Glutathione S-transferases in Festuca arundinacea: identification, characterization and inducibility by safener benoxacor

Over recent years it has emerged how certain no crop-species can be employed in phytoremediating contaminated soils or preventing herbicide pollution; in this contest Festuca arundinacea was investigated. Shoots of Festuca were submitted to fast protein liquid chromatography in order to identify their glutathione S-transferases (GST; EC 2.5.1.18), by a combination of anionic, affinity and RP-HPLC chromatography. The chromatographic procedure revealed satisfactory yield and four GSTs were identified: they were named FaGST I, FaGST II, FaGST III and FaGST IV. Among these, significant differences were observed in the chromatographic behaviours, structure, activity toward a "model" substrate, 1-chloro-2,4-dinitrobenzene, and responsiveness to the herbicide safener benoxacor. FaGST I showed the highest activity toward the above substrate, and this activity was up-regulated by the herbicide safener. Therefore, FaGST I was purified till homogeneity and was determined to be an heterodimer consisting of two subunits of 28.0 and 27.2kDa. Each subunit of FaGST I was further characterized by means of LC-ESI-MS/MS and immunoblotting analysis, which revealed that both the subunits belong to the tau subclass.     

Racemization mechanism of lithium tert-butylphenylphosphido-borane: A kinetic insight

The racemization mechanism of tert-butylphenylphosphido-borane is investigated experimentally and theoretically. Based on this converging approach, it is shown, first, that several phosphido-borane molecular species coexist at the time of the reaction and, second, that one particular of both initially assumed reactive routes most significantly contribute to the overall racemization process. From our converging modeling and experimental measurement, it comes out that the most probable species to be here encountered is a phosphido-borane-Li (THF)₂ neutral solvate, whose P-stereogenic center monomolecular inversion through a Y-shaped transition structure (Δ_rG°^≠: 81 kJ mol^?1) brings the largest contribution to the racemization process.     

Quantitative electron-microscopic analysis of the in vivo effects of tetrahydrocannabinol on rat preovulatory follicles

The ultrastructural composition of 4 regions of rat preovulatory follicles was examined using stereological techniques following an intraperitoneal injection of tetrahydrocannabinol (THC), once each day, for 3 days prior to sacrifice on proestrus. Control rats were injected with solvent alone. Changes in nuclear, mitochondrial, lipid droplet and dense-body volume density were measured. No differences in volume density were noted in the theca or antral region of the membrana granulosa. Compared to controls, the mitochondrial volume density decreased in the peripheral region and the corona radiata. The volume density of lipid droplets decreased in the peripheral region. These changes, and their relationship to the roles of these regions in follicular steroidogenesis, are discussed.     

An inquiry-based learning model for an exercise physiology laboratory course

We developed an inquiry-based learning model to better stimulate undergraduate students' cognitive development of exercise physiology laboratory concepts. The course core is the two independent research projects that students, working in small groups, complete during the last 9 wk of the semester. Student groups develop their own research question and hypothesis, design the experiment, collect and analyze the data, and report their findings to the rest of the class using presentation software. To help with success of the research projects, students are taken through a series of guided-inquiry laboratory activities during the initial 6 wk of the semester to develop laboratory skills and an understanding of the scientific process. Observations of student behaviors reflected a high level of enthusiasm and engagement in laboratory activities. Surveys, journal entries, and interviews indicated that students felt empowered by having ownership in their projects, which may be the key reason for the success of this model.     

Immunoglobulins against gp273, the ligand for sperm-egg interaction in the mollusc bivalve Unio elongatulus,are directed against charged O-linked oligosaccharide chains bearing a Lewis-like structure and interact with epitopes of the human zona pellucida

In oocytes of the mollusc bivalve Unio elongatulus, gp273 is the ligand molecule for sperm-egg interaction and binding is mediated by its O-glycans. A serum raised against this protein enabled its localization in the crater region, the area of the vitelline coat where sperm recognition occurs, and showed that after cyanogen bromide fragmentation, the anti-gp273 epitope(s) was retained by a peptide where the O-glycans are localized. In this article, we utilized purified anti-gp273 immunoglobulins to characterize the corresponding epitope by: (i) immunoblotting analysis of the protein after removal of O- and N-glycans; (ii) solid phase binding analysis of anti-gp273 IgG to gp273 N- and O-glycans; and (iii) binding analysis of the same antibody to commercially available oligosaccharides. The results showed that the epitope consists of O-glycans and contains a Lewis-like structure with fucose as determinant. Anti-gp273 IgG were then used to investigate human zona pellucida by immunoelectronmicroscopy and immunoblotting. Epitopes recognized by the antibody were demonstrated on the outer surface of the zona pellucida and shown to belong to a zona pellucida protein having electrophoretic mobility similar to human ZP3. Since human sperm specifically bind to gp273, and anti-gp273 interferes with this binding a functional role for these epitopes is suggested.     

In vitro stereoselective hydrolysis of diacylglycerols by hormone-sensitive lipase

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids in adipocytes and shows a substrate preference for the diacylglycerols (DAGs) originating from triacylglycerols. To determine whether HSL shows any stereopreference during the hydrolysis of diacylglycerols, racemic 1,2(2,3)-sn-diolein was used as a substrate and the enantiomeric excess (ee%) of residual 1,2-sn-diolein over 2,3-sn-diolein was measured as a function of DAG hydrolysis. Enantiomeric DAGs were separated by performing chiral-stationary-phase HPLC after direct derivatization from lipolysis product extracts. The fact that the ee% of 1,2-sn-diolein over 2,3-sn-diolein increased with the level of hydrolysis indicated that HSL has a preference for 2,3-sn-diolein as a substrate and therefore a stereopreference for the sn-3 position of dioleoylglycerol. The ee% of 1,2-sn-diolein reached a maximum value of 36% at 42% hydrolysis. Among the various mammalian lipases tested so far, HSL is the only lipolytic carboxylester hydrolase found to have a pronounced stereospecificity for the sn-3 position of dioleoylglycerol.     

The in vivo effects of tetrahydrocannabinol on the activity of lactate and succinate dehydrogenases in rat preovulatory follicles

Summary The effects of ⁹-tetrahydrocannabinol on the activities of lactate and succinate dehydrogenases in the theca interna and membrana granulosa of rat preovulatory follicles have been analysed microdensitometrically using the same injection regime employed in a previous study on steroidogenic enzymes. A small but statistically significant (18%) decrease in succinate dehydrogenase activity was observed in the theca interna, but none in any region of the membrana granulosa. Lactate dehydrogenase activity was unaffected by THC administration. Thus, a dosage and regimen sufficient to cause significant decreases in the activities of steroidogenic enzymes had little effect on succinate and lactate dehydrogenases in rat preovulatory follicles.