No abstract

Because of its anatomical organization, the rodent whisker-to-barrel system is an ideal model to study experience-dependent plasticity. Manipulation of sensory input causes changes in the properties of the barrels at the physiological, structural, and functional levels. However, much less is known about the molecular events underlying these changes. To explore such molecular events, we have used a genomewide approach to identify key genes and molecular pathways involved in experience-induced plasticity in the barrel cortex of adult rats. Given the natural tendency of rats to explore novel objects, exposure to an enriched environment (EE) was used to stimulate the activity of the whisker-to-barrel cortex in vivo. Microarray analysis at two different time points after EE revealed differential expression of genes encoding transcription factors, including nuclear receptors, as well as of genes involved in the regulation of synaptic plasticity, cell differentiation, metabolism, and, surprisingly, blood vessel morphogenesis. These expression differences reflect changes in somatosensory information processing because unilateral whisker clipping showed EE-induced differential expression patterns in the spared versus deprived barrel cortex. Finally, in situ hybridization revealed cortical layer patterns specific for each selected gene. Together, the present study offers the first genomewide exploration of the key genes regulated by somatosensory stimulation in the barrel cortex and thus provides a solid experimental framework for future in-depth analysis of the mechanisms underlying experience-dependent plasticity.

Recombinant human ceramide kinase (HsCERK) was analyzed with regard to dependence on divalent cations and to substrate delivery, spectrum, specificity, and stereoselectivity. Depending on the chain length of the ceramide, either albumin for short-chain ceramide or a mixed micellar form (octylglucoside/cardiolipin) for long-chain ceramide was preferred for the substrate delivery, the former resulting in higher activities. Bacterially expressed HsCERK was highly dependent on Mg 21 ions, much less on Ca 21 ions. A clear preference for the D-erythro isomer was seen. Various N-acylated amino alcohols were no substrate, but Nhexanoyl-1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol and Ntetradecanoyl-2S-amino-1-butanol were phosphorylated, suggesting that the secondary hydroxy group is not required for recognition. The properties of HsCERK, expressed in CHO cells, were similar to those of the bacterially expressed protein, including the Mg 21 dependence. In mouse, the highest activities were found in testis and cerebellum, and upon subcellular fractionation the activity was recovered mainly in the microsomal fraction. This fits with the plasma membrane localization in CHO cells, which was mediated by the N-terminal putative pleckstrin domain.No evidence for phosphorylation of ceramide by the recently described multiple lipid kinase was found. The latter kinase is localized in the mitochondria, but no firm conclusions with regard to its substrate could be drawn.

No abstract

Calcium mobilization induced by phosphorylated sphingoid bases was analyzed in calf pulmonary artery endothelial cells by confocal microscopy. A sphingenine-1-phosphate (SeP) analogue, N-acetyl-sphingenine-1-phosphate (N-C 2 -SeP), exogenously added to these cells, caused a fast and transient intracellular rise in calcium and was as potent as SeP. A minimal concentration of 0.6 nM for N-C 2 -SeP versus 1 nM for SeP was determined. The N-C 2 -SeP-induced Ca 2+ -signaling, like the response to SeP, was due to a release from thapsigargin-sensitive, ryanodine-insensitive, intracellular Ca 2+ -stores and not to a Ca 2+ -influx. N-C 2 -SeP can be considered as a truncated ceramide-phosphate, a lipid already reported to be mitogenic (Gomez-Munoz, A., Duffy, P.A., Martin, A., O'Brien, L., Byun, H.S., Bittman, R. and Brindley, D.N. (1995) Mol. Pharmacol. 47, 833^839), an effect that might be secondary to Ca 2+ -mobilization. z 1999 Federation of European Biochemical Societies.