The Topography of Non-Linear Cortical Dynamics at Rest, in Mental Calculation and Moving Shape Perception

Differential cortical activation by cognitive processing was studied using dimensional complexity, a measure derived from nonlinear dynamics that indicates the degrees of freedom (complexity) of a dynamic system. We examined the EEG of 32 healthy subjects at rest, during a visually presented calculation task, and during a moving shape perception task. As a nonlinear measure of connectivity, the mutual dimension of selected electrode pairs was used. The first Lyapunov coefficient was also calculated. Data were tested for non-linearity using a surrogate data method and compared to spectral EEG measures (power, coherence). Surrogate data testing confirmed the presence of nonlinear structure in the data. Cognitive activation led to a highly significant rise in dimensional complexity. While both tasks activated central, parietal and temporal areas, mental arithmetic showed frontal activation and an activity maximum at T3, while the moving shape task led to occipital activation and a right parietal activity maximum. Analysis of mutual dimension showed activation of a bilateral temporal-right frontal network in calculation. The Lyapunov coefficent showed clear topographic variation, but was not significantly changed by mental tasks (p<.09). While dimensional complexity was almost unrelated to power values, nonlinear (mutual dimension) and linear (coherence) measures of connectivity shared up to 37% of variance. Data are interpreted in terms of increased cortical complexity as a result of recruitment of asynchronously active, distributed neuronal assemblies in cognition. The topography of nonlinear dynamics are related to neuropsychological and neuroimaging findings on mental calculation and moving shape perception.     

Myosin VIIA gene: heterogeneity of the mutations responsible for Usher syndrome type IB

Usher syndrome is recognized as the most frequent cause of hereditary deaf-blindness. Usher syndrome type I (USH1), the most severe form of the disease, is characterized by profound congenital sensorineural deafness, constant vestibular dysfunction, and retinitis pigmentosa of prepubertal onset. This form is genetically heterogeneous and five loci (USH1A-E) have been mapped thusfar. However, only the gene responsible for USH1 B (which accounts for approximately 75% of USH1 cases) has been characterized. It encodes a long-tailed unconventional myosin, myosin VIIA, with a predicted 2215 amino acid sequence. Primers covering the complete myosin VIIA coding sequence as well as the 3' non coding sequence were designed, allowing direct sequence analysis of each of the 48 coding exons and flanking splice sites in seven patients affected by USH1. Four novel mutations were thereby identified. The possibility should now be considered of a sequence-based prenatal diagnosis in some of the families affected by this very severe form of Usher syndrome.      

Cytoskeletal modulation of the response to mechanical stimulation in human vascular endothelial cells

Possible interactions of cytoskeletal elements with mechanically induced membrane currents and Ca²⁺ signals were studied in human endothelial cells by using a combined patch-clamp and Fura II technique. For mechanical stimulation, cells were exposed to hypotonic solution (HTS). The concomitant cell swelling activates a Cl^– current, releases Ca²⁺ from intracellular stores and activates Ca²⁺ influx. To interfere with the cytoskeleton, cells were loaded either with the F-actin-stabilizing agent phalloidin (10 mol/l), or the F-actin-depolymerizing substance cytochalasin B (50 mol/l). These were administered either in the bath or the pipette solutions. The tubulin structure of the endothelial cells was modulated by taxol (50 mol/l), which supports polymerization of tubulin, or by the depolymerizing agent colcemid (10 mol/l) both applied to the bath. Immunofluorescence experiments show that under the chosen experimental conditions the cytoskeletal modifiers employed disintegrate the F-actin and microtubuli cytoskeleton. Neither of these cytoskeletal modifiers influenced the HTS-induced Cl^– current. Ca²⁺ release was not affected by cytochalasin B, taxol or colcemid, but was suppressed if the cells were loaded with phalloidin. Depletion of intracellular Ca²⁺ stores by thapsigargin renders the intracellular [Ca²⁺] sensitive to the extracellular [Ca²⁺], which is indicative of a Ca²⁺ entry pathway activated by store depletion. Neither cytochalasin B nor phalloidin affected this Ca²⁺ entry. We conclude that F-actin turnover or depolymerization is necessary for Ca²⁺ release by mechanical activation. The tubulin network is not involved. The Ca²⁺ release-activated Ca²⁺ entry is not modulated by the F-actin cytoskeleton.     

The nonselective cation channel in the basolateral membrane of rat exocrine pancreas

Nonselective Ca²⁺-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca²⁺ on the cytosolic side, the mean openstate probabilityP _o of one channel was about 0.5. In insideout oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 mol/l of NPPB or DPC decreasedP _o from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 mol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reducedP _o, but in contrast to the abov mentioned substances, it induced fast flickering. Ba²⁺ (70 mmol/l) and tetraethylammonium (TEA⁺; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and 4,4-dinitro-2,2-stilbenedisulfonate (DNDS). 10 mol/l SITS applied to the cytosolic side increasedP _o from 0.5 to 0.7 and with 100 mol/l SITS the channels remained nearly permanently in its open state (P _o1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca²⁺ concentration to 0.1 mol/l, addition of 100 mol/l of SITS had no effect. Similarly, reducing bath Ca²⁺ concentration from 1.3 mmol/l in presence of 100 mol/l SITS (channels are maximally activated) to 0.1 mol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca²⁺. SITS had no effects, when applied to the extracellular side in outside out patches. In summary, the substances DPC, NPPB and DCDPC inhibit nonselective cation channels, where DCDPC has the most potent and NPPB the smallest effect; whereas SITS, DIDS and DNDS activate the channel when applied from the cytosolic side in the presence of Ca²⁺ ions.     

The carboxyl terminus of the epithelial Ca(2+) channel ECaC1 is involved in Ca(2+)-dependent inactivation

The family of epithelial Ca(2+) channels (ECaC) is a unique group of highly Ca(2+)-selective channels consisting of two members, ECaC1 and ECaC2. We used carboxyl terminal truncations and mutants to delineate the molecular determinants of the Ca(2+)-dependent inhibition of ECaC. To this end, rabbit ECaC1 was expressed heterologously with green fluorescent protein (GFP) in human embryonic kidney 293 (HEK293) cells using a bicistronic vector. Deletion of the last 30 amino acids of the carboxyl terminus of ECaC1 (G701X) decreased the Ca(2+) sensitivity significantly. Another critical sequence for Ca(2+)-dependent inactivation of ECaC1 was found upstream in the carboxyl terminus. Analysis of truncations at amino acid 635, 639, 646, 649 and 653 disclosed a critical sequence involved in Ca(2+)-dependent inactivation at positions 650-653. C653X showed decreased Ca(2+) sensitivity, comparable to G701X, while E649X lacked Ca(2+)-dependent inactivation. Interestingly, the number of green fluorescent cells, which is an index of the number of transfected cells, was significantly smaller for cells transfected with truncations shorter than E649 than for cells transfected with wild-type ECaC. However, the expression level of GFP was restored in the presence of the ECaC blocker ruthenium red, suggesting that these truncations resulted in deleterious Ca(2+) influx. In conclusion, we have identified two domains in the carboxyl terminus of ECaC1 that control Ca(2+)-dependent inactivation.     

Cellular mechanisms of T cell mediated drug hypersensitivity

Noncovalent drug presentation leads to the activation of drug-specific T cells. In some patients with hypersensitivity, such a response occurs within hours even upon the first exposure to the drug. Thus, the reaction to the drug might not be due to a classical, primary response, but rather mediated by existing, preactivated T cells that are cross specific for the drug, and have an additional (peptide) specificity as well.     

The volume-activated chloride current in human endothelial cells depends on intracellular ATP

We have studied the effect of intracellular ATP on volume-activated Cl^–-currents in endothelial cells from human umbilical veins by means of the whole-cell patch clamp technique. The run-down of this current in ruptured patches during repetitive applications of hypotonic solutions (HTS) could be significantly reduced if the cells were internally perfused with a pipette solution that contained 4 mmol/l ATP. This run-down was much less pronounced if currents were recorded using nystatin-perforated patches. The amplitude of the current was drastically reduced and its activation became slower if the cells were superfused with a glucose-free medium with 1 mmol/l KCN. Adding 4 mmol/l ATPS, a poorly hydrolyzable ATP-analogue, to the patch pipette prevented run-down of the current during repetitive activations by HTS, even if the cells were superfused with glucose-free solution with 1 mmol/l KCN. It is concluded that activation of the mechanosensitive Cl^– conductance in human endothelial cells requires the presence of intracellular ATP, but not its hydrolysis.     

Epithelial calcium channels: from identification to function and regulation

The epithelial calcium channels TRPV5 and TRPV6 have been studied extensively in the epithelial tissues controlling Ca(2+) homeostasis and exhibit a range of distinctive properties that distinguish them from other transient receptor potential (TRP) channels. These two novel members of the superfamily of TRP channels were cloned from vitamin D-responsive epithelia: kidney, small intestine and placenta, and identified subsequently in tissues like pancreas, bone and prostate. This review addresses the unique properties of these highly Ca(2+)-selective channels and highlights their implications for the process of transepithelial Ca(2+) transport.