In vitro maturation impacts cumulus-oocyte complex metabolism and stress in cattle.

The influence of in vitro maturation (IVM) in oocytes is still not totally understood. The aim of this study was to determine the influence of IVM on the metabolism and homeostasis of bovine cumulus-oocyte complexes. In the present study, we demonstrated that IVM leads to accumulation of neutral lipids associated with differential levels of the mono-, di- and triacylglycerols in both cumulus cells and oocytes. We observed that in vitro-matured oocytes exhibited decreased glutathione and reactive oxygen species levels and a lower ATP/ADP ratio when compared to in vivo-matured oocytes, with no significant differences in metabolism and stress-related mRNA or miRNA levels. Moreover, in addition to an increase in lipids in in vitro-matured cumulus cells, fatty acid synthesis and accumulation as well as glycolysis pathway genes were upregulated, whereas those affiliated with the ß-oxidation pathway were decreased. Our gene expression data in cumulus cells suggest the disruption of endoplasmic reticulum stress, apoptosis and cellular stress response pathways during IVM. Furthermore, a total of 19 miRNAs were significantly altered by the maturation process in cumulus cells. These results indicate some new negative influences of the in vitro system in cumulus-oocyte complexes, demonstrating the occurrence of functional disruption in lipid metabolism and stress pathways and showing evidences suggesting the occurrence of altered mitochondrial activity and energy metabolism during IVM, with a massive dysregulation of the corresponding transcripts in the surrounding cumulus cells.

The multicomponent Hantzsch reaction: comprehensive mass spectrometry monitoring using charge-tagged reagents.

A novel strategy for the ESI-MS monitoring of reaction solutions involving the alternate use of permanently charge-tagged reagents has been used for comprehensive mass spectrometry monitoring of the multicomponent Hantzsch 1,4-dihydropyridine reaction. By placing a charge tag on either, or both, of the two key reactants, ion suppression effects for ESI were eliminated or minimized, and comprehensive detection of charge-tagged intermediates was achieved. The strategy allowed the trapping and characterization of the important intermediates in the mechanism for 1,4-dihydropyridine formation.

Easy dual-mode ambient mass spectrometry with Venturi self-pumping, canned air, disposable parts and voltage-free sonic-spray ionization.

An exceptionally easy to assemble source for ambient mass spectrometry is described. Based on Venturi easy ambient sonic-spray ionization (V-EASI), the source was further simplified by the use of a can of compressed air which simultaneously provides solution or solvent Venturi self-pumping and continuous, stable and abundant low-noise ion signal via voltage-free sonic-spraying. Further simplification was also attained by the use of inexpensive and readily commercially available parts: a surgical 2-way catheter, an aerosol can of compressed air, a 30 cm long fused-silica capillary and a hypodermic needle. This "Spartan" V-EASI source seems to offer one of the easiest and cheapest ways to make ions for ambient desorption/ionization mass spectrometry analysis of both liquid and solid samples.

Insecticidal action of Annona coriacea lectin against the flour moth Anagasta kuehniella and the rice moth Corcyra cephalonica (Lepidoptera: Pyralidae).

Annona coriacea lectin (ACLEC) was tested for insecticidal activity against larvae of two pyralid moths, Anagasta kuehniella and Corcyra cephalonica. ACLEC produced approximately 50% mortality and mass loss in A. kuehniella larvae when incorporated into an artificial diet at levels of 1.5% and 1.0% (w/w), respectively. In contrast, the inclusion of up to 2% ACLEC in the diet did not significantly decrease the survival or weight of C. cephalonica larvae. The nutritional indices for A. kuehniella and C. cephalonica suggested that ACLEC had a multi-mechanistic mode of action and was an antifeedant for both insects. The toxicity in A. kuehniella apparently resulted from a change in the gut membrane environment and consequent disruption of digestive enzyme recycling mechanisms. Affinity chromatography showed that ACLEC bound to midgut proteins of A. kuehniella and C. cephalonica. However, the 14 kDa subunit of ACLEC was not digested by midgut proteases of A. kuehniella, but was degraded by the corresponding C. cephalonica proteases within a few hours. These findings suggest the possibility of using ACLEC to engineer crop plants.

Insecticidal and antifungal activity of a protein from Pouteria torta seeds with lectin-like properties.

This paper describes the purification and characterization of a novel protein from the seeds of Pouteria torta (family Sapotaceae). The protein was purified by a combination of gel filtration, ion-exchange, and reverse phase chromatographies. SDS-PAGE of the purified protein resulted in a single protein band of 14 kDa in the presence and absence of DTT. The lectin-like activity of pouterin was best inhibited by glycoproteins such as fetuin, asialofetuin, heparin, orosomucoid, and ovoalbumin. Pouterin inhibited the growth of the fungi Fusarium oxysporum and Colletotrichum musae and of the yeast Saccharomyces cerevisiae. The incorporation of pouterin into an artificial diet (final concentration = 0.12%, w/w) caused 50% mortality in larvae of the insect Callosobruchus maculatus, whereas 0.08% pouterin produced an ED50.

Neutrophil migration in mice induced by a mannose-binding lectin isolated from Annona coriacea seeds.

A novel 14-kDa lectin from Annona coriacea seeds (ACLEC) with hemagglutinating activity on erythrocytes has been recently described. Since plant lectins are known to present inflammatory activity, this study aimed to investigate the leukocyte migration induced by ACLEC, and inflammatory mediators involved in this phenomenon. Male Swiss mice were intraperitoneally injected with ACLEC (3-100 microg/cavity), and at 4-96 h thereafter the leukocyte counts in peritoneal lavage fluid were evaluated. ACLEC induced a dose-dependent neutrophil accumulation, reaching maximal responses at 16 h after injection (approximately 40-fold increase for 30 microg/cavity). Significant accumulation of mononuclear cells was observed at 72 h (2.7-fold increase). The carbohydrate mannose nearly abolished the neutrophil influx, whereas sucrose, glucose and galactose had no effect. Dexamethasone, the cyclooxygenase-2 (COX-2) inhibitor celecoxib and the Platelet activating factor (PAF) receptor antagonist PCA4248 significantly reduced ACLEC-induced neutrophil influx. The tachykinin NK(1) antagonist SR140333, the tachykinin NK(2) antagonist SR48968, the non-selective NO inhibitor L-NAME, the selective inducible NOS inhibitor aminoguanidine and the lypoxygenase inhibitor AA861 all failed to modify the ACLEC-induced responses. In conclusion, ACLEC is able to attract neutrophils into the mice peritoneal cavity by mechanisms involving interactions of the lectin with cell-specific mannose recognition, leading to the release of COX-2-derived mediators and PAF.

Purification and characterization of a lectin from Annona coriacea seeds.

A novel lectin, denominated ACLEC, was isolated from Annona coriacea seeds, belonging to the Annonaceae family. The lectin presented one protein band in SDS-PAGE of 14 kDa. Of the sugars tested, Dglucose and D-mannose were the best inhibitors. A search sequence database showed that ACLEC had homology with other plant lectins, belonging to leguminous lectin family.