Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na⁺ permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na⁺. Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4''-Diisothiocyano-2,2''-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade.     

Nuclear Calmodulin-Binding Proteins in Rat Neurons

Abstract: By using a ¹²⁵I-calmodulin overlay assay, three major high-affinity calmodulin-binding proteins, showing apparent molecular masses of 135, 60, and 50 kDa, have been detected in purified nuclear fractions isolated from rat neurons. It has been shown that after extraction of the nuclei with nucleases and high salt, all these proteins remain strongly associated with the nuclear matrix. The 60- and 50-kDa proteins have been previously identified as subunits of the calmodulin-dependent protein kinase II. We report here the immunoblot identification of the 135-kDa cal modulin-binding protein as myosin light chain kinase. We also show that the calmodulin-dependent protein phosphatase calcineurin is present in the neuronal nuclei and associated with the nuclear matrix. The nuclear localization of both calcineurin and myosin light chain kinase has been confirmed by immunocytochemical studies.     

Innate Immunity Promotes Sleep through Epidermal Antimicrobial Peptides

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Interleukin-1 alpha and beta induce interleukin-1 beta gene expression in human dermal fibroblasts

The ability of the two forms of interleukin-1, IL-1 alpha and IL-1 beta, to induce IL-1 beta gene expression in human skin fibroblasts was studied in vitro, using Northern blot hybridization. Both recombinant IL-1 alpha and IL-1 beta caused a dramatic increase in IL-1 beta mRNA levels, IL-1 alpha being more efficient than IL-1 beta. Blockage of the prostaglandin synthesis by indomethacin reduced the basal level of IL-1 beta mRNA in control cultures and decreased also the stimulatory effect exerted by both IL-1s on IL-1 beta gene expression. These data suggest that IL-1 and prostaglandin (mainly PGE2) may act synergistically to stimulate IL-1 gene expression in dermal fibroblasts, contributing as a local amplifier system to the alterations of connective tissue in inflammatory processes.     

Subcellular Distribution of Tyrosine Hydroxylase in Some Catecholaminergic Rat Brain Areas Determined by a Quantitative Immunoblot Assay

The subcellular distribution of the protein tyrosine hydroxylase (TH) after fractionation of rat brain tissue was studied by a sensitive technique of immunoblot quantification in the dopaminergic nigrostriatal and the dorsal noradrenergic pathways and in the ventrolateral medulla. This repartition indicates that in all catecholaminergic regions of the cell bodies studied, the contribution of the nerve endings to the total TH amount is very low (less than 7%), in contrast to that observed in the terminal fields. The correlative subcellular determination of the TH amount and activity in the same tissue could be a useful approach for studying experimentally induced mechanisms of catecholamine synthesis modulation in different brain catecholaminergic pathways.     

Release of neuron specific enolase (NSE) in cerebrospinal fluid following experimental lesions of the rat brain

Neuron-specific enolase (NSE) levels were measured by sandwich enzymo-immunoassay as well as by enzymatic assay in rat cerebrospinal fluid (CSF), following mechanical lesions of the brain tissue. Significant increases of NSE were observed in CSF, with a peak 2 h following lesions located near the lateral ventricle. Values returned to normal around 48 h later. In another experimental group, lesions were realized further away from the lateral ventricle; the elevation of NSE in CSF reached the maximal value 11 h later. In addition, measurements which were performed following lesions at the same location but of various sizes, indicated that the quantity of NSE released is proportional to the extent of brain damage. The possible factors which govern the time course and amount of NSE release in CSF are discussed. These results suggest that NSE could be a useful and easily detected marker of neuronal damage.