Membrane potential responses controlling chemodispersal of Paramecium caudatum from quinine

Membrane potential responses of a ciliate protozoan Paramecium caudatum to the external application of quinine were investigated in relation to its motile activities. Wild-type specimens swimming in the reference solution did not enter into a quinine-containing (0.5 mM) test solution due to avoiding responses exhibited at the border between the two solutions, and therefore stayed in the reference solution (chemodispersal). Squirting of a quinine-containing test solution over a wild-type specimen evoked a train of action potentials superimposed on a depolarizing chemoreceptor potential. Squirting of a quinine-containing test solution over a CNR-mutant specimen defective in voltage-gated Ca²⁺ channel evoked only chemoreceptor potentials, which consisted of an initial transient depolarization, a following transient hyperpolarization and a sustained depolarization. A current-evoked action potential became larger in its amplitude and longer in its duration with the external application of quinine. Under the voltage-clamp condition, the fast inward current did not change whereas the delayed outward current decreased with the external application of quinine. It is concluded that quinine is a potent repellent for Paramecium because it produces a depolarizing chemoreceptor potential which evokes action potentials and prolongs the duration of the action potential.     

Dysfunction of rat liver mitochondria by selenite: induction of mitochondrial permeability transition through thiol-oxidation

Selenium is an essential trace element in mammals and is thought to play a chemopreventive role in human cancer, possibly by inducing tumor cell apoptosis. Mitochondria play a pivotal role in the induction of apoptosis in many cell types. The effects of selenite on mitochondrial function were therefore investigated. Selenite induced the oxidation and cross-linking of protein thiol groups, mitochondrial permeability transition (MPT), a decrease in the mitochondrial membrane potential, and the release of cytochrome c in mitochondria isolated from rat liver. Induction of the MPT by selenite was prevented by cyclosporin A, EGTA, or N-ethylmaleimide. These results thus indicate that selenite induces the MPT as a result of direct modification of protein thiol groups, resulting in the release of cytochrome c and a loss of mitochondrial membrane potential.     

Eco‐evolution on the edge during climate change

We urgently need to predict species responses to climate change to minimize future biodiversity loss and ensure we do not waste limited resources on ineffective conservation strategies. Currently, most predictions of species responses to climate change ignore the potential for evolution. However, evolution can alter species ecological responses, and different aspects of evolution and ecology can interact to produce complex eco‐evolutionary dynamics under climate change. Here we review how evolution could alter ecological responses to climate change on species warm and cool range margins, where evolution could be especially important. We discuss different aspects of evolution in isolation, and then synthesize results to consider how multiple evolutionary processes might interact and affect conservation strategies. On species cool range margins, the evolution of dispersal could increase range expansion rates and allow species to adapt to novel conditions in their new range. However, low genetic variation and genetic drift in small range‐front populations could also slow or halt range expansions. Together, these eco‐evolutionary effects could cause a three‐step, stop‐and‐go expansion pattern for many species. On warm range margins, isolation among populations could maintain high genetic variation that facilitates evolution to novel climates and allows species to persist longer than expected without evolution. This ‘evolutionary extinction debt’ could then prevent other species from shifting their ranges. However, as climate change increases isolation among populations, increasing dispersal mortality could select for decreased dispersal and cause rapid range contractions. Some of these eco‐evolutionary dynamics could explain why many species are not responding to climate change as predicted. We conclude by suggesting that resurveying historical studies that measured trait frequencies, the strength of selection, or heritabilities could be an efficient way to increase our eco‐evolutionary knowledge in climate change biology.     

Zipping and Unzipping of Adenylate Kinase: Atomistic Insights into the Ensemble of Open ↔ Closed Transitions

Adenylate kinase (AdK), a phosphotransferase enzyme, plays an important role in cellular energy homeostasis. It undergoes a large conformational change between an open and a closed state, even in the absence of substrate. We investigate the apo-AdK transition at the atomic level both with free-energy calculations and with our new dynamic importance sampling (DIMS) molecular dynamics method. DIMS is shown to sample biologically relevant conformations as verified by comparing an ensemble of hundreds of DIMS transitions to AdK crystal structure intermediates. The simulations reveal in atomic detail how hinge regions partially and intermittently unfold during the transition. Conserved salt bridges are seen to have important structural and dynamic roles; in particular, four ionic bonds that open in a sequential, zipper-like fashion and, thus, dominate the free-energy landscape of the transition are identified. Transitions between the closed and open conformations only have to overcome moderate free-energy barriers. Unexpectedly, the closed state and the open state encompass broad free-energy basins that contain conformations differing in domain hinge motions by up to 40°. The significance of these extended states is discussed in relation to recent experimental Förster resonance energy transfer measurements. Taken together, these results demonstrate how a small number of cooperative key interactions can shape the overall dynamics of an enzyme and suggest an “all-or-nothing” mechanism for the opening and closing of AdK. Our efficient DIMS molecular dynamics computer simulation approach can provide a detailed picture of a functionally important macromolecular transition and thus help to interpret and suggest experiments to probe the conformational landscape of dynamic proteins such as AdK.     

Pushing, pulling and trapping--modes of motor protein supported protein translocation

Protein translocation across the cellular membranes is an ubiquitous and crucial activity of cells. This process is mediated by translocases that consist of a protein conducting channel and an associated motor protein. Motor proteins interact with protein substrates and utilize the free energy of ATP binding and hydrolysis for protein unfolding, translocation and unbinding. Since motor proteins are found either at the cis- or trans-side of the membrane, different mechanisms for translocation have been proposed. In the Power stroke model, cis-acting motors are thought to push, while trans-motors pull on the substrate protein during translocation. In the Brownian ratchet model, translocation occurs by diffusion of the unfolded polypeptide through the translocation pore while directionality is achieved by trapping and refolding. Recent insights in the structure and function of the molecular motors suggest that different mechanisms can be employed simultaneously.     

Panicle Water Potential, a Physiological Trait to Identify Drought Tolerance in Rice

Two upland rice varieties （IRAT109, IAPAR9） and one lowland rice variety （Zhenshan 97B） were planted in summer and treated with both normal （full water） and drought stress in the reproductive stage. Panicle water potential （PWP） and leaf water potential （LWP） were measured every 1.0-1.5 h over 24 h on sunny days. Both PWP and LWP of upland varieties started to decrease later, maintained a higher level and recovered more quickly than that of the lowland variety. The results show that PWP can be used as an indicator of plant water status based on the parallel daily changes, and the high correlation between PWP and LWP. Similar correlations were also observed between PWP, LWP and eight traits related to plant growth and grain yield formation. PWP seemed to be more effective for distinguishing the upland rice varieties with different drought-tolerant ability. Differences in PWP and LWP between upland and lowland rice varieties were also observed at noon even under normal water conditions, implying the incorporation of the drought-tolerant mechanism to improve the photosynthesis and yield of traditional paddy rice.     

Effects of the anion transport inhibitor,SITS, on the proximal straight tubule of the rabbit perfusedin vitro

Summary Conventional microelectrodes were used to study the effects of SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonate) on the basolateral membrane potentialVbl of the superficial proximal straight tubule (PST) of the rabbit kidney perfusedin vitro. Addition of 0.1mm SITS to the bathing solution resulted in a slow and irreversible hyperpolarization ofVbl from –42.5±1.17 (37) mV to –77.3±0.83 (52) mV. The new steady-state potential was reached in 10 to 15 min and was accompanied by visible cell swelling. Associated with thisVbl hyperpolarization was: 1) an increased steady-state depolarization (from 6.2±0.77 (17) mV to 25.7±0.83 (29) mV) in response to increasing bath potassium concentration from 5 to 16.7mm (HK); 2) a decreased transient depolarization (from 19.8±1.88 (8) mV to 0.43±0.37 (8) mV) in response to decreasing bath bicarbonate concentration from 22 to 6.6mm at constant bath pH (L-HCO₃); and 3) inhibition of a depolarizing overshoot and a decreased steady-state depolarization (from 35.9±1.84 (12) mV to 4.7±1.37 (13) mV) in response to reducing bath sodium concentration from 144 to zero (0-Na). Sodium, chloride and NMDG (N-methyl-d-glucamine) were used as the substituting ions, respectively. These results are consistent with the presence of a coupled sodium-bicarbonate carrier in the basolateral membrane which is electrogenic and SITS inhibitable. Comparison of the time course of SITS effects on these ion-substitution responses suggests that the inhibition of the bicarbonate exit pathway(s) is the primary event and that the changes inVbl and in the steady-stateVbl responses to HK and 0-Na are secondary events which may be related to changes in intracellular composition and/or basolateral membrane properties.     

Relations between stomatal closure, leaf turgor and xylem vulnerability in eight tropical dry forest trees

This study examined the linkage between xylem vulnerability, stomatal response to leaf water potential (Ψ_L), and loss of leaf turgor in eight species of seasonally dry tropical forest trees. In order to maximize the potential variation in these traits species that exhibit a range of leaf habits and phenologies were selected. It was found that in all species stomatal conductance was responsive to Ψ_L over a narrow range of water potentials, and that Ψ_L inducing 50% stomatal closure was correlated with both the Ψ_L inducing a 20% loss of xylem hydraulic conductivity and leaf water potential at turgor loss in all species. In contrast, there was no correlation between the water potential causing a 50% loss of conductivity in the stem xylem, and the water potential at stomatal closure (Ψ_SC) amongst species. It was concluded that although both leaf and xylem characters are correlated with the response of stomata to Ψ_L, there is considerable flexibility in this linkage. The range of responses is discussed in terms of the differing leaf‐loss strategies exhibited by these species.     

Docking of Antizyme to Ornithine Decarboxylase and Antizyme Inhibitor using Experimental Mutant and Double-Mutant Cycle Data

Antizyme (Az) is a highly conserved key regulatory protein bearing a major role in regulating polyamine levels in the cell. It has the ability to bind and inhibit ornithine decarboxylase (ODC), targeting it for degradation. Az inhibitor (AzI) impairs the activity of Az. In this study, we mapped the binding sites of ODC and AzI on Az using Ala scan mutagenesis and generated models of the two complexes by constrained computational docking. In order to scan a large number of mutants in a short time, we developed a workflow combining high-throughput mutagenesis, small-scale parallel partial purification of His-tagged proteins and their immobilization on a tris-nitrilotriacetic-acid-coated surface plasmon resonance chip. This combination of techniques resulted in a significant reduction in time for production and measurement of large numbers of mutant proteins. The data-driven docking results suggest that both proteins occupy the same binding site on Az, with Az binding within a large groove in AzI and ODC. However, single-mutant data provide information concerning the location of the binding sites only, not on their relative orientations. Therefore, we generated a large number of double-mutant cycles between residues on Az and ODC and used the resulting interaction energies to restrict docking. The model of the complex is well defined and accounts for the mutant data generated here, and previously determined biochemical data for this system. Insights on the structure and function of the complexes, as well as general aspects of the method, are discussed.