In resting muscle, cytoplasmic Mg2+ is a potent inhibitor of Ca2+ release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites (I-sites) and to high affinity Ca2+ sites (A-sites) thus preventing Ca2+ activation. We investigate the effects of luminal and cytoplasmic Ca2+ on Mg2+ inhibition at the A-sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg2+ inhibits RyRs at the A-site in the absence of Ca2+, indicating that Mg2+ is an antagonist and does not simply prevent Ca2+ activation. Cytoplasmic Ca2+ and Cs+ decreased Mg2+ affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca2+ regulation of Ca2+ release whereby increasing luminal [Ca2+] decreases the A-site affinity for cytoplasmic Mg2+ by a noncompetitive, allosteric mechanism that is independent of Ca2+ flow. Ryanodine increases the Ca2+ sensitivity of the A-sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca2+, indicating that ryanodine activates independently of Ca2+. We describe a model for ion binding at the A-sites that predicts that modulation of Mg2+ inhibition by luminal Ca2+ is a significant regulator of Ca2+ release from the SR. We detected coupled gating of RyRs due to luminal Ca2+ permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca2+ and cytoplasmic Mg2+ did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca2+ was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively “immune” to Ca2+ emanating from their own pore but sensitive to Ca2+ from neighboring channels.

This study examined the contribution of perceptual and cognitive factors to speech-perception abilities in cochlear-implant (CI) users. Thirty CI users were tested on word intelligibility in sentences with and without semantic context, presented in quiet and in noise. Performance was compared with measures of spectral-ripple detection and discrimination, thought to reflect peripheral processing, as well as with cognitive measures of working memory and non-verbal intelligence. Thirty age-matched and thirty younger normal-hearing (NH) adults also participated, listening via tone-excited vocoders, adjusted to produce mean performance for speech in noise comparable to that of the CI group. Results suggest that CI users may rely more heavily on semantic context than younger or older NH listeners, and that non-auditory working memory explains significant variance in the CI and age-matched NH groups. Between-subject variability in spectral-ripple detection thresholds was similar across groups, despite the spectral resolution for all NH listeners being limited by the same vocoder, whereas speech perception scores were more variable between CI users than between NH listeners. The results highlight the potential importance of central factors in explaining individual differences in CI users and question the extent to which standard measures of spectral resolution in CIs reflect purely peripheral processing.

Activation of skeletal muscle ryanodine receptors (RyRs) by suramin and disulfonic stilbene derivatives (Diisothiocyanostilbene-2',2'-disulfonic acid (DIDS), 4,4'-dibenzamidostilbene-2,2'-disulfonic acid (DBDS),and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)) was investigated using planar bilayers. One reversible and two nonreversible mechanisms were identified. K(a) for reversible activation (approximately 100 micro M) depended on cytoplasmic [Ca(2+)] and the bilayer composition. Replacement of neutral lipids by negative phosphatidylserine increased K(a) fourfold, suggesting that reversible binding sites are near the bilayer surface. Suramin and the stilbene derivatives adsorbed to neutral bilayers with maximal mole fractions between 1-8% and with affinities approximately 100 micro M but did not adsorb to negative lipids. DIDS activated RyRs by two nonreversible mechanisms, distinguishable by their disparate DIDS binding rates (10(5) and 60 M(-1) s(-1)) and actions. Both mechanisms activated RyRs via several jumps in open probability, indicating several DIDS binding events. The fast and slow mechanisms are independent of each other, the reversible mechanism and ATP binding. The fast mechanism confers DIDS sensitivity approximately 1000-fold greater than previously reported, increases Ca(2+) activation and increases K(i) for Ca(2+)/Mg(2+) inhibition 10-fold. The slow mechanism activates RyRs in the absence of Ca(2+) and ATP, increases ATP activation without altering K(a), and slightly increases activity at pH < 6.5. These findings explain how different types of DIDS activation are observed under different conditions.

Summary Recently a new model of vertebrate immunity has been gaining popularity. In this new model it is hypothesized that activation of innate immunity is a prerequisite for an adaptive immune response to an antigen. Following activation the innate system induces key costimulator molecules on APC, which are essential for antigen-driven clonal expansion of T and B cells. The model largely explains the need for adjuvants in the induction of adaptive immunity, provides a possible mechanism for the immune system to perceive the biological nature of a pathogen and thereby produce the most effective immune response, and transfers much of the onus of self-non-self discrimination from the adaptive to the innate immune system. In the present article we highlight two paradoxes raised by the new model. First, by linking adaptive immunity to innate recognition the immune system is unable to take full advantage of the genetic diversity of T and B cell antigen receptors. Thus, the abihty of the immune system to combat a pathogen is totally dependent on the efficiency of" recognition by the innate system and, therefore, the germ-line mutation rate of the genes involved in the innate response. Second, if signals from the innate system induce costimulatory molecules on APC, then one would expect the accidental clonal expansion of many autoreactive T and B cells. We suggest that one means of resolving the first paradox is to propose that the major reason for the evolution of adaptive immunity wa.s to provide, via immunological memory, resistance to reinfection, rather than simply to combat the primary infection by the pathogen. In the case of autoreactivity we suggest that autodestruction is prevented by immune responses being tightly regulated at the effector T cell level. Finally, we argue that the two paradoxes, rather than undermining the new model of immunity, highlight our lack of understanding of key element.s of the vertebrate immune system.

This study assessed the impact of semantic context and talker variability on speech perception by cochlear-implant (CI) users and compared their overall performance and between-subjects variance with that of normal-hearing (NH) listeners under vocoded conditions. Thirty post-lingually deafened adult CI users were tested, along with 30 age-matched and 30 younger NH listeners, on sentences with and without semantic context, presented in quiet and noise, spoken by four different talkers. Additional measures included working memory, non-verbal intelligence, and spectral-ripple detection and discrimination. Semantic context and between-talker differences influenced speech perception to similar degrees for both CI users and NH listeners. Between-subjects variance for speech perception was greatest in the CI group but remained substantial in both NH groups, despite the uniformly degraded stimuli in these two groups. Spectral-ripple detection and discrimination thresholds in CI users were significantly correlated with speech perception, but a single set of vocoder parameters for NH listeners was not able to capture average CI performance in both speech and spectral-ripple tasks. The lack of difference in the use of semantic context between CI users and NH listeners suggests no overall differences in listening strategy between the groups, when the stimuli are similarly degraded.