Transitory gliosis in the CA3 hippocampal region in rats fed on a ketogenic diet.

The ketogenic diet (KD) is a high-fat, low-protein and low-carbohydrate diet included as medical practice against seizure disorders, particularly in children refractory to conventional anti-epileptic drug treatment. However, the molecular basis of its therapeutic effect remains unclear. Considering the growing evidence for the importance of glial cells for neuronal development, survival and plasticity, we investigated astrocyte protein markers from KD fed rats, in different regions of hippocampus, a brain structure commonly involved in seizure disorders. We found a transitory increment in GFAP in the CA3 hippocampal region, but not in the CA1 or dentate gyrus (DG). This change was not accompanied by changes in S100B content or glutamine synthetase activity. In order to evaluate possible hippocampal involvement we investigated spatial-cognitive behavior using the water-maze task. No changes were observed. This transitory gliosis in CA3 could be related to, or precede, other associated changes proposed to be involved in the attenuation of seizure disorders. These data reinforce the importance of hippocampal astrocytes as cell targets during KD feeding.

Two-dimensional polyacrylamide gel electrophoresis of bovine seminal plasma proteins and their relation with semen freezability.

The objective of this study was to evaluate the low weight (10-30 kDa) protein profile of bovine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine if any of these proteins was associated with semen freezability. Seminal plasma was collected from 16 bulls of high or low semen freezability. Twelve protein spots were identified from the 2D gel (15%); six of these were present in all samples. Of the 12 proteins found, three spots, present in all samples, 3 (15-16 kDa), 5 (16-17 kDa), and 7 (10-12 kDa) had nonsignificant variation among bulls, regardless of their freezability classification. Four proteins were more abundant (P<0.05) in seminal plasma samples collected from bulls with high semen freezability than in samples of bulls with low semen freezability: the spots 3 (15-16 kDa, pI 4.7-5.2), 7 (11-12 kDa, pI 4.8-4.9), 11 (13-14 kDa, pI 4.0-4.5), and 23 (20-22 kDa, pI 4.8-5.2). On the other hand, spot 25 (25-26 kDa, pI 6.0-6.5) was more abundant (P<0.05) on seminal plasma samples from bulls with low semen freezability. The N-terminus sequence of protein 7 was identical to the acidic seminal fluid protein (aSFP). Protein 23 (after trypsin digestion) had structural similarity to bovine clusterin. We concluded that there were differences in the seminal plasma protein profile from bulls with low and high semen freezability; aSFP, clusterin, proteins 3 and 11 may be used as semen freezability markers; and protein 25 was related to low semen freezability.

Digitonin-permeabilization of astrocytes in culture monitored by trypan blue exclusion and loss of S100B by ELISA.

The present protocol details a procedure to permeabilize astrocytes in cultures with digitonin as well as to discuss some data about factors that interfere in permeabilization, particularly divalent cations and nucleotides. Two methods to assess astrocyte permeabilization are described: trypan blue exclusion and ELISA for S100B, a specific protein expressed by these cells. Digitonin-permeabilization of astrocytes has been used to investigate intracellular pools of Ca(2+), internal stores of metabolites, phosphoinositide hydrolysis, and recently we standardized a procedure to study protein phosphorylation (Brain Res. 853 (2000) 32-40). A short incubation time (10 min) with 30 microM digitonin permeabilized at least 75% of cells. A range of media with different ionic nature can be used in cell permeabilization without affecting significantly the extent of permeabilization, but calcium and ATP of the order of 10(-5) M induced a partial resealing which deserves to be considered in assays of permeabilized preparations of astrocytes.

GFAP phosphorylation studied in digitonin-permeabilized astrocytes: standardization of conditions.

Cycles of assembly/disassembly of the intermediate filaments of astrocytes are modulated by the phosphorylation of glial fibrillary acidic protein (GFAP). The sites on GFAP are localized at the N-terminal where they are phosphorylated by cAMP-dependent and Ca(2+)-dependent protein kinases. Phosphorylation of GFAP has been investigated in brain slices, astrocyte cultures, cytoskeletal fractions and purified systems. Here we describe a different approach to study GFAP phosphorylation. We show that permeabilization of astrocytes in culture with digitonin allows direct access to the systems phosphorylating GFAP. Conditions for the permeabilization were established with an assay based on the exclusion of Trypan blue. Incubation of permeabilized cells with cAMP and Ca(2+) increased the phosphorylation state of GFAP. Immunocytochemistry with anti-GFAP showed that permeabilized astrocytes retained their typical flat, fibroblast morphology and exhibited well preserved glial filaments. On incubation with cAMP the filaments apparently condensed to form long processes. The results suggest the approach of studying structural changes in glial filaments in parallel to protein phosphorylation, in the presence of specific modulators of protein kinases and phosphatases has considerable potential.