Engineered silver nanoparticles are sensed at the plasma membrane and dramatically modify the physiology of<i>Arabidopsis thaliana</i>plants

Silver nanoparticles (Ag NPs) are the world's most important nanomaterial and nanotoxicant. The aim of this study was to determine the early stages of interactions between Ag NPs and plant cells, and to investigate their physiological roles. We have shown that the addition of Ag NPs to cultivation medium, at levels above 300 mg L-1, inhibited Arabidopsis thaliana root elongation and leaf expansion. This also resulted in decreased photosynthetic efficiency and the extreme accumulation of Ag in tissues. Acute application of Ag NPs induced a transient elevation of [Ca2+]cyt and the accumulation of reactive oxygen species (ROS; partially generated by NADPH oxidase). Whole-cell patch-clamp measurements on root cell protoplasts demonstrated that Ag NPs slightly inhibited plasma membrane K+ efflux and Ca2+ influx currents, or caused membrane breakdown; however, in excised outside-out patches, Ag NPs activated Gd3+-sensitive Ca2+ influx channels with unitary conductance of approximately 56 pS. Bulk particles did not modify the plasma membrane currents. Tests with electron paramagnetic resonance spectroscopy showed that Ag NPs were not able to catalyse hydroxyl radical generation, but that they directly oxidized the major plant antioxidant, l-ascorbic acid. Overall, the data presented shed light on mechanisms of the impact of nanosilver on plant cells, and show that these include the induction of classical stress signalling reactions (mediated by [Ca2+]cyt and ROS) and a specific effect on the plasma membrane conductance and the reduced ascorbate. Significance Statement Silver nanoparticles are known antimicrobial and antifungal agents, and also affect diverse physiological functions in animal cells, but their intracellular effects on plant cells is largely unexplored. Here we show that silver nanoparticles induce stress signalling mediated by Ca2+ and reactive oxygen species, affect plasma membrane conductance and oxidise ascorbic acid

Conformation ability test of human, rabbit and bovine plasminogens and their specific interaction with streptokinase

Human, rabbit and bovine plasminogens, having different sensitivity to streptokinase-activating action, differ, according to spectrophotometric titration, tryptophan fluorescence and circular dichroism spectroscopy, in the state of tyrosine and tryptophan residues, and in secondary and tertiary structures. Human plasminogen-streptokinase equimolar complex formation (according to gel chromatography) is accompanied by a differential ultraviolet spectrum. Difference spectroscopy is a convenient and adequate means of studying the formation of the said complexes. Streptokinase-human plasminogen complex formation is not hindered by partial substitution of water (20%) with ethanol or dimethylsulphoxide or by addition of O.OOIMsodium dodecylsulphate. The complex is not formed in 6 M urea, in solution, at pH < 2.0 or ~12.0-13.0, or with bovine plasminogen. Circular dichroism and tryptophan fluorescence spectral pattern changes during streptokinase-plasminogen complexformation enable us to conclude that streptokinase secondary and tertiary structures undergo certain rearrangements in theframework of the complex, while tryptophan-containing sites of the molecule are not drastically changed. The data obtained enable us to presuppose formation of streptokinase-rabbit plasminogen complexes which differ from human plasminogen complexes with streptokinase

Free oxygen radicals regulate plasma membrane Ca2+- and K+-permeable channels in plant root cells

Free oxygen radicals are an irrefutable component of life, underlying important biochemical and physiological phenomena in animals. Here it is shown that free oxygen radicals activate plasma membrane Ca²⁺- and K⁺-permeable conductances in Arabidopsis root cell protoplasts, mediating Ca²⁺ influx and K⁺ efflux, respectively. Free oxygen radicals generate increases in cytosolic Ca²⁺ mediated by a novel population of nonselective cation channels that differ in selectivity and pharmacology from those involved in toxic Na⁺ influx. Analysis of the free oxygen radical-activated K⁺ conductance showed its similarity to the Arabidopsis root K⁺ outward rectifier. Significantly larger channel activation was found in cells responsible for perceiving environmental signals and undergoing elongation. Quenching root free oxygen radicals inhibited root elongation, confirming the role of radical-activated Ca²⁺ influx in cell growth. Net free oxygen radical-stimulated Ca²⁺ influx and K⁺ efflux were observed in root cells of monocots, dicots, C3 and C4 plants, suggesting conserved mechanisms and functions. In conclusion, two functions for free oxygen radical cation channel activation are proposed: initialization/amplification of stress signals and control of cell elongation in root growth.Vadim Demidchik, Sergey N. Shabala, Katherine B. Coutts, Mark A. Tester and Julia M. Davie

Extracellular ATP-induced NO production and its dependence on membrane Ca2+ flux in Salvia miltiorrhiza hairy roots

Extracellular ATP (eATP) is a novel signalling agent, and nitric oxide (NO) is a well-established signal molecule with diverse functions in plant growth and development. This study characterizes NO production induced by exogenous ATP and examines its relationship with other important signalling agents, Ca2+ and H2O2 in Salvia miltiorrhiza hairy root culture. Exogenous ATP was applied at 10–500 μM to the hairy root cultures and stimulated NO production was detectable within 30 min. The NO level increased with ATP dose from 10–100 μM but decreased from 100–200 μM or higher. The ATP-induced NO production was mimicked by a non-hydrolysable ATP analogue ATPγS, but only weakly by ADP, AMP or adenosine. The ATP-induced NO production was blocked by Ca2+ antagonists, but not affected by a protein kinase inhibitor. ATP also induced H2O2 production, which was dependent on both Ca2+ and protein kinases, and also on NO biosynthesis. On the other hand, ATP induced a rapid increase in the intracellular Ca2+ level, which was dependent on NO but not H2O2. The results suggest that NO is implicated in ATP-induced responses and signal transduction in plant cells, and ATP signalling is closely related to Ca2+ and ROS signalling

The Role of Serum Neutrophil Gelatinase-associated Lipocalin in the Early Diagnosis of Nephropathy in Patients with Acute Alcohol Poisoning

AIM: In our study, we assessed the possibility of using the serum neutrophil gelatinase-associated lipocalin (NGAL) for the early detection of kidney damage in patients with acute alcohol poisoning (AAP). METHODS: The study included 89 patients and 30 healthy donors. All participants in the study were mostly represented by men (90%) aged between 20 and 40 years. The influence of alcohol poisoning severity was also taken into account in the study. The Human NGAL ELISA Kit was used for the quantitative detection of serum NGAL. We also evaluated the main laboratory indicators of kidney functions, including eGFR (calculated according to serum creatinine). RESULTS: We did not find a correlation between blood alcohol concentration and serum NGAL level; also, alcohol poisoning severity did not affect the NGAL values. The results of our study showed the possibility of using the serum NGAL in patients with AAP to detect the preclinical stage of reduced renal function, until the moment when it can be diagnosed with using only serum creatinine. CONCLUSION: We propose to consider an increase in eGFR together with an increase in serum NGAL in this group of patients as a stage, preceding nephropathy, even in the absence of clinical and laboratory signs of impaired renal function