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Autoinflammatory diseases include a number of monogenic systemic inflammatory diseases, as well as acquired autoinflammatory diseases such as gout. Here, we show that the myeloid Src-family kinases Hck, Fgr, and Lyn are critical for experimental models of gout, as well as for genetically determined systemic inflammation in the Ptpn6me-v/me-v (motheaten viable) mouse model. The Hck−/−Fgr−/−Lyn−/− mutation abrogated various monosodium urate (MSU) crystal–induced pro-inflammatory responses of neutrophils, and protected mice from the development of gouty arthritis. The Src-family inhibitor dasatinib abrogated MSU crystal–induced responses of human neutrophils and reduced experimental gouty arthritis in mice. The Hck−/−Fgr−/−Lyn−/− mutation also abrogated spontaneous inflammation and prolonged the survival of the Ptpn6me-v/me-v mice. Spontaneous adhesion and superoxide release of Ptpn6me-v/me-v neutrophils were also abolished by the Hck−/−Fgr−/−Lyn−/− mutation. Excessive activation of tyrosine phosphorylation pathways in myeloid cells may characterize a subset of autoinflammatory diseases.

Titin is a molecular spring in parallel with myosin motors in each muscle half-sarcomere, responsible for passive force development at sarcomere length (SL) above the physiological range (>2.7 μm). The role of titin at physiological SL is unclear and is investigated here in single intact muscle cells of the frog ( Rana esculenta ), by combining half-sarcomere mechanics and synchrotron X-ray diffraction in the presence of 20 μM para-nitro-blebbistatin, which abolishes the activity of myosin motors and maintains them in the resting state even during activation of the cell by electrical stimulation. We show that, during cell activation at physiological SL, titin in the I-band switches from an SL-dependent extensible spring (OFF-state) to an SL-independent rectifier (ON-state) that allows free shortening while resisting stretch with an effective stiffness of ~3 pN nm −1 per half-thick filament. In this way, I-band titin efficiently transmits any load increase to the myosin filament in the A-band. Small-angle X-ray diffraction signals reveal that, with I-band titin ON, the periodic interactions of A-band titin with myosin motors alter their resting disposition in a load-dependent manner, biasing the azimuthal orientation of the motors toward actin. This work sets the stage for future investigations on scaffold and mechanosensing-based signaling functions of titin in health and disease.

Background: Although estimates of suboptimal adherence to oral corticosteroids in asthma range from 30% to 50%, no ideal method for measurement exists; the impact of poor adherence in severe asthma is likely to be particularly high. Research Questions: What is the prevalence of suboptimal adherence detected by self-reporting and direct measures? Is suboptimal adherence associated with disease activity? Study Design and Methods: Data were included from individuals with severe asthma taking part in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) study and prescribed daily oral corticosteroids. Participants completed the Medication Adherence Report Scale, a five-item questionnaire used to grade adherence on a scale from 1 to 5, and provided a urine sample for analysis of prednisolone and metabolites by liquid chromatography-mass spectrometry. Results: Data from 166 participants were included in this study: mean (SD) age, 54.2 (± 11.9) years; FEV 1, 65.1% (± 20.5%) predicted; female, 58%; 37% completing the Medication Adherence Report Scale reported suboptimal adherence; and 43% with urinary corticosteroid data did not have detectable prednisolone or metabolites in their urine. Good adherence by both methods was detected in 49 of the 142 (35%) of participants in whom both methods were performed; adherence detection did not match between methods in 53%. Self-reported high adherers had better asthma control and quality of life, whereas directly measured high adherers had lower blood eosinophil levels. Interpretation: Low adherence is a common problem in severe asthma, whether measured directly or self-reported. We report poor agreement between the two methods, suggesting some disassociation between self-assessment of medication adherence and regular oral corticosteroid use, which suggests that each approach may provide complementary information in clinical practice.

Post-stroke muscle spasticity affects 37% of stroke survivors and disables self-supporting life management. There is a high unmet medical need for an efficient antispastic drug because current muscle relaxants are often of limited efficacy and cause severe neurological and cardiovascular side effects due to targeting the central or peripheral nervous system. We developed a new-generation anti-spastic oral drug, MPH-220, which efficiently relaxes spastic skeletal muscles and lacks cardiovascular and neurological adverse effects because it selectively targets skeletal myosin, the contractile protein of muscles. MPH-220 is an efficient skeletal muscle specific actomyosin relaxant demonstrated on human muscle samples. Orally administered MPH-220 reduces muscle force of living rats without cardiovascular side effects. Brain-damage induced spastic animals showed drastic improvement in gait disorders upon oral MPH-220 treatment resulting in significantly straightened body posture, reduced number of spontaneous falling and cramping, and more ordered limb positions. Due to selective accumulation of MPH-220 in skeletal muscle tissues, antispastic effect was maintained for more than 10 hours. Due to its mechanism of action MPH-220 does not cause complete loss of muscle tone even at high doses. Furthermore, MPH-220 has excellent ADMET properties for oral administration: it is highly absorptive, non-mutagenic and has no effects on hERG channels, kinases, nuclear hormone receptors and GPCRs. Considering these results, MPH-220 is a promising anti-spastic oral drug candidate, which provides potential nervous system-independent therapies for spasticity and muscle stiffness without cardiovascular and neuronal side effects. Phase I clinical trials are scheduled for the beginning of 2021. Funded by the Hungarian National Research, Development and Innovation Office (NVKP 16-1-2016-0051 and PIACI-KFI-2019-00488).

Subcellular dynamics of non-muscle myosin 2 (NM2) is crucial for a broad-array of cellular functions. To unveil mechanisms of NM2 pharmacological control, we determined how the dynamics of NM2 diffusion is affected by NM2′s allosteric inhibitors, i.e. blebbistatin derivatives, as compared to Y-27632 inhibiting ROCK, NM2′s upstream regulator. We found that NM2 diffusion is markedly faster in central fibers than in peripheral stress fibers. Y-27632 accelerated NM2 diffusion in both peripheral and central fibers, whereas in peripheral fibers blebbistatin derivatives slightly accelerated NM2 diffusion at low, but markedly slowed it at high inhibitor concentrations. In contrast, rapid NM2 diffusion in central fibers was unaffected by direct NM2 inhibition. Using our optopharmacological tool, Molecular Tattoo, sub-effective concentrations of a photo-crosslinkable blebbistatin derivative were increased to effective levels in a small, irradiated area of peripheral fibers. These findings suggest that direct allosteric inhibition affects the diffusion profile of NM2 in a markedly different manner compared to the disruption of the upstream control of NM2. The pharmacological action of myosin inhibitors is channeled through autonomous molecular processes and might be affected by the load acting on the NM2 proteins.

Riparian zone forests are a special type of agro-forestry systems. As buffer zones protect stream systems against stress factors. These ecosystems are diverse so ecologically valuable, on the other hand, they are valuable (because of high productivity) from an economic viewpoint as well.Riparian forests are very vulnerable because they strongly depend on surplus water (shallow groundwater or seasonally flooded condition). Long drought periods caused by changing climate induce lowering of the water table and shortening flooded periods. With reasonably designed water supply systems, these negative processes can be stopped, and valuable ecosystems can be preserved.The effect of an artificial structures (new lakes and bottom thresholds) induced water supply was evaluated in Kaszó Forest (Somogy county, Hungary). Eco-Hydrological monitoring (groundwater and phenological) was conducted on 14 regular (under the effect of water supply interventions) and 4 control plots in different forest ecosystems. The impact of water supply interventions was interpreted with spatio-temporal groundwater level difference analysis and found that surplus water had a positive effect on the riparian zone water table (40-50 cm rise in the neighbourhood of new lakes) and vegetation.A complex field monitoring (hydro-meteorological and phenological measurements) was conducted on three representative locations. 1-D Hydrus model was successfully calibrated for an alder and two common oak forest plots. Diurnal signal of groundwater levels was used for ET estimation in the model. Model results showed that groundwater uptake of forest vegetation was significantly increased (30%) regarding water supply interventions.Acknowledgements: The research was supported by EFOP-3.6.2-16-2017-00018 in University of Sopron project.Keywords: riparian forest, water supply, groundwater monitoring, 1-D Hydrus model

The thermal hydraulics of supercritical water under forced-, mixed convection and natural circulation conditions is not fully understood. In order to study the thermal hydraulic behaviour of this fluid under natural circulation conditions a small size, closed experimental loop has been designed and built. The thermal hydraulic phenomenon occurring in the loop can be measured by thermocouples mounted onto the outer surface of the heated tube wall, absolute and differential pressure transducers and a flow meter; moreover, simultaneously can be visualized by neutron radiography techniques. This paper describes the loop itself, the process of the experiment with the measurement techniques, the data acquisition system applied and the results got during the first measurement series. Based on the results of the first measurement series, it was found that the measured part of the steady state characteristic is independent from the system pressure. A slight dependence of steady state characteristic on the inlet temperature can be identified: the higher the inlet temperature the higher the mass flow rate. The total pressure drop and its components seem to be independent from the system pressure but strongly dependent on the inlet temperature due to the influence of bulk-fluid temperature on the relevant thermophysical properties (density and dynamic viscosity). The pressure drop due to acceleration of flow found to be negligible next to the two dominant components, the pressure drop due to frictional resistance and due to gravity. The coupled evaluation of the radiographic images and the thermophysical properties of water have shown that the main driving force behind the decrease of the neutron attenuation is the decreasing water density as the bulk-fluid temperature increases. The reverse of this relationship could be exploited during the validation of future Monte Carlo simulations.

BackgroundAlthough several studies link high levels of IL-6 and soluble IL-6 receptor (sIL-6R) to asthma severity and decreased lung function, the role of IL-6 trans-signaling (IL-6TS) in asthmatic patients is unclear.ObjectiveWe sought to explore the association between epithelial IL-6TS pathway activation and molecular and clinical phenotypes in asthmatic patients.MethodsAn IL-6TS gene signature obtained from air-liquid interface cultures of human bronchial epithelial cells stimulated with IL-6 and sIL-6R was used to stratify lung epithelial transcriptomic data (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes [U-BIOPRED] cohorts) by means of hierarchical clustering. IL-6TS–specific protein markers were used to stratify sputum biomarker data (Wessex cohort). Molecular phenotyping was based on transcriptional profiling of epithelial brushings, pathway analysis, and immunohistochemical analysis of bronchial biopsy specimens.ResultsActivation of IL-6TS in air-liquid interface cultures reduced epithelial integrity and induced a specific gene signature enriched in genes associated with airway remodeling. The IL-6TS signature identified a subset of patients with IL-6TS–high asthma with increased epithelial expression of IL-6TS–inducible genes in the absence of systemic inflammation. The IL-6TS–high subset had an overrepresentation of frequent exacerbators, blood eosinophilia, and submucosal infiltration of T cells and macrophages. In bronchial brushings Toll-like receptor pathway genes were upregulated, whereas expression of cell junction genes was reduced. Sputum sIL-6R and IL-6 levels correlated with sputum markers of remodeling and innate immune activation, in particular YKL-40, matrix metalloproteinase 3, macrophage inflammatory protein 1β, IL-8, and IL-1β.ConclusionsLocal lung epithelial IL-6TS activation in the absence of type 2 airway inflammation defines a novel subset of asthmatic patients and might drive airway inflammation and epithelial dysfunction in these patients.

Background The role of IL-17 immunity is well established in patients with inflammatory diseases, such as psoriasis and inflammatory bowel disease, but not in asthmatic patients, in whom further study is required. Objective We sought to undertake a deep phenotyping study of asthmatic patients with upregulated IL-17 immunity. Methods Whole-genome transcriptomic analysis was performed by using epithelial brushings, bronchial biopsy specimens (91 asthmatic patients and 46 healthy control subjects), and whole blood samples (n = 498) from the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort. Gene signatures induced in vitro by IL-17 and IL-13 in bronchial epithelial cells were used to identify patients with IL-17–high and IL-13–high asthma phenotypes. Results Twenty-two of 91 patients were identified with IL-17, and 9 patients were identified with IL-13 gene signatures. The patients with IL-17–high asthma were characterized by risk of frequent exacerbations, airway (sputum and mucosal) neutrophilia, decreased lung microbiota diversity, and urinary biomarker evidence of activation of the thromboxane B2 pathway. In pathway analysis the differentially expressed genes in patients with IL-17-high asthma were shared with those reported as altered in psoriasis lesions and included genes regulating epithelial barrier function and defense mechanisms, such as IL1B, IL6, IL8, and β-defensin. Conclusion The IL-17–high asthma phenotype, characterized by bronchial epithelial dysfunction and upregulated antimicrobial and inflammatory response, resembles the immunophenotype of psoriasis, including activation of the thromboxane B2 pathway, which should be considered a biomarker for this phenotype in further studies, including clinical trials targeting IL-17.

Background: Adult-onset severe asthma is characterized by highly symptomatic disease despite high-intensity asthma treatments. Understanding of the underlying pathways of this heterogeneous disease is needed for the development of targeted treatments. Gene set variation analysis is a statistical technique used to identify gene profiles in heterogeneous samples. Objective: We sought to identify gene profiles associated with adult-onset severe asthma. Methods: This was a cross-sectional, observational study in which adult patients with adult-onset of asthma (defined as starting at age ≥18 years) as compared with childhood-onset severe asthma (

This paper describes the first steps of the realization of a natural circulation loop cooled by supercritical water. The thermal hydraulics of supercritical water is not fully understood. The thermal hydraulic phenomenon in the loop is intended to be measured by thermocouples, absolute and differential pressure transducers, flow meter and simultaneously visualized by dynamic neutron radiography techniques. The structure of the loop, the measurement techniques applied and the data acquisition system will be presented in details.

We investigate the full randomness of the angular distribution of gamma-ray bursts (GRBs) detected by the BATSE (the Burst and Transient Source Experiment). We divided the BATSE sample into 5 subsamples (short1, short2, intermediate, long1, long2 based on their durations and peak fluxes and studied the angular distributions separately. We used three methods, the Voronoi tesselation, minimal spanning tree and multifractal spectra to search for non-randomness in the subsamples. To investigate the eventual non-randomness in the subsamples we defined 13 test-variables (9 from the Voronoi tesselation, 3 from the minimal spanning tree and one from the multifractal spectrum). We performed Monte Carlo simulations taking into account the BATSE sky-exposure function. We tested the randomness by introducing squared Euclidean distances in the parameter space of the test-variables. We concluded that the short1, short2 groups deviate significantly (99.90%, 99.98%) from the full randomness in the distribution of the squared Euclidean distances, however, it is not the case with the long subsamples. In the intermediate group, the squared Euclidean distances also yield a significant deviation (98.51%).

Zusammenfassung: In dem von Verfassern beschriebenen Fall wurden durch Stachybotrys alternans teils durch Einatmung der Pilze, teils durch unmittelbaren Kontakt mit der Haut und der Schleimhaut Mykotoxikosen hervorgerufen. Die Umstande der Erkrankung, das sie auf einmal und plotzlich aufgetreten ist, sowie die beobachteten Symptome liesen keinen Zweifel daruber, das es sich um eine Massen-Mykotoxikose handelt. Diese Hypothese wurde durch die Angaben und Zuchtungsergebnisse bestatigt, die bei der an Ort und Stelle durchgefuhrten Untersuchungen erhalten wurden. Es wird darauf aufmerksam gemacht, das schimmeliges Streu nicht nur bei Tieren, sondern auch bei Menschen Erkrankungen verursachen kann. Bisher wurde keine sich auf das Vorkommen von humanen Fallen beziehende Veroffentlichung im humanmedizinischen Schrifttum gefunden. Summary: The authors report a mass occurence of mycotoxicoses as caused by Stachybotrys alternans partly by inhalung fungi, partly by their direct contact with skin or mucous membranes. The circumstances, the sudden and simultaneous outbreak of the disease, as well as its symptoms made it clear that the disease was mycotoxicoses. This hypothesis was justified by examining the scene and the sick people, and by the results of cultivation. The authors draw attention to the fact that mildewy litter and fodder can be the cause of the disease not only among animals, but men too. Medical reference has had no reference so far conderning cases where man is involved.

This study intended to determine whether the replacement of vitamin D3 with alfacalcidol results in any bone mineral density (BMD) increase in 76 patients unresponsive to the combination of alendronate and conventional vitamin D3 treatment. In these patients the conventional vitamin D3 had been replaced with alfacalcidol (0.5 μg/day), and then the patients were followed up for a year. After treatment for 1 year, Wilcoxon test revealed a small but statistically significant (P < 0.001) increase in the BMD values of the forearm and lumbar vertebrae, in the serum calcium and urinary calcium/creatinine ratio in first-voided morning urine. However, the serum alkaline phosphatase activity, phosphorus, parathormone, osteocalcin levels and the urinary d-pyr/creatinine ratio decreased significantly (P < 0.001). As suggested by our results, combination therapy with alendronate and alfacalcidol increases bone density and improves the biochemical markers of bone turnover, without any substantial increase in the incidence of adverse effects.

This paper describes the first successful application of a preconcentration unit to increase the sensitivity of a photoacoustic ammonia concentration measuring instrument. A diode laser based near-infrared (1532 nm) photoacoustic ammonia monitoring instrument was combined with a tungsten (VI) oxide coated preconcentration unit to reach a sub-ppb detection limit with a compact, automatic measuring instrument. The system has no measurable cross-sensitivity to common atmospheric gases, most importantly to water vapor and carbon dioxide. The minimum detectable amount of ammonia is 2.9 ng, which means a minimum detectable concentration of 0.5 ppb with a 30-min measurement time. Results of intercomparison measurements carried out both under laboratory and field conditions with reference to an electrochemical AMANDA instrument prove the applicability of the system in environmental ammonia concentration monitoring.

Two sites with five wells in the Danube-Tisza interfluvial were studied in order to help understand the genesis of the high-As content of shallow ground water. Separation of As species was carried out in the field as it proved to be the best method by which to preserve the As species. Different geochemical environments could be separated by the speciation studies which correlate with the concentrations of oxyanion-forming elementss uch asV, Se, Mo, U and W.

A new variant of batch extractive distillation, the so-called inverse-fed batch extractive distillation is presented. The total amount of the entrainer is pre-loaded to the boiler, and the mixture charge to be separated is continuously fed to the column in this novel configuration. The feasibility study of conventional extractive distillation was extended and a thorough study was performed to separate a maximum boiling azeotrope with intermediate boiling entrainer. The new configuration was found more efficient than the conventional one. The results of the feasibility study was validated and completed with a sensitivity analysis performed with commercial simulator software.

A systematic comparison is presented about the separation tasks of azeotropic and close-boiling mixtures applying batch extractive distillation (BED) in rectifier. All the eight possible mixture types with at most a single azeotrope (minimum and maximum boiling azeotropes with heavy, light, and intermediate boiling entrainers; and close boiling mixtures with heavy and light entrainers) are compared. The main results of the feasibility studies on the hitherto unpublished cases are presented. All the cases are feasible in batch rectifier, applying BED. The operation steps are determined by the relative position of the azeotropic composition and entrainer in bubble point ranking. The main limiting parameters ( F / V , N , E premix ) are also determined by the mentioned relative position; only the existence of maximum number of stages in the rectifying section is determined by the type of the azeotrope. Use of residue curves maps (RCMs) for predicting feasibility is not generally satisfactory, but profiles maps can be used instead. Studying only the total reflux case can be misleading, and should be treated with great care. The theoretical results of separation variants applying intermediate boiling entrainer were proved experimentally.

The development of the thylakoid membrane was studied during illumination of dark-grown barley seedlings by using biochemical methods, and Fourier transform infrared and spin label electron paramagnetic resonance spectroscopic techniques. Correlated, gross changes in the secondary structure of membrane proteins, conformation, composition, and dynamics of lipid acyl chains, SDS/PAGE pattern, and thermally induced structural alterations show that greening is accompanied with the reorganization of membrane protein assemblies and the protein–lipid interface. Changes in overall membrane fluidity and noncovalent protein–lipid interactions are not monotonic, despite the monotonic accumulation of chlorophyll, LHCII [light-harvesting chlorophyll a / b -binding (polypeptides) associated with photosystem II] apoproteins, and 18:3 fatty acids that follow a similar time course with highest rates between 12–24 h of greening. The 18:3 fatty acid content increases 2.8-fold during greening. This appears to both compensate for lipid immobilization by membrane proteins and facilitate packing of larger protein assemblies. The increase in the amount of protein-solvating immobile lipids, which reaches a maximum at 12 h, is caused by 40% decrease in the membranous mean diameter of protein assemblies at constant protein/lipid mass ratio. Alterations in the SDS/PAGE pattern are most significant between 6–24 h. The size of membrane protein assemblies increases ≈4.5-fold over the 12–48-h period, likely caused by the 2-fold gain in LHCII apoproteins. The thermal stability of thylakoid membrane proteins increases monotonically, as detected by an increasing temperature of partial protein unfolding during greening. Our data suggest that a structural coupling between major protein and lipid components develops during greening. This protein–lipid interaction is required for the development and protection of thylakoid membrane protein assemblies.

A silicon micromachined bearing was designed and fabricated consisting of a pair of self-aligning v-grooves. The two Si v-grooves were bonded together to form the three-dimensional structure through solder bumps of 75 μm diameter that provide alignment with a crucial vertical displacement tolerance. The microbearing includes integrated diode temperature sensors that serve to monitor a safe operating range under load. Current versus temperature curves and finite element analysis simulate the range of diode sensitivity. This three-dimensional structure, fabricated by silicon technology, demonstrates that a four to ten fold reduction in surgical cutting tool size can be achieved by utilizing the micromachined silicon microbearing in a micro-electromechanical system (MEMS) device. A prototype microtool has been bench tested and compared with realistic operating conditions. Based on this comparison, the microbearing holds promise for application in microsurgical cutting tools or as a MEMS cutting tool for surgical applications.

H4[SiMo12O40] can take part in redox reactions as well as electrophilic transformations. The reduction of Mo(VI) to Mo(V) in various solvents was followed by ESR spectroscopy. The spectra show significant rhombic splitting, which can be correlated with the dielectric constants of the solvents. Coordination of solvents distorts the symmetry of MoO6 octahedra. Based on the degree of splitting it is suggested that the strength of coordination decreases in the sequence: acetone>ethanol>1,4-butanediol>acetonitrile>DMSO. The comparison of 95Mo NMR chemical shift data obtained in various solvents and the results of modelling solvent–solute interactions indicated, however, that dimethyl sulfoxide (DMSO) interacts most strongly with the Mo sites of unreduced acid.

The adsorption and dissociation of CH2I2 and C2H5I on Mo2C/Mo(111) surface have been investigated with the purpose of producing adsorbed CH2 and C2H5 species. Methods used include high resolution electron energy loss, X-ray photoelectron, Auger electron, and temperature programmed desorption spectroscopies. Independently of the coverage, CH2I2 adsorbs molecularly at 90–100 K. The dissociation of an adsorbed layer starts around 180–190 K. The primary products of thermal dissociation are adsorbed CH2 and I. The species CH2 undergoes self-hydrogenation to CH4 at Tp=300 K and dimerization into C2H4 at and above 222–280 K. Ethylene formed desorbs above 400 K. C2H5I also adsorbs molecularly on Mo2C at 90–100 K and dissociates to C2H5 and I above 150 K. The reaction of C2H5 on Mo2C/Mo(111) surface yielding C2H6 and C2H4 proceeds at a much lower temperature, above 180 K, than that of CH2. Neither the cleavage of the C–C bond nor the coupling of C2 compounds occurred to detectable extent under the reaction conditions. The ethylene formed in the reactions of both CHx species exhibited the same features as observed following C2H4 adsorption on Mo2C: the stable di-σ-bonded ethylene is transformed into ethylidyne at higher temperature. The results are discussed in relevance to the conversion of methane into benzene on Mo2C deposited on ZSM-5.

About 50 formation waters, produced from wells tapping the aquifers of the clastic Pannonian Basin and Mesozoic carbonates, were studied by GC–MS for their content of hexane soluble organic compounds. About half of the waters studied are hotter than 65°C (the regional threshold of thermal gas generation) and always contain a great variety of naturally occurring polycyclic aromatic hydrocarbons (PAH) consisting of aromatic hydrocarbons, heteroaromatics and in some cases phenols. These waters are not known to be connected with oil pools. The most characteristic compound classes of six PAH-rich water samples are discussed here. Mono-, di-, tri- and tetracyclic aromatic and heterocyclic fused aromatic hydrocarbons are present in these waters but in rather varying relative proportion. The absence of phenols in two of these waters, one produced from a carbonate and one from a sandy aquifer, is explained by the absence of terrestrial kerogen and insufficient maturity (R0

Gramicidin S was incorporated into dimyristoylphosphatidylcholine dispersions and the observed two-component EPR spectra of spin-labelled lipids at 30 °C were analysed by a two-stage algorithm, including spectral subtractions and two-site exchange simulations. A limited range of temperatures around 30 °C was found suitable for such measurements. It has been found that negatively charged labelled lipids display a selectivity towards the intramembranous part of the peptide. The relative association constants for spin-labelled stearic acid (14-SASL) and phosphatidylserine (14-PSSL) were Kr = 2.08± 0.10 and 1.18±0.08, respectively, when compared with the zwitterionic phosphatidylcholine label (14-PCSL, Kr≡ 1). The lateral diffusion of spin-labelled lipids in peptide-free regions causes exchange between those labels in the bulk fluid lipid phase and motionally restricted boundary labelled lipids at the apolar interface of gramicidin S. Owing to exchange, the spectral anisotropy of labelled lipids giving rise to the slow-motion spectral component was gradually decreased, and there was an augmentation of spectral intensity in between the motionally restricted (slow motion) and the fast tumbling (motionally averaged) labelled lipid components. Two-component exchange simulations allowed the determination of off-rates of labelled phospholipids, showing an inverse proportionality with lipid-protein selectivity. Spin-labelled procaine exhibited limited selectivity and fast exchange similar to the on-coming non-specifically associated lipids.

The specific effects exerted by different heavy metals on both the function and the structure of the photosynthetic apparatus were addressed. The functional analysis performed via the fluorescence induction kinetics revealed that the applied toxic heavy metals can be classified into two groups: Cd and Ni had no significant effect on the photosynthetic electron transport, while Cu, Pb and Zn strongly inhibited the Photosystem II (PS II) activity, as evidenced by the dramatic decreases in both the variable (Fv) and the maximal (Fm) fluorescence. The structural effects of the heavy metal ions on the thylakoid membranes were considered in three relations: (1) lipids, (2) proteins — studied by Fourier transform infrared (FTIR) spectroscopy, and (3) lipid—protein interactions — investigated by electron spin resonance (ESR) spectroscopy using spin-labeled probe molecules. The studied heavy metal ions had only a non-specific rigidifying effect on the thylakoid lipids. As regards proteins, Cd and Ni had no effect on the course of their heat denaturation. The heat denaturation of the proteins was accompanied by a decrease in the α-helix content (1656 cm-1), a parallel increase in the disordered segments (1651 cm-1), a decrease in the intramolecular β-sheet (1636 cm-1) content and the concomitant appearance of an intermolecular β-structure (1621 cm-1). In contrast with Cd and Ni, Cu and Zn blocked the appearance of the intermolecular β-structure. Pb represented an intermediate case. It seems that these heavy metals alter the native membrane structure in such a way that heat-induced aggregation becomes more limited. The ESR data revealed that certain heavy metals also affect the lipid—protein interactions. While Cd and Ni had hardly any effect on the solvation fraction of thylakoid lipids, Cu, Pb and Zn increased the fraction of lipids solvating the proteins. On the basis of the FTIR and ESR data, it seems that Cu, Pb, and Zn increase the surfaces available for lipid—protein interactions by dissociating membrane protein complexes, and that these ‘lipidated’ proteins have a smaller chance to aggregate upon heat denaturation. The data presented here indicate that the damaging effects of poisonous heavy metals are element-specific, Cu, Pb and Zn interact directly with the thylakoid membranes of the photosynthetic apparatus, while Cd and Ni interfere rather with other metabolic processes of plants.

Implications of the data on lipid-protein interactions involving integral proteins that are obtained from EPR spectroscopy with spin-labelled lipids in membranes are reviewed. The lipid stoichiometry, selectivity and exchange dynamics at the lipid-protein interface can be determined, in addition to information on the configuration and rotational dynamics of the protein-associated lipid chains. These parameters, particularly the stoichiometry and selectivity, are directly related to the intramembranous structure and degree of oligomerisation of the integral protein, and conversely may be used to study the state of assembly of such proteins in the membrane. Insertion of proteins into membranes can be studied by analogous methods. Comparison with the results obtained from integral proteins helps to define the extent of membrane penetration and degree of transmembrane crossing that are relevant to protein translocation mechanisms.

We examined whether the inhibition of nitric oxide (NO) synthesis by NG-nitro-L-arginine (lNNA) abolished pacing-induced preconditioning, and if prolonged exposure to cholesterol-enriched diet led to the loss of preconditioning due to decreased cardiac NO formation. Therefore, Wistar rats fed 2% cholesterol-enriched diet or standard diet for 24 weeks were treated with a single dose of 1 mg/kg lNNA or its solvent at the end of the week 24, respectively. Isolated hearts from all groups were subjected to either preconditioning induced by three consecutive periods of pacing at 600 beats/min for 5 min, with 5-min interpacing periods, or time-matched non-preconditioning perfusion, followed by a 10-min coronary occlusion, respectively. In the control group, coronary occlusion after a non-preconditioning protocol decreased aortic flow (AF) from 45.4+/-2.4 to 15.6+/-1.5 ml/min, and resulted in a lactate dehydrogenase (LDH) release of 219+/-55 mU/min/g, however, preconditioning attenuated the consequences of coronary occlusion [AF: 27.3+/-1.7 ml/min (P0.05); LDH: 44+/-14 mU/min/g (P0.05)]. Preconditioning did not confer protection in the lNNA-treated (AF: 17.4+/-1.5 ml/min; LDH: 151+/-21 mU/min/g), and/or in the high-cholesterol-fed groups (AF: 15.7+/-1.2 ml/min; LDH: 168+/-22 mU/min/g). Preconditioning was preserved however, when hearts were treated with lNNA after the preconditioning protocol [AF: 29.6+/-2.2 ml/min (P0.05); LDH: 48+/-17 mU/min/g (P0.05)]. Both lNNA treatment and cholesterol-enriched diet markedly decreased cardiac NO content assayed by electron spin resonance spectroscopy. We conclude that NO may be involved in the triggering mechanism of pacing-induced preconditioning, the protective effect of which is blocked by sustained exposure to dietary cholesterol, possibly by influencing cardiac metabolism of NO.

Acidic properties of Al-, Zr- and Cr- pillared montmorillonites prepared from Jelsový Potok bentonite (Central Slovakia) have been investigated using temperature-programmed desorption of ammonia. The number of acid sites varied with different interlayer cations from 0.06 to 0.23 mmol g−1. The character of acid sites was similar in the case of Al- and Zr- forms. It is obvious that the chromium analogue contains fewer acid sites, nevertheless, they seem to be stronger compared to Al- and Zr- pillared montmorillonites.

Among several mediators, nitric oxide (NO) and calcitonin gene-related peptide (CGRP) were suggested to be involved in the mechanism of preconditioning. We examined the possible role of the cardiac capsaicin-sensitive sensory innervation in pacing-induced preconditioning, as well as in the cardiac NO and CGRP content. Wistar rats were treated subcutaneously with capsaicin or its solvent in the sequence of 10, 30, and 50 mg/kg increasing single daily doses for 3 days to deplete neurotransmitters of the sensory innervation. Isolated hearts from both groups were then subjected to either preconditioning induced by three consecutive periods of pacing at 600 beats per minute for 5 min with 5 min interpacing periods, or time-matched non-preconditioning perfusion, followed by a 10-min coronary occlusion. NO content of left ventricular tissue samples was assayed by electron-spin resonance, and CGRP release was determined by radioimmunoassay. CGRP immunohistochemistry was also performed. In the non-preconditioned, solvent-treated group, coronary occlusion decreased cardiac output (CO) from 68.1 to 32.1 mL/min, increased left ventricular end-diastolic pressure (LVEDP) from 0.58 to 1.90 kPa, and resulted in 200 mU/min/g LDH release. Preconditioning significantly increased ischaemic CO to 42.9 mL/min (P < 0.05), decreased ischaemic LVEDP to 1.26 kPa (P < 0.05) and decreased LDH release to 47 mU/min/g (P < 0.05) in the solvent-treated group. Preconditioning did not confer protection in the capsaicin-pretreated group (ischaemic CO: 35.6 mL/min; LVEDP: 1.76 kPa; LDH 156 mU/min/g). Capsaicin-treatment markedly decreased cardiac NO content, CGRP release, and CGRP-immunoreactivity. Conclusions: (i) The presence of an intact local sensory innervation is a prerequisite to elicit pacing-induced preconditioning in the rat heart. (ii) A significant portion of cardiac basal NO content may be of neural origin. (iii) Release of NO and CGRP from capsaicin-sensitive nerves may be involved in the mechanism of pacing-induced preconditioning.

Primary and secondary forms of Photorhabdus luminescens Hm and Xenorhabdus nematophilus N2-4 were grown at 18 and 28(deg)C for 24 to 96 h, and we made determinations of the fatty-acid compositions of total lipids and of the fluidity measured by 5-doxyl-stearic acid embedded in liposomes made from total lipids. The levels of the unsaturated fatty acids 16:1 and 18:1 (those with chain lengths of 16 or 18 and one double bond) generally were higher in primary-phase variants of P. luminescens grown at 18(deg)C than in those grown at 28(deg)C. Prolonged culture at 18(deg)C caused the level of 18:1 to fall and reach that observed at 28(deg)C. The ratio of saturated to unsaturated fatty acids rose with prolonged culture times in variants of each species at both phases. When grown at 18(deg)C, the proportion of 16:1 in X. nematophilus was lower than in P. luminescens; the patterns of temperature-induced changes were similar in these species. X. nematophilus contained a greater percentage of short-chain fatty acids (i.e., with chain lengths of

3-(2-Methenylindane-1,3-dione)-1-oxyl-2,2,5,5-tetramethylpyrroline (5-InVSL) is an effective nitroxyl spin label of biological macromolecules, attached rigidly due to its bulky planar side group and the anchoring capability of its oxo groups to protein via hydrogen bonding. Comparing an integral membrane protein, the Ca 2+ -ATPase of sarcoplasmic reticulum (M.W. 113 kD) and the water-soluble hemoglobin (M.W. 64 kD) several notable differences were observed in the labeling processes. Hemoglobin has a single sulfhydryl group in the protein interior (β93), whereas Ca 2+ -ATPase has two classes of labeling sites which have different accessibilities to the aqueous phase. Since the single labeling site of hemoglobin is not exposed to the aqueous phase the labeling efficiency depends on steric hindrance arising due to the bulky side group which, on the other hand, is rather useful for limiting segmental motion of this label in Ca 2+ -ATPase. As a consequence, paramagnetic quenching is proposed for the calibration of spectral parameters used in saturation-transfer ESR of spin-labeled hemoglobin with 5-InVSL, to mask the signals of nitroxyls released via the retro-Michael reaction.

The rotational diffusion of Ca2+-ATPase [Ca2+,Mg2+-activated ATP phosphohydrolase E.C. 3.6.1.38] was studied in native sarcoplasmic reticulum membrane by saturation transfer ESR spectroscopy after covalent labelling of intramembranous sulfhydryl groups with nitroxyl derivative of maleimide (5-MSL) as a function of sucrose and glycerol in the suspending medium. The relative enzymatic activity of sarcoplasmic reticulum was followed by increasing the viscosity of the aqueous phase. The ATP hydrolysing activity of the enzyme decreased differently on adding sucrose and glycerol. In the case of sucrose the reciprocal of power dependence of viscosity was observed, whereas for glycerol an exponential decay law was obtained, indicating solvent-protein interaction. On increasing the viscosity of the aqueous phase by either sucrose or glycerol, no changes were observed in the intramembranous viscosity as measured using intercalated spin-labelled stearic acid (16-SASL). The effective rotational correlation time of the protein was measured, as a mobility parameter, using saturation transfer ESR spectroscopy and found to be increased linearly with the viscosity of the sucrose containing medium and for the extramembranous size a height of 6.8 nm was obtained, indicating that approx. 82% of the volume of Ca2+-ATPase protein is external to the sarcoplasmic reticulum. The addition of glycerol probably promoted protein–protein interaction, as indicated by the larger changes in rotational diffusion and non-linear viscosity dependence.

The cellular distribution of the motor protein non-muscle myosin 2 (NM2) leads to different forms of intracellular strain driving cell motility, cytokinesis and morphogenetic processes including axonal growth and retraction. However, it remains elusive how these cellular processes are governed by the dynamic changes in NM2 localization and supramolecular assembly. To address this problem, we determined the effect of different types of NM2 inhibition on the dynamics of load-bearing stress fibers and unloaded inner cytoplasmatic NM2 structures in live HeLa cells. We followed NM2 redistribution via FRAP, applied also in combination with our recently developed optopharmacological tool, Molecular Tattoo, which allows subcellular confinement of drug effects via 2-photon induced photocrosslinking to targets. We found that the Rho-kinase inhibitor, Y-27632, dramatically accelerates NM2 redistribution and induces stress fiber dissolution, due to NM2 filament disassembly resulting from myosin light chain dephosphorylation. When NM2 was inhibited by para-nitroblebbistatin (pNBleb) or locally by tattooed azidoblebbistatin, in the stress fibers a significant acceleration and suppression of NM2 redistribution was detected at moderate and high inhibitor concentrations, respectively. The observed effects were local and specific for load-bearing peripheral stress fibers, implying the role of mechanical load in NM2 redistribution. Furthermore, in these tests stress fibers remained intact, contrary to that seen upon Rho-kinase inhibition. These results highlight that variations in the localization and/or pharmacological mechanism of NM2 inhibition produce distinct effects on intracellular strain and cellular morphogenesis.Supported by Hungarian Research and Innovation Fund (VKSZ_14-1-2015-0052).

Background: Neuroendorcin tumors are groups of neoplastic neuroendocrin cells located anywhere in the body. They can be divided into two subgroups according to their origin: 1. pancreas, 2. gastrointestinal and bronchopulmonary system of origin (formerly called carcinoid tumors). Recent classification is based on clinicopathology: 1. well differenciated neuroendocrin tumors, 2. well differentiated neuroendocrin carcinoma, 3. poorly differentiated neuroendocrin carcinoma, against former classification based on embrional origin (foregut, midgut, hintgut). Those producing bioactive agents (8–10% of NETs) cause symptoms of carcinoid syndrome (flush, diarrhoea, abdominal pain, bronchospasm, heart disease, pellagra-like skin symptoms). The incidence of NETs increased fivefold in the past 30 years owing to the better and widely available diagnostic methods and our focusing on the disease formerly considered rare. Results: In our Department of Gastroenterology there were 9 cases diagnosed with NET during one year from November 2010 to November 2011. (From 2006 to 2008 one case per year.) We place greater emphasy on the symptoms of carcinoid syndrome which appear more often in the individuals with hepatic metastases and tumors of midgut origin. Cases without special symptoms were diagnosed by histopathology. Diagnosis is getting easier due to the availability of Chromogranin A test and octreoscan. Last year we followed up 12 patients treated with octreotid. One of them died due to co-morbidity (CHF). 11 patients' condition improved, which manifested in decreasing of chromogranin A level, radiological regression and diminution of symptoms. Conclusions: The better the availability of diagnosis is, the more patients we find and due to the uptodate treatment possibilities the patients survive longer