Alessia Gallo

Synergistic effects of neurons and astrocytes on the differentiation of brain capillary endothelial cells in culture

Brain capillary endothelial cells form a functional barrier between blood and brain, based on the existence of tight junctions that limit paracellular permeability. Occludin is one of the major transmembrane proteins of tight junctions and its peripheral localization gives indication of tight junction formation. We previously reported that RBE4.B cells (brain capillary endothelial cells), cultured on collagen IV, synthesize occludin and correctly localize it at the cell periphery only when cocultured with neurons. In the present study, we describe a three‐cell type‐culture system that allowed us to analyze the combined effects of neurons and astrocytes on differentiation of brain capillary endothelial cells in culture. In particular, we found that, in the presence of astrocytes, the neuron‐induced synthesis and localization of occludin is precocious as compared to cells cocultured with neurons only.

Permeability properties of a three-cell type in vitro model of blood-brain barrier

We previously found that RBE4.B brain capillary endothelial cells (BCECs) form a layer with blood‐brain barrier (BBB) properties if co‐cultured with neurons for at least one week. As astrocytes are known to modulate BBB functions, we further set a culture system that included RBE4.B BCECs, neurons and astrocytes. In order to test formation of BBB, we measured the amount of 3H‐sucrose able to cross the BCEC layer in this three‐cell type model of BBB. Herein we report that both neurons and astrocytes induce a decrease in the permeability of the BCEC layer to sucrose. These effects are synergic as if BCECs are cultured with both neurons and astrocytes for 5 days, permeability to sucrose decreases even more. By Western analysis, we also found that, in addition to the canonical 60 kDa occludin, anti‐occludin antibodies recognize a smaller protein of 48 kDa which accumulates during rat brain development. Interestingly this latter protein is present at higher amounts in endothelial cells cultured in the presence of both astrocytes and neurons, that is in those conditions in which sucrose permeation studies indicate formation of BBB.

Screening of subtelomeric rearrangements in autistic disorder: Identification of a partial trisomy of 13q34 in a patient bearing a 13q;21p translocation

Within the framework of a FISH screening protocol to detect cryptic subtelomeric rearrangements in autistic disorder (AD), a patient bearing three copies of the subtelomeric portion of the q arm of chromosome 13 has been identified. Beside AD, the patient also has severe mental retardation and displays several dysmorphic features. Further FISH analyses revealed that the trisomy was caused by the translocation of a 13q subtelomeric fragment to the acrocentric tip of one chromosome 21 [46,XY.ish der(21) t(13;21) (q34;p13)(D13S1825+)]. Gene dosage experiments carried out with three multiallelic polymorphisms of the subtelomeric region of chromosome 13q showed that the putative length of the triplicate region does not exceed 300 kb about, that is, the distance from telomere to the first normally inherited marker. In addition, gene dosage analysis performed on the derivative chromosome 21, did not reveal loss of the most telomeric protein‐encoding genes on 21p. The potential relationship between a postulated increased expression of genes on 13q34 and the complex phenotype in this trisomic patient is discussed.

Analysis of the gastrin-releasing peptide receptor gene in Italian patients with autism spectrum disorders†

The gastrin‐releasing peptide receptor (GRPR) was implicated for the first time in the pathogenesis of Autism spectrum disorders (ASD) by Ishikawa‐Brush et al. [Ishikawa‐Brush et al. (1997): Hum Mol Genet 6: 1241–1250]. Since this original observation, only one association study [Marui et al. (2004): Brain Dev 26: 5–7] has further investigated, though unsuccessfully, the involvement of the GRPR gene in ASD. With the aim of contributing further information to this topic we have sequenced the entire coding region and the intron/exon junctions of the GRPR gene in 149 Italian autistic patients. The results of this study led to the identification of four novel point mutations, two of which, that is, C6S and L181F, involve amino acid changes identified in two patients with ASD and Rett syndrome, respectively. Both the leucine at position 181 and the cysteine at position 6 are strongly conserved in vertebrates. C6S and L181F mutant proteins were expressed in COS‐7 and BALB/3T3 cells, but they did not affect either GRPs binding affinity or its potency for stimulating phospholipase C‐mediated production of inositol 1,4,5‐trisphosphate. In summary, our results do not provide support for a major role of the GRPR gene in ASD in the population of patients we have studied. However, there is a potential role of C6S and L181F mutations on GRPR function, and possibly in the pathogenesis of the autistic disorders in the two patients.

Yeast vectors for the integration/expression of any sequence at the TYR1 locus

We have constructed new yeast vectors for targeted integration and conditional expression of any sequence at the Saccharomyces cerevisiae TYR1 locus which becomes disrupted. We show that vector integration is not neutral, causing prototrophy for tyrosine and auxotrophy for the vectors selectable marker (uracil or leucine, depending on the vector used). This feature allows a double screening of transformed yeast cells, improving the identification of colonies with the desired chromosomal structure. The GAL10 gene promoter has been added to drive conditional expression of cloned sequences. Using these vectors, chromosomal structure verification of recombinant clones is no longer necessary, since the noise of non‐homologous recombination, as well as spontaneous reversion of the selected phenotype, can easily be identified. The ability of the vector to conditionally control gene expression has been confirmed using the gene for the green fluorescent protein (GFP) as a reporter. GenBank Accession Nos EF202083–202086. Copyright

Ras-pathway has a dual role in yeast galactose metabolism

In the yeast Saccharomyces cerevisiae the genes involved in galactose metabolism (GAL1,7,10) are transcriptionally activated more than a 1000‐fold in the presence of galactose as the sole carbon source in the culture media. In the present work, we monitored the activity of the GAL10 gene promoter in different Ras‐cAMP genetic backgrounds. We demonstrate that overexpression of C‐terminus of the nucleotide exchange factor Cdc25p stimulates GAL10 transcription in yeast strains carrying the contemporary deletion of both RAS genes. Moreover, the deletion of the chromosomal CDC25 gene provokes impaired growth on galactose based media in yeast strain lacking both RAS genes and adenylate cyclase (whose viability is assured by the presence of the Bcy1‐11 allele). Surprisingly, reconstitution of the Ras‐pathway inhibits GAL10‐promoter activation. Activation of GAL10 gene promoter is indeed possible in the presence of Ras protein but only in strains with chromosomal deletion of adenylate cyclase. These results indicate a dual role of Ras‐pathway on galactose metabolism and suggest that Cdc25p has a Ras‐independent role in cellular metabolism.