María Teresa González-Garza

Differential effects of the (+)- and (–)-gossypol enantiomers upon Entamoeba histolytica axenic cultures

Abstract— The in‐vitro anti‐amoebic effects of (±)‐, (+)‐, (–)‐gossypol and emetine were tested against axenic trophozoites from five Entamoeba histolytica strains. The (–)‐isomer was more active than the racemate and the (+)‐isomer. These results indicate that the gossypol anti‐amoebic activity is mainly due to its content of (–)‐gossypol in all strains tested.

Identification ofEntamoeba histolytica intracellular phospholipase A and lysophospholipase L1 activities

Entamoeba histolytica phospholipase A and lysophospholipase activities from a vesicular subcellular fraction (P30) were analyzed. The products, obtained using specific substrates labeled with14C or3H, indicated the presence of phospholipase A1 and A2 as well as lysophospholipase L1 activities. The enzymes detected could participate in phospholipid metabolism and the alkaline phospholipase A2 may contribute toE. histolytica cytopathogenicity.

A multipurpose solid-phase method for protein determination with Coomassie brilliant blue G-250

The multipurpose method for protein determination (MMPD) is based in the Coomassie brilliant blue G-250 binding to immobilized and washed proteins in paper filter disks, and the A600 measurement of the eluted protein-dye complexes. The analysis requires 5-microliters samples, put in 7-mm paper filter disks, which can be stored for up to 2 weeks, without sensible changes in their protein content. The A600 is a logarithmic function of the log of bovine serum albumin quantity, between 2.5 and 250 micrograms with two linear segments used as standard curves: one between 2.5 to 20 micrograms and the other between 20 and 80 micrograms. Results with the MMPD were quantitatively comparable with those obtained with the method of Lowry et al., and those reported by other workers, assaying the following material: (i) bovine serum albumin, in doubly distilled water or in presence of several substances that interfere with the current methods; and (ii) total homogenates and their respective trichloroacetic acid-insoluble extracts, which were obtained from several phenol-rich vegetal specimens and complex animal products. The MMPD is proposed as an alternative for protein determination for its versatility and reliability, in both vegetal or animal products, especially when the analysis with traditional methods is made difficult by the presence of natural or added interfering substances and the sample volume is too small to discard them, or when the material must be stored for relatively long periods of time, prior to its processing.

Entamoeba histolytica cysts with a defective wall formed under axenic conditions

Axenic HK9Entamoeba histolytica strain amoebae, maintained in PEHS medium, displayed several cystic characteristics that involve an active process of cystic wall formation, cellular volume and density diminution, and one or two nuclear divisions. The differentiation process was asynchronic, beginning after the logarithmic growth phase. The axenic cysts, which were maintained in a 50 mOsm/kg medium at 4°C for 72 h, produced growing trophozoites within 1–7 days of incubation at 36°C in fresh medium. Negative results were obtained with trophozoites submitted to the above treatment, and with axenic cysts maintained in double-distilled water at 4°C for 24 h, or in 0.1% sarkosyl, for 10 min at room temperature instead of 55 mosmol/kg medium. Thus, the HK9E. histolytica strain, cultured in PEHPS, produced under axenic conditions a small proportion of mature, metabolically active cysts, but with an immature or abnormal wall.

Auxotrophy to lipoproteins of Entamoeba histolytica cultivated under axenic conditions

Entamoeba histolytica grows in media without serum but with a mixture of aminoacids, vitamins, lipoproteins, free cholesterol, phospholipids and fatty acids called PACSR. The ability of lipoproteins and free lipids to support growth of three E. histolytica strains (HK9, HMI:IMSS and HM3:IMSS) was analysed. Tubes containing 5 ml culture medium, amino acids, vitamins and either 120–1,200 μg lipoproteins/ml or 0.017–0.10 mg free lipids/ml (predissolved in absolute ethanol) were inoculated with 1 × 104 trophozoites/ml and incubated at 37 °C for 72 h. Amoebae died within 12 h in the presence of any free lipid combination, while those having 240–480 mg lipoproteins/ml reached densities similar to or higher than those of controls (depending on strain). The addition of ethanol (0.1%) to the media produced stable lipid solutions and did not show significant adverse effects. Accordingly, E. histolytica is auxotrophic to lipoproteins and unable to use free cholesterol, phospholipids or fatty acids.

Cytotoxic effect of two legumes in epithelial cells of the small intestine

The previously known high toxicity and hemagglutinin concentration found in Escumite bean (Phaseolous acutifolius), widely consumed in the south-east of Mexico, induced us to study the cytotoxic effect of these beans on the epithelial cells of the small intestine of rats. Another, less toxic leguminosae, chick-pea (Cicer arietinum), was used for comparison. In vitro tests showed that the viability of the cells was much more affected by the Escumite bean extract than by the chick-pea extract (P<0.001). The cellularity was much diminished when the epithelial cells were treated with Escumite extract (P<0.001), but not with chick-pea extract. In vivo experiments confirmed these findings. The present results suggest that the deleterious effect of some toxicants present in the raw legume is due to an interaction with intestinal epithelium, which, in turn, may cause a reduced absorption of nutrients.

Differential cytotoxicity of the isolated protein fraction of Escumite bean (Phaseolus acutifolius)

The Escumite bean (Phaseolus acutifolius) is an edible legume which in raw form is highly toxic to rats. Proteins were separated by DEAE cellulose and affinity chromatography. Hemagglutinating activity, trypsin inhibitory effect, cytotoxicity on human peripheral lymphocytes and on intestinal epithelial cells of rats, and mitogenic activity were assayed with each protein fraction. Hemagglutinins and trypsin inhibitory fractions showed differential toxicity with lymphocytes as compared to intestinal epithelial cells. A protein fraction without the previous activities was cytotoxic mainly to intestinal epithelial cells.

Lactic dehydrogenase activity in various organs of rats fed cottonseed flour

Abstract Male rats fed cottonseed meal diet or protein-free diet for four weeks showed a decreased lactic dehydrogenase (LDH) activity in the liver, heart and testicle, but not in skeletal muscle. At the end of this treatment, the animals were fed with Purina Chow control diet for 14 weeks of recovery. After ten weeks of recovery, the animals fed cottonseed meal showed normal LDH values for the testicle and heart. In the liver, the low value remained so during the 14 weeks of recovery. Normal values were reached in the animals fed protein-free diet at six weeks of recovery. The decreased LDH activity in animals fed cottonseed diet may be due to the gossypol present in it.

Effect of Clofibric Acid on Desmin and Vimentin Contents in Rat Myocardiocytes

The aim of this experimental study was to analyze in vitro effects of clofibric acid on vimentin and desmin contents in rat myocardiocytes, which was carried out in primary myocardiocyte cells that were treated only with clofibric acid at 0.1 mM. The measurement of vimentin and desmin were done by Western blotting and densitometry. This study showed that myocardiocytes exposed to clofibric acid exhibit a 26.3% decrease in vimentin and a 42.1% decrease in desmin. Considering the role that these intermediate filaments play in the anchorage and cellular organization of myocardiocytes, the decrease of desmin and vimentin observed in cells treated with clofibric acid may be partially responsible for the adverse effects observed in patients. In conclusion, the alteration of cytoskeletal proteins may be a cause of cardiopathy in patients treated with these compounds.