iLight2: A near-infrared optogenetic tool for gene transcription with low background activation.

Optogenetic tools (OTs) operating in the far-red and near-infrared (NIR) region offer advantages for light-controlling biological processes in deep tissues and spectral multiplexing with fluorescent probes and OTs acting in the visible range. However, many NIR OTs suffer from background activation in darkness. Through shortening linkers, we engineered a novel NIR OT, iLight2, which exhibits a significantly reduced background activity in darkness, thereby increasing the light-to-dark activation contrast. The resultant optimal configuration of iLight2 components suggests a molecular mechanism of iLight2 action. Using a biliverdin reductase knock-out mouse model, we show that iLight2 exhibits advanced performance in mouse primary cells and deep tissues in vivo. Efficient light-controlled cell migration in wound healing cellular model demonstrates the possibility of using iLight2 in therapy and, overall, positions it as a valuable addition to the NIR OT toolkit for gene transcription applications.

On the Importance of Low-Frequency Signals in Functional and Molecular Photoacoustic Computed Tomography.

In photoacoustic computed tomography (PACT) with short-pulsed laser excitation, wideband acoustic signals are generated in biological tissues with frequencies related to the effective shapes and sizes of the optically absorbing targets. Low-frequency photoacoustic signal components correspond to slowly varying spatial features and are often omitted during imaging due to the limited detection bandwidth of the ultrasound transducer, or during image reconstruction as undesired background that degrades image contrast. Here we demonstrate that low-frequency photoacoustic signals, in fact, contain functional and molecular information, and can be used to enhance structural visibility, improve quantitative accuracy, and reduce spare-sampling artifacts. We provide an in-depth theoretical analysis of low-frequency signals in PACT, and experimentally evaluate their impact on several representative PACT applications, such as mapping temperature in photothermal treatment, measuring blood oxygenation in a hypoxia challenge, and detecting photoswitchable molecular probes in deep organs. Our results strongly suggest that low-frequency signals are important for functional and molecular PACT.

On the importance of low-frequency signals in functional and molecular photoacoustic computed tomography.

In photoacoustic computed tomography (PACT) with short-pulsed laser excitation, wideband acoustic signals are generated in biological tissues with frequencies related to the effective shapes and sizes of the optically absorbing targets. Low-frequency photoacoustic signal components correspond to slowly varying spatial features and are often omitted during imaging due to the limited detection bandwidth of the ultrasound transducer, or during image reconstruction as undesired background that degrades image contrast. Here we demonstrate that low-frequency photoacoustic signals, in fact, contain functional and molecular information, and can be used to enhance structural visibility, improve quantitative accuracy, and reduce spare-sampling artifacts. We provide an in-depth theoretical analysis of low-frequency signals in PACT, and experimentally evaluate their impact on several representative PACT applications, such as mapping temperature in photothermal treatment, measuring blood oxygenation in a hypoxia challenge, and detecting photoswitchable molecular probes in deep organs. Our results strongly suggest that low-frequency signals are important for functional and molecular PACT.

Brain vitamin D3-auto/paracrine system in relation to structural, neurophysiological, and behavioral disturbances associated with glucocorticoid-induced neurotoxicity.

Introduction: Vitamin D3 (VD3) is a potent para/autocrine regulator and neurosteroid that can strongly influence nerve cell function and counteract the negative effects of glucocorticoid (GC) therapy. The aim of the study was to reveal the relationship between VD3 status and behavioral, structural-functional and molecular changes associated with GC-induced neurotoxicity. Methods: Female Wistar rats received synthetic GC prednisolone (5 mg/kg b.w.) with or without VD3 (1000 IU/kg b.w.) for 30 days. Behavioral, histological, physiological, biochemical, molecular biological (RT-PCR, Western blotting) methods, and ELISA were used. Results and discussion: There was no difference in open field test (OFT), while forced swim test (FST) showed an increase in immobility time and a decrease in active behavior in prednisolone-treated rats, indicative of depressive changes. GC increased the perikaryon area, enlarged the size of the nuclei, and caused a slight reduction of cell density in CA1-CA3 hippocampal sections. We established a GC-induced decrease in the long-term potentiation (LTP) in CA1-CA3 hippocampal synapses, the amplitude of high K+-stimulated exocytosis, and the rate of Ca2+-dependent fusion of synaptic vesicles with synaptic plasma membranes. These changes were accompanied by an increase in nitration and poly(ADP)-ribosylation of cerebral proteins, suggesting the development of oxidative-nitrosative stress. Prednisolone upregulated the expression and phosphorylation of NF-κB p65 subunit at Ser311, whereas downregulating IκB. GC loading depleted the circulating pool of 25OHD3 in serum and CSF, elevated VDR mRNA and protein levels but had an inhibitory effect on CYP24A1 and VDBP expression. Vitamin D3 supplementation had an antidepressant-like effect, decreasing the immobility time and stimulating active behavior. VD3 caused a decrease in the size of the perikaryon and nucleus in CA1 hippocampal area. We found a recovery in depolarization-induced fusion of synaptic vesicles and long-term synaptic plasticity after VD3 treatment. VD3 diminished the intensity of oxidative-nitrosative stress, and suppressed the NF-κB activation. Its ameliorative effect on GC-induced neuroanatomical and behavioral abnormalities was accompanied by the 25OHD3 repletion and partial restoration of the VD3-auto/paracrine system. Conclusion: GC-induced neurotoxicity and behavioral disturbances are associated with increased oxidative-nitrosative stress and impairments of VD3 metabolism. Thus, VD3 can be effective in preventing structural and functional abnormalities in the brain and behavior changes caused by long-term GC administration.

N-Stearoylethanolamine Inhibits Integrin-Mediated Activation, Aggregation, and Adhesion of Human Platelets.

N-stearoylethanolamine (NSE), a lipid mediator that belongs to the N-acylethanolamine (NAE) family, has anti-inflammatory, antioxidant, and membranoprotective actions. In contrast to other NAEs, NSE does not interact with cannabinoid receptors. The exact mechanism of its action remains unclear. The aim of this study is to evaluate the action of NSE on activation, aggregation, and adhesion of platelets that were chosen as a model of cellular response. Aggregation of platelets was measured to analyze the action of NSE (10-6-10-10 M) on platelet reactivity. Changes in granularity and shape of resting platelets and platelets stimulated with ADP in the presence of NSE were monitored by flow cytometry, and platelet deganulation was monitored by spectrofluorimetry. In vivo studies were performed using obese insulin-resistant rats. Binding of fibrinogen to the GPIIb/IIIa receptor was estimated using indirect ELISA and a scanning electron microscopy (SEM). It was found that NSE inhibits the activation and aggregation of human platelets. Our results suggest that NSE may decrease the activation and subsequent aggregation of platelets induced by ristocetin, epinephrine, and low doses of ADP. NSE also reduced the binding of fibrinogen to GPIIb/IIIa on activated platelets. These effects could be explained by the inhibition of platelet activation mediated by integrin receptors: the GPIb-IX-V complex for ristocetin-induced activation and GPIIb/IIIa when epinephrine and low doses of ADP were applied. The anti-platelet effect of NSE complements its anti-inflammatory effect and allows us to prioritize studies of NSE as a potent anti-thrombotic agent. SIGNIFICANCE STATEMENT: N-stearoylethanolamine (NSE) was shown to possess inhibitory action on platelet activation, adhesion, and aggregation. The mechanism of inhibition possibly involves integrin receptors. This finding complements the known anti-inflammatory effects of NSE.

Design and Initial Characterization of a Small Near-Infrared Fluorescent Calcium Indicator.

Near-infrared (NIR) genetically encoded calcium indicators (GECIs) are becoming powerful tools for neuroscience. Because of their spectral characteristics, the use of NIR GECIs helps to avoid signal loss from the absorption by body pigments, light-scattering, and autofluorescence in mammalian tissues. In addition, NIR GECIs do not suffer from cross-excitation artifacts when used with common fluorescent indicators and optogenetics actuators. Although several NIR GECIs have been developed, there is no NIR GECI currently available that would combine the high brightness in cells and photostability with small size and fast response kinetics. Here, we report a small FRET-based NIR fluorescent calcium indicator iGECInano. We characterize iGECInano in vitro, in non-neuronal mammalian cells, and primary mouse neurons. iGECInano demonstrates the improvement in the signal-to-noise ratio and response kinetics compared to other NIR GECIs.

Transgenic mice encoding modern imaging probes: Properties and applications.

Modern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

Optogenetic manipulation and photoacoustic imaging using a near-infrared transgenic mouse model.

Optogenetic manipulation and optical imaging in the near-infrared range allow non-invasive light-control and readout of cellular and organismal processes in deep tissues in vivo. Here, we exploit the advantages of Rhodopseudomonas palustris BphP1 bacterial phytochrome, which incorporates biliverdin chromophore and reversibly photoswitches between the ground (740-800 nm) and activated (620-680 nm) states, to generate a loxP-BphP1 transgenic mouse model. The mouse enables Cre-dependent temporal and spatial targeting of BphP1 expression in vivo. We validate the optogenetic performance of endogenous BphP1, which in the activated state binds its engineered protein partner QPAS1, to trigger gene transcription in primary cells and living mice. We demonstrate photoacoustic tomography of BphP1 expression in different organs, developing embryos, virus-infected tissues and regenerating livers, with the centimeter penetration depth. The transgenic mouse model provides opportunities for both near-infrared optogenetics and photoacoustic imaging in vivo and serves as a source of primary cells and tissues with genomically encoded BphP1.

Three-Dimensional Deep-Tissue Functional and Molecular Imaging by Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT).

Non-invasive small-animal imaging technologies, such as optical imaging, magnetic resonance imaging and x -ray computed tomography, have enabled researchers to study normal biological phenomena or disease progression in their native conditions. However, existing small-animal imaging technologies often lack either the penetration capability for interrogating deep tissues (e.g., optical microscopy), or the functional and molecular sensitivity for tracking specific activities (e.g., magnetic resonance imaging). To achieve functional and molecular imaging in deep tissues, we have developed an integrated photoacoustic, ultrasound and acoustic angiographic tomography (PAUSAT) system by seamlessly combining light and ultrasound. PAUSAT can perform three imaging modes simultaneously with complementary contrast: high-frequency B-mode ultrasound imaging of tissue morphology, microbubble-enabled acoustic angiography of tissue vasculature, and multi-spectral photoacoustic imaging of molecular probes. PAUSAT can provide three-dimensional (3D) multi-contrast images that are co-registered, with high spatial resolutions at large depths. Using PAUSAT, we performed proof-of-concept in vivo experiments on various small animal models: monitoring longitudinal development of placenta and embryo during mouse pregnancy, tracking biodistribution and metabolism of near-infrared organic dye on the whole-body scale, and detecting breast tumor expressing genetically-encoded photoswitchable phytochromes. These results have collectively demonstrated that PAUSAT has broad applicability in biomedical research, providing comprehensive structural, functional, and molecular imaging of small animal models.

Targeting hippocampal amyloidogenesis with SV2A protein modulator levetiracetam.

Cerebral amyloid ß (Aß) proteostasis is compromised under neuronal overexcitation, long-term neuroinflammation and brain aging. Using the animal model of LPS-induced neuroinflammation we demonstrated that treatment with levetiracetam, a specific modulator of synaptic vesicle glycoprotein SV2A, rescues abnormal synaptic vesicle (SV) fusion and neurotransmitter release, decreasing elevated hippocampal APP levels in vivo. Therapy with levetiracetam upregulates the SV2A in hippocampus and restores the level of apolipoprotein E, involved in brain Aß aggregation/clearance and resolution of inflammation. We demonstrated that oligomers of Aß1-42 and Aß1-40 peptides promote SV clustering, which reduces the rate and plateau level of subsequent homo- and heterotypic SNARE-mediated SV fusion. Oligomeric Aß1-42 lowered ΔpH gradient across the vesicular membrane, thus affecting their neurotransmitter storage capacity. In contrast, monomers of Aß1-42 and Aß1-40 had negligible impact on studied processes. Our data suggests that in the course of progression of neuroinflammation oligomeric forms of Aß1-42 and Aß1-40 can compromise the SV fusion machinery and that antiepileptic agent levetiracetam, acting on SV recycling and restricting overexcitation, is able to affect APP processing and Aß generation within the hippocampus in vivo.

Neuroprotective and Immunomodulatory Action of the Endocannabinoid System under Neuroinflammation.

Endocannabinoids (eCBs) are lipid-based retrograde messengers with a relatively short half-life that are produced endogenously and, upon binding to the primary cannabinoid receptors CB1/2, mediate multiple mechanisms of intercellular communication within the body. Endocannabinoid signaling is implicated in brain development, memory formation, learning, mood, anxiety, depression, feeding behavior, analgesia, and drug addiction. It is now recognized that the endocannabinoid system mediates not only neuronal communications but also governs the crosstalk between neurons, glia, and immune cells, and thus represents an important player within the neuroimmune interface. Generation of primary endocannabinoids is accompanied by the production of their congeners, the N-acylethanolamines (NAEs), which together with N-acylneurotransmitters, lipoamino acids and primary fatty acid amides comprise expanded endocannabinoid/endovanilloid signaling systems. Most of these compounds do not bind CB1/2, but signal via several other pathways involving the transient receptor potential cation channel subfamily V member 1 (TRPV1), peroxisome proliferator-activated receptor (PPAR)-α and non-cannabinoid G-protein coupled receptors (GPRs) to mediate anti-inflammatory, immunomodulatory and neuroprotective activities. In vivo generation of the cannabinoid compounds is triggered by physiological and pathological stimuli and, specifically in the brain, mediates fine regulation of synaptic strength, neuroprotection, and resolution of neuroinflammation. Here, we review the role of the endocannabinoid system in intrinsic neuroprotective mechanisms and its therapeutic potential for the treatment of neuroinflammation and associated synaptopathy.

Nicotinic acetylcholine receptors regulate clustering, fusion and acidification of the rat brain synaptic vesicles.

The brain nicotinic acetylcholine receptors (nAChRs) expressed in pre-synaptic nerve terminals regulate neurotransmitter release. However, there is no evidence for the expression of nAChRs in synaptic vesicles, which deliver neurotransmitter to synaptic cleft. The aim of this paper was to investigate the presence of nAChRs in synaptic vesicles purified from the rat brain and to study their possible involvement in vesicles life cycle. According to dynamic light scattering analysis, the antibody against extracellular domain (1-208) of α7 nAChR subunit inhibited synaptic vesicles clustering. Sandwich ELISA with nAChR subunit-specific antibodies demonstrated the presence of α4ß2, α7 and α7ß2nAChR subtypes in synaptic vesicles and showed that α7 and ß2 nAChR subunits are co-localized with synaptic vesicle glycoprotein 2A (SV2A). Pre-incubation with either α7-selective agonist PNU282987 or nicotine did not affect synaptic vesicles clustering but delayed their Ca2+-dependent fusion with the plasma membranes. In contrast, nicotine but not PNU282987 stimulated acidification of isolated synaptic vesicles, indicating that α4ß2 but not α7-containing nAChRs are involved in regulation of proton influx and neurotransmitter refilling. Treatment of rats with levetiracetam, a specific modulator of SV2A, increased the content of α7 nAChRs in synaptic vesicles accompanied by increased clustering but decreased Ca2+-dependent fusion. These data for the first time demonstrate the presence of nAChRs in synaptic vesicles and suggest an active involvement of cholinergic regulation in neurotransmitter release. Synaptic vesicles may be an additional target of nicotine inhaled upon smoking and of α7-specific drugs widely discussed as anti-inflammatory and pro-cognitive tools.

Stearoylethanolamide interferes with retrograde endocannabinoid signalling and supports the blood-brain barrier integrity under acute systemic inflammation.

Neuroinflammation plays a prominent role in the onset of demyelinating diseases, major depressive disorder and delayed neurodegeneration. An open question remains whether pharmacological suppression of inflammation can effectively reduce the progression of these states. Bioactive lipid mediators such as N-acylethanolamines (NAEs) have an anti-inflammatory activity and are of pharmacological interest due to their endogenous on-demand production and the existence of distinct biological targets in humans and animals. Here we demonstrate for the first time, that treatment with stearoylethanolamide (SEA), a prevailing endogenously formed NAE, is neuroprotective against LPS-induced neuroinflammation in C57BL/6 male mice. SEA restricted the spreading of peripheral inflammation to the brain, and averted the activation of resident microglia and leukocyte trafficking to the brain parenchyma. Treatment with SEA per se increased the neuronal expression of cannabinoid receptors CB1/2 and brain levels of the most potent endogenous CB1/2 agonist 2-arachidonoylglycerol in vivo. SEA enhanced the amplitude of synaptic vesicle release, supported the balanced signal-to-noise ratio in glutamate- and GABAergic neurotransmission and decreased the excitotoxic risk associated with higher extracellular glutamate levels under neuroinflammation. The interference of SEA with the endocannabinoid system and presynaptic neurotransmitter release may represent an intrinsic neuroprotective mechanism that is triggered by inflammation and glutamate excitotoxicity. Thus, our data allows to consider SEA for the preventive therapy of acute and late-onset neuroinflammation-associated synaptic dysfunction and neurodegeneration.

Vitamin D deficiency induces the excitation/inhibition brain imbalance and the proinflammatory shift.

Vitamin D3 is among the major neurosteroids whose role in developing and adult brain is intensively studied now. Its active form 1,25(OH)2D3 regulates the expression and functioning of a range of brain-specific proteins, which orchestrate the neurotransmitter turnover, neurogenesis and neuroplasticity. Despite numerous studies of the vitamin D role in normal and pathological brain function, there is little evidence on the mechanisms of alterations in excitatory and inhibitory neurotransmission under vitamin D deficiency (VDD). Using the animal model we characterized the dysfunction of excitatory and inhibitory neurotransmission under alimentary VDD. The shift between unstimulated and evoked GABA release under VDD was largely reversed after treatment of VDD, whereas the impairments in glutamatergic system were only partially recovered after 1-month vitamin D3 supplementation. The increase of the external glutamate level and unstimulated GABA release in brain nerve terminals was associated with intensified ROS production and higher [Ca2+]i in presynapse. The negative allosteric modulation of presynaptic mGlu7 receptors significantly enhanced exocytotic GABA release, which was decreased under VDD, thereby suggesting the neuroprotective effect of such modulation of inhibitory neurotransmission. Synaptic plasma membranes and cytosolic proteins contribute to the decreased stimulated release of neurotransmitter, by being the crucial components, whose functional state is impaired under VDD. The critical changes with synaptic vesicles occurred at the docking step of the process, whereas malfunctioning of synaptic cytosolic proteins impacted the fusion event foremost. The decreased amplitude of exocytosis was inherent for non-excitable cells as well, as evidenced by lower platelet degranulation. Our data suggest the presynaptic dysfunction and proinflammatory shift as the early events in the pathogenesis of VDD-associated disorders and provide evidences for the neuroprotective role of vitamin D3.

Modulation of neurosecretion and approaches for its multistep analysis.

BACKGROUND: Neurosecretion is the multistep process occurring in separate spatial and temporal cellular boundaries which complicates its comprehensive analysis. Most of the research are focused on one distinct stage of synaptic vesicle recycling. Here, we describe approaches for complex analysis of synaptic vesicle (SV) endocytosis and separate steps of exocytosis at the level of presynaptic bouton and highly purified SVs. METHODS: Proposed fluorescence-based strategies and analysis of neurotransmitter transport provided the advantages in studies of exocytosis steps. We evaluated SV docking/tethering, their Ca2+-dependent fusion and release of neurotransmitters gamma-aminobutyric acid (GABA) and glutamate in two animal models. RESULTS: Approaches enabled us to study: 1) endocytosis/Ca2+-dependent release of fluorescent carbon nanodots (CNDs) during stimulation of nerve terminals; 2) the action of levetiracetam, modulator of SV glycoprotein SV2, on fusion competence of SVs and stimulated release of GABA and glutamate; 3) impairments of several steps of neurosecretion under vitamin D3 deficiency. CONCLUSIONS: Our algorithm enabled us to verify the method validity for multidimensional analysis of SV turnover. By increasing SV docking and the size of readily releasable pool (RRP), levetiracetam is able to selectively enhance the stimulated GABA secretion in hippocampal neurons. Findings suggest that SV2 regulates RRP through impact on the number of docked/primed SVs. GENERAL SIGNIFICANCE: Methodology can be widely applied to study the stimulated neurosecretion in presynapse, regulation of SV docking, their Ca2+-dependent fusion with target membranes, quantitative analysis of expression of neuron-specific proteins, as well as for testing the efficiency of pre-selected designed neuroactive substances.

Antiplatelet and anti-proliferative action of disintegrin from Echis multisquamatis snake venom.

AIM: To purify the platelet aggregation inhibitor from Echis multisquamatis snake venom (PAIEM) and characterize its effect on platelet aggregation and HeLa cell proliferation. METHODS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) were used for PAIEM identification. Platelet aggregation in the presence of PAIEM was studied on aggregometer Solar-AP2110. The changes of shape and granularity of platelets in the presence of PAIEM were studied on flow cytometer COULTER EPICS XL, and degranulation of platelets was estimated using spectrofluorimetry. Indirect enzyme-linked immunosorbent assay was used for the determination of target of PAIEM on platelet surface. An assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to evaluate the effect of PAIEM on the proliferation of HeLa cells in cell culture. RESULTS: The molecular weight of the protein purified from Echis multisquamatis venom was 14.9 kDa. Half-maximal inhibitory concentration (IC50) of PAIEM needed to inhibit adenosine diphosphate (ADP)-induced platelet aggregation was 7 µM. PAIEM did not affect thrombin- or ADP-induced platelet activation, but it did prevent binding of the anti-IIb antibody to glycoprotein IIb/IIIa (GPIIbIIIa)-receptor of adhered platelets and inhibited the viability of HeLa cells by 54%. CONCLUSION: As a member of the disintegrin family, PAIEM inhibited platelet aggregation and cell proliferation possibly by blocking integrin-mediated interactions. However, it did not impair cellular signaling causing any changes in platelet shape and granularity and did not affect ADP-induced platelet degranulation. This disintegrin was shown to be a potent inhibitor of integrin-mediated cellular interactions including platelet aggregation or cancer cell proliferation.

4-Аminopyridine sequesters intracellular Ca2+ which triggers exocytosis in excitable and non-excitable cells.

4-aminopyridine is commonly used to stimulate neurotransmitter release resulting from sustained plasma membrane depolarization and Ca2+-influx from the extracellular space. This paper elucidated unconventional mechanism of 4-aminopyridine-stimulated glutamate release from neurons and non-neuronal cells which proceeds in the absence of external Ca2+. In brain nerve terminals, primary neurons and platelets 4-aminopyridine induced the exocytotic release of glutamate that was independent of external Ca2+ and was triggered by the sequestration of Ca2+ from intracellular stores. The initial level of 4-aminopyridine-stimulated glutamate release from neurons in the absence or presence of external Ca2+ was subequal and the difference was predominantly associated with subsequent tonic release of glutamate in Ca2+-supplemented medium. The increase in [Ca2+]i and the secretion of glutamate stimulated by 4-aminopyridine in Ca2+-free conditions have resulted from Ca2+ efflux from endoplasmic reticulum and were abolished by intracellular free Ca2+ chelator BAPTA. This suggests that Ca2+ sequestration plays a profound role in the 4-aminopyridine-mediated stimulation of excitable and non-excitable cells. 4-Aminopyridine combines the properties of depolarizing agent with the ability to sequester intracellular Ca2+. The study unmasks additional mechanism of action of 4-aminopyridine, an active substance of drugs for treatment of multiple sclerosis and conditions related to reduced Ca2+ efflux from intracellular stores.

Anion carrier formation by calix[4]arene-bis-hydroxymethylphosphonic acid in bilayer membranes.

The action of calix[4]arenes C-91, C-97, C-99, C-107 and C-160 on solvent-containing planar bilayer membranes made of cholesterol and egg phosphatidylcholine (egg PC) or synthetic 18-carbon-tail phospholipid DOPC has been investigated in a voltage-clamp mode. Within the range of calix[4]arenes tested, a steady-state voltage-dependent transmembrane current was achieved only after addition of calix[4]-arene C-99 (calix[4]arene-bis-hydroxymethylphosphonic acid) from the side of the membrane the positive potential was applied to. This current exhibited anion selectivity passing more chloride at negative potentials applied from the side of the membrane to which calix[4]arene C-99 was introduced. The kinetics and temperature-dependence determined for calix[4]arene C-99-mediated ionic transport suggest a carrier mode of facilitated diffusion.

Glutamate release from platelets: exocytosis versus glutamate transporter reversal.

Platelets express neuronal and glial glutamate transporters EAAT 1-3 in the plasma membrane and vesicular glutamate transporters VGLUT 1,2 in the membrane of secretory granules. This study is focused on the assessment of non-exocytotic glutamate release, that is, the unstimulated release, heteroexchange and glutamate transporter reversal in platelets. Using the glutamate dehydrogenase assay, the absence of unstimulated release of endogenous glutamate from platelets was demonstrated, even after inhibition of glutamate transporters and cytoplasmic enzyme glutamine synthetase by dl-threo-ß-benzyloxyaspartate and methionine sulfoximine, respectively. Depolarization of the plasma membrane by exposure to elevated [K(+)] did not induce the release of glutamate from platelets that was shown using the glutamate dehydrogenase assay and radiolabeled l-[(14)C]glutamate. Glutamate efflux by means of heteroexchange with transportable inhibitor of glutamate transporters dl-threo-ß-hydroxyaspartate (dl-THA) was not observed. Furthermore, the protonophore cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and inhibitor of V-type H(+)-ATPase bafilomycin A1 also failed to stimulate the release of glutamate from platelets. However, exocytotic release of glutamate from secretory granules in response to thrombin stimulation was not prevented by elevated [K(+)], dl-THA, FCCP and bafilomycin A1. In contrast to nerve terminals, platelets cannot release glutamate in a non-exocytotic manner. Heteroexchange, transporter-mediated and unstimulated release of glutamate are not inherent to platelets. Therefore, platelets may be used as a peripheral marker/model for the analysis of glutamate uptake by brain nerve terminals only (direct function of transporters), whereas the mechanisms of glutamate release are different in platelets and nerve terminals. Glutamate is released by platelets exclusively by means of exocytosis. Also, reverse function of vesicular glutamate transporters of platelets is rather ambiguous.

Neurotoxic potential of lunar and martian dust: influence on em, proton gradient, active transport, and binding of glutamate in rat brain nerve terminals.

The harmful effects of lunar dust (LD) on directly exposed tissues are documented in the literature, whereas researchers are only recently beginning to consider its effects on indirectly exposed tissues. During inhalation, nano-/microsized particles are efficiently deposited in nasal, tracheobronchial, and alveolar regions and transported to the central nervous system. The neurotoxic potential of LD and martian dust (MD) has not yet been assessed. Glutamate is the main excitatory neurotransmitter involved in most aspects of normal brain function, whereas disturbances in glutamate homeostasis contribute to the pathogenesis of major neurological disorders. The research was focused on the analysis of the effects of LD/MD simulants (JSC-1a/JSC, derived from volcanic ash) on the key characteristics of glutamatergic neurotransmission. The average size of LD and MD particles (even minor fractions) before and after sonication was determined by dynamic light scattering. With the use of radiolabeled l-[(14)C]glutamate, it was shown that there is an increase in l-[(14)C]glutamate binding to isolated rat brain nerve terminals (synaptosomes) in low [Na(+)] media and at low temperature in the presence of LD. MD caused significantly lesser changes under the same conditions, whereas nanoparticles of magnetite had no effect at all. Fluorimetric experiments with potential-sensitive dye rhodamine 6G and pH-sensitive dye acridine orange showed that the potential of the plasma membrane of the nerve terminals and acidification of synaptic vesicles were not altered by LD/MD (and nanoparticles of magnetite). Thus, the unique effect of LD to increase glutamate binding to the nerve terminals was shown. This can have deleterious effects on extracellular glutamate homeostasis in the central nervous system and cause alterations in the ambient level of glutamate, which is extremely important for proper synaptic transmission. During a long-term mission, a combination of constant irritation due to dust particles, inflammation, stress, low gravity and microgravity, radiation, UV, and so on may consequently change the effects of the dust and aggravate neurological consequences.