Ligand-insensitive State of Cardiac ATP-sensitive K+ Channels : Basis for Channel Opening

The mechanism by which ATP-sensitive K⁺ (K_ATP) channels open in the presence of inhibitory concentrations of ATP remains unknown. Herein, using a four-state kinetic model, we found that the nucleotide diphosphate UDP directed cardiac K_ATP channels to operate within intraburst transitions. These transitions are not targeted by ATP, nor the structurally unrelated sulfonylurea glyburide, which inhibit channel opening by acting on interburst transitions. Therefore, the channel remained insensitive to ATP and glyburide in the presence of UDP. “Rundown” of channel activity decreased the efficacy with which UDP could direct and maintain the channel to operate within intraburst transitions. Under this condition, the channel was sensitive to inhibition by ATP and glyburide despite the presence of UDP. This behavior of the K_ATP channel could be accounted for by an allosteric model of ligand-channel interaction. Thus, the response of cardiac K_ATP channels towards inhibitory ligands is determined by the relative lifetime the channel spends in a ligand-sensitive versus -insensitive state. Interconversion between these two conformational states represents a novel basis for K_ATP channel opening in the presence of inhibitory concentrations of ATP in a cardiac cell.     

Inhibition of Membrane Lipid Peroxidation by a Radical Scavenging Mechanism: a Novel Function for Hydroxyl-Containing Ionophores

In the present study we show that K⁺/H⁺ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe²⁺-citrate and 2,2'-azobis(2-amidinopropane) di-hydrochloride (ABAP)-induced lipid peroxidation in intact rat liver mitochondria and in egg phosphatidyl-choline (PC) liposomes containing negatively charged lipids—dicetyl phosphate (DCP) or cardiolipin (CL)—and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-containing ionophore with higher affinity for Na⁺ than for K⁺, promotes a similar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-insensitive O₂ consumption, formation of thiobarbituric acid-reactive substances (TBARS), conjugated dienes, and electron paramagnetic resonance (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid peroxidation induced by both inductor systems in liposomes. Nitroxide destruction is inhibited by buty-lated hydroxytoluene, a known antioxidant, and by the hydroxyl-containing ionophores. In contrast, vali-nomycin (VAL), which does not possess alcoholic groups, does not display this protective effect. Effective order parameters (S_eff), determined from the spectra of an incorporated spin label are larger in the presence of salt and display a small increase upon addition of the ionophores, as a result of the increase of counter ion concentration at the negatively charged bilayer surface. This condition leads to increased formation of the ion-ionophore complex, the membrane binding (uncharged) species. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution (in the absence of membranes) showed that NIG and LAS, but not VAL, decrease the Fe²⁺-citrate-induced production of radicals derived from piperazine-based buffers, demonstrating their property as radical scavengers. Both Fe²⁺-citrate and ABAP promote a much more pronounced decrease of LAS fluorescence in PC/CL liposomes than in dimyristoyl phosphatidyl-choline (DMPC, saturated phospholipid)-DCP liposomes, indicating that the ionophore also scavenges lipid peroxyl radicals. A slow decrease of fluorescence is observed in the latter system, for all lipid compositions in sucrose medium, and in the absence of membranes, indicating that the primary radicals stemming from both inductors also attack the ionophore. Altogether, the data lead to the conclusion that the membrane-incorporated cation complexes of NIG, LAS and MON inhibit lipid peroxidation by blocking initiation and propagation reactions in the lipid phase via a free radical scavenging mechanism, very likely due to the presence of alcoholic hydroxyl groups in all three molecules and to the attack of the aromatic moiety of LAS.     

ESR Studies of a Series of Phthalocyanines. Mechanism of Phototoxicity. Comparative Quantitation of O2-using ESR Spin-Trapping and Cytochrome c Reduction Techniques

ESR experiments with 2,2,6,6-tetramethyl-4-piperi-done (4-oxo-TEMP) and the spin-trap 5,5-dimethyl pyrroline-N-oxide (DMPO) have been performed on a series of new phthalocyanines: the bis(tri-n-hexyl-siloxy) silicon phthalocyanine ([(nhex)₃SiO]₂SiPc), the hexadecachloro zinc phthalocyanine (ZnPcCl₁₆), the hexadecachloro aluminum phthalocyanine (AlPcCl₁₆), the hexadecachloro aluminum phthalocyanine sulfate (HSO₄A1PcCl₁₆), whose photocytotoxicity has been studied against various leukemic and melanotic cell lines. Type I and Type II pathways occur simultaneously in DMF although the Type II seems to be prevalent. These results are not changed when the bis(tri-n-hexylsiloxy) silicon phthalocyanine is entrapped into liposomes. By contrast, the Type I process is favored in membrane models for all the perchlori-nated phthalocyanines. This modified behavior may be accounted on a possible stacking of phthalocyanines in membranes and a preventing effect of axial ligands against aggregation in the case of the bis(tri-n-hexyl-siloxy) silicon phthalocyanine. The photodynamic action of zinc perchlorinated phthalocyanine is not dependent on singlet oxygen, phototoxicity of this molecule being essentially mediated by oxygen free radicals. Quantitation of the superoxide radical was accomplished, with good agreement, by two techniques: the cytochrome c reduction and the ESR quantitation based on the double integration of the first derivative of the ESR signal. The disproportionation of the superoxide radical or degradation of the spin-trap seem to be avoided in aprotic solvents such as DMF.     

Discrete consumers,small scale resource heterogeneity,and population stability

We present a consumer-resource model in which individual consumers subsist on a continuum of resource distributed over a very large number of small “bite-sized” patches, each patch being sufficiently small that all its resource is eaten whenever a consumer visits. This form of consumer–resource interaction forces a heterogeneous distribution of resource among the patches, and may dampen out the large amplitude, consumer-resource cycles that are predicted by traditional models of well-mixed, spatially homogeneous systems. The resource equilibrium does not increase with enrichment, a prediction that distinguishes this model from models that invoke direct or indirect consumer density dependence as a stabilizing mechanism.     

Rib cage muscle interaction in airway pressure generation

We have previously demonstrated in dogs that the change inairway opening pressure (Pao) produced by isolated maximumactivation of the parasternal intercostal or triangularis sterni musclein a single interspace, the sternomastoids, and the scalenes isproportional to the product of muscle mass and the fractional change inmuscle length per unit volume increase of the relaxed chest wall. In the present study, we have assessed the interactions between these muscles by comparing the Pao obtained during simultaneous activation of a pair of muscles (measured Pao) to the sum of the Pao values obtained during their separate activation (predicted Pao). Measured and predicted Pao values were compared for the following pairs ofmuscles: the parasternal intercostals in two interspaces, the parasternal intercostals in one interspace and either thesternomastoids or the scalenes, two segments of the triangularissterni, and the interosseous intercostals in two contiguousinterspaces. For all these pairs, the measured Pao was within~10% of the predicted value. We therefore conclude that1) the pressure changes generated bythe rib cage muscles are essentially additive; and2) measurements of the mass of aparticular muscle and of its fractional change in length during passiveinflation can be used to estimate the potential pressure-generatingability of the muscle during coordinated activity as well as duringisolated activation.

     

Evidence for Direct Physical Association between a K+ Channel (Kir6.2) and an ATP-Binding Cassette Protein (SUR1) Which Affects Cellular Distribution and Kinetic Behavior of an ATP-Sensitive K+ Channel

Structurally unique among ion channels, ATP-sensitive K⁺ (K_ATP) channels are essential in coupling cellular metabolism with membrane excitability, and their activity can be reconstituted by coexpression of an inwardly rectifying K⁺ channel, Kir6.2, with an ATP-binding cassette protein, SUR1. To determine if constitutive channel subunits form a physical complex, we developed antibodies to specifically label and immunoprecipitate Kir6.2. From a mixture of Kir6.2 and SUR1 in vitro-translated proteins, and from COS cells transfected with both channel subunits, the Kir6.2-specific antibody coimmunoprecipitated 38- and 140-kDa proteins corresponding to Kir6.2 and SUR1, respectively. Since previous reports suggest that the carboxy-truncated Kir6.2 can form a channel independent of SUR, we deleted 114 nucleotides from the carboxy terminus of the Kir6.2 open reading frame (Kir6.2ΔC37). Kir6.2ΔC37 still coimmunoprecipitated with SUR1, suggesting that the distal carboxy terminus of Kir6.2 is unnecessary for subunit association. Confocal microscopic images of COS cells transfected with Kir6.2 or Kir6.2ΔC37 and labeled with fluorescent antibodies revealed unique honeycomb patterns unlike the diffuse immunostaining observed when cells were cotransfected with Kir6.2-SUR1 or Kir6.2ΔC37-SUR1. Membrane patches excised from COS cells cotransfected with Kir6.2-SUR1 or Kir6.2ΔC37-SUR1 exhibited single-channel activity characteristic of pancreatic K_ATP channels. Kir6.2ΔC37 alone formed functional channels with single-channel conductance and intraburst kinetic properties similar to those of Kir6.2-SUR1 or Kir6.2ΔC37-SUR1 but with reduced burst duration. This study provides direct evidence that an inwardly rectifying K⁺ channel and an ATP-binding cassette protein physically associate, which affects the cellular distribution and kinetic behavior of a K_ATP channel.     

Estimating the cumulative incidence of COVID-19 in the United States using influenza surveillance,virologic testing,and mortality data: Four complementary approaches

Effectively designing and evaluating public health responses to the ongoing COVID-19 pandemic requires accurate estimation of the prevalence of COVID-19 across the United States (US). Equipment shortages and varying testing capabilities have however hindered the usefulness of the official reported positive COVID-19 case counts. We introduce four complementary approaches to estimate the cumulative incidence of symptomatic COVID-19 in each state in the US as well as Puerto Rico and the District of Columbia, using a combination of excess influenza-like illness reports, COVID-19 test statistics, COVID-19 mortality reports, and a spatially structured epidemic model. Instead of relying on the estimate from a single data source or method that may be biased, we provide multiple estimates, each relying on different assumptions and data sources. Across our four approaches emerges the consistent conclusion that on April 4, 2020, the estimated case count was 5 to 50 times higher than the official positive test counts across the different states. Nationally, our estimates of COVID-19 symptomatic cases as of April 4 have a likely range of 2.3 to 4.8 million, with possibly as many as 7.6 million cases, up to 25 times greater than the cumulative confirmed cases of about 311,000. Extending our methods to May 16, 2020, we estimate that cumulative symptomatic incidence ranges from 4.9 to 10.1 million, as opposed to 1.5 million positive test counts. The proposed combination of approaches may prove useful in assessing the burden of COVID-19 during resurgences in the US and other countries with comparable surveillance systems.     

Electronic Tagging of Atlantic Bluefin Tuna (Thunnus thynnus,L.) Reveals Habitat Use and Behaviors in the Mediterranean Sea

We analyzed the movements of Atlantic tuna (Thunnus thynnus L.) in the Mediterranean Sea using data from 2 archival tags and 37 pop-up satellite archival tags (PAT). Bluefin tuna ranging in size from 12 to 248 kg were tagged on board recreational boats in the western Mediterranean and the Adriatic Sea between May and September during two different periods (2000 to 2001 and 2008 to 2012). Although tuna migrations between the Mediterranean Sea and the Atlantic Ocean have been well reported, our results indicate that part of the bluefin tuna population remains in the Mediterranean basin for much of the year, revealing a more complex population structure. In this study we demonstrate links between the western Mediterranean, the Adriatic and the Gulf of Sidra (Libya) using over 4336 recorded days of location and behavior data from tagged bluefin tuna with a maximum track length of 394 days. We described the oceanographic preferences and horizontal behaviors during the spawning season for 4 adult bluefin tuna. We also analyzed the time series data that reveals the vertical behavior of one pop-up satellite tag recovered, which was attached to a 43.9 kg tuna. This fish displayed a unique diving pattern within 16 days of the spawning season, suggesting a use of the thermocline as a thermoregulatory mechanism compatible with spawning. The results obtained hereby confirm that the Mediterranean is clearly an important habitat for this species, not only as spawning ground, but also as an overwintering foraging ground.     

Glucocorticoid Stress Responses of Reintroduced Tigers in Relation to Anthropogenic Disturbance in Sariska Tiger Reserve in India

Tiger (Panthera tigris), an endangered species, is under severe threat from poaching, habitat loss, prey depletion and habitat disturbance. Such factors have been reported causing local extermination of tiger populations including in one of the most important reserves in India, namely Sariska Tiger Reserve (STR) in northwestern India. Consequently, tigers were reintroduced in STR between 2008 and 2010, but inadequate breeding success was observed over the years, thus invoking an investigation to ascertain physiological correlates. In the present study, we report glucocorticoid stress responses of the reintroduced tigers in relation to anthropogenic disturbance in the STR from 2011 to 2013. We found anthropogenic disturbance such as encounter rates of livestock and humans, distance to roads and efforts to kill domestic livestock associated with an elevation in fecal glucocorticoid metabolite (fGCM) concentrations in the monitored tigers. In this regard, female tigers seem more sensitive to such disturbance than males. It was possible to discern that tiger’s fGCM levels were significantly positively related to the time spent in disturbed areas. Resulting management recommendations include relocation of villages from core areas and restriction of all anthropogenic activities in the entire STR.