Our data revealed that channel trafficking may contribute to the pathogenesis of LQT1.

The calcineurin (CaN) a and b catalytic subunit isoforms are coexpressed within almost all cell types. The enzymatic properties of CaN heterodimers comprised of the regulatory B subunit (CnB) with either the a or b catalytic subunit were compared using in vitro phosphatase assays. CaN containing the a isoform (CnAa) has lower K m and higher V max values than CaN containing the b isoform (CnAb) toward the PO 4 -RII, PO 4 -DARPP-32(20-38) peptides, and p-nitrophenylphosphate (pNPP). CaN heterodimers containing the a or b catalytic subunit isoform displayed identical calmodulin dissociation rates. Similar inhibition curves for each CaN heterodimer were obtained with the CaN autoinhibitory peptide (CaP) and cyclophilin A/cyclosporin A (CyPA/CsA) using each peptide substrate at K m concentrations, except for a five-to ninefold higher IC 50 value measured for CaN containing the b isoform with p-nitrophenylphosphate as substrate. No difference in stimulation of phosphatase activity toward p-nitrophenylphosphate by FKBP12/FK506 was observed. At low concentrations of FKBP12/FK506, CaN containing the a isoform is more sensitive to inhibition than CaN containing the b isoform using the phosphopeptide substrates. Higher concentrations of FKBP12/FK506 are required for maximal inhibition of b CaN using PO 4 -DARPP-32(20-38) as substrate. The functional differences conferred upon CaN by the a or b catalytic subunit isoforms suggest that the a:b and CaN:substrate ratios may determine the levels of CaN phosphatase activity toward specific substrates within tissues and specific cell types. These findings also indicate that the a and b catalytic subunit isoforms give rise to substrate-dependent differences in sensitivity toward FKBP12/FK506.

Abstract-ATP-sensitive K ϩ (K ATP ) channels are broadly distributed in the vasculature and regulate arterial tone. These channels are inhibited by intracellular ATP ([ATP] i ) and vasoconstrictor agents and can be activated by vasodilators. It is widely assumed that K ATP channels are insensitive to Ca 2ϩ , although regulation has not been examined in the intact cell where cytosolic regulatory processes may be important. Thus we investigated the effects of Ca 2ϩ on whole-cell K ATP current in rat aortic smooth muscle cells recorded in a physiological [ATP] i and K ϩ gradient. Under control recording conditions, cells had a resting potential of ϷϪ40 mV when bathed in 1.8 mmol/L Ca 2ϩ . The K ATP channel inhibitor glibenclamide caused membrane depolarization (9 mV) and inhibited a small, time-independent background current. Reducing [ATP] i from 3 to 0.1 mmol/L hyperpolarized cells to ϷϪ60 mV and increased glibenclamide-sensitive current by 2-to 4-fold. Similar effects were observed when Ca 2ϩ levels were decreased either externally or internally by increasing EGTA from 1 to 10 mmol/L. Dialysis with solutions containing different free [Ca 2ϩ ] i showed that K ATP current was maximally activated at 10 nmol/L [Ca 2ϩ ] i and almost totally inhibited at 300 nmol/L. Moreover, under control conditions, when rat aortic smooth muscle cells were dialyzed with either cyclosporin A, FK-506, or calcineurin autoinhibitory peptide (structurally unrelated inhibitors of Ca 2ϩ -dependent protein phosphatase, type 2B), glibenclamide-sensitive currents were large and the resting potential was hyperpolarized by Ϸ20 to 25 mV. We report for the first time that K ATP channels can be modulated by Ca 2ϩ at physiological [ATP] i and conclude that modulation occurs via protein phosphatase type 2B.

KCNE1 associates with the pore-forming alpha-subunit KCNQ1 to generate the slow (I(Ks)) current in cardiac myocytes. Mutations in either KCNQ1 or KCNE1 can alter the biophysical properties of I(Ks) and mutations in KCNE1 underlie cases of long QT syndrome type 5 (LQT5). We previously investigated a mutation in KCNE1, T58P/L59P, which causes severe attenuation of I(Ks). However, how T58P/L59P acts to disrupt I(Ks) has not been determined. In this study, we investigate and compare the effects of T58P/L59P with three other LQT5 mutations (G52R, S74L, and R98W) on the biophysical properties of the current, trafficking of KCNQ1, and assembly of the I(Ks) channel. G52R and T58P/L59P produce currents that lack the kinetic behavior of I(Ks). In contrast, S74L and R98W both produce I(Ks)-like currents but with rightward shifted voltage dependence of activation. All of the LQT5 mutants express protein robustly, and T58P/L59P and R98W cause modest, but significant, defects in the trafficking of KCNQ1. Despite defects in trafficking, in the presence of KCNQ1, T58P/L59P and the other LQT5 mutants are present at the plasma membrane. Interestingly, in comparison to KCNE1 and the other LQT5 mutants, T58P/L59P associates only weakly with KCNQ1. In conclusion, we identify the disease mechanisms for each mutation and reveal that T58P/L59P causes disease through a novel mechanism that involves defective I(Ks) complex assembly.

1 Endotoxin-induced vascular hyporeactivity to phenylephrine (PE) is well described in rodent aorta, but has not been investigated in smaller vessels in vitro. 2 Segments of rat superior mesenteric artery were incubated in culture medium with or without foetal bovine serum (10%) for 6, 20 or 46 h in the presence or absence of bacterial lipopolysaccharide (LPS; 1 ± 100 mg ml 71 ). 3 Contractions to PE were measured with or without nitric oxide synthase (NOS) inhibitors: L-NAME (300 mM), aminoguanidine (AMG; 400 mM) 1400W (10 mM) and GW273629 (10 mM); the guanylyl cyclase inhibitor, ODQ (3 mM); the COX-2 inhibitor, NS-398 (10 mM). Contractile responses to the thromboxane A 2 mimetic, U46619 were also assessed. 4 In the presence of serum, LPS induced hyporeactivity at all time points. In its absence, hyporeactivity only occurred at 6 and 20 h. 5 L-NAME and AMG fully reversed hyporeactivity at 6 h, whereas they were only partially e ective at 20 h and not at all at 46 h. In contrast partial reversal of peak contraction was observed with 1400W (62% at 46 h), GW273629 (57% at 46 h) and ODQ (75% at 46 h). COX-2 inhibition produced no reversal. 6 In contrast to PE, contractions to U46619 were substantially less a ected by LPS. 7 We describe a well-characterized reproducible model of LPS-induced hyporeactivity, which is largely mediated by the NO-cyclic GMP-dependent pathway. Importantly, long-term (2-day) production of NO via iNOS is demonstrated. Moreover, conventional doses of L-NAME and AMG became increasingly ine ective over time. Thus, the choice of inhibitor merits careful consideration in long-term models.

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Here, we describe the first use of thromboelastography (TEG) in the management of 2 cases of Ebola virus disease. Early in their illness, both patients had evidence of a consumptive coagulopathy. As this resolved, TEG demonstrated that both developed a marked hypercoagulable state, which was treated with low-molecular-weight heparin.

This book examines how national and ethnic identities are being reforged in the post-Soviet borderland states. The first chapter provides a conceptual and theoretical context for examining national identities, drawing in particular upon post-colonial theory. The rest of the book is divided into three parts. In Part I, the authors examine how national histories of the borderland states are being rewritten especially in relation to new nationalising historiographies, around myths of origin, homeland, and descent. Part II explores the ethnopolitics of group boundary construction and how such a politics has led to nationalising policies of both exclusion and inclusion. Part III examines the relationship between nation-building and language, especially with regard to how competing conceptions of national identity have informed the thinking of both political decision-takers and nationalising intellectuals, and the consequences for ethnic minorities. Such perspectives on nation-building are illustrated with substantive studies drawn from the Baltic states, Ukraine, and Belarus, Transcaucasia, and Central Asia.