Elongating modified conserved peptides eliminates their immunogenicity and protective efficacy against P. falciparum malaria

Plasmodium falciparum malaria protein peptides were synthesised in the search for more effective routes for inducing a protective immune response against this deadly parasite and this information has been associated with such molecules' three-dimensional structure. These peptides had high red blood cell binding activity and their carboxy- and amino-terminal extremes were elongated for determining their immunogenic and protection-inducing activity against this disease in the Aotus monkey experimental model. 1H-NMR was used for analysing their three-dimensional structure; FAST ELISA, immunofluorescence antibody test, and Western blot were used for identifying their antibody inducing capacity and these previously immunised Aotus were inoculated with a highly infective P. falciparum strain to determine whether these elongated peptides were able to induce protection. This was aimed at establishing an association or correlation between long peptides' three-dimensional structure and their immunogenic and protection-inducing response in these monkeys. Peptides 20026 (25 residue), 20028 (30 residue), and 20030 (35 residues) were synthesised based on elongating the amino-terminal region of the 10022 highly immunogenic and protection-inducing modified peptide. 1H-NMR studies revealed that the first three had Classical type III beta-turn structures, different from the 20-amino acid long modified peptide 10022 which had a distorted type III beta-turn. Humoral immune response analysis showed that even when some antibodies could be generated against the parasite, none of the immunised Aotus could be protected with elongated peptides suggesting that elongating them eliminated modified peptide 10022 immunogenic and protection-inducing capacity.     

A comparative study of MHC Class-II HLA-DRbeta1*0401-Col II and HLA-DRbeta1*0101-HA complexes: a theoretical point of view

A study was performed on the HLA-DRbeta1*0401-collagen II peptide complex using the computation of electronic multipolar variables proposed by us previously. Furthermore, these results were compared with those obtained for the HLA-DRbeta1*0101-haemaglutinin peptide complex studied by us with the same tools, confirming that Pocket 1 for this new complex is also the most important pocket for the interaction between the presenting molecule and the presented peptide. The pocket hierarchy established for HLA-DRbeta1*0401 allele was P1 > P9 approximately P7 > P6 > P4, whilst a P1 > P4 > P9 approximately P7>P6 pocket hierarchy was found for HLA-DRbeta1*0101, showing how the relative importance of the pockets distinguishes the two alleles. There are high correlation levels with experimental results (when possible), again confirming the validity of using calculated values for electronic multipolar variables as a useful tool for studying interactions between immune system molecules and peptides.     

Development of a microplate-based, electrophoretic fluorescent protein kinase a assay: comparison with filter-binding and fluorescence polarization assay formats

A microplate-based electrophoretic assay has been developed for the serine/threonine kinase protein kinase A (PKA). The ElectroCapture PKA assay developed uses a positively charged, lissamine-rhodamine-labeled kemptide peptide substrate for the kinase reaction and Nanogen's ElectroCapture HTS Workstation and 384-well laminated membrane plates to electrophoretically separate the negatively charged phosphorylated peptide product from the kinase reaction mix. After the electrophoretic separation, the amount of rhodamine-labeled phosphopeptide product was quantified using a Tecan Ultra384 fluorescence reader. The ElectroCapture PKA assay was validated with both known PKA inhibitors and library compounds. The pK(iapp) results obtained in the ElectroCapture PKA assay were comparable to those generated with current radioactive filter-binding assay and antibody-based competitive fluorescence polarization PKA assay formats.     

Effects of repeated administration with CP-55,940, a cannabinoid CB1 receptor agonist on the metabolism of the hepatic heme

Drugs metabolised by cytochrome P450 (CYP) such as analgesics may induce acute attacks in patients with hepatic porphyrias. In recent years, preclinical and clinical studies have suggested that cannabinoid pharmaceutical preparations may be potentially useful in the treatment of pain. The purpose of the study was to examine the effects of CP-55,940, a cannabinoid CB1 receptor agonist, on the hepatic heme metabolism in mice. To this end, hepatic activities of aminolevulinic acid synthase (ALAS), heme oxygenase (HO) and CYP levels were determined in mice treated with CP-55,940 (0.5 mg/kg/day; i.p.; 5 or 24 days). Results showed that treatment with CP-55,940 decreased CYP concentrations by 80% and increased HO activity by 158%. However, ALAS activity also decreased by 37%, suggesting that regulatory free heme pool was not modified. Our findings indicate that CP-55,940 and its metabolites do not behave as porphyrinogenic drugs and may potentially be safe for treating pain in patients with acute porphyrias.     

Modulation of carbohydrate metabolism and peptide hormones by soybean isoflavones and probiotics in obesity and diabetes

Soybean and its isoflavones have been shown to have beneficial effects on carbohydrate and lipid metabolism and on renal function. Probiotics may potentiate the beneficial effects of isoflavones by converting the inactive isoflavone glycoside to aglycones, which are biologically active, thereby producing a synergistic effect. We therefore studied the effects of soybean isoflavones in the presence and absence of probiotics on glucose and triglyceride metabolism and the peptide hormones involved in their metabolism. Lean and obese SHR/N-cp rats were fed AIN-93 diets containing 0.1% soybean isoflavone mixture, 0.1% probiotics mixture or both. Plasma was analyzed for glucose, triglycerides, parameters of renal function and peptide hormones -- insulin, leptin, glucagon and ACTH -- that are involved in glucose and lipid metabolism. Isoflavones given alone lowered plasma glucose in both phenotypes while triglyceride was decreased only in lean animals. Isoflavones also lowered aspartate amino transferase and alanine amino transferase in both phenotypes. Isoflavones had significant effect on plasma insulin, leptin and glucagon in lean rats but not in obese rats. Thus, our data show that in lean animals, isoflavones have hypoglycemic and hypolipidemic effect, and the effect is mediated by changes in peptide hormones. When lipid levels are very high as in obese rats, isoflavones fail to lower plasma triglyceride levels. Probiotics do not appear to enhance the effect of isoflavones.     

Viability of the Endangered Little Bustard <Emphasis Type="Italic">Tetrax tetrax</Emphasis> Population of Western France

Stochastic computer simulations are used to evaluate the sensitivity of Little bustard population parameters, estimating the survival probabilities of the seven endangered Little bustard populations of central-western France for which conservation actions are currentlybeing or have been implemented. Different scenarios of parameter compensation for those nuclei to establish parameter levels assuring population viability are discussed. Adult survival, productivity per female, initial population size and carrying capacity were the most sensitive parameters in a hypothetical, isolated population. Juvenile survival also affected population survival, although its sensitivity was lower. Sex ratio did not have a linear effect on population survival, but probability of extinction increased for extreme values. Productivity per female and initial population size, varied strongly among the populations studied, determining their average time of extinction and growth rate. When a metapopulation scenario was simulated, the survival probabilities of each population and the metapopulations stayed close to 1.0 if no mortality was associated to migration. When mortality during migration was included in the simulations, the metapopulation's probability of survival significantly decreased under 90%. This approach may help managers to correctly address conservation measures and design effective strategies, which should be directed mainly to improve productivity, enhance female survival, and minimise mortality during migration (e.g. promoting insect-rich nesting substrates, avoiding female killing and nest destruction at harvesting, reducing the risk of collision with powerlines, or controlling poaching).     

Identifying Plasmodium falciparum cytoadherence-linked asexual protein 3 (CLAG 3) sequences that specifically bind to C32 cells and erythrocytes

Adhesion of mature asexual stage Plasmodium falciparum parasite-infected erythrocytes (iRBC) to the vascular endothelium is a critical event in the pathology of Plasmodium falciparum malaria. It has been suggested that the clag gene family is essential in cytoadherence to endothelial receptors. Primers used in PCR and RT-PCR assays allowed us to determine that the gene encoding CLAG 3 (GenBank accession no. NP_473155) is transcribed in the Plasmodium falciparum FCB2 strain. Western blot showed that antisera produced against polymerized synthetic peptides from this protein recognized a 142-kDa band in P. falciparum schizont lysate. Seventy-one 20-amino-acid-long nonoverlapping peptides, spanning the CLAG 3 (cytoadherence-linked asexual protein on chromosome 3) sequence were tested in C32 cell and erythrocyte binding assays. Twelve CLAG peptides specifically bound to C32 cells (which mainly express CD36) with high affinity, hereafter referred to as high-affinity binding peptides (HABPs). Five of them also bound to erythrocytes. HABP binding to C32 cells and erythrocytes was independent of peptide charge or peptide structure. Affinity constants were between 100 nM and 800 nM. Cross-linking and SDS-PAGE analysis allowed two erythrocyte binding proteins of around 26 kDa and 59 kDa to be identified, while proteins of around 53 kDa were identified as possible receptor sites for C-32 cells. The HABPs' role in Plasmodium falciparum invasion inhibition was determined. Such an approach analyzing various CLAG 3 regions may elucidate their functions and may help in the search for new antigens important for developing antimalarial vaccines.