hormone that stimulates insulin secretion and reduces postprandial glycaemic excursions. The glucose-dependent action of GIP on pancreatic beta-cells has attracted attention towards its exploitation as a potential drug for type 2 diabetes. Use of NMR or X-ray crystallography is vital to determine the three-dimensional structure of the peptide. Therefore, to understand the basic structural requirements for the biological activity of GIP, the solution structure of the major biologically active fragment, GIP(1-30)amide, was investigated by proton NMR spectroscopy and molecular modelling. The structure is characterised by a full length alpha-helical conformation between residues F(6) and A(28). This structural information could play an important role in the design of therapeutic agents based upon GIP receptor agonists.     

Influence of osmolytes on in vivo glucose monitoring using optical coherence tomography

Diabetes mellitus and its complications are the third leading cause of death in the world, exceeded only by cardiovascular disease and cancer. Tighter monitoring and control of blood glucose could minimize complications associated with diabetes. Recently, optical coherence tomography (OCT) for noninvasive glucose monitoring was proposed and tested in vivo. The aim of this work was to investigate the influence of changes in blood glucose concentration ([glu]) and sodium concentration ([Na+]) on the OCT signal. We also investigated the influence of other important analytes on the sensitivity of glucose monitoring with OCT. The experiments were carried out in anesthetized female pigs. The OCT images were acquired continuously from skin, while [glu] and [Na+] were experimentally varied within their physiological ranges. Correlations of the OCT signal slope with [glu] and [Na+] were studied at different tissue depths. The tissue area probed with OCT was marked and cut for histological examination. The correlation of blood [glu] and [Na+] with the OCT signal slope was observed in separate tissue layers. On average, equimolar changes in [glu] produced 2.26 +/- 1.15 greater alterations of the OCT signal slope than changes in [Na+]. Variation of concentrations of other analytes did not influence the OCT signal slope. The influence of [Na+] on relative changes in the OCT signal slope was generally less than [glu]-induced changes. OCT is a promising method for noninvasive glucose monitoring because of its ability to track the influence of changing [glu] on individual tissue layers.