Homeostatic control of slow vacuolar channels by luminal cations and evaluation of the channel-mediated tonoplast Ca2+ fluxes in situ

Ca2+, Mg2+, and K+ activities in red beet (Beta vulgaris L.) vacuoles were evaluated using conventional ion-selective microelectrodes and, in the case of Ca2+, by non-invasive ion flux measurements (MIFE) as well. The mean vacuolar Ca2+ activity was ∼0.2 mM. Modulation of the slow vacuolar (SV) channel voltage dependence by Ca2+ in the absence and presence of other cations at their physiological concentrations was studied by patch-clamp in excised tonoplast patches. Lowering pH at the vacuolar side from 7.5 to 5.5 (at zero vacuolar Ca2+) did not affect the channel voltage dependence, but abolished sensitivity to luminal Ca2+ within a physiological range of concentrations (0.1–1.0 mM). Aggregation of the physiological vacuolar Na+ (60 mM) and Mg2+ (8 mM) concentrations also results in the SV channel becoming almost insensitive to vacuolar Ca2+ variation in a range from nanomoles to 0.1 mM. At physiological cation concentrations at the vacuolar side, cytosolic Ca2+ activates the SV channel in a voltage-independent manner with Kd=0.7–1.5 μM. Comparison of the vacuolar Ca2+ fluxes measured by both the MIFE technique and from estimating the SV channel activity in attached patches, suggests that, at resting membrane potentials, even at elevated (20 μM) cytosolic Ca2+, only 0.5% of SV channels are open. This mediates a Ca2+ release of only a few pA per vacuole (∼0.1 pA per single SV channel). Overall, our data suggest that the release of Ca2+ through SV channels makes little contribution to a global cytosolic Ca2+ signal

Верификация систем с параллелизмом поведения на основе графа достижимых состояний

Considered problem of model based verification of control systems is the checking whether the system behavior satisfies the requirements fixed in the design specification The testing includes the experiments consisting in simulation of investigated system to see input-output correspondence to the model. The test sequence is generated on the basis of the model that describes the desired behavior of the system. The method to construct a test sequence for verification of hardware (or software) implementation of a control system with behavior parallelism is suggested that is based on traversal of the graph of the states that are reachable in system functioning. A method for constructing the set of reachable global states for a parallel algorithm of the control system behavior and a method to obtain the test sets are described. The description of the system functioning, which is given by the design specification, is assumed to be correct. The hardware (or software) implementation that must conform to this specification is to be verified.Рассматривается задача верификации систем управления на основе моделей их поведения, которая состоит в проверке соответствия поведения системы требованиям, предъявляемым спецификацией на ее проектирование. Тестирование предполагает выполнение экспериментов, заключающихся в моделировании исследуемой системы, в ходе которого она проверяется на вход-выходное соответствие модели. Тестовая последовательность генерируется на основе модели, описывающей желаемое поведение системы. Предлагается метод построения тестовой последовательности для верификации схемной (или программной) реализации системы управления с параллелизмом поведения, который основан на обходе графа состояний, достижимых при функционировании системы. Описывается метод построения множества достижимых полных состояний для параллельного алгоритма описания поведения системы управления и получения тестовых наборов. Полагается, что описание функционирования системы, заданное спецификацией на проектирование, корректно; проверке подлежит схемная (или программная) реализация, которая должна соответствовать этой спецификации

Tubulin Binds to the Cytoplasmic Loop of TRESK Background K+ Channel In Vitro.

The cytoplasmic loop between the second and third transmembrane segments is pivotal in the regulation of TRESK (TWIK-related spinal cord K+ channel, K2P18.1, KCNK18). Calcineurin binds to this region and activates the channel by dephosphorylation in response to the calcium signal. Phosphorylation-dependent anchorage of 14-3-3 adaptor protein also modulates TRESK at this location. In the present study, we identified molecular interacting partners of the intracellular loop. By an affinity chromatography approach using the cytoplasmic loop as bait, we have verified the specific association of calcineurin and 14-3-3 to the channel. In addition to these known interacting proteins, we observed substantial binding of tubulin to the intracellular loop. Successive truncation of the polypeptide and pull-down experiments from mouse brain cytosol narrowed down the region sufficient for the binding of tubulin to a 16 amino acid sequence: LVLGRLSYSIISNLDE. The first six residues of this sequence are similar to the previously reported tubulin-binding region of P2X2 purinergic receptor. The tubulin-binding site of TRESK is located close to the protein kinase A (PKA)-dependent 14-3-3-docking motif of the channel. We provide experimental evidence suggesting that 14-3-3 competes with tubulin for the binding to the cytoplasmic loop of TRESK. It is intriguing that the 16 amino acid tubulin-binding sequence includes the serines, which were previously shown to be phosphorylated by microtubule-affinity regulating kinases (MARK kinases) and contribute to channel inhibition. Although tubulin binds to TRESK in vitro, it remains to be established whether the two proteins also interact in the living cell

Using jasmonates and salicylates to reduce losses within the fruit supply chain

The fresh produce industry is constantly growing, due to increasing consumer demand. The shelf-life of some fruit, however, is relatively short, limited by microbial contamination or visual, textural and nutritional quality loss. Thus, techniques for reducing undesired microbial contamination, spoilage and decay, as well as maintaining product’s visual, textural and nutritional quality are in high demand at all steps within the supply chain. The postharvest use of signalling molecules, i.e. jasmonates and salicylates seems to have unexplored potential. The focus of this review is on the effects of treatment with jasmonates and salicylates on the fresh produce quality, defined by decay incidence and severity, chilling injury, maintenance of texture, visual quality, taste and aroma, and nutritional content. Postharvest treatments with jasmonates and salicylates have the ability to reduce decay by increasing fruit resistance to diseases and reducing chilling injury in numerous products. These treatments also possess the ability to improve other quality characteristics, i.e. appearance, texture maintenance and nutritional content. Furthermore, they can easily be combined with other treatments, e.g. heat treatment, ultrasound treatment. A good understanding of all the benefits and limitations related to the postharvest use of jasmonates and salicylates is needed, and relevant information has been reviewed in this paper

Ion homeostasis in the Chloroplast

peer reviewedThe chloroplast is an organelle of high demand for macro- and micro-nutrient ions, which are required for the maintenance of the photosynthetic process. To avoid deficiency while preventing excess, homeostasis mechanisms must be tightly regulated. Here, we describe the needs for nutrient ions in the chloroplast and briefly highlight their functions in the chloroplastidial metabolism. We further discuss the impact of nutrient deficiency on chloroplasts and the acclimation mechanisms that evolved to preserve the photosynthetic apparatus. We finally present what is known about import and export mechanisms for these ions. Whenever possible, a comparison between cyanobacteria, algae and plants is provided to add an evolutionary perspective to the description of ion homeostasis mechanisms in photosynthesis

Verification of systems with behavior parallelism on the basis of the graph of reachable states

Considered problem of model based verification of control systems is the checking whether the system behavior satisfies the requirements fixed in the design specification The testing includes the experiments consisting in simulation of investigated system to see input-output correspondence to the model. The test sequence is generated on the basis of the model that describes the desired behavior of the system. The method to construct a test sequence for verification of hardware (or software) implementation of a control system with behavior parallelism is suggested that is based on traversal of the graph of the states that are reachable in system functioning. A method for constructing the set of reachable global states for a parallel algorithm of the control system behavior and a method to obtain the test sets are described. The description of the system functioning, which is given by the design specification, is assumed to be correct. The hardware (or software) implementation that must conform to this specification is to be verified