Ubiquitin of Entamoeba histolytica induces antibody response in patients with invasive amoebiasis.

Entamoeba histolytica causes amoebic liver abscess (ALA) in humans. The injury of target cells by E. histolytica includes processes controlled by the ubiquitin Ehub. Previously, we found immunodominance of Ehub glycan moieties using immunized rabbits. In this work, we analysed dominance of antibodies to the glycoprotein Ehub in the sera from 52 patients with ALA. Controls were sera from 20 healthy people living in endemic areas with a high seroprevalence of antibodies to amoebas, and 20 patients with alcoholic hepatitis (AH) to rule out the cross-reaction of Ehub with autoantibodies induced by liver damage. Antigens were trophozoite extract, glycoprotein Ehub and the recombinant protein E. histolytica recombinant ubiquitin (rEhub). The sera from healthy volunteers and patients with AH do not have antibodies to glycoprotein Ehub. Surprisingly, only the antibodies from patients with ALA recognized the glycoprotein Ehub, and some sera gave a faint reaction with the recombinant protein, especially because evolutionarily, the ubiquitin is conserved between species. This is the first report demonstrating that antibodies to ubiquitin Ehub are induced exclusively in patients with invasive amoebiasis, and the antibody response is mainly to the glycoprotein, indicating glycans are immunodominant. Inhibitors of the Ehub glycans could be potential treatment for amoebiasis by selectively damaging trophozoites.

Characterization of N-glycosylations in Entamoeba histolytica ubiquitin.

Entamoeba histolytica harbors an extensive intracellular distribution of ubiquitin-proteasome systems important for numerous cellular processes. However, glycosylation studies of ubiquitin-proteasome components have not yet been elucidated. Here we report the partial characterization of N-linked glycosylation profile in E. histolytica ubiquitin by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE), Nanoelectrospray Ionization-Tandem Mass Spectrometry (NSI-MS), Matrix-Assisted Laser-Desorption time-of-flight Mass Spectrometry (MALDI-TOF MS) and Gas Chromatography-Mass Spectrometry (GC-MS) analysis. To our knowledge, the data presented in this report represents the first structural glycomics analysis of E. histolytica ubiquitin, while most of the reports are performed on whole parasitic glycan profiles. The glycan profile of E. histolytica ubiquitin has high mannose N-glycan structures. The N-linked glycan profile showed fragments from Hex3HexNAc2 to Hex9HexNAc2. Based in our findings and ubiquitin function, we hypothesize that the same ubiquitin Asn-Asp-Ser sequon carries heterogenic glycosylations, at different metabolic pathway stages according to ubiquitin functional requirements. Finally, we propose a set of possible high mannose N-glycan structures that will help to elucidate the ubiquitin biochemical composition and may well represent good targets for anti-amoebic drugs.

Ethidium bromide inhibits rat brain acetylcholinesterase activity in vitro.

Ethidium bromide (EtBr), a fluorescent dark red compound and stain for double-stranded DNA and RNA was used to study acetylcholinesterase (AChE) activity in vitro together with kinetic parameters of this enzyme in the striatum (ST), hippocampus (HP), cerebral cortex (CC) and cerebellum (CB) of adult rats. AChE activity in vitro in the ST, HP, CC and CB was significantly reduced (p<0.05) in the presence of EtBr at concentrations of 0.00625, 0.0125, 0.025, 0.05 and 0.1 mM. For the analysis of the kinetic three concentrations of EtBr were tested (0.00625, 0.025 and 0.1 mM). An uncompetitive inhibition type was observed in the ST, HP and CC, whereas in the CB the inhibition type was mixed. These data indicate that EtBr should be considered a strong inhibitor of AChE activity demonstrating that there is an interaction between this compound and the cholinergic system.

Effects per se of organic solvents in the cerebral acetylcholinesterase of rats.

Acetylcholinesterase (AChE) was studied in different rat brain regions (cerebellum, hypothalamus, striatum, hippocampus and cortex) in the presence of different organic solvents normally used in the in vitro assay. The organic solvents used were acetone (C3H6O), acetonitrile (C2H3N), ethyl alcohol (C2H6O), isopropyl alcohol (C3H8O), methyl alcohol (CH4O), tert-butyl alcohol (C4H10O) and dimethyl sulfoxide (DMSO, C2H6OS) ranging from 0.6 to 10%. Ethyl and methyl alcohol presented no effect on AChE activity at any of the concentrations and brain structures tested. In the hippocampus, isopropyl alcohol did not demonstrate a significant inhibitory effect, even at high concentrations. Tert-butyl alcohol presented an interesting result, increased AChE activity (P < .05) in the hypothalamus (1.8%), cortex (1.8 and 2.5) and striatum (1.2, 1.8 and 2.5%) and decreased activity at a concentration of 10% in the cortex (P < .05) and striatum (P < .01). Acetone and acetonitrile presented similar results, both significantly inhibiting AChE in all structures (5%, P < .05 and 10%, P < .01). DMSO exhibited a highly inhibitory effect at practically all concentrations tested (P < .01). In conclusion, for testing new compounds on AChE activity in vitro, methyl and ethyl alcohol may be the best organic solvent choice.