Biomedical subjects

G L Smith

Publications and source records attributed to G L Smith.

At least 19 recordsLinked to original sources

PubMed2005

S100A1 increases the gain of excitation-contraction coupling in isolated rabbit ventricular cardiomyocytes.

The effect of S100A1 protein on cardiac excitation-contraction (E-C) coupling was studied using recombinant human S100A1 protein (0.01-10 microM) introduced into single rabbit ventricular cardiomyocytes via a patch pipette. Voltage clamp experiments (20 degrees C) indicated that 0.1 microM S100A1 increased Ca(2+) transient amplitude by approximately 41% but higher or lower S100A1 concentrations had no significant effect. L-type Ca(2+) current amplitude or Ca(2+) efflux rates via the Na(+)/Ca(2+) exchanger (NCX) were unaffected. The rate of Ca(2+) uptake associated with the SR Ca(2+)-ATPase (SERCA2a) was increased by approximately 22% with 0.1 microM S100A1, but not at other S100A1 concentrations. Based on the intracellular Ca(2+) and I(NCX) signals in response to 10 mM caffeine, no significant change in SR Ca(2+) content was observed with S100A1 (0.01-10 microM). Therefore, 0.1 microM S100A1 appeared to increase the fractional Ca(2+) release from the SR. This result was confirmed by measurements of Ca(2+) transient amplitude at a range of SR Ca(2+) contents. The hyperbolic relationship between these two parameters was shifted to the left by 0.1 microM S100A1. [(3)H]-ryanodine binding studies indicated that S100A1 increased ryanodine receptor (RyR) activity at 0.1 and 0.3 microM Ca(2). As with the effects on E-C coupling, 0.1 microM S100A1 produced the largest effect. Co-immunoprecipitation studies on a range of Ca(2+)-handling proteins support the selective interaction of S100A1 on SERCA2a and RyR. In summary, S100A1 had a stimulatory action on RyR2 and SERCA2a in rabbit cardiomyocytes. Under the conditions of this study, the net effect of this dual action is to enhance the Ca(2+) transient amplitude without significantly affecting the SR Ca(2+) content.

Animals↗

PubMed2005

Knowledge of blood pressure levels and targets in patients with coronary artery disease in the USA.

Little is known about patient awareness of nationally recommended blood pressure targets, especially among patients with cardiac disease. To examine this issue, we interviewed 738 patients hospitalized with coronary artery disease to assess their knowledge of their systolic and diastolic blood pressure levels as well as corresponding national targets. We used bivariate and multivariate analyses to determine if any patient demographic or clinical characteristics were associated with blood pressure knowledge. Only 66.1% of patients could recall their own systolic and diastolic blood pressure levels. Only 48.9% of all patients could correctly name targets for these values. Knowledge of target blood pressure levels was particularly poor among patients who were female (odds ratio (OR) 0.69; 95% confidence interval (CI) 0.49-0.98), aged > or =60 years (OR 0.70, CI 0.51-0.97), without any college education (OR 0.48, CI 0.35-0.65), without a documented history of hypertension (OR 0.57, CI 0.39-0.84), and with known diabetes (OR 0.46, CI 0.33-0.66). Patients in the highest risk group, according to Joint National Committee guidelines stratification, were no more knowledgeable about their blood pressure levels and targets than lower risk patients. A significant proportion of patients hospitalized with coronary artery disease do not know their own blood pressure levels or targets. Current blood pressure education efforts appear inadequate, particularly for certain patient subgroups in which hypertension is an important modifiable risk factor.

Aged↗

PubMed2004

Comparison of Ca2+ release and uptake characteristics of the sarcoplasmic reticulum in isolated horse and rabbit cardiomyocytes.

Both the cardiac action potential duration (APD) (0.6-1 s) and resting heart rate (30-40 beats/min) in the horse are significantly different from humans and smaller mammals, including the rabbit. This would be anticipated to have consequences for excitation-contraction (EC) coupling and require adaptation of the individual processes involved. The sarcoplasmic reticulum (SR) is one of the main components involved in EC coupling. This study examines and compares the activity of this organelle in the horse with that of the rabbit. In particular, the study focuses on SR Ca2+ release via the Ca2+ release channel/ryanodine receptor (RyR2) and Ca2+ uptake via the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump. Isolated cardiomyocytes from both horse and rabbit hearts were permeabilized, bathed in a mock intracellular solution, and exposed to a specified [Ca2+]. Rabbit cardiomyocytes exposed to 260 nM [Ca2+] produced spontaneous Ca2+ release and propagated Ca2+ waves. Horse cells failed to produce Ca2+ waves; instead, only local release in the form of Ca2+ sparks was evident. However, at 550 nM [Ca2+], Ca2+ waves were produced in both species. Ca2+ waves were four times less frequent yet approximately 1.5 times greater in amplitude in the horse compared with the rabbit. Ca2+ wave velocity was comparable between the species. The reason for this disparity in Ca2+ wave characteristics is unknown. Separate measurements of oxalate-supported Ca2+ uptake into the SR suggest that both horse and rabbit cardiomyocytes have comparable levels SERCA activity. The possible reasons for the observed differences in SR Ca2+ release between the horse and rabbit are discussed.

Animals↗

PubMed2004

The electrophysiological and mechanical effects of 2,3-butane-dione monoxime and cytochalasin-D in the Langendorff perfused rabbit heart.

UNLABELLED: Procedures that reduce contraction are used to facilitate optical measurements of membrane potential, but it is unclear to what extent they affect the excitability of the heart. This study has examined the electrophysiological consequences of a range of extracellular [Ca2+] (0.7-2.5 mmol l(-1)), 2,3-butane-dione monoxime (BDM; 1-20 mmol l(-1)) and cytochalasin-D (Cyto-D; 1-5 micromol l(-1)). METHODS: Monophasic action potentials (MAPs) were recorded from the basal epicardial surface of the left ventricle of isolated rabbit hearts. Conduction delay (CD) and time to 90% repolarisation of the monophasic action potential (MAPD90) were measured. The effects of BDM and Cyto-D on restitution were studied at a [Ca2+] of 1.9 mmol l(-1). Restitution curves for MAPD90 were generated using a standard S1-S2 protocol. RESULTS: All manoeuvres decreased left ventricular developed pressure (LVDP): 0.7 mmol l(-1) Ca2+ to 74.0 +/- 6.1%, 20 mmol l(-1) BDM to 4.5 +/- 1.0%, and 5 micromol l(-1) Cyto-D to 12.8 +/- 3.5% of control value. CD decreased from a control value (33.3 +/- 1.0 ms, n= 16) to 93.0 +/- 2.2% in 0.7 mmol l(-1) Ca2+, but increased to 133.7 +/- 10.5% in 20 mmol l(-1) BDM and 127.4 +/- 10.6% in 5 micromol l(-1) Cyto-D. At 350 ms pacing cycle length, MAPD90 (control = 119.6 +/- 1.7 ms n= 16) was prolonged by reduced extracellular [Ca2+]. BDM had no effects on MAPD90 at control pacing rates. Cyto-D caused a significant prolongation (to 115.0 +/- 3.0% of control, n= 6) at the highest concentration studied (5 micromol l(-1)). Both BDM (20 mmol l(-1)) and Cyto-D (3 micromol l(-1)) flattened the restitution curves but neither agent altered maximum MAPD90. CONCLUSIONS: Extracellular [Ca2+] of 1.9 mmol l(-1) in conjunction with a moderate dose of Cyto-D (3 micromol l(-1)) reduced contractility with minimal effects on action potential duration and conduction at a fixed pacing cycle length. However, both BDM and Cyto-D had pronounced effects on electrical restitution.

Action Potentials↗

PubMed2004

Over-expression of FK506-binding protein FKBP12.6 alters excitation-contraction coupling in adult rabbit cardiomyocytes.

This study investigated the function of FK506-binding protein (FKBP12.6) using adenoviral-mediated gene transfer to over-express FKBP12.6 (Ad-FKBP12.6) in adult rabbit ventricular cardiomyocytes. Infection with a beta-galactosidase-expressing adenovirus (Ad-LacZ) was used as a control. Peak-systolic intracellular [Ca(2+)] (measured with Fura-2) was higher in the Ad-FKBP12.6 group compared to Ad-LacZ (1 Hz field stimulation at 37 degrees C). The amplitude of caffeine-induced Ca(2+) release was also greater, indicating a higher SR Ca(2+) content in the Ad-FKBP12.6 group. Voltage clamp experiments indicated that FKBP12.6 over-expression did not change L-type Ca(2+) current amplitude or Ca(2+) efflux rates via the Na(+)-Ca(2+) exchanger. Ca(2+) transients comparable to those after Ad-FKBP12.6 transfection could be obtained by enhancing SR Ca(2+) content of Ad-LacZ infected cells with periods of high frequency stimulation. Line-scan confocal microscopy (Fluo-3 fluorescence) of intact cardiomyocytes stimulated at 0.5 Hz (20-21 degrees C) revealed a higher degree of synchronicity of SR Ca(2+) release and fewer non-responsive Ca(2+) release sites in the Ad-FKBP12.6 group compared to control. Ca(2+) spark morphology was measured in beta-escin-permeabilized cardiomyocytes at a free [Ca(2+)](i) of 150 nm. The average values of the spark parameters (amplitude, duration, width and frequency) were reduced in the Ad-FKBP12.6 group. Increasing [Ca(2+)](i) to 400 nm caused coherent propagating Ca(2+) waves in the Ad-FKBP12.6 group but only limited Ca(2+) release events were recorded in the control group. These data indicate that FKBP12.6 over-expression enhances Ca(2+) transient amplitude predominately by increasing SR Ca(2+) content. Moreover, there is also evidence that FKBP12.6 can enhance the coupling between SR Ca(2+) release sites independently of SR content.

Adenoviridae↗

PubMed2004

Characterization of a range of fura dyes with two-photon excitation.

Two-photon excitation (TPE) spectra of Fura-2, -4F, -6F, -FF, and Furaptra were characterized using a tunable (750-850 nM) ultra-short pulse laser. Two-photon fluorescence of these dyes was studied in free solution and in the cytosol of isolated rabbit ventricular cardiomyocytes. The TPE spectra of the Ca(2+)-free and Ca(2+)-bound forms of the dyes were measured in free solution and expressed in terms of the two-photon fluorescence cross section (Goppert-Meyer units). The Fura dyes displayed the same Ca(2+)-free TPE spectrum in the intracellular volume of permeabilized and intact cardiomyocytes. Fluorescence measurements over a range of laser powers confirmed the TPE of both Ca(2+)-free and Ca(2+)-bound forms of the dyes. Single-wavelength excitation at 810 nM was used to determine the effective dissociation constants (K(eff)) and dynamic ranges (R(f)) of Fura-2, -4F, -6F, -FF, and Furaptra dyes (K(eff) = 181 +/- 52 nM, 1.16 +/- 0.016 micro M, 5.18 +/- 0.3 micro M, 19.2 +/- 1 micro M, and 58.5 +/- 2 micro M; and R(f) = 22.4 +/- 3.8, 12.2 +/- 0.34, 6.3 +/- 0.17, 16.1 +/- 2.8, and 25.4 +/- 4, respectively). Single-wavelength excitation of intracellular Fura-4F resolved diastolic and peak [Ca(2+)] in isolated stimulated cardiomyocytes after calibration of the intracellular signal using reversible exposure to low (100 micro M) extracellular [Ca(2+)]. Furthermore, TPE of Fura-4F allowed continuous, long-term (5-10 min) Ca(2+) imaging in ventricular cardiomyocytes using laser-scanning microscopy without significant cellular photodamage or photobleaching of the dye.

Animals↗

PubMed2003

Two-photon microscopy of fura-2-loaded cardiac myocytes with an all-solid-state tunable and visible femtosecond laser source.

We report a robust and reliable platform source for visible-wavelength multiphoton microscopy that is based on nonlinear optical methods. We demonstrate a synchronously pumped, singly resonant optical parametric oscillator with simultaneous intracavity third-order quasi-phase matching in a single crystal that generates continuously tunable, visible, and femtosecond-pulsed radiation. The application of the system is demonstrated by two-photon laser-scanning fluorescence microscopy of rabbit cardiac myocytes loaded with the fluorescent Ca2+ indicator fura-2.

Animals↗

PubMed2003

Myocardial infarction causes increased expression but decreased activity of the myocardial Na+-Ca2+ exchanger in the rabbit.

Na+-Ca2+ exchanger (NCX) protein levels and activity were measured in myocardium from the basal region of the left ventricle of rabbit hearts with significant left ventricular dysfunction (LVD), 8-9 weeks after an apical infarction. NCX protein abundance was higher in the tissue homogenates (121 +/- 11%) and purified membrane fractions (143 +/- 12%) in the LVD compared to the sham-operated (sham) group. NCX mRNA was also higher in the LVD group (126%). Lower NCX protein expression was observed in the membrane fractions from the epicardium compared to the endocardium in both the sham and LVD groups. Transmembrane currents were recorded in isolated cardiomyocytes by single-electrode voltage clamp; [Ca2+]i was measured using Fura-2. Rapid application of 10 mmol l-1 caffeine was used to induce Ca2+ release from the sarcoplasmic reticulum. The subsequent NCX-mediated Ca2+ efflux rate constant was lower (70% of sham) in the LVD group. NCX currents were measured in cardiomyocytes dialysed with 250 nM Ca2+ (50 mmol l-1 EGTA). A lower NCX current (75% of sham) was observed in the LVD group. Lower NCX activity was also observed in cardiomyocytes isolated from the epicardium compared to the endocardium; a transmural difference that was also seen in the LVD group. Reduced activity despite increased protein expression may result from reduced Ca2+ sensitivity of the allosteric regulation of NCX. However, measurements indicated increased Ca2+ sensitivity in the LVD group. Cardiomyocytes from LVD hearts displayed a marked reduction in the transverse tubule area (59% of sham) and the surface area/volume ratio (80% of sham). Disrupted transverse tubule structure may contribute to the decrease in NCX activity despite increased protein expression in LVD.

Algorithms↗

PubMed2003

Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics.

The Ca(2+) dissociation constant (K(d)) of Fluo-3 was determined using confocal fluorescence microscopy in two different situations: (i) within the cytosol of a permeabilised cardiomyocyte; and (ii) in an intact cardiomyocyte after incubation with the acetoxymethyl ester form of Fluo-3 (AM). Measurements were made on isolated rabbit ventricular cardiomyocytes after permeabilisation by a brief treatment with beta-escin (0.1mg/ml) and equilibration with 10 microM Fluo-3. The K(d) of Fluo-3 within the cytosol was not significantly different from that in free solution (558 +/- 15 nM, n=6). Over a range of cytoplasmic [Ca(2+)], the minimum [Ca(2+)] values between Ca(2+) waves was relatively constant despite changes in wave frequency. After loading intact cardiomyocytes with Fluo-3 by incubation with the -AM, spontaneous Ca(2+) waves were produced by incubation with strophanthidin (10 microM). By assuming a common minimum [Ca(2+)] in permeabilised and intact cells, the intracellular K(d) of Fluo-3 in intact myocytes was estimated to be 898 +/-64 nM (n=6). Application of this K(d) to fluorescence records shows that Ca(2+) waves in intact cells have similar amplitudes to those in permeabilised cells. Stimulation of cardiac myocytes at 0.5 Hz in the absence of strophanthidin (room temperature) resulted in a Ca(2+) transient with a maximum and minimum [Ca(2+)] of 1190 +/- 200 and 158 +/- 30 nM (n=11), respectively.

Aniline Compounds↗

PubMed2003

Human brain regions required for the dividing and switching of attention between two features of a single object.

This combined PET and ERP study was designed to identify the brain regions activated in switching and divided attention between different features of a single object using matched sensory stimuli and motor response. The ERP data have previously been reported in this journal [64]. We now present the corresponding PET data. We identified partially overlapping neural networks with paradigms requiring the switching or dividing of attention between the elements of complex visual stimuli. Regions of activation were found in the prefrontal and temporal cortices and cerebellum. Each task resulted in different prefrontal cortical regions of activation lending support to the functional subspecialisation of the prefrontal and temporal cortices being based on the cognitive operations required rather than the stimuli themselves.

Adult↗

PubMed2003

Ca(2+)-handling proteins and heart failure: novel molecular targets?

Calcium (Ca(2+)) ions are the currency of heart muscle activity. During excitation-contraction coupling Ca(2+) is rapidly cycled between the cytosol (where it activates the myofilaments) and the sarcoplasmic reticulum (SR), the Ca(2+) store. These fluxes occur by the transient activity of Ca(2+)-pumps and -channels. In the failing human heart, changes in activity and expression profile of Ca(2+)-handling proteins, in particular the SR Ca(2+)-ATPase (SERCA2a), are thought to cause an overall reduction in the amount of SR-Ca(2+) available for contraction. In the steady state, the Ca(2+)-content of the SR is essentially a balance between Ca(2+)-uptake via SERCA2a pump and Ca(2+)-release via the cardiac SR Ca(2+)-release channel complex (Ryanodine receptor, RyR2). This review discusses current pharmacological options available to enhance cardiac SR Ca(2+) content and the implications of this approach as an inotropic therapy in heart failure. Two options are considered: (i) activation of the SERCA2a pump to increase SR Ca(2+)-uptake, and (ii) reduction of SR Ca(2+)-leakage through RyR2. RyR2 forms a macromolecular complex with a number of regulatory proteins that either remain permanently bound or that interact in a time- and/or Ca(2+)-dependant manner. These regulatory proteins can dramatically affect RyR2 function, e.g. over-expression of the accessory protein FK 506-binding protein 12.6 (FKBP12.6) has recently been shown to reduce SR Ca(2+)-leak. Recent attempts to design positive inotropes for chronic administrations have focussed on the use of phosphodiesterase III inhibitors (PDE III inhibitors). These compounds, which increase intracellular cAMP-levels, have failed in clinical trials. Therefore medical researchers are seeking new drugs that act through alternative pathways. Novel cardiac inotropes targeting SR Ca(2+)-cycling proteins may have the potential to fill this gap.

Calcium↗

PubMed2002

The relationship between intracellular [Ca(2+)] and Ca(2+) wave characteristics in permeabilised cardiomyocytes from the rabbit.

Spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and propagated intracellular Ca(2+) waves are a consequence of cellular Ca(2+) overload in cardiomyocytes. We examined the relationship between average intracellular [Ca(2+)] and Ca(2+) wave characteristics. The amplitude, time course and propagation velocity of Ca(2+) waves were measured using line-scan confocal imaging of beta-escin-permeabilised cardiomyocytes perfused with 10 microM Fluo-3 or Fluo-5F. Spontaneous Ca(2+) waves were evident at cellular [Ca(2+)] > 200 nM. Peak [Ca(2+)] during a wave was 2.0-2.2 microM; the minimum [Ca(2+)] between waves was 120-160 nM; wave frequency was approximately 0.1 Hz. Raising mean cellular [Ca(2+)] caused increases in all three parameters, particularly Ca(2+) wave frequency. Increases in the rate of SR Ca(2+) release and Ca(2+) uptake were observed at higher cellular [Ca(2+)], indicating calcium-sensitive regulation of these processes. At extracellular [Ca(2+)] > 2 microM, the mean [Ca(2+)] inside the permeabilised cell did not increase above 2 microM. This extracellular-intracellular Ca(2+) gradient could be maintained for periods of up to 5 min before the cardiomyocyte developed a sustained and irreversible hypercontraction. Inclusion of mitochondrial inhibitors (2 microM carbonyl cyanide m-chlorophenylhydrazone and 2 microM oligomycin) while perfusing with > 2 microM Ca(2+) abolished the extracellular-intracellular Ca(2+) gradient through the generation of Ca(2+) waves with a higher peak [Ca(2+)] compared to control conditions. Under these conditions, cardiomyocytes rapidly (< 2 min) developed a sustained and irreversible contraction. These results suggest that mitochondrial Ca(2+) uptake acts to delay an increase in [Ca(2+)] by blunting the peak of the Ca(2+) wave.

Animals↗

PubMed2001

S100A1: a regulator of myocardial contractility.

S100A1, a Ca(2+) binding protein of the EF-hand type, is preferentially expressed in myocardial tissue and has been found to colocalize with the sarcoplasmic reticulum (SR) and the contractile filaments in cardiac tissue. Because S100A1 is known to modulate SR Ca(2+) handling in skeletal muscle, we sought to investigate the specific role of S100A1 in the regulation of myocardial contractility. To address this issue, we investigated contractile properties of adult cardiomyocytes as well as of engineered heart tissue after S100A1 adenoviral gene transfer. S100A1 gene transfer resulted in a significant increase of unloaded shortening and isometric contraction in isolated cardiomyocytes and engineered heart tissues, respectively. Analysis of intracellular Ca(2+) cycling in S100A1-overexpressing cardiomyocytes revealed a significant increase in cytosolic Ca(2+) transients, whereas in functional studies on saponin-permeabilized adult cardiomyocytes, the addition of S100A1 protein significantly enhanced SR Ca(2+) uptake. Moreover, in Triton-skinned ventricular trabeculae, S100A1 protein significantly decreased myofibrillar Ca(2+) sensitivity ([EC(50%)]) and Ca(2+) cooperativity, whereas maximal isometric force remained unchanged. Our data suggest that S100A1 effects are cAMP independent because cellular cAMP levels and protein kinase A-dependent phosphorylation of phospholamban were not altered, and carbachol failed to suppress S100A1 actions. These results show that S100A1 overexpression enhances cardiac contractile performance and establish the concept of S100A1 as a regulator of myocardial contractility. S100A1 thus improves cardiac contractile performance both by regulating SR Ca(2+) handling and myofibrillar Ca(2+) responsiveness.

Actin Cytoskeleton↗

PubMed2001

A prime-boost immunisation regimen using DNA followed by recombinant modified vaccinia virus Ankara induces strong cellular immune responses against the Plasmodium falciparum TRAP antigen in chimpanzees.

Two chimpanzees were vaccinated intramuscularly against malaria using plasmid DNA expressing the pre-erythrocytic antigens thrombospondin related adhesion protein (PfTRAP) and liver stage specific antigen-1 (PfLSA-1) of Plasmodium falciparum together with GM-CSF protein. A recombinant modified vaccinia virus Ankara (MVA) expressing PfTRAP was injected intramuscularly 6 weeks later to boost the immune response. This sequence of antigen delivery induced a specific and long-lasting T cell and antibody response to PfTRAP as detected by ELISPOT assay and ELISA. Antibody responses were detected after four DNA injections, and were boosted by injection of recombinant MVA expressing PfTRAP. Interferon-gamma secreting antigen-specific T cells were detected in both animals, but only after boosting with recombinant MVA. By screening a panel of PfTRAP-derived peptides, an epitope was identified that was recognized by cytotoxic T lymphocytes in one of the chimpanzees studied. T cells specific for this epitope were present in PBMCs and liver-infiltrating lymphocytes at a frequency of between 1 in 200 and 1 in 500. The high immunogenicity of this prime-boost regimen in chimpanzees supports further assessment of this delivery strategy for the induction of protection against P. falciparum malaria in humans.

Animals↗

PubMed2001

Vaccinia virus utilizes microtubules for movement to the cell surface.

Vaccinia virus (VV) egress has been studied using confocal, video, and electron microscopy. Previously, intracellular-enveloped virus (IEV) particles were proposed to induce the polymerization of actin tails, which propel IEV particles to the cell surface. However, data presented support an alternative model in which microtubules transport virions to the cell surface and actin tails form beneath cell-associated enveloped virus (CEV) particles at the cell surface. Thus, VV is unique in using both microtubules and actin filaments for egress. The following data support this proposal. (a) Microscopy detected actin tails at the surface but not the center of cells. (b) VV mutants lacking the A33R, A34R, or A36R proteins are unable to induce actin tail formation but produce CEV and extracellular-enveloped virus. (c) CEV formation is inhibited by nocodazole but not cytochalasin D or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP1). (d) IEV particles tagged with the enhanced green fluorescent protein fused to the VV B5R protein moved inside cells at 60 microm/min. This movement was stop-start, was along defined pathways, and was inhibited reversibly by nocodazole. This velocity was 20-fold greater than VV movement on actin tails and consonant with the rate of movement of organelles along microtubules.

Actin Cytoskeleton↗

PubMed2001

A comparison of the effects of ATP and tetracaine on spontaneous Ca(2+) release from rat permeabilised cardiac myocytes.

1. Fluo-3 fluorescence measurements were made in isolated beta-escin permeablised rat cardiac myocytes using confocal microscopy. Perfusion of a mock intracellular solution containing 0.22-0.23 microM Ca(2+) and 5 mM ATP elicited regular waves of Ca(2+) (approximately every 5 s) due to spontaneous release of Ca(2+) from the sarcoplasmic reticulum (SR). 2. An approximately linear relationship was noted between Ca(2+) wave velocity (v) and amplitude (sigma). Under the control conditions the ratio of velocity to amplitude (v/sigma) varied little and was 99.8 +/- 2.5 m s(-1) microM(-1) (n = 78). 3. Reduction of [ATP] in the bathing solution to 0.5 and 0.2 mM ATP progressively decreased Ca(2+) wave frequency and propagation velocity while increasing the amplitude. The changes in Ca(2+) wave characteristics in 0.5 mM ATP were similar to those observed during perfusion with 50 microM tetracaine. In 0.2 mM ATP the decline of [Ca(2+)] during a Ca(2+) wave was slowed suggesting a lowered rate of Ca(2+) re-uptake by the SR Ca(2+) pump. 4. Reduction of [ATP] to 0.1 mM abolished Ca(2+) waves after 15-20 s. Returning the [ATP] to 5 mM caused a burst of high frequency and large amplitude waves. Mean velocity of the first wave on returning to 5 mM ATP was larger than normal but the v/sigma value was 32 +/- 6 % of control (n = 6). In the similar burst on removal of 100 microM tetracaine v/sigma was higher than control (166 +/- 9 %, n = 6). 5. Rapid application of caffeine (10 mM) was used to assess the SR Ca(2+) content. This showed that SR Ca(2+) increased as [ATP] was reduced or [tetracaine] was increased. The highest SR Ca(2+) content was observed after perfusion with 0.1 mM ATP, which was 245 +/- 15 % of control values. 6. Returning [ATP] from 0.1 mM to 5 mM caused a burst of high frequency, large amplitude Ca(2+) waves. But recovery after incubation with 300 microM tetracaine resulted in SR Ca(2+) release with no coherent wave pattern. The reason for this discrepancy is discussed.

Adenosine Triphosphate↗

PubMed2001

The genome sequence of Yaba-like disease virus, a yatapoxvirus.

The genome sequence of Yaba-like disease virus (YLDV), an unclassified member of the yatapoxvirus genus, has been determined. Excluding the terminal hairpin loops, the YLDV genome is 144,575 bp in length and contains inverted terminal repeats (ITRs) of 1883 bp. Within 20 nucleotides of the termini, there is a sequence that is conserved in other poxviruses and is required for the resolution of concatemeric replicative DNA intermediates. The nucleotide composition of the genome is 73% A+T, but the ITRs are only 63% A+T. The genome contains 151 tightly packed open reading frames (ORFs) that either are > or =180 nucleotides in length or are conserved in other poxviruses. ORFs within 23 kb of each end are transcribed toward the termini, whereas ORFs within the central region of the genome are encoded on either DNA strand. In the central region ORFs have a conserved position, orientation, and sequence compared with vaccinia virus ORFs and encode many enzymes, transcription factors, or structural proteins. In contrast, ORFs near the termini are more divergent and in seven cases are without counterparts in other poxviruses. The YLDV genome encodes several predicted immunomodulators; examples include two proteins with similarity to CC chemokine receptors and predicted secreted proteins with similarity to MHC class I antigen, OX-2, interleukin-10/mda-7, poxvirus growth factor, serpins, and a type I interferon-binding protein. Phylogenic analyses indicated that YLDV is very closely related to yaba monkey tumor virus, but outside the yatapoxvirus genus YLDV is more closely related to swinepox virus and leporipoxviruses than to other chordopoxvirus genera.

Adjuvants, Immunologic↗

PubMed2001

Overexpression of FK506-binding protein FKBP12.6 in cardiomyocytes reduces ryanodine receptor-mediated Ca(2+) leak from the sarcoplasmic reticulum and increases contractility.

The FK506-binding protein FKBP12.6 is tightly associated with the cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel (ryanodine receptor type 2 [RyR2]), but the physiological function of FKBP12.6 is unclear. We used adenovirus (Ad)-mediated gene transfer to overexpress FKBP12.6 in adult rabbit cardiomyocytes. Western immunoblot and reverse transcriptase-polymerase chain reaction analysis revealed specific overexpression of FKBP12.6, with unchanged expression of endogenous FKBP12. FKBP12.6-transfected myocytes displayed a significantly higher (21%) fractional shortening (FS) at 48 hours after transfection compared with Ad-GFP-infected control cells (4.8+/-0.2% FS versus 4+/-0.2% FS, respectively; n=79 each; P:=0.001). SR-Ca(2+) uptake rates were monitored in beta-escin-permeabilized myocytes using Fura-2. Ad-FKBP12.6-infected cells showed a statistically significant higher rate of Ca(2+) uptake of 0.8+/-0.09 nmol/s(-)(1)/10(6) cells (n=8, P:<0.05) compared with 0.52+/-0.1 nmol/s(-)(1)/10(6) cells in sham-infected cells (n=8) at a [Ca(2+)] of 1 micromol/L. In the presence of 5 micromol/L ruthenium red to block Ca(2+) efflux via RyR2, SR-Ca(2+) uptake rates were not significantly different between groups. From these measurements, we calculate that SR-Ca(2+) leak through RyR2 is reduced by 53% in FKBP12.6-overexpressing cells. Caffeine-induced contractures were significantly larger in Ad-FKBP12.6-infected myocytes compared with Ad-GFP-infected control cells, indicating a higher SR-Ca(2+) load. Taken together, these data suggest that FKBP12.6 stabilizes the closed conformation state of RyR2. This may reduce diastolic SR-Ca(2+) leak and consequently increase SR-Ca(2+) release and myocyte shortening.

Adenoviridae↗