Acute Hypoxia Differentially Affects the NMDA Receptor NR1, NR2A and NR2B Subunit mRNA Levels in the Developing Chick Optic Tectum: Stage-Dependent Plasticity in the 2B–2A Ratio

It is known that the NMDA-R NR1 subunit is needed for the receptor activity and that under hypoxia the evolution toward apoptosis or neuronal survival depends on the balance NR2A/NR2B subunits. This paper analyzes the effect of acute hypoxia on the above mentioned subunits mRNAs during development. The mean percentage of NR1+ neurons displayed the higher plasticity during development while the NR2A+ neurons the higher stability. Acute hypoxia increased the mean percentage of NR1+ and NR2B+ neurons at ED12 but only that of NR1+ neurons at ED18. Acute hypoxia increased the levels of expression of NR1 and NR2B mRNAs at ED12 without changes in the NR2A mRNA. During early stages there is a higher sensitivity to change the subunits mRNA levels under a hypoxic treatment. At ED12 acute hypoxia increased the probability of co-expression of the NR1–NR2A and NR1–NR2B subunits combinations, the level of NR1 and NR2B and the ratio NR2B/NR2A. These conditions facilitate the evolution towards apoptosis.     

Hypoxia-induced Cell Death and Activation of Pro- and Anti-apoptotic Proteins in Developing Chick Optic Lobe

Exposure of the CNS to hypoxia is associated with cell death. Our aim was to establish a temporal correlation between cellular and molecular alterations induced by an acute hypoxia evaluated at different post-hypoxia (p-h) times and at two stages of chick optic lobe development: embryonic days (ED) 12 and 18. TUNEL assays at ED12 disclosed a significant increase (300%) in pyknotic cells at 6 h p-h, while at ED18 no morphological changes were observed in hypoxic versus controls. At ED12 there was a significant increase (48%) in Bcl-2 levels at the end of the hypoxic treatment, followed by a significant increase of active caspase-9 (49%) and active caspase-3 (58%) at 30 and 60 min p-h, respectively, while at ED18 no significant changes were observed. These findings indicate that prenatal hypoxia produces an equilibrated imbalance in both pro- and anti-apoptotic proteins that culminates in a process of cell death, present at earlier stages of development.     

A microsatellite-based consensus linkage map for species of Eucalyptus and a novel set of 230 microsatellite markers for the genus

Background  

Eucalypts are the most widely planted hardwood trees in the world occupying globally more than 18 million hectares as an important source of carbon neutral renewable energy and raw material for pulp, paper and solid wood. Quantitative Trait Loci (QTLs) in Eucalyptus have been localized on pedigree-specific RAPD or AFLP maps seriously limiting the value of such QTL mapping efforts for molecular breeding. The availability of a genus-wide genetic map with transferable microsatellite markers has become a must for the effective advancement of genomic undertakings. This report describes the development of a novel set of 230 EMBRA microsatellites, the construction of the first comprehensive microsatellite-based consensus linkage map for Eucalyptus and the consolidation of existing linkage information for other microsatellites and candidate genes mapped in other species of the genus.     

Resolving mismatches in the flexible production of ethanol and butanol from eucalyptus wood with vacuum fermentation

Bioprocess and Biosystems Engineering - In flexible ethanol-butanol plants, low tolerance to butanol by solventogenic clostridia (and resulting dilute fermentation) results in considerable number...     

Epipregnanolone Acts as a Partial Agonist on a Common Neurosteroid Modulatory Site of the GABAA Receptor Complex in Avian CNS

Neurosteroid modulatory sites present in the GABA_A receptor complex in chick optic lobe were investigated, in order to evaluate whether allopregnanolone and alphaxalone act through a common site of action. Results showed that either allopregnanolone or alphaxalone present a single-component enhancement of [³H]flunitrazepam binding with EC₅₀ of 1.18 ± 0.12 and 6.56 ± 0.86 M and E_max of 82.18 ± 5.80 and 62.98 ± 3.73 %, respectively. Epipregnanolone behaved as a partial agonist of these steroid modulatory sites with EC₅₀ of 0.49 ± 0.15 M and E_max 12.34 ± 1.03%. Moreover, the addition of 16 M epipregnanolone to either allopregnanolone or alphaxalone decreased EC₅₀ values to 0.54 ± 0,09 and 1.24 ± 0.25 M respectively, while E_max values were not significantly affected. On the other hand, additivity experiments disclosed that a maximal concentration (16 M) of alphaxalone in the presence of allopregnanolone failed to enhance [³H]flunitrazepam binding in excess of that produced by allopregnanolone alone. Results indicate that not only allopregnanolone and alphaxalone act through a common site of action, but such site is highly stereospeciflc with regard to the neurosteroid spatial configuration.     

Presence of an Endogenous Factor in Avian CNS with Agonistic Action on Benzodiazepine Receptors

In the present paper we describe the presence in avian CNS of an endogenous inhibitor of [³H]flunitrazepam binding. This compound was extracted from a synaptic membrane fraction isolated from chick optic lobe and brain using an exhaustive aqueous washing procedure, then purified by means of solid-phase extraction with C₁₈ cartridges and several HPLC steps until an homogeneous peak was obtained. Its chemical structure was studied by size-exclusion chromatography of the purified material which indicated that it possesses a molecular weight below 1350. Although its inhibitory activity was lost by HCl treatment, its peptidic nature was ruled out by an amino acid and N-terminal sequence analyses. Ultraviolet absorption spectrum showed two main peaks at 230 and 280 nm. The endogenous compound was found to inhibit competitively [³H]flunitrazepam binding to its recognition site without affecting [³H]GABA binding to the same receptor complex. The behavior of the endogenous factor in an in vitro GABA shift test and GABA-dependent chloride flux experiments were similar to that of benzodiazepine receptor agonists. In conclusion, these results demonstrate the existence in avian CNS of a competitive endogenous inhibitor of benzodiazepine binding with agonistic action on benzodiazepine receptors.     

Comparative Modulation by 3α,5α and 3β,5β Neurosteroids of GABA Binding Sites During Avian Central Nervous System Development

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABA_A receptor complex. The presence of a 3-hydroxyl group and a 5-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [³H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3-hydroxy,5-pregnan-20-one (3,5-P) and 3-hydroxy,5-pregnan-20-one (3,5-P). Both steroids were found able to enhance [³H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3,5-P had greater potency (EC₅₀ 0.22 M) and enhancing effect (E_max: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABA_A receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3,5-P by its 3,5 isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5-reduced progesterone metabolites present since early development, while 3,5-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5-steroids in early avian development, we propose that 3,5-P, instead of the classical potent 3,5-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.