Alejandra Hidalgo

Inhibition of brain ST8SiaIII sialyltransferase leads to impairment of procedural memory in mice.

Several glycoproteins in mammalian brains contain α2,8-linked disialic acid residues. We previously showed a constant expression of disialic acid (DiSia) in the hippocampus, olfactory bulb and cortex, and a gradual decrease of expression in the cerebellum from neonatal to senile mice. Previous publications indicate that neurite extension of neuroblastoma-derived Neuro2A cells is inhibited in the presence of DiSia antibody. Based on this, we treated Neuro2A cell cultures with RNA interference for ST8SiaIII mRNA, the enzyme responsible for DiSia formation. We observed that neurite extension was inhibited by this treatment. Taking this evidence into consideration and the relationship of the cerebellum with learning and memory, we studied the role of DiSia expression in a learning task. Through delivery of pST8SiaIII into the brains of C57BL/6 neonatal mice, we inhibited the expression of ST8SiaIII. ST8SiaIII mRNA and protein expressions were analyzed by real-time PCR and western blot, respectively. In this work, we showed that pST8SiaIII-treated mice presented a significantly reduced level of ST8SiaIII mRNA in the cerebellum (p<0.01) in comparison to control mice at 8 days after treatment. It is also noted that these levels returned to baseline values in the adulthood. Then, we evaluated behavioural performance in the T-Maze, a learning task that estimates procedural memory. At all ages, pST8SiaIII-treated mice showed a lower performance in the test session, being most evident at older ages (p<0.001). Taken all together, we conclude that gene expression of ST8SiaIII is necessary for some cognitive tasks at early postnatal ages, since reduced levels impaired procedural memory in adult mice.

Differential expression of glycans in the hippocampus of rats trained on an inhibitory learning paradigm

The glycan chains of glycoconjugates play important roles in cell–cell and cell–matrix interactions. In the CNS, previous studies on learning and memory suggest the importance of oligosaccharides attached to glycoconjugates in the modulation of synaptic connections. We studied the hippocampal glycan distribution of rats subject to an inhibitory avoidance task. The expression of glycans was examined by lectin‐histochemistry using Vicia villosa lectin (VVL) for terminal α/β N‐acetylgalactosamine (α/β GalNAc); Galanthus nivalus lectin (GNL) for terminal mannose α‐1,3 (Man α‐1,3); Peanut agglutinin (PNA) for galactose β‐1,3N‐acetylgalactosamine (Gal β‐1,3 GalNAc); Erythrina cristagalli lectin (ECL) for galactose β‐1,4 N‐acetylglucosamine (Gal β‐1,4 GlcNAc); Sambucus nigra lectin (SNA) for sialic acid α‐2.6 galactose (SA α‐2,6 Gal); Maackia amurensis lectin II (MAL II) for sialic acid α‐2,3 (SA α‐2,3); Wheat germ agglutinin (WGA) for terminal N‐acetylglucosamine with/without sialic acid (GlcNAc wo SA); succynilated WGA (sWGA) for terminal N‐acetylglucosamine without sialic acid (terminal GlcNAc without SA); Griffonia simplicifolia lectin II (GSL II) for terminal α/β N‐acetylglucosamine (α/β GlcNAc terminal); and Lotus tetragonolobus lectin (LTL) α–fucose. Two groups of 10 animals were examined: non‐trained (Control) and Trained rats. ECL, sWGA and GSL II were negative for both groups in all the hippocampal subfields studied. For both groups, VVL was negative in CA4 and granular cells of the Dentate Gyrus (DG) and LTL was negative in the CA4 subfield. Expression of α/β GalNAc, α ‐fucose and GlcNAc in other hippocampal subfields was positive, with no differences between groups. However, expression of Man α‐1,3 was significantly higher in the CA1, CA2, CA3, and CA4 subfields in the Trained group. On the other hand, expression of Gal β‐1,3 GalNAc was significantly low in CA4 and DG in the Trained group. In conclusion, the results here presented indicate that the exposure of rats to an associative behavioral paradigm related to declarative memory, involves some regulatory mechanism/s for the differential patterns of glycan expression.

CULTURE AND CHARACTERIZATION OF HUMAN HEPATOCYTES OBTAINED AFTER GRAFT REDUCTION FOR LIVER TRANSPLANTATION: A RELIABLE SOURCE OF CELLS FOR A BIOARTIFICIAL LIVER

This article describes results obtained when human liver cells obtained from reduced grafts are cultured in a chemically defined medium. Remnants of livers after reduction for pediatric transplantation were processed by a multiple cannulation system through the existing vasculature, which allowed the homogeneous perfusion of collagenase. The graft weight ranged between 55 and 1000 g (median value: 145.6 g). The yield ranged between 0.13 x 10(6) and 38 x 10(6) cells/g of tissue (median value 14.73 x 10(6) cells/g), and the viability was 61.17 +/- 27.43%. The total number of cells ranged between 57.6 x 10(6) and 12 150 x 10(6) cells (median value: 740 x 10(6) cells). Cells were cultured for 30 days. Albumin synthesis was observed during the first 2 weeks, with a peak value at day 6 (27.85 +/- 1.77 micro g/mL). Urea production was detected during the first week (peak value at day 6: 17.12 +/- 2.11 mg/dL). Light microscopy showed the presence of cells in a monolayer. Biliary pigments were observed at day 20. By immunohistochemistry, positive cells for albumin, for hepatocyte marker, cytokeratin 19, CD 34, CD 68, and for alpha actin for smooth muscle, were observed. Our results showed that hepatocytes obtained from reduced liver grafts are easily cultured and are able to maintain viability and functionality in vitro. This alternative source of human cells maintained under controlled culture conditions may play an important role in the development of a bioartificial liver.