Optimization of Free Phytoprostane and Phytofuran Production by Enzymatic Hydrolysis of Pea Extracts Using Esterases.

Given the growing interest in phytoprostanes (PhytoPs) and phytofurans (PhytoFs) in the fields of plant physiology, biotechnology, and biological function, the present study aims to optimize a method of enzymatic hydrolysis that utilizes bacterial and yeast esterases that allow the appropriate quantification of PhytoPs and PhytoFs. To obtain the highest concentration of PhytoPs and PhytoFs, a response surface methodology/Box-Behnken design was used to optimize the hydrolysis conditions. Based on the information available in the literature on the most critical parameters that influence the activity of esterases, the three variables selected for the study were temperature (°C), time (min), and enzyme concentration (%). The optimal hydrolysis conditions retrieved differed between PhytoPs (21.5 °C, 5.7 min, and 0.61 µg of enzyme per reaction) and PhytoFs (20.0 °C, 5.0 min, and 2.17 µg of enzyme per reaction) and provided up to 25.1- and 1.7-fold higher contents relative to nonhydrolyzed extracts. The models were validated by comparing theoretical and experimental values for PhytoP and PhytoF yields (1.01 and 1.06 theoretical/experimental rates, respectively). The optimal conditions were evaluated for their relative influence on the yield of individual nonesterified PhytoPs and PhytoFs to define the limitations of the models for obtaining the highest concentration of most considered compounds. In conclusion, the models developed provided valuable alternatives to the currently applied methods using unspecific alkaline hydrolysis to obtain free nonesterified PhytoPs and PhytoFs, which give rise to more specific hydrolysis of PhytoP and PhytoF esters, reducing the degradation of free compounds by classical chemical procedures.

Anion channel blockers differentially affect T-type Ca(2+) currents of mouse spermatogenic cells, alpha1E currents expressed in Xenopus oocytes and the sperm acrosome reaction.

The direct electrophysiological characterization of sperm Ca(2+) channels has been precluded by their small size and flat shape. An alternative to study these channels is to use spermatogenic cells, the progenitors of sperm, which are larger and easier to patch-clamp. In mouse and rat, the only voltage-dependent Ca(2+) currents displayed by these cells are of the T type. Because compounds that block these currents inhibit the zona pellucida-induced Ca(2+) uptake and the sperm acrosome reaction (AR) at similar concentrations, it is likely that they are fundamental for this process. Recent single channel recordings in mouse sperm demonstrated the presence of a Cl(-) channel. This channel and the zona pellucida (ZP)-induced AR were inhibited by niflumic acid (NA), an anion channel blocker [Espinosa et al. (1998): FEBS Lett 426:47-51]. Because NA and other anion channel blockers modulate cationic channels as well, it became important to determine whether they affect the T-type Ca(2+) currents of spermatogenic cells. These currents were blocked in a voltage-dependent manner by NA, 1, 9-dideoxyforskolin (DDF), and 5-nitro-2-(3-phenylpropylamine)benzoic acid (NPPB). The IC(50) values at -20 mV were 43 microM for NA, 28 microM for DDF, and 15 microM for NPPB. Moreover, DDF partially inhibited the ZP-induced AR (40% at 1 microM) and NPPB displayed an IC(50) value of 6 microM for this reaction. These results suggest that NA and DDF do not inhibit the ZP-induced AR by blocking T-type Ca(2+) currents, while NPPB may do so. Interestingly 200 microM NA was basically unable to inhibit alpha1E Ca(2+) channels expressed in Xenopus oocytes, questioning that this alpha subunit codes for the T-type Ca(2+) channels present in spermatogenic cells. Evidence for the presence of alpha1C, alpha1G, and alpha1H in mouse pachytene spematocytes and in round and condensing spermatids is presented.