Linda J. Van Eldik

Secretion of S-100 from rat C6 glioma cells

Conditioned media from rat C6 glioma cells contain significant levels of S-100 immunoreactivity as determined by radioimmunoassay. The levels of extracellular S-100 detected could not be accounted for by the release of intracellular S-100 into the media from lysed cells. The extracellular form of S-100 exhibits fractionation properties and immunological characteristics that are different from those of the intracellular form of S-100 in C6 cells. While the intracellular S-100 levels increase as a function of days in culture, the extracellular S-100 levels are high until the cells reach confluency and are lower in postconfluent cultures. Altogether, our data suggest that C6 glioma cells secrete S-100, and that the quantitative levels of the intracellular and secreted forms of S-100 are differentially regulated.

Calcium-Binding Proteins and the Molecular Basis of Calcium Action

Publisher Summary This chapter discusses the ways in which calcium is bound to a protein as well as the role of calcium in protein function. Proteins that contain γ-carboxyglutamic acid (Gla) are discussed as an example of proteins that bind calcium mainly by bidentate chelation. Prothrombin is the prototypical Gla-containing plasma protein, especially with regard to structural characteristics and calcium- and phospholipid-binding properties. Lectins are proteins that have no known enzymatic activity, but exhibit numerous biological activities that are related to their ability to bind carbohydrates in the presence of divalent cations. The proposed roles for calcium and the postulated calcium-binding sites are based on homology with concanavalin A (Con A). A variety of enzymes that catalyze the hydrolysis of ester, phosphodiester, and peptide bonds bind calcium ions. The calcium-binding hydrolytic enzymes include many hydrolytic enzymes, but mechanistic and crystallographic data are available only for phospholipase A 2 , staphylococcal nuclease, thermolysin, trypsin, and chymotrypsin. The vitamin D-dependent calcium-binding protein structure indicates that intracellular calcium-modulated proteins may utilize other structures for calcium-binding.

Disulfide-linked S100β dimers and signal transduction

Abstract S100β is a calcium-binding protein with neurotrophic and mitogenic activities, both of which may be mediated by the proteins ability to stimulate an increase in intracellular free calcium ([Ca 2+ ]). These extracellular trophic activities of S100β require a disulfide-linked, dimeric form of the protein. In this chapter, we present a minireview on the current state of knowledge concerning extracellular functions of S100β, with emphasis on the potential relevance of these activities to neuropathological disorders. We also report a simplified procedure for preparation of pharmacological amounts of biologically active S100β dimers, based on the finding that formation of disulfide-linked S100β dimers can be stimulated by the presence of calcium or lipid.

Neurotrophic activity of S-100β in cultures of dorsal root ganglia from embryonic chick and fetal rat

We report here that S-100 beta, a protein with neurotrophic activity on central nervous system neurons, stimulates neuritic outgrowth from cultures of dorsal root ganglia (DRG). S-100 beta elicited neurites from explant and dissociated cell cultures of embryonic chick DRG, and the extent of the response varied with the age of the embryo. Specificity was demonstrated by the observation that incubation of S-100 beta with antibodies directed against S-100 beta reduced the neurite outgrowth, whereas incubation of S-100 beta with normal rabbit serum had little effect. S-100 beta also stimulated the area of neuritic outgrowth from organotypic cultures of fetal rat DRG, showing that the activity of the protein is not restricted to a particular species or culture condition. A mutant S-100 beta lacking neurotrophic activity on cerebral cortex neurons was unable to effectively stimulate neurite outgrowth from DRG cultures. These studies suggest that S-100 beta may play a role in neuronal growth and/or maintenance in the peripheral nervous system.

Conditions for reproducible detection of calmodulin and S100β in immunoblots

Abstract The conditions used in some immunoblot procedures can fail to detect calmodulin, S100 proteins, and other proteins with similar physical properties. We describe here some of the basis of this difficulty, and provide an immunoblot protocol that allows the rapid and reproducible detection of calmodulin and S100β in crude biological samples. These proteins are rapidly transferred from sodium dodecyl sulfate-polyacrylamide gels to membrane matrices, and retention on the matrix is enhanced by a glutaraldehyde fixation step. Either nitrocellulose or a positively charged membrane filter (ZetaProbe) can be used as the immobilizing matrix. By combining microslab gel electrophoresis, 30 min electrophoretic transfer, and glutaraldehyde fixation of nitrocellulose paper, an immunoblot analysis can be done in an 8-hr day.

Immunohistochemical localization of the S-100β protein in postnatal cat visual cortex: spatial and temporal patterns of expression in cortical and subcortical glia

The ontogenic expression of the glial-specific protein S100 beta was examined in postnatal cat visual cortex using immunocytochemical methods. Astrocytes in visual cortex and oligodendrocytes in the subcortical white matter exhibited distinct spatio-temporal gradients in their expression of the S100 beta protein. In the visual cortex, S100 beta-immunoreactivity was detected in astroglial cytoplasm, as well as in the extracellular interstitium, in a lamina-specific manner throughout postnatal development. Using double labeling procedures, the S100 beta protein was found to be strictly colocalized with GFAP-immunoreactive astrocytes when GFAP was present. The glial fibrillary acidic protein (GFAP), a marker of mature astrocytes, was not present at high levels until the 4th postnatal week. From the 2nd through 5th postnatal weeks, the expression of S100 beta was highest in the thalamocortical recipient, layer IV, of visual cortical areas 17 and 18. At ages beyond 6 postnatal weeks, S100 beta-immunoreactivity increased disproportionately in supra- and infragranular layers such that areas 17 and 18 were demarcated from adjacent cortices by lower levels in layer IV. The S100 beta protein was also highly expressed in oligodendroglial somata and processes in the subcortical white matter between the 2nd and 6th postnatal weeks. The levels of S100 beta in the subcortical white matter progressively diminished to adult levels, where it was localized only to a few remaining oligodendroglial somata. The differential laminar expression of the S100 beta protein in astrocytes during the period within which the visual cortex exhibits input- and experience-dependent synaptic modifications suggests that astrocytes, possibly via their release of S100 beta, may play a special role in mediating plasticity in visual cortical development. A consistent feature of the appearance of the S100 beta protein was its expression in immature astroglia and oligodendroglia, well before they are considered morphologically mature. This characteristic underscores the potential of S100 beta as a marker of distinct populations of glial cells and of their role in normal and abnormal development.

Interaction of calmodulin and other calcium-modulated proteins with mammalian and arthropod junctional membrane proteins

Calmodulin and other calcium-modulated proteins bind in vitro to purified junctional polypeptides from rat liver gap junctions, bovine lens fiber junctions, a chymotryptic fragment from bovine lens junctions, and crayfish hepatopancreas gap junctions. The potential biological relevance of the interaction of calmodulin with junctional proteins is suggested by immunocytochemical localization of endogenous calmodulin in cortical regions of the cell where gap junctions exist. These observations provide a molecular basis for understanding the potential regulatory role of calmodulin on cell-cell communication channels in vivo. In addition, the calmodulin binding represents the first molecular homology that has been found for junctional channel proteins from mammalian and arthropod tissues.

Inhibition of protein kinase C- and casein kinase II-mediated phosphorylation of GAP-43 by S100β

Abstract The effect of the glial-derived protein, S100β, on the in vitro phosphorylation of the growth-associated protein GAP-43 was investigated. S100β inhibited in a dose dependent manner the phosphorylation of GAP-43 by protein kinase C (PKC) or by casein kinase II (CKII). S100β appeared to slow down the rate and the degree to which GAP-43 can be phosphorylated by either kinase. The specificity of the inhibition was demonstrated by the observation that the phosphorylation of two other CKII substrates, casein and a selective peptide substrate, was not inhibited by S100β. The marked inhibitory effect of S100β required the presence of calcium in the phosphorylation reactions. In addition, S100β inhibition of GAP-43 phosphorylation was seen with GAP-43 purified under a variety of conditions that alter acylation, suggesting that the acylation state of GAP-43 does not affect the ability of S100β to modulate CKII- or PKC-mediated phosphoryation of GAP-43.

Chapter 39: S100β and serotonin: a possible astrocytic-neuronal link to neuropathology of Alzheimer's disease

Publisher Summary The chapter discusses a possible role for Sl00β and serotonin (5-HT) as an astrocytic (S100β)/ neuronal (serotonin; 5-HT) link to neuropathologies associated with Alzheimers disease. Sl00β is a protein produced and released from astrocytes in the brain. Extracellular Sl00β can function as a neurotrophic factor to select central and peripheral neurons and a gliotrophic factor, inducing mitosis and morphogenesis of astrocytes. Release of S100β from astrocytes can be stimulated by serotonin acting through 5-HT1, receptors on astrocytes, and S100β functions as a serotonergic neurite extension factor. The abnormal regulation of Sl00β levels or of serotonin-stimulated release of Sl00β in Alzheimers disease contributes to the cascade of events that with aging lead to neuropathologies. It is also suggested that this astrocytic (S100β)/ neuronal (serotonin) link may contribute to neuropathological changes observed not only in Alzheimers disease, but also in Down syndrome.

[36] Interaction of calmodulin and other calcium-modulated proteins with gap junctions

Publisher Summary This chapter focuses on the interaction of calmodulin with gap junctions. Gap junctions are plasma membrane specializations formed between contacting cells consisting of an array of subunits, connexons; and containing an aqueous channel between the cells, which is insulated from the extracellular space. Hence, cells containing gap junctions are able to communicate directly with each other by the intercellular diffusion of hydrophilic molecules limited in size by the diameter of the channel. The interaction of calmodulin and other calcium-modulated proteins with purified gap junction proteins from rat liver and bovine lens is examined by the gel overlay procedure. The gap junction polypeptides bind to other calcium-modulated proteins, including troponin C, S-100β, and parvalbumin. The binding of iodinated troponin C and S-100β shows no calcium dependence, whereas the binding of iodinated parvalbumin is enhanced in the presence of chelator. The observed interaction of the junction proteins with these calcium-modulated proteins is consistent with data in other systems where it has been found that these proteins can sometimes substitute for calmodulin in modulating activity of proteins.