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BACKGROUND: Pneumonia is a leading cause of childhood mortality globally. Children with severe pneumonia associated with hypoxaemia require oxygen (O2) therapy, which is scarce across resource-constrained countries. Solar-powered oxygen (SPO2) is a novel technology developed for delivering therapeutic O2 in resource-constrained environments. RESEARCH QUESTION: Is the introduction of SPO2 associated with a reduction in mortality, relative to the existing practice? STUDY DESIGN: This was a pragmatic, quasi-experimental study comparing mortality amongst children < 5 years of age with hypoxaemic respiratory illness before and after the installation of SPO2 in two resource-constrained hospitals. METHODS: Participants were children < 5 years old admitted with acute hypoxaemic respiratory illness. The intervention was SPO2, installed at two resource-constrained hospitals. The primary outcome was 30-day mortality. Secondary outcomes included in-hospital mortality (time to death), length of hospital stay among survivors, duration of O2 therapy (time to wean O2), and O2 delivery system failure(s). RESULTS: Mortality amongst children admitted with acute hypoxaemic respiratory illness decreased from 30/50 (60%) pre-SPO2 to 15/50 (30%) post-SPO2 (relative risk reduction 50%, 95%CI 19 - 69, p = 0.0049). The post-SPO2 period was consistently associated with decreased mortality in statistical models adjusting for potential confounding factors. Likewise, survival curves pre- and post- SPO2 differed significantly (hazard ratio 0.39, 95% CI 0.20 - 0.74, p = 0.0043). A reduction in the frequency of O2 delivery interruptions due to fuel shortages and multiple patients needing the concentrator at once was observed, explaining the mortality reduction. INTERPRETATION: Solar-powered oxygen installation was associated with decreased mortality in resource-constrained settings.

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Ebola virus (EBOV) causes outbreaks of lethal febrile illness in Africa, the largest of which resulted in over 11,000 deaths and represented a global public health threat. A new biomedical countermeasure, the recombinant vesicular stomatitis virus expressing EBOV glycoprotein (rVSV-EBOV) has been licensed (Ervebo; Merck & Co.). rVSV-EBOV is a replicative viral vaccine engineered to express EBOV antigen. Following rapid development stimulated by the large West African epidemic, an open-label, cluster-randomized ring vaccination trial called Ebola Ça Suffit! in Guinea and Sierra Leone demonstrated strong efficacy. The vaccine has a good safety profile, but is associated with self-limited arthritis and rash in a minority of recipients. rVSV-EBOV is highly immunogenic after a single intramuscular dose with antibody titers persisting for at least 2 years. In the recent outbreak in the Democratic Republic of the Congo, rVSV-EBOV was administered to more than 300,000 individuals and may have contributed, at least in part, to controlling the epidemic.

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BACKGROUND: Stroke burden is highest and is still rising in low- and middle-income countries. Epidemiologic stroke data are lacking in many of these countries. Stroke prevalence in Argentina has been unexplored for almost three decades. AIM: This population-based study aims to determine prevalence of stroke in a representative sample of the Argentinean population. METHODS: We performed a door-to-door survey of randomly selected households in a city of 18,650 inhabitants. A structured questionnaire screening for potential stroke cases was used. All subjects screened positive were then evaluated by stroke neurologists for final adjudication. Data about stroke subtypes, neurological status, vascular risk factors, medications, and diagnostic tests were also collected. RESULTS: Among 2156 surveys, 294 were screened positive for a possible stroke. After neurological evaluation, there were 41 confirmed cases. The adjusted stroke prevalence was 1,974/100,000 inhabitants older than 40 years, and it was higher in men than in women (26.3‰ vs 13.2‰, p<0.01). Prevalence of ischemic stroke, intracranial hemorrhage, and transient ischemic attack were 15.8‰, 2.93‰, and 2.93‰, respectively. The most prevalent vascular risk factors in stroke survivors were hypertension, obstructive sleep apnea, and dyslipidemia. CONCLUSION: Approximately 2 in every 100 subjects older than 40 years in this population are stroke survivors. Stroke prevalence in Argentina has remained stable over the last 30 years; it is higher than in most Latin American countries and similar to western populations.

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BACKGROUND: Premature development of cardiovascular disease in children living with HIV-1 (CLWH) may be associated with compromised gut barrier function, microbial translocation, immune activation, systemic inflammation and endothelial activation. Biomarkers of these pathways may provide insights into pathogenesis of atherosclerotic disease in CLWH. METHODS: This was a cross-sectional study of CLWH enrolled in the multicentre Early Pediatric Initiation-Canadian Child Cure Cohort (EPIC4 ) who were on antiretroviral therapy (ART) with undetectable viral load. Plasma biomarkers of intestinal epithelial injury [intestinal fatty acid binding protein-1 (IFABP)], systemic inflammation [tumour necrosis factor (TNF) and interleukin-6 (IL-6)] and endothelial activation [angiopoietin-2 (Ang2), soluble vascular endothelial growth factor-1 (sVEGFR1) and soluble endoglin (sEng)] were quantified by enzyme-linked immunosorbent assay. Correlation and factor analysis of biomarkers were used to examine associations between innate immune pathways. RESULTS: Among 90 CLWH, 16% of Ang2, 15% of sVEGFR1 and 23% of sEng levels were elevated relative to healthy historic controls. Pairwise rank correlations between the three markers of endothelial activation were statistically significant (ρ = 0.69, ρ = 0.61 and ρ = 0.65, P < 0.001 for all correlations). An endothelial activation index, derived by factor analysis of the three endothelial biomarkers, was correlated with TNF (ρ = 0.47, P < 0.001), IL-6 (ρ = 0.60, P < 0.001) and intestinal fatty acid binding protein-1 (ρ = 0.67, P < 0.001). Current or past treatment with ritonavir-boosted lopinavir (LPV/r) was associated with endothelial activation (odds ratio = 5.0, 95% CI: 1.7-17, P = 0.0020). CONCLUSIONS: Endothelial activation is prevalent in CLWH despite viral suppression with combination ART and is associated with intestinal epithelial injury, systemic inflammation and treatment with LPV/r.

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BACKGROUND AND PURPOSE: The long-term outcomes and stroke recurrence after basilar artery occlusion (BAO) are largely unknown. We aimed to assess these variables in a comparatively large series of consecutive patients. METHODS: Adults with acute BAO were retrospectively identified from 1976 to 2011. Post-discharge records were reviewed to assess for stroke recurrences, mortality and disability. Exploratory analysis of survival was carried out using Kaplan-Meier and log-rank tests. Factors associated with survival time were determined using Cox models. RESULTS: A total of 86 patients (34% female, median age 72 [interquartile range (IQR), 60-79] years) with a median National Institutes of Health Stroke Scale score of 11 (IQR, 6-27) were included. Twenty-nine patients (34%) died during the initial hospitalization. Median modified Rankin Scale (mRS) score at discharge among survivors was 4 (IQR, 2.5-5.5). At 1 and 5 years, 70% of survivors ad a mRS ≤3. Seventeen patients had recurrent strokes during the hospitalization and 12 patients had 19 recurrent strokes after discharge. The median survival time was 52 days (IQR, 6-1846). Older age per decade on admission [adjusted hazard ratios (aHR), 1.32; 95% confidence interval (CI), 1.05-1.66, P = 0.02] and a higher mRS at discharge (aHR, 4.48; 95% CI, 2.72-7.39, P < 0.0001) were associated with mortality. Patients who were not treated with any reperfusion therapy had a trend towards reduced mortality (aHR, 0.39; 95% CI, 0.14-1.08, P = 0.07). CONCLUSIONS: Survivors from BAO had severe short-term functional disability. Most deaths and stroke recurrences occurred within the first year following the initial event. The risk of death was higher in older and more disabled survivors. However, favorable long-term recovery was possible.

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The seemingly transparent wings of many insects have recently been found to display unexpected structural coloration. These structural colours (wing interference patterns: WIPs) may be involved in species recognition and mate choice, yet little is known about the evolutionary processes that shape them. Furthermore, to date investigations of WIPs have not fully considered how they are actually perceived by the viewers' colour vision. Here, we use multispectral digital imaging and a model of Drosophila vision to compare WIPs of male and female Drosophila simulans from replicate populations forced to evolve with or without sexual selection for 68 generations. We show that WIPs modelled in Drosophila vision evolve in response to sexual selection and provide evidence that WIPs correlate with male sexual attractiveness. These findings add a new element to the otherwise well-described Drosophila courtship display and confirm that wing colours evolve through sexual selection.

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The BA allele of the Drosophila cytochrome P450 gene Cyp6g1 confers resistance to a range of insecticides. It is also subject to intralocus sexual conflict when introgressed into the Canton-S background, whose collection predates the widespread use of insecticides. In this genetic background, the allele confers a pleiotropic fitness benefit to females but a cost to males, and exhibits little sexual dimorphism in conferred insecticide resistance. It is unclear whether these sexually antagonistic effects also exist in current populations that have naturally evolved with insecticides, where genetic modifiers that offset male costs might be expected to evolve. Here, we explore these issues using Drosophila melanogaster caught recently from an Australian population in which the BA allele naturally segregates. While we find increased fecundity in insecticide-resistant BA females and no consistent evidence of fitness costs in males, experimental evolution indicates balancing selection at the locus. We suggest that this apparent discrepancy may be due to reduced investment in reproduction in resistant males. Our results at the population level are consistent with previous work, and suggest that individual-level fitness assays do not always capture sexually antagonistic fitness effects that emerge in a population context.

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SETTING: A resource-limited paediatric hospital in Uganda. OBJECTIVE: Pneumonia is a leading cause of child mortality worldwide. Access to life-saving oxygen therapy is limited in many areas. We designed and implemented a solar-powered oxygen delivery system for the treatment of paediatric pneumonia. DESIGN: Proof-of-concept pilot study. A solar-powered oxygen delivery system was designed and piloted in a cohort of children with hypoxaemic illness. RESULTS: The system consisted of 25 × 80 W photovoltaic solar panels (daily output 7.5 kWh [range 3.8-9.7kWh]), 8 × 220 Ah batteries and a 300 W oxygen concentrator (output up to 5 l/min oxygen at 88% [±2%] purity). A series of 28 patients with hypoxaemia were treated with solar-powered oxygen. Immediate improvement in peripheral blood oxygen saturation was documented (median change +12% [range 5-15%], P < 0.0001). Tachypnoea, tachycardia and composite illness severity score improved over the first 24 h of hospitalisation (P < 0.01 for all comparisons). The case fatality rate was 6/28 (21%). The median recovery times to sit, eat, wean oxygen and hospital discharge were respectively 7.5 h, 9.8 h, 44 h and 4 days. CONCLUSION: Solar energy can be used to concentrate oxygen from ambient air and oxygenate children with respiratory distress and hypoxaemia in a resource-limited setting.

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Sexual selection is responsible for the evolution of many elaborate traits, but sexual trait evolution could be influenced by opposing natural selection as well as genetic constraints. As such, the evolution of sexual traits could depend heavily on the environment if trait expression and attractiveness vary between environments. Here, male Drosophila simulans were reared across a range of diets and temperatures, and we examined differences between these environments in terms of (i) the expression of male cuticular hydrocarbons (CHCs) and (ii) which male CHC profiles were most attractive to females. Temperature had a strong effect on male CHC expression, whereas the effect of diet was weaker. Male CHCs were subject to complex patterns of directional, quadratic and correlational sexual selection, and we found differences between environments in the combination of male CHCs that were most attractive to females, with clearer differences between diets than between temperatures. We also show that genetic covariance between environments is likely to cause a constraint on independent CHC evolution between environments. Our results demonstrate that even across the narrow range of environmental variation studied here, predicting the outcome of sexual selection can be extremely complicated, suggesting that studies ignoring multiple traits or environments may provide an over-simplified view of the evolution of sexual traits.

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SETTING: Chest clinic of a national referral hospital in a resource-limited country. OBJECTIVES: To determine the level of asthma control, factors influencing asthma control and the accuracy of the Asthma Control Test (ACT). DESIGN: We collected demographic and clinical data and administered the Global Initiative for Asthma (GINA) criteria test and the ACT. The proportions of patients in each of the GINA and ACT control categories (uncontrolled, partly controlled and well controlled) were calculated. Multivariate analysis was performed to identify factors associated with asthma control. Diagnostic test parameters for the ACT using GINA criteria as gold standard were calculated. RESULTS: Of 88 asthma patients enrolled, 67% were female. The median age was 34 years (range 12-85). Using GINA criteria, respectively 59 (67%), 17 (19%) and 12 (14%) patients had uncontrolled, partly controlled and well controlled asthma; per ACT, the corresponding figures were respectively 40% (35/88), 43% (38/88) and 17% (15/88). ACT sensitivity, specificity, positive predictive and negative predictive value were respectively 95%, 92%, 99% and 73%. Nasal congestion was associated with uncontrolled asthma (P = 0.031). CONCLUSION: The majority of the patients at the Mulago Hospital have inadequately controlled asthma, and this is associated with nasal congestion. A simple symptom questionnaire, the ACT, can correctly classify asthma control.

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Genotype-by-environment interactions (G × Es) describe genetic variation for phenotypic plasticity. Recent interest in the role of these interactions in sexual selection has identified G × Es across a diverse range of species and sexual traits. Additionally, theoretical work predicts that G × Es in sexual traits could help to maintain genetic variation, but could also disrupt the reliability of these traits as signals of mate quality. However, empirical tests of these theoretical predictions are scarce. We reared iso-female lines of Drosophila simulans across two axes of environmental variation (diet and temperature) in a fully factorial design and tested for G × Es in the expression of cuticular hydrocarbons (CHCs), a multivariate sexual trait in this species. We find sex-specific environmental, genetic and G × E effects on CHC expression, with G × Es for diet in both male and female CHC profile and a G × E for temperature in females. We also find some evidence for ecological crossover in these G × Es, and by quantifying variance components, genetic correlations and heritabilities, we show the potential for these G × Es to help maintain genetic variation and cause sexual signal unreliability in D. simulans CHC profiles.

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OBJECTIVE: To describe the viral etiology and epidemiology of nosocomial viral gastroenteritis (NVG) at a tertiary care pediatric hospital and identify any changes over the past two decades. METHODS: Retrospective review of all patients with laboratory-confirmed NVG at The Hospital for Sick Children (Toronto, Ontario), from January 1, 2004, to December 31, 2005. RESULTS: One hundred forty-two episodes of NVG were found among 133 patients, occurring in 0.48 of 100 admissions. The median age was two years; 42% were <1 year of age and 41% were immunocompromised. The most commonly detected pathogen was torovirus (67% of episodes), followed by rotavirus (19%) and adenovirus (9%). Seventy-five cases (53%) were epidemiologically linked in 32 separate clusters (median cluster size two, range two to four). The NVG rate fell from 0.63 of 100 to 0.22 of 100 admissions after March 2005 (P<0.001) when enhanced infection control precautions were instituted in response to an outbreak of vancomycin-resistant Enterococcus. CONCLUSIONS: Torovirus remains the most commonly identified cause of NVG at The Hospital for Sick Children. Most NVG cases were epidemiologically linked, and a significant reduction in cases occurred after the institution of enhanced infection control practices following an outbreak of vancomycin-resistant Enterococcus. Improved education and surveillance for NVG should lead to further reduction in this problem.

HISTORIQUE: Les chercheurs ont effectué la présente étude pour décrire l'étiologie virale et l'épidémiologie de la gastroentérite virale nosocomiale (GVN) dans un hôpital de pédiatrie tertiaire et déterminer les changements constatés depuis vingt ans. MÉTHODOLOGIE: Analyse rétrospective de tous les patients ayant une GVN confirmée en laboratoire à The Hospital for Sick Children (HSC) de Toronto, en Ontario, entre le 1er janvier 2004 et le 31 décembre 2005. RÉSULTATS: Les chercheurs ont retracé 142 épisodes de GVN chez 133 patients, dans 0,48 pour 100 des admissions. Ils avaient un âge médian de deux ans, 42 % avaient moins d'un an et 41 % étaient immunocompromis. Le torovirus était le pathogène le plus décelé (67 % des épisodes), suivi du rotavirus (19 %) et de l'adénovirus (9 %). Soixante-quinze cas (53 %) étaient liés à 32 grappes distinctes (grappe médiane de deux, plage de deux à quatre) sur le plan épidémiologique. Le taux de GVN a fléchi de 0,63 à 0,22 pour 100 des admissions après le 5 mars 2005 (P<0,001), lorsqu'on a renforcé les précautions de contrôle des infections en réponse à une épidémie d'entérocoques résistant à la vancomycine (ERV). CONCLUSIONS: Le torovirus demeure la principale cause de GVN diagnostiquées à l'HSC. La plupart des cas de GVN étaient liés sur le plan épidémiologique, et on a constaté une importante diminution du nombre de cas après le renforcement des pratiques de contrôle des infections découlant d'une éclosion d'ERV. Une meilleure éducation et une plus grande surveillance à l'égard du GVN devraient favoriser l'atténuation plus marquée de ce problème.

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BACKGROUND: Juvenile onset recurrent respiratory papillomatosis (JORRP) results from HPV transmission. Cervical cancer, also transmitted via HPV, is known to be correlated with socioeconomic status (SES). This study aims to determine if an association exists between SES and severity of JORRP. METHODS: Cross-sectional study of all active JORRP patients at the Hospital for Sick Children in Toronto in 2005. SES information from Hollingshead surveys, Postal walk Census data, and Low Income Cutoff Data were compared with Derkay-Wiatrak disease severity scores, peak annual surgical frequency, and age of diagnosis. Statistical analysis was performed using Spearman, Mann-Whitney, and linear regression analyses. RESULTS: Twenty-one patients were surveyed. Hollingshead results were as follows: two patients (10%) were class I (major business and professional); 11 patients (52%) were class II (medium business, minor professional, technical); 4 patients (19%) were class III (skilled craftsmen, clerical, sales workers); 4 patients (19%) were class IV (machine operators, semiskilled workers); 0% were from class V (unskilled laborers, menial service workers). Interestingly, based on postal code data nine patients (45%) were below the low income cutoff as compared to the Toronto (metropolitan) and Ontario (provincial) rates of low income (17% and 14%, respectively). There was significant correlation between each of the SES measures and between disease severity measures. However, analysis of the SES measures versus disease severity measures did not demonstrate any significant relationship. CONCLUSIONS: Though almost half the patients lived below the low income cutoff, this study did not demonstrate a significant correlation between socioeconomic status and severity of disease in JORRP. One possible explanation is that universal access to the Canadian health care system is able to provide support despite a large proportion of patients being socioeconomically vulnerable. A national level study is underway to further detect any relationship between SES and JORRP severity in the general population.

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OBJECTIVE: To document the clinical presentation and prevalence of stool viruses among children presenting with symptoms of acute gastroenteritis (AGE) at Gertrude's Garden Children's Hospital, Nairobi, Kenya. DESIGN: Retrospective case-control study. SETTING: A private paediatric clinic in Nairobi. RESULTS: Viral antigen was detectable in the stool samples of 21 (rotavirus alone in ten cases, adenovirus alone in seven cases, and both viruses in four cases). Diarrhoea was almost universally present (20/21 cases) and was reported more frequently than in a control group of ten children with clinical acute gastroenteritis whose stools tested negative for viruses. Fever, an elevated total leukocyte count, and neutrophilia were commonly observed in patients with viral gastroenteritis. Eight children with viral AGE were treated with antibiotics and eight children were admitted to hospital. CONCLUSION: A viral etiology can frequently be identified among children in Nairobi with AGE. Fever, an elevated leukocyte count, and neutrophilia were not helpful in differentiating viral from non-viral AGE in this series. Supportive management consisting of outpatient oral rehydration therapy without antibiotic treatment should be considered in the non-toxic child with AGE.

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Samples of aqueous fluid were obtained from 35 "controls" who were people undergoing routine cataract surgery. Similar samples were taken from seven patients with clinically definite multiple sclerosis (MS) and a previous history of optic neuritis, either at cataract surgery or as an elective procedure. Oligoclonal bands were found in only one subject who suffered from the MS-uveitis syndrome.

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The defect in malignant hyperthermia (MH) alters the binding of [3H]ryanodine to the Ca(2+)-release channel by increasing its apparent affinity for the binding site. In sarcoplasmic reticulum (SR) membranes from both normal and mutant pigs the apparent Kd is dependent on a number of parameters. Adenosine 5'-(beta,gamma-methylene)triphosphate, ionic strength, and Ca2+ each increase the apparent affinity of the binding site for [3H]ryanodine. Equilibrium and kinetic evaluation of the binding of [3H]ryanodine to these membranes demonstrates that the MH defect in pigs increases the apparent affinity of the membranes for [3H]ryanodine by increasing the amount of high affinity relative to low affinity binding sites. Both the association and dissociation of [3H]ryanodine with all three types of membranes (normal, heterozygous MH, homozygous MH) are characterized by two or more components, with the relative ratios of these components altered by the MH defect. These findings suggest that the observed Kd is the weighted average of the binding of ryanodine to two or more interconvertible states of the channel. Dilution of [3H]ryanodine bound to normal membranes at high Ca2+ into low Ca2+ solutions enhances the rate of dissociation. This conversion occurs to a much lesser extent with MH membranes, suggesting that the MH defect may alter the rate at which the high affinity form of the protein converts to the low affinity form.

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The alpha subunits of heterotrimeric GTP-binding (G) proteins act upon ion channels through both cytoplasmic and membrane-delimited pathways (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213). The membrane pathway may involve either a direct interaction between G protein and ion channel or an indirect interaction involving a membrane-delimited second messenger. To distinguish between the two possibilities, we tested whether a purified G protein could interact with a purified channel protein in a defined system to produce changes in channel currents. We selected the alpha subunit of Gs and the dihydropyridine (DHP)-sensitive Ca2+ channel of skeletal muscle T-tubules, the DHP binding protein (DHPBP), because: 1) a membrane-delimited interaction between the two has been shown (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213; Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895); and 2) at the present time, these Ca2+ channels are the only putative G protein channel effectors which, following purification, still retain channel function. We used a defined system in which purified components were studied by direct reconstitution in planar lipid bilayers. Just as we had found in crude skeletal muscle T-tubule membranes (Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895), alpha*s but not alpha*i-3 stimulated Ca2+ currents. However, in the reconstituted system, this probably represents a direct interaction between Gs alpha and Ca2+ channels. To establish whether the two proteins were physically associated in the native T-tubule membrane, we examined the ability of either endogenous G proteins or exogenous alpha*s to purify with detergent-solubilized DHPBP through a wheat germ agglutinin affinity column and a sucrose gradient. Small amounts of a labeled G protein were found to co-purify with DHPBP. In addition, partially purified DHPBP increased the sedimentation rate of purified alpha*s but not alpha*i-3. G proteins were immunoprecipitated with an antibody to the alpha 1 subunit of the DHPBP, and, in addition, both alpha s and the beta subunit of Gs were detected in Western blots of the partially purified DHPBP. The results suggest that Gs and Ca2+ channels are closely associated in the T-tubule plasma membrane, and we conclude that skeletal muscle Ca2+ channels are direct effectors for Gs.

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To assess the role of oestrogen regulation in the growth of ovarian cancer, we examined the effects of an oestrogen, 17 beta-oestradiol, and an anti-oestrogen, tamoxifen, on oestrogen receptor (ER) -positive and -negative human ovarian carcinoma cell lines. As measured by a dextran-coated charcoal adsorption assay, cell lines PEO1, PEO4 and PEO6 possessed moderate concentrations of ER (96-132 fmol mg-1 protein), PEA1 and PEA2 had low values (12-23 fmol mg-1 protein) and PEO14, TO14, PEO23 and PEO16 were ER-negative. Addition of 17 beta-oestradiol (10 nM or 0.1 nM) to the ER +ve cell line, PEO4, increased the growth rate. This oestrogen stimulation could be blocked by 1 microM tamoxifen. In contrast, the growth rate of the ER -ve cell line PEO14 was unaffected by the addition of 17 beta-oestradiol or tamoxifen. Concentrations of tamoxifen in excess of 8 microM were required to produce complete cytostasis in all lines. This concentration of tamoxifen over 72 hours also inhibited 50% colony formation when cells were plated on plastic. These data indicate that some ovarian carcinoma cell lines contain ER and their growth can be sensitive to oestrogen and anti-oestrogen modulation.

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In this paper, we study the modulation of the rabbit fast twitch skeletal muscle calcium release channel by assaying the kinetics of [3H]ryanodine binding, 45Ca2+ flux, and single-channel activity. The effects of modulators of the Ca2+ release channel (confirmed here with both flux and single-channel data) were examined for effects on [3H]ryanodine binding to terminal cisternae vesicles. We find that activators of the release channel, such as adenine nucleotides (1 mM) and caffeine (1 mM), enhance the rate of association of [3H]ryanodine, whereas inhibitors, such as Mg2+ (1 mM) and ruthenium red (100 nM), decrease the rate of association. High concentrations of either ryanodine or ruthenium red, which close the channel, slow the dissociation of [3H]ryanodine, suggesting that at these concentrations the inhibitory effects of both ryanodine and ruthenium red occur as the result of binding at a site distinct from but interacting cooperatively with the high affinity site. Our data are consistent with a model in which the high affinity ryanodine binding site is within a conformationally sensitive area of the channel, such that conditions that open the channel (ATP, caffeine, etc.) enhance the rate at which [3H]ryanodine reaches its binding site and other conditions that close the channel (the binding of ryanodine and ruthenium red to a low affinity site) slow the dissociation of [3H]ryanodine from the high affinity site. Some conditions that inhibit channel activity (high concentrations of Mg2+ and Ca2+) slow association but do not affect dissociation of bound [3H]ryanodine, suggesting a completely different state of the channel from that which is inactive in the presence of high concentrations of ryanodine or ruthenium red. In summary, the functional state of the fast twitch skeletal muscle calcium release channel can be characterized by the changes in the kinetics of [3H]ryanodine binding. Different modulators (activators/inhibitors) affect different aspects of ryanodine binding (association/dissociation).

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