Sergei G. Sokolovski

Nitric oxide regulates K+ and Cl- channels in guard cells through a subset of abscisic acid-evoked signaling pathways

Abscisic acid (ABA) triggers a complex sequence of signaling events that lead to concerted modulation of ion channels at the plasma membrane of guard cells and solute efflux to drive stomatal closure in plant leaves. Recent work has indicated that nitric oxide (NO) and its synthesis are a prerequisite for ABA signal transduction in Arabidopsis and Vicia guard cells. Its mechanism(s) of action is not well defined in guard cells and, generally, in higher plants. Here we show directly that NO selectively regulates Ca2+-sensitive ion channels of Vicia guard cells by promoting Ca2+ release from intracellular stores to raise cytosolic-free [Ca2+]. NO-sensitive Ca2+ release was blocked by antagonists of guanylate cyclase and cyclic ADP ribose-dependent endomembrane Ca2+ channels, implying an action mediated via a cGMP-dependent cascade. NO did not recapitulate ABA-evoked control of plasma membrane Ca2+ channels and Ca2+-insensitive K+ channels, and NO scavengers failed to block the activation of these K+ channels evoked by ABA. These results place NO action firmly within one branch of the Ca2+-signaling pathways engaged by ABA and define the boundaries of parallel signaling events in the control of guard cell movements.

Nitric Oxide Block of Outward-Rectifying K+ Channels Indicates Direct Control by Protein Nitrosylation in Guard Cells

Recent work has indicated that nitric oxide (NO) and its synthesis are important elements of signal cascades in plant pathogen defense and are a prerequisite for drought and abscisic acid responses in Arabidopsis (Arabidopsis thaliana) and Vicia faba guard cells. Nonetheless, its mechanism(s) of action has not been well defined. NO regulates inward-rectifying K+ channels of Vicia guard cells through its action on Ca2+ release from intercellular Ca2+ stores, but alternative pathways are indicated for its action on the outward-rectifying K+ channels (IK,out), which are Ca2+ insensitive. We report here that NO affects IK,out when NO is elevated above approximately 10 to 20 nm. NO action on IK,out was consistent with oxidative stress and was suppressed by several reducing agents, the most effective being British anti-Lewisite (2,3-dimercapto-1-propanol). The effect of NO on the K+ channel was mimicked by phenylarsine oxide, an oxidizing agent that cross-links vicinal thiols. Neither intracellular pH buffering nor the phosphotyrosine kinase antagonist genistein affected NO action on IK,out, indicating that changes in cytosolic pH and tyrosine phosphorylation are unlikely to contribute to NO or phenylarsine oxide action in this instance. Instead, our results strongly suggest that NO directly modifies the K+ channel or a closely associated regulatory protein, probably by nitrosylation of cysteine sulfhydryl groups.

A Tripartite SNARE-K+ Channel Complex Mediates in Channel-Dependent K+ Nutrition in Arabidopsis

A few membrane vesicle trafficking (SNARE) proteins in plants are associated with signaling and transmembrane ion transport, including control of plasma membrane ion channels. Vesicle traffic contributes to the population of ion channels at the plasma membrane. Nonetheless, it is unclear whether these SNAREs also interact directly to affect channel gating and, if so, what functional impact this might have on the plant. Here, we report that the Arabidopsis thaliana SNARE SYP121 binds to KC1, a regulatory K+ channel subunit that assembles with different inward-rectifying K+ channels to affect their activities. We demonstrate that SYP121 interacts preferentially with KC1 over other Kv-like K+ channel subunits and that KC1 interacts specifically with SYP121 but not with its closest structural and functional homolog SYP122 nor with another related SNARE SYP111. SYP121 promoted gating of the inward-rectifying K+ channel AKT1 but only when heterologously coexpressed with KC1. Mutation in any one of the three genes, SYP121, KC1, and AKT1, selectively suppressed the inward-rectifying K+ current in Arabidopsis root epidermal protoplasts as well as K+ acquisition and growth in seedlings when channel-mediated K+ uptake was limiting. That SYP121 should be important for gating of a K+ channel and its role in inorganic mineral nutrition demonstrates an unexpected role for SNARE–ion channel interactions, apparently divorced from signaling and vesicle traffic. Instead, it suggests a role in regulating K+ uptake coordinately with membrane expansion for cell growth.

Functional Interaction of the SNARE Protein NtSyp121 in Ca2+ Channel Gating, Ca2+ Transients and ABA Signalling of Stomatal Guard Cells

There is now growing evidence that membrane vesicle trafficking proteins, especially of the superfamily of SNAREs, are critical for cellular signalling in plants. Work from this laboratory first demonstrated that a soluble, inhibitory (dominant-negative) fragment of the SNARE NtSyp121 blocked K+ and Cl- channel responses to the stress-related hormone abscisic acid (ABA), but left open a question about functional impacts on signal intermediates, especially on Ca2+-mediated signalling events. Here, we report one mode of action for the SNARE mediated directly through alterations in Ca2+ channel gating and its consequent effects on cytosolic-free [Ca2+] ([Ca2+]i) elevation. We find that expressing the same inhibitory fragment of NtSyp121 blocks ABA-evoked stomatal closure, but only partially suppresses stomatal closure in the presence of the NO donor, SNAP, which promotes [Ca2+]i elevation independently of the plasma membrane Ca2+ channels. Consistent with these observations, Ca2+ channel gating at the plasma membrane is altered by the SNARE fragment in a manner effective in reducing the potential for triggering a rise in [Ca2+]i, and we show directly that its expression in vivo leads to a pronounced suppression of evoked [Ca2+]i transients. These observations offer primary evidence for the functional coupling of the SNARE with Ca2+ channels at the plant cell plasma membrane and, because [Ca2+]i plays a key role in the control of K+ and Cl- channel currents in guard cells, they underscore an important mechanism for SNARE integration with ion channel regulation during stomatal closure.

Individual variability analysis of fluorescence parameters measured in skin with different levels of nutritive blood flow

Fluorescence spectroscopy has recently become more common in clinical medicine. However, there are still many unresolved issues related to the methodology and implementation of instruments with this technology. In this study, we aimed to assess individual variability of fluorescence parameters of endogenous markers (NADH, FAD, etc.) measured by fluorescent spectroscopy (FS) in situ and to analyse the factors that lead to a significant scatter of results. Most studied fluorophores have an acceptable scatter of values (mostly up to 30%) for diagnostic purposes. Here we provide evidence that the level of blood volume in tissue impacts FS data with a significant inverse correlation. The distribution function of the fluorescence intensity and the fluorescent contrast coefficient values are a function of the normal distribution for most of the studied fluorophores and the redox ratio. The effects of various physiological (different content of skin melanin) and technical (characteristics of optical filters) factors on the measurement results were additionally studied. The data on the variability of the measurement results in FS should be considered when interpreting the diagnostic parameters, as well as when developing new algorithms for data processing and FS devices.

Infrared laser pulse triggers increased singlet oxygen production in tumour cells

Photodynamic therapy (PDT) is a technique developed to treat the ever-increasing global incidence of cancer. This technique utilises singlet oxygen (1O2) generation via a laser excited photosensitiser (PS) to kill cancer cells. However, prolonged sensitivity to intensive light (6–8 weeks for lung cancer), relatively low tissue penetration by activating light (630 nm up to 4 mm), and the cost of PS administration can limit progressive PDT applications. The development of quantum-dot laser diodes emitting in the highest absorption region (1268 nm) of triplet oxygen (3O2) presents the possibility of inducing apoptosis in tumour cells through direct 3O2 → 1O2 transition. Here we demonstrate that a single laser pulse triggers dose-dependent 1O2 generation in both normal keratinocytes and tumour cells and show that tumour cells yield the highest 1O2 far beyond the initial laser pulse exposure. Our modelling and experimental results support the development of direct infrared (IR) laser-induced tumour treatment as a promising approach in tumour PDT.

Nitric Oxide and Plant Ion Channel Control

Nitric oxide (NO) has profound effects on the regulation of ion channels in plants. Although direct evidence to date comes exclusively from electrophysiological studies of guard cells, there is good reason to expect similar patterns of action in other plant cell types as well. As in animals, NO appears to act through two distinct mechanisms. One mechanism is mediated via stimulation of guanylate cyclase, which leads to a rise in cyclic ADP-ribose and, in turn, an increase in the efficacy of Ca2+ release triggered by Ca2+ entry across the plasma membrane. This signal cascade underpins intracellular Ca2+ release and the elevation of cytosolic-free [Ca2+] by the water-stress hormone abscisic acid and leads to profound changes in K+ and Cl− channel activities, to facilitate the ion fluxes for stomatal closure. The second mechanism appears to arise from direct, covalent modification of ion channels by NO, notably of the outward-rectifying K+ channel at the guard cell plasma membrane. The physiological significance of this process of S-nitrosylation has yet to be explored in depth, but almost certainly is allied to plant cell responses to pathogen attack and apoptosis. Both processes, and ion transport in guard cells generally, are now sufficiently well-defined for a full description with accurate kinetics and flux equations in which all of the key parameters are constrained by experimental data. Thus, guard cells are now a prime focus for integrative (so-called systems biology) approaches. Applications of integrative analysis have already demonstrated the potential for accurately predicting physiological behaviours and signal interactions with membrane ion transport.

Substantiation of medical and technical requirements for noninvasive spectrophotometric diagnostic devices

Abstract. A scientific approach to the formulation of medical and technical requirements (MTRs) for noninvasive spectrophotometric diagnostic devices using optical technologies such as laser Doppler flowmetry and absorption spectroscopy is proposed. The theoretical modeling framework, metrological certification, and testing of these devices are still in the early stages of development. The theoretical estimation of the received signal levels for wavelengths between 514 and 940 nm is highly dependent on the blood volume level in the subject tissue. The proposed approach allows, in particular, the calculation of technical and metrological performance constraints of the instruments, such as the ranges of the sensitivity and power-related signal-to-noise ratios for different spectral channels and different biomedical (biochemical and physiological) parameters. Substantiation of specialized MTRs for the noninvasive spectrophotometric diagnostic devices can enable them to develop to the level of standardized measurement techniques.

Basic principles of design and functioning of multifunctional laser diagnostic system for non-invasive medical spectrophotometry

The devising of a general engineering theory of multifunctional diagnostic systems for non-invasive medical spectrophotometry is an important and promising direction of modern biomedical engineering. We aim in this study to formalize in scientific engineering terms objectives for multifunctional laser non-invasive diagnostic system (MLNDS). The structure-functional model as well as a task-function of generalized MLNDS was formulated and developed. The key role of the system software for MLNDS general architecture at steps of ideological-technical designing has been proved. The basic principles of block-modules composition of MLNDS hardware are suggested as well.

Laser reflectance oximetry and Doppler flowmetry in assessment of complex physiological parameters of cutaneous blood microcirculation

The integration of multiple optical techniques within a single diagnostic device is used to address the difficulties in standardising measurement of cutaneous blood micro-dynamics caused by high variability. We demonstrate the benefits of simultaneous assessment of blood relative volume (Vb), microcirculation index (Im) and tissue oxygen saturation (StO2), during long-term examination of healthy volunteers. Consequently, five rhythmic components: endothelial, neurogenic, myogenic, breath and heart pulses were established showing high variability up to 30 – 50% as well as in initial parameters around 16%. All rhythmic components were synchronous with some latency between Im and StO2 in the myogenic component supports the hypothesis of strong correlation between peripheral hemodynamics and oxygen utilisation in tissues.