Effect of cysteine,glutamate and glycine supplementation to in vitro fertilization medium during bovine early embryo development

Glutathione (GSH) is an antioxidant synthesized from three constitutive amino acids (CAA): cysteine (Cys), glycine (Gly) and glutamate (Glu). Glutathione plays an important role in oocyte maturation, fertilization and early embryo development. This study aimed to investigate the effect of Cys (0.6 mM), Gly (0.6 mM) and Glu (0.9 mM) supplementation during in vitro fertilization (IVF) of cattle oocytes. In a Pilot Experiment, de novo synthesis of GSH in bovine zygote was evaluated using a modified TALP medium prepared without MEM-essential and MEM-non-essential amino acids (mTALP): mTALP + CAA (constitutive amino acids); mTALP + CAA+5 mMBSO (buthionine sulfoximide); mTALP + Cys + Gly; mTALP + Cys + Glu and mTALP + Gly + Glu. This evidence led us to investigate the impact of CAA supplementation to TALP medium (with essential and non-essential amino acids) on zygote viability, lipid peroxidation, total intracellular GSH content (include reduced and oxidized form; GSH-GSSG), pronuclear formation in zygotes and subsequent embryo development. IVF media contained a) TALP; b) TALP + Cys + Gly + Glu (TALP + CAA); c) TALP + Cys + Gly; d) TALP + Cys + Glu; e) TALP + Gly + Glu, were used. Total GSH-GSSG concentration was increased in TALP, TALP + CAA, and TALP + Cys + Gly. The viability of zygote was similar among treatments. Lipid peroxidation was increased in zygote fertilized with TALP + Cys + Gly; TALP + Cys + Glu; TALP + Gly + Glu and TALP + CAA. The percentage of penetrated oocytes decreased in TALP + CAA and TALP + Cys + Gly. The cleavage rate was lower in TALP + CAA and TALP + Gly + Glu. The percentage of embryos developing to the blastocyst stage was lower in TALP + Cys + Glu and TALP + CAA. In conclusion, we have demonstrated the synthesis of GSH during IVF. However, Cys, Gly and Glu supplementation to TALP medium had negative effects on embryonic development.     

Across island and continents, mammals are more successful invaders than birds (Reply)

Aim  The establishment success of exotic species is calculated as the fraction of introduced species that have become established, and invasion success is estimated as the fraction of established species that have spread significantly from their points of introduction. Records on species introductions are highly incomplete, so strong conclusions about the tens rule and invasibility of island and continents cannot be drawn.
Location  Global.
Methods  Using Jeschke (2008) as an example, we explain the issue of the inadequacy of data to draw conclusions about the tens rule and invasibility of island and continents.
Results  Lack of adequate data.
Main Conclusion  Jeschke (2008) probably overestimates the establishment and invasion success rates, so his conclusions about violation of the tens rule and that islands are not more susceptible to invasion than continents are misleading.     

Specificity and Plasticity of Thalamocortical Connections in Sema6A Mutant Mice

The establishment of connectivity between specific thalamic nuclei and cortical areas involves a dynamic interplay between the guidance of thalamocortical axons and the elaboration of cortical areas in response to appropriate innervation. We show here that Sema6A mutants provide a unique model to test current ideas on the interactions between subcortical and cortical guidance mechanisms and cortical regionalization. In these mutants, axons from the dorsal lateral geniculate nucleus (dLGN) are misrouted in the ventral telencephalon. This leads to invasion of presumptive visual cortex by somatosensory thalamic axons at embryonic stages. Remarkably, the misrouted dLGN axons are able to find their way to the visual cortex via alternate routes at postnatal stages and reestablish a normal pattern of thalamocortical connectivity. These findings emphasize the importance and specificity of cortical cues in establishing thalamocortical connectivity and the spectacular capacity of the early postnatal cortex for remapping initial sensory representations.     

Characterization of a nitric oxide synthase from the plant kingdom: NO generation from the green alga Ostreococcus tauri is light irradiance and growth phase dependent

The search for a nitric oxide synthase (NOS) sequence in the plant kingdom yielded two sequences from the recently published genomes of two green algae species of the Ostreococcus genus, O. tauri and O. lucimarinus. In this study, we characterized the sequence, protein structure, phylogeny, biochemistry, and expression of NOS from O. tauri. The amino acid sequence of O. tauri NOS was found to be 45% similar to that of human NOS. Folding assignment methods showed that O. tauri NOS can fold as the human endothelial NOS isoform. Phylogenetic analysis revealed that O. tauri NOS clusters together with putative NOS sequences of a Synechoccocus sp strain and Physarum polycephalum. This cluster appears as an outgroup of NOS representatives from metazoa. Purified recombinant O. tauri NOS has a K(m) for the substrate l-Arg of 12 ± 5 μM. Escherichia coli cells expressing recombinant O. tauri NOS have increased levels of NO and cell viability. O. tauri cultures in the exponential growth phase produce 3-fold more NOS-dependent NO than do those in the stationary phase. In O. tauri, NO production increases in high intensity light irradiation and upon addition of l-Arg, suggesting a link between NOS activity and microalgal physiology.     

Preparation of a very stable immobilized biocatalyst of glucose oxidase from Aspergillus niger

Glucose oxidase (GOX) has been immobilized on different activated supports, including glyoxyl agarose, epoxy sepabeads and glutaraldehyde-activated supports. Immobilization onto supports pre-activated with glutaraldehyde rendered the most thermo-stable preparation of GOX. Therefore, as the glutaraldehyde chemistry gave a high stabilization of the enzyme, we proposed another technique for improving the multipoint attachment through glutaraldehyde: the enzyme was ionically adsorbed on cationic supports with primary amino groups and then the immobilized preparation was treated with a glutaraldehyde solution. The decrease on enzyme activity was <20%. Following this methodology, we achieved the highest stability of all the immobilization systems analyzed, showing a half-life 100 times higher than the soluble enzyme. Moreover, this derivative showed a higher stability in the presence of organic solvents (for instance methanol) or hydrogen epoxide than the ionically adsorbed enzyme or the soluble one. Therefore, the adsorption of GOX on aminated cationic support and subsequent treatment with glutaraldehyde was presented as a very successful methodology for achieving a very stable biocatalyst.     

Effectiveness of Ethylenediamine-N(o-hydroxyphenylacetic)-N′(p-hydroxyphenylacetic) acid (o,p-EDDHA) to Supply Iron to Plants

The Fe chelate o,p-EDDHA/Fe³⁺, in addition to o,o-EDDHA/Fe³⁺, was found recently to be a component of commercial EDDHA/Fe³⁺ chelates. The European Regulation on fertilisers has included o,p-EDDHA as an authorized chelating agent. The efficacy of o,o-EDDHA/Fe³⁺, o,p-EDDHA/Fe³⁺ and EDTA/Fe³⁺ chelates as Fe sources in plant nutrition was studied. Iron-chelate reductase (FC-R) in young cucumber plants (Cucumis sativus L.) roots reduced o,p-EDDHA/Fe³⁺ faster than o,o-EDDHA/Fe³⁺, EDTA/Fe³⁺ and a commercial source of EDDHA/Fe³⁺. The o,p-EDDHA/Fe³⁺ chelate was also more effective than the o,o-EDDHA/Fe³⁺ in decreasing the severity of Fe-deficiency chlorosis in leaves of young soybean (Glycine max L.) plants grown hydroponically. The o,p-EDDHA ligand was more effective in the short-term than the EDTA and o,o-EDDHA ligands at dissolving Fe from selected Fe minerals and soils. However, the ultimate quantity of dissolve Fe was greatest with the o,o-EDDHA ligand.     

A Kazal prolyl endopeptidase inhibitor isolated from the skin of Phyllomedusa sauvagii.

Searching for bioactive peptides, we analyzed acidic extracts of Phyllomedusa sauvagii skin and found two new proteins, PSKP-1 and PSKP-2, of 6.7 and 6.6 kDa, respectively, which, by sequence homology, belong to the Kazal family of serine protease inhibitors. PSKP-1 and PSKP-2 exhibit the unprecedented feature of having proline at P(1) and P(2) positions. A gene encoding PSKP-1 was synthesized and expressed in Escherichia coli. Recombinant PSKP-1 was purified from inclusion bodies, oxidatively refolded to the native state, and characterized by chemical, hydrodynamic and optical studies. PSKP-1 shows inhibitory activity against a serum prolyl endopeptidase, but is unable to inhibit trypsin, chymotrypsin, V8 protease, or proteinase K. In addition, PSKP-1 can be rendered active against trypsin by active-site site-specific mutagenesis, has bactericidal activity, and induces agglutination of red cells at micromolar concentrations. PSKP-1 might protect P. sauvagii teguments from microbial invasion, by acting as an inhibitor of an as-yet unidentified prolyl endopeptidase or directly as a microbicidal compound.     

Preparation of a robust biocatalyst of d-amino acid oxidase on sepabeads supports using the glutaraldehyde crosslinking method

In this work different protocols to immobilize d-amino acid oxidase (DAAO) on sepabeads were assayed (ionic adsorption on different supports and covalent attachment using glutaraldehyde), studying the stability of the final preparations. The highest stability was achieved by the treatment with glutaraldehyde of DAAO adsorbed on Sepabeads EA (a commercial aminated support having ethylendiamine groups). In fact, this derivative was six times more stable than the enzyme adsorbed only by ionic interaction and much more stable than the soluble enzyme. The effect of the nature of the amino groups in the support was then analyzed. DAAO adsorbed on sepabeads coated with polyethylenimine (PEI) yielded a higher stability than the preparation on Sepabeads EA. The treatment with glutaraldehyde of DAAO adsorbed on Sepabeads PEI yielded the best results in terms of stability, being 200 times more stable than DAAO adsorbed onto Sepabeads EA. The effects of polyethylenimine size and glutaraldehyde concentration were also studied. sepabeads coated with 25 kDa polyethylenimine and treatment with 0.5% glutaraldehyde solution were the optimal parameters regarding the stability (the half life time was 9 h at 56° C, 600 times more stable than the soluble enzyme). Moreover, the optimal derivative showed a maximum load capacity of 15 mg/g of support. This derivative seems to fulfill the requirements for industrial applications.     

Selection for environmental variation: a statistical analysis and power calculations to detect response

Data from uterine capacity in rabbits (litter size) were analyzed to determine whether the environmental variance was partly genetically determined. The fit of a classical homogeneous variance mixed linear (HOM) model and that of a genetically structured heterogeneous variance mixed linear (HET) model were compared. Various methods to assess the quality of fit favor the HET model. The posterior mean (95% posterior interval) of the additive genetic variance affecting the environmental variance was 0.16 (0.10; 0.25) and the corresponding number for the coefficient of correlation between genes affecting mean and variance was -0.74 (-0.90;-0.52). It is argued that stronger support for the HET model than that derived from statistical analysis of data would be provided by a successful selection experiment designed to modify the environmental variance. A simple selection criterion is suggested (average squared deviation from the mean of repeated records within individuals) and its predicted response and variance under the HET model are derived. This is used to determine the appropriate size and length of a selection experiment designed to change the environmental variance. Results from the analytical expressions are compared with those obtained using simulation. There is good agreement provided selection intensity is not intense.     

The ovary of the gestating South American plains vizcacha (Lagostomus maximus): suppressed apoptosis and corpora lutea persistence

The South American plains vizcacha, Lagostomus maximus, displays an exceptional ovulation rate of up to 800 eggs per cycle, the highest rate recorded for a mammal. Massive polyovulation arises from the overexpression of the apoptosis-inhibiting BCL2 gene leading to a suppression of apoptotic pathways responsible for follicular atresia in mammals. We analyzed the ovarian histology, ovarian apoptosis, and apoptosis-related protein expression with special emphasis in corpora lutea throughout the 5-mo-long gestation period, at parturition day and early postpartum, in L. maximus. Corpora lutea were abundant throughout gestation with no sign of structural regression even at the end of gestation. Both immunohistochemistry and Western blot analysis showed strong signals for apoptosis-inhibiting BCL2 protein, whereas the proapoptotic BAX protein was just detected in isolated luteal cells in gestating females and postpartum females. Apoptosis-associated DNA fragmentation detected by TUNEL was very scarce and occasional and correlated with BAX detection in luteal cells. Marked expression of progesterone and alpha-estrogen receptors in luteal cells was found at early, mid-, and late gestation as well as at parturition day and early postpartum samples. Additionally, serum level of progesterone increased markedly to reach maximal values at late gestation and decreasing at parturition to levels found at early gestation, suggesting that corpora lutea remained functional throughout gestation. These results point out that the unusual ovarian environment of L. maximus in which germ cell demise is abolished through antiapoptotic BCL2 gene overexpression also preserves structural integrity and functionality of corpora lutea during the whole gestation. Overexpression of antiapoptotic BCL2 gene may represent a strategy for an essential need of ovary and corpora lutea in order to maintain pregnancy until term.