Primary astrocyte cultures prepared from neonatal hippocampus, cerebral cortex and cerebellum were morphologically distinct. Cells from hippocampus and cortex were almost entirely protoplasmic, whereas cerebellar astrocytes had many processes; in the absence of serum these differences were accentuated. We compared the immunocontent and secretion of the mitogenic protein S100B in these cultures. Immunocontent was 2.5 times higher in cerebellar astrocytes than in hippocampal or cortical astrocytes. Cells from all three regions secreted S100B under basal conditions, but the secretion rate was higher in cerebellar astrocytes. Secretion depended on protein synthesis and was increased by incubation with forskolin or lysophosphatidic acid in mechanisms which were additive. The stellate morphology induced by forskolin was reversed by lysophosphatidic acid in hippocampal but not in cerebellar cultures, suggesting that S100B secretion was not associated with a process-bearing phenotype of astrocytes. ß

S100B is a calcium binding protein expressed and secreted by astrocytes. Extracellular S100B stimulates the proliferation of astroglial cells and the survival of neurons. Extracellular signal regulated kinases (ERK) are involved in the transduction of proliferating signals in astrocytes. Here we report that S100B significantly increases the activity of ERK in primary cultures of astrocytes, a result which may be related to previous observations of the effect of this protein on glial proliferation. We further confirm that conversion of S100B to its covalent dimer by oxidation of cysteine residues increases its extracellular activity. Although we cannot exclude S100B involvement in other mechanisms of signal transduction, these results suggest that ERK activity in astrocytes is modulated by extracellular S100B.

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Several molecules have been shown to be involved in glial-neuronal communication, including S100B, an astrocyte-derived neurotrophic cytokine. Extracellular S100B protects hippocampal neurons from excitotoxic damage, whilst toxic levels of glutamate to neurons have been shown to reduce S100B secretion in astrocytes and brain slices, by an unknown mechanism. Here, we investigate which mechanisms are possibly involved in this effect in primary cultures of hippocampal astrocytes using glutamate agonists and glutamate uptake inhibitors. DCG-IV, an agonist of group II metabotropic glutamate receptors, caused a smaller decrease in S100B secretion when compared to 1 mM glutamate. D: -aspartate partially reverted the glutamate effect on S100B release and two other inhibitors, PDC and DIDS, reverted it completely. These findings suggest that S100B secretion is inversely coupled to glutamate uptake. Decrease in S100B secretion may be considered as direct excitotoxic damage, but a beneficial mechanism effect cannot be ruled out, because S100B elevation could cause an additional cell death.

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ATP, acting via P2Y, G protein-coupled receptors (GPCRs), is a mitogenic signal and also synergistically enhances ®broblast growth factor-2 (FGF-2)-induced proliferation in astrocytes. Here, we have examined the effects of ATP and FGF-2 cotreatment on the main components of the extracellularsignal regulated protein kinase (ERK) cascade, cRaf-1, MAPK/ERK kinase (MEK) and ERK, key regulators of cellular proliferation. Surprisingly, ATP inhibited activation of cRaf-1 by FGF-2 in primary cultures of rat cortical astrocytes. The inhibitory effect did not diminish MEK and ERK activation; indeed, cotreatment resulted in a greater initial activation of ERK. ATP inhibition of cRaf-1 activation was not mediated by an increase in cyclic AMP levels or by protein kinase C activation. ATP also inhibited the activation of cRaf-1 by other growth factors, epidermal growth factor and platelet-derived growth factor, as well as other MEK1 activators stimulated by FGF-2, MEK kinase 1 (MEKK1) and MEKK2. Serotonin, an agonist of another GPCR coupled to ERK, did not inhibit FGF-2-induced cRaf-1 activation, thereby indicating speci®city in the ATP-induced inhibitory cross-talk. These ®ndings suggest that ATP stimulates an inhibitory activity that lays upstream of MEK activators and inhibits growth factor-induced activation of cRaf-1 and MEKKs. Such a mechanism might serve to integrate the actions of receptor tyrosine kinases and P2Y-GPCRs. Signal transduction pathways have been generally treated as a linear sequence of events, from a receptor, through a second messenger, to an effector, resulting in a speci®c set of effects on the cell. With the discovery of new pathways and regulatory mechanisms, it became clear that these linear pathways actually were part of large and extremely complex systems, with signaling capabilities much beyond the simple linear transference of information from the plasma membrane to cellular components (Bhalla and Iyengar 1999). One of the most important characteristics of these complex signaling systems is the numerous points of cross-talk and feedback, providing them with multiple and dynamic features for signal integration.Mitogen-activated protein kinase (MAPK) cascades are examples of complex signaling systems. These cascades are comprised of at least three protein kinases. In the case of the extracellular signal regulated protein kinase (ERK) cascade, Raf phosphorylates MEK1/2 which in turn phosphorylates ERK1/2; these key enzymes are involved

Infection of wMel Wolbachia in Aedes aegypti imparts two signature features that enable its application for biocontrol of dengue. First, the susceptibility of mosquitoes to viruses such as dengue and Zika is reduced. Second, a reproductive manipulation is caused that enables wMel introgression into wild-type mosquito populations. The long-term success of this method relies, in part, on evolution of the wMel genome not compromising the critical features that make it an attractive biocontrol tool. This study compared the wMel Wolbachia genome at the time of initial releases and 1–7 years post-release in Cairns, Australia. Our results show the wMel genome remains highly conserved up to 7 years post-release in gene sequence, content, synteny and structure. This work suggests the wMel genome is stable in its new mosquito host and, therefore, provides reassurance on the potential for wMel to deliver long-term public-health impacts.