Interrelations between the parasitophorous vacuole of Toxoplasma gondii and host cell organelles.

Toxoplasma gondii, the causative agent of toxoplasmosis, is capable of actively penetrating and multiplying in any nucleated cell of warm-blooded animals. Its survival strategies include escape from fusion of the parasitophorous vacuole with host cell lysosomes and rearrangement of host cell organelles in relation to the parasitophorous vacuole. In this article we report the rearrangement of host cell organelles and elements of the cytoskeleton of LLCMK2 cells, a lineage derived from green monkey kidney epithelial cells, in response to infection by T. gondii tachyzoites. Transmission electron microscopy made on flat embedded monolayers cut horizontally to the apical side of the cells or field emission scanning electron microscopy of monolayers scraped with scotch tape before sputtering showed that association of mitochondria to the vacuole is much less frequent than previously described. On the other hand, all parasitophorous vacuoles were surrounded by elements of the endoplasmic reticulum. These data were complemented by observations by laser scanning microscopy using fluorescent probes from mitochondria and endoplasmic reticulum and reinforced by three-dimensional reconstruction from serial sections observed by transmission electron microscopy and labeling of mitochondria and endoplasmic reticulum by fluorescent probes.     

Influence of trace elements supplementation on the production of recombinant frutalin by Pichia pastoris KM71H in fed-batch process

Frutalin, a galactose-specific lectin used to detect specific tumour markers, is a protein with low expression level in breadfruit. In the present study, fed-batch fermentation in a stirred tank bioreactor was used as a strategy to enhance protein production by a recombinant Pichia pastoris KM71H. By using this process, the production of recombinant frutalin was 4-fold higher than the value obtained in shaker flasks batch assays. Supplementation of the fermentation medium with trace elements (Pichia trace minerals, PTM) was also evaluated in order to stimulate production of the recombinant protein. The addition of PTM to the minimum medium afforded a recombinant protein production of 13.4 mg L^?1, which was 2.5-fold higher than that achieved from the culture medium without PTM supplementation. These results are significant as the development of strategies to improve the production of recombinant frutalin may broaden its application in cancer diagnosis.     

Biogenesis of the reservosomes of Trypanosoma cruzi.

Reservosomes are endocytic compartments found in the posterior region of epimastigotes of Trypanosoma cruzi. In the differentiation from trypomastigotes to epimastigotes (reverse metacyclogenesis in vitro), one has the rare opportunity of following the biogenesis of an endocytic compartment. Metacyclic trypomastigotes incubated in LIT medium highly enriched with fetal calf serum differentiated directly to epimastigotes. In recently differentiated epimastigotes, acidic organelles were found in round compartments spread along the cell body, whereas in control epimastigotes they were found in reservosomes located in the posterior region. Ultrastructural analysis of intermediate forms showed that the cytostome and reservosomes appeared before differentiation to epimastigotes was completed. Many polymorphic reservosomes, with or without lipid inclusions, were observed from the anterior portion of the cell body, in close relationship with the Golgi complex, to the posterior region. Endocytic tracers were observed in the cytostome, flagellar pocket, vesicles, and newly formed reservosomes. Cruzipain, the main protease of T. cruzi, was localized in newly formed reservosomes and in vesicles budding from the trans-Golgi network that seem to fuse with reservosomes. Ingested gold-labeled albumin and cruzipain colocalized in recently formed reservosomes. Endocytosis and immunocytochemical analysis suggested that the endocytic and the secretory pathways may contribute to reservosome formation.     

Using environmental scanning electron microscopy (ESEM) as a quantitative method to analyse the attachment of Giardia duodenalis to epithelial cells

Giardia duodenalis is a human parasite that colonises the small intestine. In some studies, it may be necessary to estimate the parasite-host adhesion index. When working in vitro, it is very difficult to determine the relative number of parasites adhered to intestinal cells because parasites might be removed from enterocytes during specimen preparation. We have encountered such difficulties with sample analyses when using light and conventional scanning electron microscopy, including the loss of adherent cells during the processing steps. In this study, we showed that environmental scanning electron microscopy (ESEM), which allows the examination of specimens at normal atmospheric pressure or in very low vacuum without any previous treatment, can be used effectively in quantitative studies of the parasite-host relationship.     

Comparative computational studies of 3,4-dihydro-2,6-diaryl-4-oxo-pyrimidine-5-carbonitrile derivatives as potential antinociceptive agents

In this study, the antinociceptive properties of 3,4-dihydro-2,6-diaryl-4-oxo-pyrimidine-5-carbonitrile derivatives 5a-i at doses of 25 and 50 mg/kg were evaluated in mice, using the abdominal constriction test. Molecular modeling studies were also performed using density functional theory calculations. These data provided information about the electrostatic and ionization potentials and were used to compare the antinociceptive activity of the title compounds. The most active compounds were 3,4-dihydro-2-(4-chlorophenyl)-6-(4-methoxyphenyl)-4-oxo-pyrimidine-5-carbonitrile (5b) and 3,4-dihydro-2,6-diphenyl-4-oxo-pyrimidine-5-carbonitrile (5i), which inhibited the number of abdominal constrictions, at 50 mg/kg dose, in 88.6% and 88% of the sample, respectively. A preliminary SAR study demonstrated that halogen replacement in the phenyl rings of the compounds under study reduces the antinociceptive activity. DFT calculations showed that there is a high correlation between the ionization potentials and the analgesic properties of the compounds. It was found that compounds with a positive ionization potential (compounds 5b and 5i) were found to be the best analgesic drugs in this series.     

Simultaneous determination of polyether ionophores, macrolides and lincosamides in hen eggs by liquid chromatography-electrospray ionization tandem mass spectrometry using a simple solvent extraction

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) method was developed and validated for the determination of residues of 6 polyether ionophores (lasalocid, maduramicin, monensin, narasin, salinomycin, semduramicin), 3 macrolides (erythromycin, tylosin, clarithromycin) and 1 lincosamide (lincomycin) in eggs. Nigericin was used as qualitative internal standard. Samples were deproteinizated/extracted with acetonitrile without pH adjustments. Aliquots of the extracts were evaporated and reconstituted for injection in the instrument operated in positive multiple reaction monitoring (MRM) mode. The stability of the antibiotics and the intensity of the formed ions were considered in order to select a suitable solvent for the reconstitution of the obtained dry extracts. No clean-up steps were required and matrix effects were controlled by sample dilution, selection of appropriate chromatographic conditions and reduced injection volume. Good within-laboratory reproducibility was obtained, with relative standard deviations (RSD(R)) from 4.0 (semduramicin at 5 μgkg(-1)) to 18.6 (erythromycin at 25 μgkg(-1)) for the ionophores and macrolides. Lincomycin showed the least precise results, with a maximum RSD(R) of 20.2% at 75 μgkg(-1)). Satisfactory decision limits (CCα) and detection capabilities (CCβ) were also attained. Method limits of detection (LODs) from 0.04 (salinomycin) to 1.6 μgkg(-1) (lincomycin) were achieved. Method limits of quantification (LOQs) were from 0.14 to 5.3 μgkg(-1) for the same drugs, respectively. All the LOQs, except that obtained for maduramicin were remarkably below the lowest validation level. The proposed method is suitable for routine application in commercial egg samples.     

Vermiculite-aliphatic amine interactions at the solid/liquid interface: a thermodynamic approach

Polar n-alkylmonoamines of general formula H₃C(CH₂) _n NH₂ (n = 1, 3, 5) interacted with layered silicate vermiculite at the solid/liquid interface. The maximum amount of amine intercalated (N _f ) inside the interlamellar space were 0.62, 0.46, and 0.38 mmol g⁻¹, to give the following order of intercalation ethyl → butyl → hexylamines. The layered vermiculite solid was suspended in deionized water and calorimetrically titrated with this series of amines, to give favorable thermodynamic data, such as exothermic enthalpy, negative Gibbs free energy and positive entropy data.