The Ca 2؉ channel ␣1A-subunit is a voltage-gated, pore-forming membrane protein positioned at the intersection of two important lines of research: one exploring the diversity of Ca 2؉ channels and their physiological roles, and the other pursuing mechanisms of ataxia, dystonia, epilepsy, and migraine. ␣1A-Subunits are thought to support both P-and Q-type Ca 2؉ channel currents, but the most direct test, a null mutant, has not been described, nor is it known which changes in neurotransmission might arise from elimination of the predominant Ca 2؉ delivery system at excitatory nerve terminals. We generated ␣1A-deficient mice (␣1A ؊͞؊ ) and found that they developed a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying Ϸ3-4 weeks after birth. P-type currents in Purkinje neurons and P-and Q-type currents in cerebellar granule cells were eliminated completely whereas other Ca 2؉ channel types, including those involved in triggering transmitter release, also underwent concomitant changes in density. Synaptic transmission in ␣1A ؊͞؊ hippocampal slices persisted despite the lack of P͞Q-type channels but showed enhanced reliance on N-type and R-type Ca 2؉ entry. The ␣1A ؊͞؊ mice provide a starting point for unraveling neuropathological mechanisms of human diseases generated by mutations in ␣1A. The ␣ 1A -subunit, the most abundant ␣ 1 -subunit in vertebrate brain (1), mediates Ca 2ϩ influx across presynaptic and somatodendritic membranes, thereby triggering fast neurotransmitter release and other key neuronal responses (2-5). Because of its high expression levels in the brain, the ␣ 1A -subunit was the first representative of its subclass to be isolated by cDNA cloning (1, 6). This predominantly neuronal subclass also includes ␣ 1B (N-type Ca 2ϩ channel) and ␣ 1E [possibly R-type Ca 2ϩ channel (7-9)] and is referred to as ABE or Ca V 2. There is no information to date on the behavioral or electrophysiological consequences of deleting a member of the ABE subfamily.␣ 1A Transcripts are widely distributed in rat (10) and human brain (11), most prominently in cell body layers in cerebellum and hippocampus. At the subcellular level, ␣ 1A immunoreactivity has been found in cell bodies, dendrites, and presynaptic terminals (12). Less clear has been the role of ␣ 1A in supporting Ca 2ϩ channel components defined by biophysical and pharmacological criteria. In either Xenopus oocytes (13, 14) or HEK293 cells (15), expression of ␣ 1A -subunits along with ancillary ␣ 2 ͞␦-and ␤-subunits generated currents with properties closely resembling the Q-type current found in cerebellar granule cells (8) and much less the P-type current first described in cerebellar Purkinje neurons by Llinás and colleagues (16,17). Unlike native P-type channels (18), the expressed currents showed pronounced inactivation during sustained depolarizations and responded to -agatoxin IVA (-Aga-IVA) at half-blocking doses of Ϸ100 nM, not Ϸ1 nM (13). Various explanations for the discrepancies have been advanced...

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental pollutant with many toxic effects, including endocrine disruption, reproductive dysfunction, immunotoxicity, liver damage, and cancer. These are mediated by TCDD binding to and activating the aryl hydrocarbon receptor (AhR), a basic helix-loop-helix transcription factor. In this regard, targeting the AhR using novel small molecule inhibitors is an attractive strategy for the development of potential preventive agents. In this study, by screening a chemical library composed of approximately 10,000 compounds, we identified a novel compound, 2-methyl-

Human adipose tissue-derived mesenchymal stem cells (hASCs) are useful for regeneration of inflamed or injured tissues. To identify secreted hASC proteins during inflammation, hASCs were exposed to tumor necrosis factor-alpha (TNF-alpha) and conditioned media derived from hASCs were analyzed by liquid chromatography coupled with tandem mass spectrometry. We identified 187 individual proteins as secreted proteins (secretome) in hASC-conditioned media; 118 proteins were secreted at higher levels upon TNF-alpha treatment. The TNF-alpha-induced secretome included a variety of cytokines and chemokines such as interleukin-6 (IL-6), IL-8, chemokine (C-X-C motif) ligand 6, and monocyte chemotactic protein-1 (MCP-1). TNF-alpha also increased expression of various proteases including cathepsin L, matrix metalloproteases and protease inhibitors, and induced secretion of long pentraxin 3, a key inflammatory mediator implicated in innate immunity. TNF-alpha-conditioned media stimulated migration of human monocytes, which play a key role in inflammatory responses. This migration was abrogated by pretreatment with neutralizing anti-IL-6, anti-IL-8, and anti-MCP-1 antibodies, suggesting that IL-6, IL-8, and MCP-1 are involved in migration of monocytes. Taken together, these results suggest that TNF-alpha-induced secretome may play a pivotal role in inflammatory responses and that shotgun proteomic analysis will be useful for elucidation of the paracrine functions of mesenchymal stem cells.