Search

We found that sparse and confluent C6 glioma cells differ both in GM3 content, which increases with cell density, and in endothelin-1 (ET-1)-induced phosphoinositide hydrolysis, which was markedly higher in the sparse cells than in the confluent. Also after manipulation of the cellular GM3 content through treatment with exogenous GM3 or with drugs known to affect GM3 metabolism, the ET-1 effect was inversely related to GM3 cellular levels. Cell treatment with an anti-GM3 mAb resulted in the enhancement of ET-1-induced phospholipase C activation and restored the capacity of GM3-treated cells to respond to ET-1. These findings suggest that the GM3 ganglioside represents a physiological modulator of ET-1 signaling in glial cells.

The possible relation between nitric oxide synthase (NOS) activity and neural differentiation was investigated using primary cultures of rat cerebellar granule cells differentiating in culture. NOS activity was measured in the cytosolic and particulate fractions obtained from cell homogenate. In the experimental conditions used the optimal pH for NOS activity was about 6.4, the activity being about 3-fold higher than at pH 7.4. Cerebellar granule cell differentiation was associated with marked increases in NOS activity. In undifferentiated cells the enzyme was almost evenly distributed between the cytosolic and particulate fractions, during differentiation there was a 12-fold increase in activity in the cytosolic enzyme and a 3-fold increase in the particulate one. This indicates a marked preferential enrichment of the cytosolic enzyme during differentiation. Cerebellar granule cells produced and released NO in the culture medium; NO formation being markedly higher in differentiated cells (7–12 DIC) than in undifferentiated (2–3 DIC) ones. These data demonstrate a relationship between NOS expression and NO production and the differentiation of cerebellar granule cells, supporting the notion that NO may play a role in this process.

Mammalian neurodevelopment is characterized by qualitative and quantitative changes in plasma membrane glycosphingolipids due to a fine regulation of their metabolic pathways. While the biosynthetic pathway is largely studied scant is the information available on the catabolic one. For this reason, I studied the activity of the main glycohydrolases expressed in the central nervous system during neuronal differentiation. To this purpose the activities of the principal glycohydrolases involved in glycosphingolipid catabolism have been evaluated in different experimental models such as brains and cerebella of mouse at different ages and neuronal cell cultures (immortalized mouse neuronal cell lines GN11 and GT1-7, primary cultures of mouse cerebellar granule cells and human neuroblastoma cells SH-SY5Y). The results obtained indicate that the process of neuronal differentiation is associated with a marked increase in the activities of all the glycohydrolases evaluated, in particular, the activity of the non-lysosomal β-glucosylceramidase GBA2 undergoes the most relevant increase representing the prevalent form of β-glucocerebrosidase in mature neurons. In order to evaluate the possible role of GBA2 in the neuronal differentiation, SH-SY5Y cells have been stably trasfected for GBA2 overexpression. Cells overexpressing GBA2 acquired a neuronal phenotype and showed a significant increase in ceramide levels. These results are in line with literature data that demonstrate the involvement of ceramide in the neuronal differentiation. Therefore, it is possible to hypothesize that the hydrolysis of glucosylceramide to ceramide, catalyzed by GBA2 at the plasma membrane level, has a functional role in the neuronal differentiation process. Collectively these findings suggest that GBA2 may represent a possible neuronal marker and demonstrate for the first time its direct involvement in the neuronal differentiation.

DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular patterns that increase activation of the innate immune system. Importantly, viral-vectored vaccines that act through the induction of one or more of these factors also may benefit from cytokine coadministration and increased antigen presentation. In order to increase immunogenicity to the level achieved with viral-vectored vaccines, various synergistic components may need to be incorporated into DNA vaccines. From the perspective of the future clinical use of DNA vaccines, it has been suggested that antigen presentation should be improved and cytokine coadministration attempted. However, even with these modifications, it is likely that the primary use of DNA vaccines may be as primers for viral-vectored vaccines, rather than as single agents. This review discusses the approaches used to enhance DNA vaccine immunogenicity, with a primary focus on fusion strategies that enhance antigen presentation.