Search

Signal transduction depends critically on the spatial localization of protein constituents. A key question in odor transduction is whether chemotransduction proteins organize into discrete molecular complexes throughout olfactory cilia or distribute homogeneously along the ciliary membrane. Our recordings of Ca(2+) changes in individual cilia with unprecedented spatial and temporal resolution, by the use of two-photon microscopy, provide solid evidence for Ca(2+) microdomains (transducisomes). Dissociated frog olfactory neurons were preloaded with caged-cAMP and fluo-4 acetoxymethyl ester probe Ca(2+) indicator. Ca(2+) influx through cyclic nucleotide-gated (CNG) channels was evoked by uniformly photoreleasing cAMP, while ciliary Ca(2+) was measured. Discrete fluorescence events were clearly resolved. Events were missing in the absence of external Ca(2+) , consistent with the absence of internal Ca(2+) sources. Fluorescence events at individual microdomains resembled single-CNG channel fluctuations in shape, mean duration and kinetics, indicating that transducisomes typically contain one to three CNG channels. Inhibiting the Na(+) /Ca(2+) exchanger or the Ca(2+) -ATPase prolonged the decay of evoked intraciliary Ca(2+) transients, supporting the participation of both transporters in ciliary Ca(2+) clearance, and suggesting that both molecules localize close to the CNG channel. Chemosensory transducisomes provide a physical basis for the low amplification and for the linearity of odor responses at low odor concentrations., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Odour-mediated signal transduction is a complex process that occurs in the cilia of olfactory sensory neurons. To gain insight in to the molecular organization of the odour transduction machinery, we developed a procedure to purify olfactory cilia membranes by differential centrifugation of rat olfactory epithelium extracts. We tested whether known scaffolding proteins that might participate in the assembly of the complex chemotransduction apparatus are present in the purified membrane fraction. Utilizing immunoblotting and immunohistochemistry, we show that the multidomain scaffolding proteins ProSAP/Shanks and calcium/calmodulin-dependent serine protein kinase CASK are present in the olfactory cilia. Ion channels involved in chemotransduction could be reconstituted into planar lipid bilayers for electrophysiological recordings. Our procedure should allow the identification of further chemotransduction-related proteins.

Olfactory sensory neurons respond to odorants increasing Ca(2+) concentrations in their chemosensory cilia. Calcium enters the cilia through cAMP-gated channels, activating Ca(2+)-dependent chloride or potassium channels. Calcium also has a fundamental role in odour adaptation, regulating cAMP turnover rate and the affinity of the cyclic nucleotide-gated channels for cAMP. It has been shown that a Na(+)/Ca(2+) exchanger (NCX) extrudes Ca(2+) from the cilia. Here we confirm previous evidence that olfactory cilia also express plasma membrane Ca(2+)-ATPase (PMCA), and show the first evidence supporting a role in Ca(2+) removal. Both transporters were detected by immunoblot of purified olfactory cilia membranes. The pump was also revealed by immunocytochemistry and immunohistochemistry. Inside-out cilia membrane vesicles transported Ca(2+) in an ATP-dependent fashion. PMCA activity was potentiated by luminal Ca(2+) (K(0.5) = 670 nm) and enhanced by calmodulin (CaM; K(0.5) = 31 nm). Both carboxyeosin (CE) and calmidazolium reduced Ca(2+) transport, as expected for a CaM-modulated PMCA. The relaxation time constant (tau) of the Ca(2+)-dependent Cl(-) current (272 +/- 78 ms), indicative of luminal Ca(2+) decline, was increased by CE (2181 +/- 437 ms), by omitting ATP (666 +/- 49 ms) and by raising pH (725 +/- 65 ms), suggesting a role of the pump on Ca(2+) clearance. Replacement of external Na(+) by Li(+) had a similar effect (tau = 442 +/- 8 ms), confirming the NCX involvement in Ca(2+) extrusion. The evidence suggests that both Ca(2+) transporters contribute to re-establish resting Ca(2+) levels in the cilia following olfactory responses.

Recent evidence suggests that reactive oxygen species function as second messenger molecules in normal physiological processes. For example, activation of N-Methyl-D-Aspartate receptor results in the production of ROS, which appears to be critical for synaptic plasticity, one of the cellular mechanisms that underlie learning and memory. In this work, we studied the effect of iron in the activation of MAPK/ERK pathway and on Ca2+ signaling in neuronal PC12 cells. We found that iron-dependent generation of hydroxyl radicals is likely to modulate Ca2+ signaling through RyR calcium channel activation, which, in turn, activates the MAPK/ERK pathway. These findings underline the relevance of iron in normal neuronal function.

Olfactory cilia contain cyclic nucleotide-gated and Ca2+-dependent Cl- conductances that underlie excitatory chemotransduction, and a Ca2+-dependent K+ (KCa) conductance, apparently involved in inhibitory transduction. Previous single-channel patch-clamp studies on olfactory cilia revealed four different KCas, with different conductances and kinetics. Here, we further characterized these channels in planar bilayers, where blockers could be properly tested. All four ciliary KCas were observed: The 16 pS channel, K0.5,Ca=40 microM and apamin-sensitive; the 30 and 50 pS channel, K0.5,Ca=59 microM, clotrimazole-sensitive and charybdotoxin-insensitive; the 60 pS channel, clotrimazole-sensitive and charybdotoxin-insensitive; and the 210 pS channel, K0.5,Ca=63 microM, blocked by charybdotoxin and iberiotoxin. The presence of the 16 and 210 pS channels was confirmed by immunoblotting.

Background: Psoriasis is a chronic inflammatory skin disease. Probably autoimmune in nature, and associated with streptococcal throat infections as a triggering factor. Although many groups have associated the disease with other pathogens, Streptococcus pyogenes seems to be the most important microorganism related to this disease. Therefore, it is necessary to identify the streptococcal antigens involved in the process., Methods: In this work IgG class antibodies to soluble antigens obtained from Staphyloccus aureus, Candica albicans or S. pyogenes before and after heat shock induction, were analyzed by ELISA in 28 psoriatic patients and 30 healthy donors., Results: In all cases, the patients and the controls had IgG class antibodies to the four antigens. Nevertheless, the IgG levels to the heat shock-induced S. pyognes were statistically different between the patients and the controls (P < 0.001). There was no difference between the groups when the IgG antibodies to the other antigens, including the noninduced streptococcal extract, were analyzed. Additionally, anti-streptolysin O titers and throat cultures were carried out in all patients and controls. No differences between ASO titers were found but the patients were more frequently colonized by pyogenes., Conclusion: Results obtained in this study suggest that heat shock-induced proteins from S. pyogenes are associated with psoriasis.

Objective: Lispro is a newly FDA-approved analog of human insulin that will be widely used in patients with IDDM. This insulin, however, may have an increased potential for hypoglycemia because of its very rapid subcutaneous absorption, especially in a setting of decreased carbohydrate intake. Using a short-term prospective randomized parallel group-study design, we studied the incidence of hypoglycemia when lispro was given before breakfast compared with regular human insulin. Since carbohydrate intake is a determinant of postprandial glycemia, we administered three isocaloric meals characterized by low, average, and high carbohydrate content., Research Design and Methods: Two groups of six IDDM subjects were randomized to receive 0.15 U/kg of lispro or regular human insulin subcutaneously before the ingestion of three 500-kcal breakfast meals of differing carbohydrate content on separate days. Lispro was administered at mealtime, and regular insulin was administered 30 min before mealtime., Results: Postprandial plasma glucose concentrations were decreased in the lispro group compared with the regular-insulin group for all three meal types (P < 0.05), and hypoglycemia developed more frequently and rapidly in the lispro group, compared with the regular-insulin group by survival analysis. Additionally, peak insulin concentrations were higher (P < 0.001) and peaked more rapidly (P < 0.05) in the lispro group, compared with the regular-insulin group., Conclusions: We conclude that lispro has a tendency for early postprandial hypoglycemia compared with regular insulin in the setting of reduced carbohydrate intake. This fact should be told to patients who decide to switch from regular insulin to lispro. Health care professionals should instruct their IDDM patients to monitor glucose levels frequently after switching to lispro since adjustments in their carbohydrate intake and/or their lispro dosage may be necessary to avoid hypoglycemia.