The slow-channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular junction caused by gain-of-function mutations to the muscle nicotinic acetylcholine (ACh) receptor (AChR). Although it is clear that the slower deactivation time course of the ACh-elicited currents plays a central role in the etiology of this disease, it has been suggested that other abnormal properties of these mutant receptors may also be critical in this respect. We characterized the kinetics of a panel of five SCCMS AChRs (αS269I, βV266M, εL221F, εT264P, and εL269F) at the ensemble level in rapidly perfused outside-out patches. We found that, for all of these mutants, the peak-current amplitude decreases along trains of nearly saturating ACh pulses delivered at physiologically relevant frequencies in a manner that is consistent with enhanced entry into desensitization during the prolonged deactivation phase. This suggests that the increasingly reduced availability of activatable AChRs upon repetitive stimulation may well contribute to the fatigability and weakness of skeletal muscle that characterize this disease. Also, these results emphasize the importance of explicitly accounting for entry into desensitization as one of the pathways for burst termination, if meaningful mechanistic insight is to be inferred from the study of the effect of these naturally occurring mutations on channel function. Applying a novel single-channel–based approach to estimate the contribution of Ca2+ to the total cation currents, we also found that none of these mutants affects the Ca2+-conduction properties of the AChR to an extent that seems to be of physiological importance. Our estimate of the Ca2+-carried component of the total (inward) conductance of wild-type and SCCMS AChRs in the presence of 150 mM Na+, 1.8 mM Ca2+, and 1.7 mM Mg2+ on the extracellular side of cell-attached patches turned out be in the 5.0–9.4 pS range, representing a fractional Ca2+ current of ∼14%, on average. Remarkably, these values are nearly identical to those we estimated for the NR1-NR2A N-methyl-d-aspartate receptor (NMDAR), which has generally been considered to be the main neurotransmitter-gated pathway of Ca2+ entry into the cell. Our estimate of the rat NMDAR Ca2+ conductance (using the same single-channel approach as for the AChR but in the nominal absence of extracellular Mg2+) was 7.9 pS, corresponding to a fractional Ca2+ current of 13%.

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Introduction Numerous thermal free radical stabilization techniques are used in the production of highly cross-linked polyethylene (HXLPE) to improve oxidative stability. Little knowledge exists on the effects of in vivo time on the mechanical properties of HXLPE. The purpose of this study was to determine if free radical stabilization of HXLPE impacts mechanical properties as well as oxidative stability of acetabular liner rims after extended in vivo time. Methods Retrieved and control remelted, single annealed and sequentially annealed HXLPE liner rims were tested for mechanical properties. Oxidation was measured with FTIR spectroscopy and crystalline phase composition measured with Raman spectroscopy. Results No correlation was found between in vivo, ex vivo time and hardness for annealed groups. A statistically significant difference in hardness was identified between free radical stabilization groups. No correlation between maximum rim oxidation and in vivo time was found. Detectable levels of rim oxidation were present in 100% of single annealed, 75% of sequentially annealed, and 25% of remelted retrieved liners. Single and sequentially annealed liners demonstrated oxidation and increased crystallinity. Rim mechanical properties change in vivo for implant types. With in vivo time, retrieved remelted HXLPE demonstrated decreased mechanical properties, whereas retrieved single and sequentially annealed HXLPE properties remained stable. All liner cohorts demonstrated evidence of rim oxidation. Subsequent changes in crystallinity were only observed in oxidized annealed liners. Conclusion HXLPE acetabular liner rims show evidence of in vivo mechanical property degradation, notably in remelted HXLPE, which may be a risk factor in rim fracture and catastrophic implant failure.

The splicing mutation in intron 2 (nucleotide 656) of the 21-hydroxylase gene (CYP21B) is the most common mutation causing congenital adrenal hyperplasia (CAH). Homozygosity for nt656G is associated with the classical phenotype of CAH. In several studies, a number of clinically asymptomatic relatives of CAH-patients were genotyped as nt656G homozygotes. We have proposed that the putative asymptomatic nt656G/G individuals are incorrectly typed due to dropout of one allele (in most cases nt656C) during PCR amplification. Here, we report the successful amplification of all alleles at nt656 with a Taq/Pwo DNA polymerase mixture in the primary PCR reaction. The results were independent from the type of polymerase used for sequencing reactions as the second step in mutation analysis.