Epidemiology and risk factors for hepatitis C virus infection in a high-prevalence population.

To understand increasing rates of hepatitis C virus (HCV) infection in Tennessee, we conducted testing, risk factor analysis and a nested case-control study among persons who use drugs. During June-October 2016, HCV testing with risk factor assessment was conducted in sexually transmitted disease clinics, family planning clinics and an addiction treatment facility in eastern Tennessee; data were analysed by using multivariable logistic regression. A nested case-control study was conducted to assess drug-using risks and behaviours among persons who reported intranasal or injection drug use (IDU). Of 4753 persons tested, 397 (8.4%) were HCV-antibody positive. HCV infection was significantly associated with a history of both intranasal and IDU (adjusted odds ratio (aOR) 35.4, 95% confidence interval (CI) 24.1-51.9), IDU alone (aOR 52.7, CI 25.3-109.9), intranasal drug use alone (aOR 2.6, CI 1.8-3.9) and incarceration (aOR 2.7, CI 2.0-3.8). By 4 October 2016, 574 persons with a reported history of drug use; 63 (11%) were interviewed further. Of 31 persons who used both intranasal and injection drugs, 26 (84%) reported previous intranasal drug use, occurring 1-18 years (median 5.5 years) before their first IDU. Our findings provide evidence that reported IDU, intranasal drug use and incarceration are independent indicators of risk for past or present HCV infection in the study population.

Optically pumped 5 microm IV-VI VECSEL with Al-heat spreader.

Mid-infrared vertical external cavity surface emitting lasers (VECSELs) for 5 microm in wavelength have been realized. The active parts are of a simple structure, either a 2 microm thick PbTe gain layer or two 150 nm PbTe layers embedded in Pb(1-x)Eu(x)Te barriers. Epitaxial 2.5 pair Pb(1-y)Eu(y)Te/BaF(2) Bragg mirrors are employed to form the cavity, and an Al layer is deposited for improved heat dissipation. Emission up to 300 mW(p) is observed with microsecond pulses or 3 mW cw at 100 K is obtained. Quantum efficiency is up to 14%, and lasing occurs up to 175 K when pumped with a 1.55 microm wavelength pump laser.

Differential Ca2+ and Sr2+ regulation of intracellular divalent cations release in ventricular myocytes.

The regulation of the Ca2+ -induced Ca2+ release (CICR) from intracellular stores is a critical step in the cardiac cycle. The inherent positive feedback of CICR should make it a self-regenerating process. It is accepted that CICR must be governed by some negative control, but its nature is still debated. We explore here the importance of the Ca2+ released from sarcoplasmic reticulum (SR) on the mechanisms that may control CICR. Specifically, we compared the effect of replacing Ca2+ with Sr2+ on intracellular Ca2+ signaling in intact cardiac myocytes as well as on the function of single ryanodine receptor (RyR) Ca2+ release channels in panar bilayers. In cells, both CICR and Sr2+ -induced Sr2+ release (SISR) were observed. Action potential induced Ca2+ -transients and spontaneous Ca2+ waves were considerably faster than their Sr2+ -mediated counterparts. However, the kinetics of Ca2+ and Sr2+ sparks was similar. At the single RyR channel level, the affinities of Ca2+ and Sr2+ activation were different but the affinities of Ca2+ and Sr2+ inactivation were similar. Fast Ca2+ and Sr2+ stimuli activated RyR channels equally fast but adaptation (a spontaneous slow transition back to steady-state activity levels) was not observed in the Sr2+ case. Together, these results suggest that regulation of the RyR channel by cytosolic Ca2+ is not involved in turning off the Ca2+ spark. In contrast, cytosolic Ca2+ is important in the propagation global Ca2+ release events and in this regard single RyR channel sensitivity to cytosolic Ca2+ activation, not low-affinity cytosolic Ca2+ inactivation, is a key factor. This suggests that the kinetics of local and global RyR-mediated Ca2+ release signals are affected in a distinct way by different divalent cations in cardiac muscle cells.

Differential activation by Ca2+, ATP and caffeine of cardiac and skeletal muscle ryanodine receptors after block by Mg2+.

The block of rabbit skeletal ryanodine receptors (RyR1) and dog heart RyR2 by cytosolic [Mg2+], and its reversal by agonists Ca2+, ATP and caffeine was studied in planar bilayers. Mg2+ effects were tested at submaximal activating [Ca2+] (5 microM). Approximately one third of the RyR1s had low open probability ("LA channels") in the absence of Mg2+. All other RyR1s displayed higher activity ("HA channels"). Cytosolic Mg2+ (1 mM) blocked individual RyR1 channels to varying degrees (32 to 100%). LA channels had residual P(o) <0.005 in 1 mM Mg2+ and reactivated poorly with [Ca2+] (100 microM), caffeine (5 mM), or ATP (4 mM; all at constant 1 mM Mg2+). HA channels had variable activity in Mg2+ and variable degree of recovery from Mg2+ block with Ca2+, caffeine or ATP application. Nearly all cardiac RyR2s displayed high activity in 5 microM [Ca2+]. They also had variable sensitivity to Mg2+. However, the RyR2s consistently recovered from Mg2+ block with 100 microM [Ca2+] or caffeine application, but not when ATP was added. Thus, at physiological [Mg2+], RyR2s behaved as relatively homogeneous Ca2+/caffeine-gated HA channels. In contrast, RyR1s displayed functional heterogeneity that arises from differential modulatory actions of Ca2+ and ATP. These differences between RyR1 and RyR2 function may reflect their respective roles in muscle physiology and excitation-contraction coupling.

Sarcoplasmic reticulum calcium release is stimulated and inhibited by daunorubicin and daunorubicinol.

Cardiac effects of anthracyclines or their metabolites may include both the stimulation and inhibition of Ca(2+) release from sarcoplasmic reticulum. In this study, the ability of daunorubicin and its primary metabolite, daunorubicinol, to stimulate and inhibit Ca(2+) release from canine sarcoplasmic reticulum (SR) vesicles was investigated. It was observed that both daunorubicin and daunorubicinol were several fold more potent at inhibiting than they were at stimulating SR Ca(2+) release. Respective IC50 inhibition of daunorubicin and daunorubicinol for caffeine-induced calcium release was 1.2 and 0.6 microM, and for spontaneous Ca(2+) release was 3 and 1 microM. EC50's for daunorubicin- and daunorubicinol-induced calcium release were 30 and 15 microM, respectively. Inhibition of either spontaneous or caffeine-induced SR Ca(2+) release was inversely related to the amount of Ca(2+) loaded into the SR before exposure to daunorubicin or daunorubicinol. The free-radical scavenger dithiothreitol did not attenuate the ability of anthracyclines to inhibit SR Ca(2+) release. A nonquinone daunorubicin derivative, 5-iminodaunorubicin, was less potent than daunorubicin at inhibiting caffeine-induced Ca(2+) release. These data suggest anthracyclines and their metabolites may produce cardiotoxicity through free-radical independent, concentration-dependent effects on SR Ca(2+) release. These effects involve either inhibition or stimulation of SR Ca(2+) release and are partly dependent upon the presence of the quinone moiety.

Single-channel function of recombinant type 2 inositol 1,4, 5-trisphosphate receptor.

A full-length rat type 2 inositol 1,4,5-trisphosphate (InsP(3)) receptor cDNA construct was generated and expressed in COS-1 cells. Targeting of the full-length recombinant type 2 receptor protein to the endoplasmic reticulum was confirmed by immunocytochemistry using isoform specific affinity-purified antibodies and InsP(3)R-green fluorescent protein chimeras. The receptor protein was solubilized and incorporated into proteoliposomes for functional characterization. Single-channel recordings from proteoliposomes fused into planar lipid bilayers revealed that the recombinant protein formed InsP(3)- and Ca(2+)-sensitive ion channels. The unitary conductance ( approximately 250 pS; 220/20 mM Cs(+) as charge carrier), gating, InsP(3), and Ca(2+) sensitivities were similar to those previously described for the native type 2 InsP(3)R channel. However, the maximum open probability of the recombinant channel was slightly lower than that of its native counterpart. These data show that our full-length rat type 2 InsP(3)R cDNA construct encodes a protein that forms an ion channel with functional attributes like those of the native type 2 InsP(3)R channel. The possibility of measuring the function of single recombinant type 2 InsP(3)R is a significant step toward the use of molecular tools to define the determinants of isoform-specific InsP(3)R function and regulation.

Spatial Ca(2+) distribution in contracting skeletal and cardiac muscle cells.

The spatiotemporal distribution of intracellular Ca(2+) release in contracting skeletal and cardiac muscle cells was defined using a snapshot imaging technique. Calcium imaging was performed on intact skeletal and cardiac muscle cells during contractions induced by an action potential (AP). The sarcomere length of the skeletal and cardiac cells was approximately 2 micrometer. Imaging Rhod-2 fluorescence only during a very brief (7 ns) snapshot of excitation light minimized potential image-blurring artifacts due to movement and/or diffusion. In skeletal muscle cells, the AP triggered a large fast Ca(2+) transient that peaked in less than 3 ms. Distinct subsarcomeric Ca(2+) gradients were evident during the first 4 ms of the skeletal Ca(2+) transient. In cardiac muscle, the AP-triggered Ca(2+) transient was much slower and peaked in approximately 100 ms. In contrast to the skeletal case, there were no detectable subsarcomeric Ca(2+) gradients during the cardiac Ca(2+) transient. Theoretical simulations suggest that the subsarcomeric Ca(2+) gradients seen in skeletal muscle were detectable because of the high speed and synchrony of local Ca(2+) release. Slower asynchronous recruitment of local Ca(2+) release units may account for the absence of detectable subsarcomeric Ca(2+) gradients in cardiac muscle. The speed and synchrony of local Ca(2+) gradients are quite different in AP-activated contracting cardiac and skeletal muscle cells at normal resting sarcomere lengths.

Location of the permeation pathway in the recombinant type 1 inositol 1,4,5-trisphosphate receptor.

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) forms ligand-regulated intracellular Ca(2+) release channels in the endoplasmic reticulum of all mammalian cells. The InsP(3)R has been suggested to have six transmembrane regions (TMRs) near its carboxyl terminus. A TMR-deletion mutation strategy was applied to define the location of the InsP(3)R pore. Mutant InsP(3)Rs were expressed in COS-1 cells and single channel function was defined in planar lipid bilayers. Mutants having the fifth and sixth TMR (and the interceding lumenal loop), but missing all other TMRs, formed channels with permeation properties similar to wild-type channels (gCs = 284; gCa = 60 pS; P(Ca)/P(Cs) = 6.3). These mutant channels bound InsP(3), but ligand occupancy did not regulate the constitutively open pore (P(o) > 0.80). We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398-2589) near the COOH terminus of the protein forms the InsP(3)R pore. Further, we have produced a constitutively open InsP(3)R pore mutant that is ideal for future site-directed mutagenesis studies of the structure-function relationships that define Ca(2+) permeation through the InsP(3)R channel.

Unitary Ca2+ current through cardiac ryanodine receptor channels under quasi-physiological ionic conditions.

Single canine cardiac ryanodine receptor channels were incorporated into planar lipid bilayers. Single-channel currents were sampled at 1-5 kHz and filtered at 0.2-1.0 kHz. Channel incorporations were obtained in symmetrical solutions (20 mM HEPES-Tris, pH 7.4, and pCa 5). Unitary Ca2+ currents were monitored when 2-30 mM Ca2+ was added to the lumenal side of the channel. The relationship between the amplitude of unitary Ca2+ current (at 0 mV holding potential) and lumenal [Ca2+] was hyperbolic and saturated at approximately 4 pA. This relationship was then defined in the presence of different symmetrical CsCH3SO3 concentrations (5, 50, and 150 mM). Under these conditions, unitary current amplitude was 1.2 +/- 0.1, 0.65 +/- 0.1, and 0.35 +/- 0.1 pA in 2 mM lumenal Ca2+; and 3.3 +/- 0.4, 2.4 +/- 0. 2, and 1.63 +/- 0.2 pA in 10 mM lumenal Ca2+ (n > 6). Unitary Ca2+ current was also defined in the presence of symmetrical [Mg2+] (1 mM) and low [Cs+] (5 mM). Under these conditions, unitary Ca2+ current in 2 and 10 mM lumenal Ca2+ was 0.66 +/- 0.1 and 1.52 +/- 0.06 pA, respectively. In the presence of higher symmetrical [Cs+] (50 mM), Mg2+ (1 mM), and lumenal [Ca2+] (10 mM), unitary Ca2+ current exhibited an amplitude of 0.9 +/- 0.2 pA (n = 3). This result indicates that the actions of Cs+ and Mg2+ on unitary Ca2+ current were additive. These data demonstrate that physiological levels of monovalent cation and Mg2+ effectively compete with Ca2+ as charge carrier in cardiac ryanodine receptor channels. If lumenal free Ca2+ is 2 mM, then our results indicate that unitary Ca2+ current under physiological conditions should be <0.6 pA.

Bcl-xL regulates apoptosis by heterodimerization-dependent and -independent mechanisms.

A hydrophobic cleft formed by the BH1, BH2 and BH3 domains of Bcl-xL is responsible for interactions between Bcl-xL and BH3-containing death agonists. Mutants were constructed which did not bind to Bax but retained anti-apoptotic activity. Since Bcl-xL can form an ion channel in synthetic lipid membranes, the possibility that this property has a role in heterodimerization-independent cell survival was tested by replacing amino acids within the predicted channel-forming domain with the corresponding amino acids from Bax. The resulting chimera showed a reduced ability to adopt an open conductance state over a wide range of membrane potentials. Although this construct retained the ability to heterodimerize with Bax and to inhibit apoptosis, when a mutation was introduced that rendered the chimera incapable of heterodimerization, the resulting protein failed to prevent both apoptosis in mammalian cells and Bax-mediated growth defect in yeast. Similar to mammalian cells undergoing apoptosis, yeast cells expressing Bax exhibited changes in mitochondrial properties that were inhibited by Bcl-xL through heterodimerization-dependent and -independent mechanisms. These data suggest that Bcl-xL regulates cell survival by at least two distinct mechanisms; one is associated with heterodimerization and the other with the ability to form a sustained ion channel.

Bay K 8644 increases resting Ca2+ spark frequency in ferret ventricular myocytes independent of Ca influx: contrast with caffeine and ryanodine effects.

Bay K 8644, an L-type Ca2+ channel agonist, was shown previously to increase resting sarcoplasmic reticulum (SR) Ca2+ loss and convert post-rest potentiation to decay in dog and ferret ventricular muscle. Here, the effects of Bay K 8644 on local SR Ca2+ release events (Ca2+ sparks) were measured in isolated ferret ventricular myocytes, using laser scanning confocal microscopy and the fluorescent Ca2+ indicator fluo-3. The spark frequency under control conditions was fairly constant during 20 s of rest after interruption of electrical stimulation. Bay K 8644 (100 nmol/L) increased the spark frequency by 466+/-90% of control at constant SR Ca2+ load but did not change the spatial and temporal characteristics of individual sparks. The increase in spark frequency was maintained throughout the period of rest. The increase in Ca2+ spark frequency induced by Bay K 8644 was not affected by superfusion with Ca2+-free solution (with 10 mmol/L EGTA) but was suppressed by the addition of 10 micromol/L nifedipine (which by itself did not alter resting Ca2+ spark frequency). This suggests that the effect of Bay K 8644 on Ca2+ sparks is mediated by the sarcolemmal dihydropyridine receptor but is also independent of Ca2+ influx. Low concentrations of caffeine (0.5 mmol/L) increased both the average frequency and duration of sparks. Ryanodine (50 nmol/L) increased the spark frequency and also induced long-lasting Ca2+ signals. This may indicate long-lasting openings of SR Ca2+ release channels and a lack of local SR Ca2+ depletion. In lipid bilayers, Bay K 8644 had no effect on either single-channel current amplitude or open probability of the cardiac ryanodine receptor. It is concluded that Bay K 8644 activates SR Ca2+ release at rest, independent of Ca2+ influx and perhaps through a functional linkage between the sarcolemmal dihydropyridine receptor and the SR ryanodine receptor. In contrast, caffeine and ryanodine modulate Ca2+ sparks by a direct action on the SR Ca2+ release channels.

Single channel function of recombinant type-1 inositol 1,4,5-trisphosphate receptor ligand binding domain splice variants.

In this study we describe the expression and function of the two rat type-1 inositol 1,4,5-trisphosphate receptor (InsP3R) ligand binding domain splice variants (SI+/-/SII+). Receptor protein from COS-1 cells transfected with the type-1 InsP3R expression plasmids (pInsP3R-T1, pInsP3R-T1ALT) or control DNA were incorporated into planar lipid bilayers and the single channel properties of the recombinant receptors were defined. The unitary conductance of the two splice variants were approximately 290 pS with Cs+ as charge carrier and approximately 65 pS with Ca2+ as charge carrier. Both InsP3R expression products consistently behaved like those of the native type-1 receptor isoform isolated from cerebellum in terms of their InsP3, Ca2+, and heparin sensitivity. An InsP3 receptor ligand binding domain truncation lacking the 310 amino-terminal amino acids (pInsP3R-DeltaT1ALT) formed tetrameric complexes but failed to bind InsP3 with high affinity, and did not form functional Ca2+ channels when reconstituted in lipid bilayers. These data suggest that 1) the ligand binding alternative splice site is functionally inert in terms of InsP3 binding and single channel function, and 2) the single channel properties of the expressed recombinant type-1 channel are essentially identical to those of the native channel. This work establishes a foundation from which molecular/biophysical approaches can be used to define the structure-function properties of the InsP3 receptor channel family.

Isoform-specific function of single inositol 1,4,5-trisphosphate receptor channels.

The inositol 1,4,5-trisphosphate receptor (InsP3R) family of Ca2+ release channels is central to intracellular Ca2+ signaling in mammalian cells. The InsP3R channels release Ca2+ from intracellular compartments to generate localized Ca2+ transients that govern a myriad of cellular signaling phenomena (Berridge, 1993. Nature. 361:315-325; Joseph, 1996. Cell Signal. 8:1-7; Kume et al., 1997. Science. 278:1940-1943; Berridge, 1997. Nature. 368:759-760). express multiple InsP3R isoforms, but only the function of the single type 1 InsP3R channel is known. Here the single-channel function of single type 2 InsP3R channel is defined for the first time. The type 2 InsP3R forms channels with permeation properties similar to that of the type 1 receptor. The InsP3 regulation and Ca2+ regulation of type 1 and type 2 InsP3R channels are strikingly different. Both InsP3 and Ca2+ are more effective at activating single type 2 InsP3R, indicating that single type 2 channels mobilize substantially more Ca2+ than single type 1 channels in cells. Furthermore, high cytoplasmic Ca2+ concentrations inactivate type 1, but not type 2, InsP3R channels. This indicates that type 2 InsP3R channel is different from the type 1 channel in that its activity will not be inherently self-limiting, because Ca2+ passing through an active type 2 channel cannot feed back and turn the channel off. Thus the InsP3R identity will help define the spatial and temporal nature of local Ca2+ signaling events and may contribute to the segregation of parallel InsP3 signaling cascades in mammalian cells.

Identification and functional reconstitution of the type 2 inositol 1,4,5-trisphosphate receptor from ventricular cardiac myocytes.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ release channel that mediates the rise in cytoplasmic calcium in response to receptor-activated production of InsP3. The InsP3R-mediated signaling pathway appears to be ubiquitous and is involved in many cellular processes including cell division, smooth muscle contraction, and neuronal signaling. Different regions of the heart also express InsP3 receptors. We report here that acutely dissociated ventricular myocytes from ferret and rat hearts express significant levels of InsP3R as indicated by immunoblotting with a receptor consensus antibody. InsP3 binding experiments (KD = 23.6 nM and Bmax = 0.46 pmol/mg) suggest the myocytes contain the high affinity type 2 InsP3 receptor. Exhaustive mRNA screening by polymerase chain reaction, RNase protection, and subsequent DNA sequencing positively identify the InsP3R as type 2. The type 2 receptor from ferret heart was then incorporated into planar lipid bilayers and formed Ca2+-selective, InsP3-activated, heparin-blocked ion channels. We conclude that the predominant InsP3 receptor isoform expressed in cardiac myocytes is type 2 and that it forms a functional InsP3-gated Ca2+ channel when reconstituted in planar lipid bilayers.

Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis.

We describe a high temporal resolution confocal spot microfluorimetry setup which makes possible the detection of fluorescence transients elicited by Ca2+ indicators in response to large (50-200 microM), short duration (< 100 ns), free [Ca2+] transients generated by laser flash photolysis of DM-nitrophen (DM-n; caged Ca2+). The equilibrium and kinetic properties of the commercially available indicators Fluo-3, Rhod-2, CalciumOrange-5N (COr-5N) and CalciumGreen-2 (CGr-2) were determined experimentally. The data reveal that COr-5N displays simple, fast response kinetics while, in contrast, Fluo-3, Rhod-2 and CGr-2 are characterized by significantly slower kinetic properties. These latter indicators may be unsuitable for tracking Ca2+ signaling events lasting only a few milliseconds. A model which accurately predicts the time course of fluorescence transients in response to rapid free [Ca2+] changes was developed. Experimental data and model predictions concur only when the association rate constant of DM-n is approximately 20 times faster than previously reported. This work establishes a quantitative theoretical framework for the study of fast Ca2+ signaling events and the use of flash photolysis in cells and model systems.

Adaptation of single cardiac ryanodine receptor channels.

Single cardiac ryanodine receptor (RyR) channel adaptation was previously defined with Ca2+ stimuli produced by flash photolysis of DM-nitrophen (caged-Ca+2). Photolysis of DM-nitrophen induced a very fast Ca+2 overshoot (Ca+2 spike) at the leading edge of the Ca+2 stimuli. It has been suggested that adaptation (tau approximately 1.3 s) may reflect Ca+2 slowly coming off the RyR Ca+2 activation sites following the faster Ca+2 spike (tau approximately 1 ms). This concern was addressed by defining the Ca2+ deactivation kinetics of single RyR channels in response to a rapid reduction in free Ca2+ concentration ([Ca2+]FREE). The [Ca2+]FREE was lowered by photolysis of Diazo-2. Single RyR channels deactivated (tau approximately 5.3 ms) quickly in response to the photolytically induced [Ca2+]FREE reduction. Improved estimates of the Ca2+ spike time course indicate that the Ca2+ spike is considerably faster (10-100-fold) than previously thought. Our data suggest that single RyRs are not significantly activated by fast Ca2+ spikes and that RyR adaptation is not due to deactivation following the fast Ca2+ spike. Thus, RyR adaptation may have an important impact on Ca2+ signaling in heart.

Bcl-x(L) forms an ion channel in synthetic lipid membranes.

Bcl-2-related proteins are critical regulators of cell survival that are localized to the outer mitochondrial, outer nuclear and endoplasmic reticulum membranes. Despite their physiological importance, the biochemical function of Bcl-2-related proteins has remained elusive. The three-dimensional structure of Bcl-xL, an inhibitor of apoptosis, was recently shown to be similar to the structures of the pore-forming domains of bacterial toxins. A key feature of these pore-forming domains is the ability to form ion channels in biological membranes. Here we demonstrate that Bcl-xL shares this functional feature. Like the bacterial toxins, Bcl-xL can insert into either synthetic lipid vesicles or planar lipid bilayers and form an ion-conducting channel. This channel is pH-sensitive and becomes cation-selective at physiological pH. The ion-conducting channel(s) formed by Bcl-xL display multiple conductance states that have identical ion selectivity. Together, these data suggest that Bcl-xL may maintain cell survival by regulating the permeability of the intracellular membranes to which it is distributed.