Mario Morales-Vallarta

ACTIVITY OF INTRACELLULAR PHOSPHOLIPASE A1 AND A2 IN GIARDIA LAMBLIA

Neither phospholipase A1 (PLA A1) nor phospholipase A2 (PLA A2), nor their respective genes, have been identified in Giardia lamblia, even though they are essential for lipid metabolism in this parasite. A method to identify, isolate, and characterize these enzymes is needed. The activities of PLA A1 and PLA A2 were analyzed in a total extract (TE) and in vesicular (P30) and soluble (S30) subcellular fractions of G. lamblia trophozoites; the effects of several chemical and physicochemical factors on their activities were investigated. The assays were performed using substrate labeled with 14C, and the mass of the 14C-product was quantified. PLA A1 and PLA A2 activity was present in the TE and the P30 and S30 fractions, and it was dependent on pH and the concentrations of protein and Ca2+. In all trophozoite preparations, PLA A1 and PLA A2 activities were inhibited by ethylenediaminetetraacetic acid and Rosenthals inhibitor. These results suggest that G. lamblia possesses several PLA A1 and PLA A2 isoforms that may be soluble or associated with membranes. In addition to participating in G. lamblia phospholipid metabolism, PLA A1 and PLA A2 could play important roles in the cytopathogenicity of this parasite.

In vitro Amoebicidal Activity of Borage (Borago officinalis) Extract on Entamoeba histolytica

Borage (Borago officinalis) is a plant with nutritional value that is also used in traditional medicine to treat gastrointestinal disease. This study investigated the amoebicidal activity of a methanol extract of borage. The 50% inhibitory concentration (IC₅₀) of the extract for Entamoeba histolytica was 33 μg/mL. The 50% lethal dose of the extract for brine shrimp was greater than 1,000 μg/mL. The IC₅₀ of the extract for Vero cells was 203.9 μg/mL. These results support the use of borage to prevent diseases associated with E. histolytica infection.

Identification and characterization of microRNAs from Entamoeba histolytica HM1-IMSS.

Background Entamoeba histolytica is the causative agent of amebiasis, a disease that is a major source of morbidity and mortality in the developing world. MicroRNAs (miRNAs) are a large group of non-coding RNAs that play important roles in regulating gene expression and protein translation in animals. Genome-wide identification of miRNAs is a critical step to facilitating our understanding of genome organization, genome biology, evolution, and post-transcriptional regulation. Methodology/Principal Findings We sequenced a small RNA library prepared from a culture of trophozoites of Entamoeba histolytica Strain HM1-IMSS using a deep DNA sequencing approach. Deep sequencing yielded 16 million high-quality short sequence reads containing a total of 5 million non-redundant sequence reads. Based on a bioinformatics pipeline, we found that only 0.5% of these non-redundant small RNA reads were a perfect match with the drafted E. histolytica genome. We did not find miRNA homologs in plant or animal miRNAs. We discovered 199 new potential Entamoeba histolytica miRNAs. The expression and sequence of these Ehi-miRNAs were further validated through microarray by µParaflo Microfluidic Biochip Technology. Ten potential miRNAs were additionally confirmed by real time RT-PCR analysis. Prediction of target genes matched 32 known genes and 34 hypothetical genes. Conclusions/Significance These results show that there is a number of regulatory miRNAs in Entamoeba histolytica. The collection of miRNAs in this parasite could be used as a new platform to study genomic structure, gene regulation and networks, development, and host-parasite interactions.

Trichomonas vaginalis acidic phospholipase A2: isolation and partial amino acid sequence

Sexually transmitted diseases are a major cause of acute disease worldwide, and trichomoniasis is the most common and curable disease, generating more than 170 million cases annually worldwide. Trichomonas vaginalis is the causal agent of trichomoniasis and has the ability to destroy in vitro cell monolayers of the vaginal mucosa, where the phospholipases A2 (PLA2) have been reported as potential virulence factors. These enzymes have been partially characterized from the subcellular fraction S30 of pathogenic T. vaginalis strains. The main objective of this study was to purify a phospholipase A2 from T. vaginalis, make a partial characterization, obtain a partial amino acid sequence, and determine its enzymatic participation as hemolytic factor causing lysis of erythrocytes. Trichomonas S30, RF30 and UFF30 sub-fractions from GT-15 strain have the capacity to hydrolyze [2-14C-PA]-PC at pH 6.0. Proteins from the UFF30 sub-fraction were separated by affinity chromatography into two eluted fractions with detectable PLA A2 activity. The EDTA-eluted fraction was analyzed by HPLC using on-line HPLC-tandem mass spectrometry and two protein peaks were observed at 8.2 and 13 kDa. Peptide sequences were identified from the proteins present in the eluted EDTA UFF30 fraction; bioinformatic analysis using Protein Link Global Server charged with T. vaginalis protein database suggests that eluted peptides correspond a putative ubiquitin protein in the 8.2 kDa fraction and a phospholipase preserved in the 13 kDa fraction. The EDTA-eluted fraction hydrolyzed [2-14C-PA]-PC lyses erythrocytes from Sprague-Dawley in a time and dose-dependent manner. The acidic hemolytic activity decreased by 84% with the addition of 100 μM of Rosenthal’s inhibitor.

Effect of agents that modify cAMP levels on growth and differentiation of Entamoeba invadens.

Amebiasis is a disease that extends worldwide, and is an important public health problem in developing countries. The infective form of the parasite is the cyst, but unfortunately, very little is known concerning the molecular and cell biology of Entamoeba histolytica encystation. This is mainly due to the absence of an axenic medium for E. histolytica that promotes in vitro mass encystation. E. invadens cyst formation has, therefore, been used as an encystation alternative model in amebiasis studies. Although the cyst is the parasitic infective form, little is known about the process of differentiation. 3 9 -5 9 -Cyclic adenosin monophosphate (cAMP) plays an important role in the control of growth and differentiation of many organisms. In Dictyostelium discoideum , cAMP acts as a differentiation promoting factor. cAMP is necessary in chemotaxis mobility that forms ameba aggregates and in the spore formation of this organism (1). Two inhibitory agents of the cAMP-phosphodiesterase are caffeine and theophylline, which increase cAMP levels and reduce growth of BHK cells in both normal and transformed cells. Theophylline induces encystation in Hartmanella culbertsoni (2). In Trypanosoma cruzi , activation of adenylyl cyclase or cAMP analogues such as dbcAMP, and 8-BrcAMP, have an important function in inducing differentiation (3). In Entamoeba , changes in the organization of the cytoskeleton and on transcription of actin mRNA are also affected by cAMP (4), but there are no reports concerning the effect of cAMP on the growth and differentiation of Entamoeba. The objective of this work was to understand how growth and differentiation are affected by substances (caffeine, theophylline, NaF, and L-epinephrine) that could modify cAMP levels in E. invadens.