Trypanosoma cruzi: Impact of dual-clone infections on parasite biological properties in BALB/c mice

Herein, we have analyzed major biological properties following dual-clone Trypanosoma cruzi infections in BALB/c mice. Eight T. cruzi clonal stocks, two of each principal genotype, including genotype 19 and 20 (T. cruzi I), hybrid genotype 39 (T. cruzi) and 32 (T. cruzi II) were combined into 24 different dual-clone infections. Special attention was given to characterize biological parameters assayed including: prepatent period, patent period, maximum of parasitemia, day of maximum parasitemia, area under the parasitemia curve, infectivity, mortality, and hemoculture positivity. Our findings clearly demonstrated that features resultant of dual-clone infections of T. cruzi clonal stocks did not display either the characteristics of the corresponding monoclonal infections or the theoretical mixture based on the respective monoclonal infections. Significant changes in the expected values were observed in 4.2-79.2% of the mixtures considering the eight biological parameters studied. A lower frequency of significant differences was found for mixtures composed by phylogenetically distant clonal stocks. Altogether, our data support our hypothesis that mixed T. cruzi infections have a great impact on the biological properties of the parasite in the host and re-emphasizes the importance of considering the possible occurrence of natural mixed infections in humans and their consequences on the biological aspects of ongoing Chagas' disease.     

Biochemical,physiological and molecular evaluation of rice cultivars differing in salt tolerance at the seedling stage

Changes in the antioxidant enzymes, lipid peroxidation, sodium and potassium, chlorophyll, H₂O₂ and proline content were monitored in the leaves of 42 rice varieties which were not yet well-documented for the salinity tolerance under different salinity levels. The tolerant varieties (FL478, Hassani, Shahpasand, Gharib and Nemat) showed signs of tolerance (lower Na⁺/K⁺ ratio, high proline accumulation, less membrane damage, lower H₂O₂ production, and higher superoxide dismutase and catalase activity) very well. The positive relationship between the level of salt tolerance and the amount of proline accumulation in the rice varieties support the important role of proline under the salt stress. The varieties were genotyped for 12 microsatellite markers that were closely linked to SalTol QTL. The results of association analysis indicated that RM1287, RM8094, RM3412 and AP3206 markers had the high value of R² for the regression models of the studied traits. It shows the important role of SalTol in controlling physio-biochemical traits. The results can be used in the future marker assisted selection (MAS) directly, if the results are confirmed.     

Antigenicity and immunogenicity of the C-terminal peptide of human thyroglobulin

Thyroglobulin (Tg) is cleaved into several peptides during thyroid hormone synthesis, an oxidative process. P40, an iodinated C-terminal peptide from human Tg, has a molecular weight of about 40 kDa and contains two hormonogenic sites. P40 is the smallest peptide that is still recognized by monoclonal antibodies from mice immunized with human Tg directed against its immunodominant region. Since P40 also contains several T-cell epitopes, it is a good candidate for studying the primary events involved in the process of hormone synthesis leading to thyroid autoimmunity. The present results show that P40 is recognized by Tg antibodies from patients with thyroid disorders and induces Tg antibodies in CBA mice. P40 may therefore be involved in the autoimmune process, thus providing a useful tool for diagnostic and therapeutic purposes.     

Genotoxic effects of rotenone on cultured lymphocytes

Rotenone is a heterocyclic compound widely used as an insecticide, acaricide and piscicide. Its toxicity is mainly caused by the inhibition of mitochondrial respiratory processes and ATP production, resulting in the generation of reactive oxygen species. Reactive oxygen species can interact with DNA, RNA and proteins, leading to cell damage, followed by death. We used the Comet assay, and we analyzed chromosome aberrations, in order to evaluate the genotoxic and clastogenic effects of rotenone on the different phases of the cell cycle. Cultured human lymphocytes were treated with 1.0, 1.5 and 2.0 microg/mL rotenone during the G1, G1/S, S (pulses of 1 and 6 h), and G2 phases of the cell cycle. Rotenone induced DNA damage and was clastogenic, but the clastogenicity was detected only with treatments conducted during the G1/S and S phases of the cell cycle. Rotenone also induced endoreduplication and polyploidy in treatments made during G1, while it significantly reduced the mitotic index in all phases of the cell cycle.     

Thy1.2 YFP-16 Transgenic Mouse Labels a Subset of Large-Diameter Sensory Neurons that Lack TRPV1 Expression

The Thy1.2 YFP-16 mouse expresses yellow fluorescent protein (YFP) in specific subsets of peripheral and central neurons. The original characterization of this model suggested that YFP was expressed in all sensory neurons, and this model has been subsequently used to study sensory nerve structure and function. Here, we have characterized the expression of YFP in the sensory ganglia (DRG, trigeminal and vagal) of the Thy1.2 YFP-16 mouse, using biochemical, functional and anatomical analyses. Despite previous reports, we found that YFP was only expressed in approximately half of DRG and trigeminal neurons and less than 10% of vagal neurons. YFP-expression was only found in medium and large-diameter neurons that expressed neurofilament but not TRPV1. YFP-expressing neurons failed to respond to selective agonists for TRPV1, P2X_2/3 and TRPM8 channels in Ca²⁺ imaging assays. Confocal analysis of glabrous skin, hairy skin of the back and ear and skeletal muscle indicated that YFP was expressed in some peripheral terminals with structures consistent with their presumed non-nociceptive nature. In summary, the Thy1.2 YFP-16 mouse expresses robust YFP expression in only a subset of sensory neurons. But this mouse model is not suitable for the study of nociceptive nerves or the function of such nerves in pain and neuropathies.     

Indirect synchronous fluorescence spectroscopy and direct high‐performance thin‐layer chromatographic methods for enantioseperation of zopiclone and determination of chiral‐switching eszopiclone: Evaluation of thermodynamic quantities of chromatographic separation

Economic and enantioselective synchronous fluorescence spectroscopy and high‐performance thin‐layer chromatography methods have been developed and validated as per ICH guidelines for the separation of zopiclone enantiomers using L‐(+)‐tartaric acid as a chiral selector, followed by determination of the chiral‐switching eszopiclone. Synchronous fluorescence spectroscopy was successfully applied for chiral recognition of R & S enantiomers of zopiclone at ?λ = 110 nm based on creating of diastereomeric complexes with 0.06M tartaric acid in an aqueous medium containing 0.2M disodium hydrogen orthophosphate. Synchronous fluorescence intensities of eszopiclone were recorded at 296 nm in concentration range 0.2‐ to 4‐μg/mL eszopiclone. High‐performance thin‐layer chromatography method depends on resolution of zopiclone enantiomers on achiral HPTLC silica‐gel plates using acetonitrile:methanol:water (8:2:0.25, v/v/v) containing L‐(+)‐tartaric acid as a chiral mobile‐phase additive followed by densitometric measurements at 304 nm in concentration range of 1 to 10 μg/band of eszopiclone. The effect of chiral‐selector concentration, pH, and temperature on the resolution have been studied and optimized for the proposed methods. The cited procedures were successfully applied to determine eszopiclone in commercial tablets of pure and racemic forms. Enantiomeric excess was evaluated using optical purity test and integrated peak area to describe the enantiomeric ratio. Thermodynamics of chromatographic separation, enthalpy, and entropy were evaluated using the Van't Hoff equation. The proposed methods were found to be selective for identification and determination of the eutomer in drug substances and products.     

Helicobacter pylori Genotypes Associated with Gastric Histo-Pathological Damages in a Moroccan Population

H. pylori persistent infection induces chronic gastritis and is associated with peptic ulcer disease and gastric carcinoma development. The severity of these diseases is related to human’s genetic diversity, H. pylori genetic variability and environmental factors. To identify the prevalence of histo-pathological damages caused by H. pylori infection in Moroccan population, and to determine their association to H. pylori genotypes, a prospective study has been conducted during 3 years on patients attending the gastroenterology department of Hassan II University Hospital (CHU) of Fez, Morocco. A total of 801 Moroccan adults’ patients were recruited; H. pylori was diagnosed and genotyped by PCR in biopsy specimens and histological exam was performed. We found a high rate of glandular atrophy. Chronic inflammation, neutrophil activity and glandular atrophy showed statistically significant association with H. pylori infection. However, intestinal metaplasia was inversely associated to this infection and no association was observed with gastric cancer cases. A statistically significant association was found between intestinal metaplasia and vacAs1 and vac Am1 genotypes in patients aged 50 years and more but not in younger. This last genotype is also associated to gastric cancer. In this study, gastric cancer showed no significant association with H. pylori. Further studies are warranted to determine the role of other etiological agents such as Epstein-Barr virus, human papillomavirus and possibly environmental and dietetic factors in the occurrence of this pathology.     

Temperature-dependent induction of an acid-inducible stimulon of Escherichia coli in broth.

The induction of the inducible lysyl-tRNA synthetase, LysU, and the inducible lysine and arginine decarboxylases of Escherichia coli K-12 grown in AC broth to a pH of 5.5 or less is temperature dependent, being distinctly lower at 24 than at 37 degrees C. This induction does not appear to be under HtpR control.     

Crystal and molecular structure of two geometrically restricted chemotactic tripeptides, analogues of formyl-methionine-leucine-phenylalanine

The crystal structures of HCO-Met-Leu-Phe-OC(CH3)3, (CH25H39N3O5S), fMLP-OtBu, and HCO-Met psi [CSNH]-Leu-Phe-OCH3, (C22H33N3O4S2), fMS LP-OMe, have been determined by single crystal X-ray diffraction, and their conformational properties investigated by molecular mechanics energy calculations. Crystals of fMLP-OtBu are monoclinic, space group P2(1), a = 12.027(4), b = 9.492(3), c = 12.660(4) A, beta = 101.99(3) degrees, Z = 2; those of fMS LP-OMe are orthorhombic, space group P2(1)2(1)2(1), a = 7.130(1), b = 12.097(2), c = 31.060(5) A, Z = 4. The first compounds fMLP-OtBu is the t-butyl ester of the tripeptide fMLP that represents one of the most potent compounds in inducing the lysozyme release from human neutrophils that reflects the chemotactic activity. From the crystal structure, it is shown that the orientation of the phenylalanine side chain is largely affected by the presence of the bulky group. fMSLP-OMe was shown to be inactive after thionation of the methionine residue in the original tripeptide. Nevertheless, the crystal structure does not reveal any influence of the presence of the thionated peptidic bond on the backbone conformation. The X-ray results have been used to generate parameters for empirical energy calculations. Subsequently, a strategy based on random generation of conformations followed by energy-minimization was applied to investigate the conformational space of thiopeptides, in comparison with normal peptides. From molecular free energy calculations, it is shown that the main influence of the introduction of a thioamide bond on the molecular structure is to prevent the existence of C7(eq) conformations involving the thiomethionine residue. Consequently, a larger number of conformers are found to form intramolecular hydrogen bonds involving the formyl group, reducing its availability to interact with the receptor. For the first time, the theoretical prediction of the existence of C7eq conformations for fMLP is made. The resulting conformers are compared to previously active structures of these chemotactic agents.