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Articles by E. Miranda-Miranda
Total Records ( 3 ) for E. Miranda-Miranda
  E. Miranda-Miranda , A. Zamora-Ruiz and R. Cossio-Bayugar
  The present study reports the cloning and expression of a Caenorhabditis elegans Cathepsin B-like Protease (CBP), with the objective of obtaining a recombinant enzyme bearing biochemical properties similar to natural CBP reported in the literature for C. elegans and parasitic nematodes. The gene was isolated by PCR from C. elegans cDNA, resulting amplicon was cloned into a baculovirus expression plasmid, insect cells were used for assembly of a recombinant baculovirus containing the C. elegans CBP gene. Thirty five and 45 kDa recombinant proteins were identified from baculovirus infected crude cells containing a His-tag antigenic marker identified by a specific polyclonal antibody in a Western blot assay, both of these recombinant proteins were capable of digesting gelatin in a SDS-PAGE-gelatin assay. Affinity chromatography purified fractions of this recombinant protease, were assayed for peptidase activity against synthetic fluorogenic peptides, including specific cathepsin B substrates and a caspase tetra peptide substrate, maximum cathepsin activity was detected at pH 6.0 for all synthetic substrates and total inhibition was achieved by cystein protease inhibitor E-64 but not by EDTA, pepstatin or PMSF protease inhibitors. Recombinant C. elegans cathepsin B-like protease can be obtained in large amounts from the infected insect cell culture.
  R. Cossio-Bayugar , E. Miranda-Miranda , D. Portilla-Salgado and J. Osorio-Miranda
  This study was done with the objective of developing a methodology for cholinesterase and carboxylesterase expression measurement in acaricide resistant R. microplus, for that purpose the expression levels for these enzymes were measured by real time PCR quantification of mRNA specific detection, comparing acaricide sensitive with multiple acaricide resistant strains of ticks known as Mora and San Alfonso. Acaricide susceptible ticks were used as standard cholinesterase and carboxylesterase expression level and adjusted as a baseline of 1 Relative Expression Units (REU). A statistical significance was observed in cholinesterase gene expression level as 13.07 ± 3.49 REU for Mora strain and 10.81 ± 2.98 REU for San Alfonso strain compared to the susceptible strain. Also, carboxylestrase expression found statistically significant for Mora and San Alfonso strains (6.9 ± 1.14 and 12.11 ± 1.81 REU, respectively) compared to the susceptible strain. Present results proved that the carboxylestrase and cholinesterase genes expression increased by acaricide pesticide exposure in Mora and San Alfonso, R. microplus, which explained as an overexpression of AchE2 at the singanglion level for OP resistant ticks, as well as increased levels of esterase gene CzEST9 implicated in pyrethroid resistant strains.
  E. Miranda-Miranda , R. Cossio-Bayugar , M.D.R. Quezada-Delgado , F. Olvera-Valencia and S. Neri-Orantes
  An acaricide-resistant and an acaricide-susceptible strain of Rhipicephalus microplus ticks, were evaluated during a 40 day larval development period for carboxylesterase expression on SDS-PAGE, with the objective of establishing the optimal larval age for carboxylesterase specific activity measurement. Experimental results demonstrated that carboxylesterase expression is located at 62 and 55 kDa polypeptides that exhibited enzymatic specific activity reaching a maximum between day 20 and 30 of larval development, larvae of this age showed carboxylesterase activity statistically higher (p<0.001 unpaired t-test) in acaricide resistant ticks when compared both strains, additionally, carboxylesterase activity in both strains was found to coincide with the vitellin degradation process. The assessment of the results suggested that carboxylesterase active enzymes are mature polypeptide products originating from higher mass protein precursors associated with the yolk nutrient storage reserves in preparation to pre-starving conditions. Experimental data obtained during this study demonstrated that in order to obtain reproducible results of carboxylesterase expression levels associated to acaricide resistance by zymograms, the enzymatic assays should be performed on 20 to 30 day old larvae.
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