Spermine Induces Autophagy in Plants: Possible Role of NO and Reactive Oxygen Species.

This is the first study to show that polyamine spermine, a low-molecular-weight nitrogen-containing compound, can induce autophagy in plants. This process is accompanied by an increased generation of reactive oxygen species and nitric oxide, which play a signal role and are required for triggering autophagy.

Intracellular origin and ultrastructure of platelet-derived microparticles.

Essentials Platelet microparticles play a major role in pathologies, including hemostasis and thrombosis. Platelet microparticles have been analyzed and classified based on their ultrastructure. The structure and intracellular origin of microparticles depend on the cell-activating stimulus. Thrombin-treated platelets fall apart and form microparticles that contain cellular organelles. SUMMARY: Background Platelet-derived microparticles comprise the major population of circulating blood microparticles that play an important role in hemostasis and thrombosis. Despite numerous studies on the (patho)physiological roles of platelet-derived microparticles, mechanisms of their formation and structural details remain largely unknown. Objectives Here we studied the formation, ultrastructure and composition of platelet-derived microparticles from isolated human platelets, either quiescent or stimulated with one of the following activators: arachidonic acid, ADP, collagen, thrombin or calcium ionophore A23187. Methods Using flow cytometry, transmission and scanning electron microscopy, we analyzed the intracellular origin, structural diversity and size distributions of the subcellular particles released from platelets. Results The structure, dimensions and intracellular origin of microparticles depend on the cell-activating stimulus. The main structural groups include a vesicle surrounded by one thin membrane or multivesicular structures. Thrombin, unlike other stimuli, induced formation of microparticles not only from the platelet plasma membrane and cytoplasm but also from intracellular structures. A fraction of these vesicular particles having an intracellular origin contained organelles, such as mitochondria, glycogen granules and vacuoles. The size of platelet-derived microparticles depended on the nature of the cell-activating stimulus. Conclusion The results obtained provide a structural basis for the qualitative differences of various platelet activators, for specific physiological and pathological effects of microparticles, and for development of advanced assays.

[STRUCTURAL CHARACTERIZATION OF PLATELETS AND PLATELET-DERIVED MICROVESICLES].

Platelets are the anucleated blood cells, wich together with the fibrin stop bleeding (hemostasis). Cellular microvesicles are membrane-surrounded microparticles released into extracellular space upon activation and/or apoptosis of various cells. Platelet-derived macrovesicles from the major population of circulating blood microparticles that play an important role in hemostasis and thrombosis. Despite numerous studies on the pathophysiology of platelet-derived macrovesicles, mechanisms of their formation and structural details remain poorly understood. Here we investigated the ultrastructure of parental platelets and platelet-derived microvesicles formed in vitro by quiescent cells as well as by cells stimulated with one of the following activators: arachidonic acid, ADP, thrombin, calcium ionophore A23187. Using transmission electron microscopy of human platelets and isolated microvesicles, we analyzed the intracellular origin, steps of formation, structural diversity, and size distributions of the subcellular particles. We have revealed that thrombin, unlike other stimuli, not only induced vesiculation of the plasma membrane but also caused break-up of the cells followed by formation of microparticles that are comparable with microvesicles by size. A fraction of these microparticles contained cellular organelles surrounded by a thin membrane. The size of platelet-derived macrovesicles varied from 30 nm to 500 nm, however, the size distributions depended on the nature of a cell-activating stimulus. The results obtained provide new information about the formation of platelet-derived macrovesicles and their structural diversity, wich is important to understand their multiple functions in normal and disease states.

[Circulating microRNAs in lung cancer: prospects for diagnostics, prognosis and prediction of antitumor treatment efficiency].

The major methods of microRNA extraction from different biological fluids (particularly, serum and plasma), approaches to the analysis of microRNA concentration and composition, normalization methods used in data analysis are outlined in the review. The advantages and disadvantages of the described methodological approaches are being highlighted. Special attention is given to microRNAs, circulating in blood, which could be used as the markers for minimally invasive lung cancer diagnostics, prediction of antitumor treatment efficiency and disease prognosis. Prospects and limitations arising from the evaluation of clinical significance of microRNAs as the potential tumor markers, and emerging as roles of various microRNAs in the pathogenesis of lung cancer become known, are discussed.

Nitrate reductase from Triticum aestivum leaves: regulation of activity and possible role in production of nitric oxide.

Nitrate reductase (NR) and peroxidase (POX) are important enzymes involved in the metabolism of reactive oxygen (ROS) and nitrogen species in leaves of wheat (Triticum aestivum L.) seedlings. It has been confirmed that NR activity in wheat leaves depends on the light conditions and the presence of nitrates during the cultivation of the seedlings, and it is regulated by the molybdenum cofactor and phosphorylation. In the present study, confocal microscopy and EPR spectroscopy studies showed that the addition of nitrite, a product of NR, increased the level of nitric oxide (NO). This increase was prevented by the addition of sodium azide, an inhibitor of NR. The results suggest that in wheat leaves one of the key functions of NR is the formation of the signaling NO molecule. Cultivation of green plants under conditions of prolonged (4 days) darkness, a strong stress factor for photosynthesizing cells, decreased the activity of NR. Moreover, darkness induced significant elevation of the POX activity that was prevented by the addition of nitrate to the growth medium. It is proposed that the changes in light conditions result in the competition between nitrate- and ROS-metabolizing activities of POX in leaves, and a possible interaction between NR and POX controls the levels of NO and ROS in the leaf tissue.

[Comparative characteristic of craniometric and computed tomography studies of the mandible structure in applied aspect of dental implantation].

The series of 60 computed tomography scans of maxillofacial area performed in frontal projection, were used to study the peculiarities of mandible structure. The values of mandible morphometric parameters obtained with craniometric method and by computed tomography, were compared. The scope of computer-aided tomography in the evaluation of mandible structure variability was examined. The method of computer-aided tomography makes it possible to receive the data on mandibular corpus height and thickness and on the inclination angle of its alveolar part in the area of prospective surgical operation, as well as on the anatomic-topographical interrelations between teeth root apical portions and mandibular canal.

[Assay of methylated gene RARbeta2 in circulating DNA of blood from patients with lung cancer as a potential prognostic marker].

Blood-based methylated DNA gene RARbeta2 in circulating plasma (cir DNA) and one associated with blood cell surface were assayed in patients with non small cell lung cancer before and after combined treatment. The levels in both appeared to be significantly higher than in healthy subjects. Enhanced levels prior to treatment were associated with greater advancement of the disease and unfavorable prognosis (overall survival). After two courses of neoadjuvant therapy plus surgery methylation indices fell down to match those in healthy subjects. Our data may be instrumental in working out additional criteria to be used in diagnosis, prognosis and follow-up of patients with non small cell lung cancer.

[Molecular-genetic markers in lung cancer diagnostics].

The major approaches to different lung cancer marker development are outlined in the review, including genetic, epigenetic, protein, transcryptomic, proteomic, metabolic, and miRNA markers. As far as epigenetic changes are among the earliest events in malignant transformation, methylated markers are thoroughly discussed. Special attention is given to minimally invasive tumor markers, which could be detected in easily accessible biological fluids, because they can be useful for screening and early diagnostics of cancer (before its clinical manifestation) as well as for verification of standard methods of diagnostics. Extracellular nucleic acids, circulating in blood (cirNA), are highlighted as the potential source of material for the early lung cancer diagnostics, prediction of antitumor treatment efficiency, post-treatment monitoring and disease prognosis.

[Stromules-like protrusions of the membrane environment of plastids in the root cells].

The results of visualization of the stromules-like protrusions of the membrane environment of plastids in the root cells with the help of an electronic microscope are submitted. The cases of occurrence of long narrow protrusion of the external membrane with more short protrusion of the internal membrane of plastid environment inside it are discussed. The possible role of cytoskeleton and plastoskeleton in formation, accordingly, of "external" and "internal" protrusions is considered. The conclusion that the structure and functions of stromules in plant cells should be considered in unity with the structure and functions of the endoplasmic reticulum internal space is made.

[Membrane contacts of the endoplasmic reticulum and their possible functions in the plant cell].

Close contacts of the endoplasmic reticulum membrane and plasmalemma have been visualized inside plant cells by means of electron microscopy. The qualitative similarity of these contacts to high-permeable intercellular contacts in animals has been shown. New data confirming the hypothesis of the identity of stromules, i.e., dynamic tubular protuberances of the plastid membrane of the plant cell, and tubular elements of the endoplasmic reticulum have been presented. New possible functions of the contacts of the endoplasmic reticulum membrane with other membranes inside the cell have been discussed on the basis of this hypothesis.

[Protonophores as inducers of energy dependent changes in the ultrastructure of wheat root cells mitochondria].

The effects of protonophores, 2, 4-DNP (2, 4-dinitrophenol) and CCCP (carbonyl cyanide m-chlorophenyl-hydrazone), on plasma membrane potential, release of K+ into incubation medium, respiratory metabolism, ATP content, and changes in the ultrastructure of cells from excised roots of wheat seedlings were studied. Dissipation of the plasma membrane potential, release of K+ ions and inhibition of the rate of oxygen consumption by the cells were observed following 1 h incubation of roots with the protonophores. Mitochondrial had condensed appearance with numerous sharply defined and slightly swollen cristae. The evidence for cytoplasmic acidification was provided by an increase in plasma membrane potential, a decrease in K+ release into the incubation medium and an increase in ATP content in the cells after 4 h treatment with the protonophores. The protonophores caused unusual spatial arrangements of cristae in mitochondria, e. g. stacked on the top of each other, or having the shape of propellers or florets. Such re-organization of cristae might be of adaptive significance in response to increased concentration of H+ in cytoplasm. After 6 h exposure of the cells to the protonophores, cells ultrastructure destruction started. It is suggested that observed ultrastructural changes in the mitochondria reflect changes in their functional activity and the physiological state of cells during their long-term exposure to the protonophores.

[Membrane contacts of endoplasmic reticulum with plasmalemma in plant cells].

Close contacts of endoplasmatic reticulum membrane with plasmalemma have been shown in common wheat root cells by means of electron microscopy. Qualitative analogy of these contacts with high-permeable intercellular contacts in animals has been preliminary established.

[The effect of apoptosis inductor dexamethasone on the energy exchange and ultrastructure of plant cells].

Changes in respiration and cell ultrastructure induced by long-term incubation with dexamethasone (DM) in excised roots of 5-day old wheat (Triticum aestivum L.) seedlings were investigated. During 5 h incubation of roots with DM, oxygen consumption was inhibited by 20-30%, while respiratory coefficient did not change and its value was about 1. DM prevented from glucose-induced activation of respiration, which indicated blockade of glycolysis and decrease in oxygen uptake by this apoptotic inductor. It has been suggested that the respiratory inhibition by DM might be also connected with the influence of DM on the 1st segment ofmitochondrial electron transport chains. This suggestion is supported by the fact that succinate prevented DM-induced inhibition of respiration. Furthermore, stabilization of intracellular pH by dipeptide carnosine abolished inhibitory effect of DM on respiration. Probably depression of oxygen consumption by DM is also due to acidification of cytoplasm. Strong vacuolization of cytoplasm, one of the characteristics of cell death, occurred in 5 h after treatment of roots with DM. Vacuolization was to a great extent prevented by carnosine. The ultrastructure of root cells after long-term (23 h) treatment with DM was disturbed, and oxygen consumption was also dramatically decreased. These effects of DM were in part prevented by carnosine. The data obtained suggest that DM causes acidification of cytoplasm, disturbance of energy exchange and cytoplasm vacuolization in root cells, and induces death of these cells.

[Wheat root cells functioning under inhibition of I and II complexes of mitochondrial respiratory chain].

A joint effect of rotenone and malonate on the intensity of respiration, output of K+ and ultrastructure of wheat root cells treated for 6 h was studied. The addition of malonate to rotenone containing solution, in which wheat roots had been incubated for an hour, caused further decrease in respiration intensity and K+ output into external medium. Many mitochondria acquired torus shape in 2h after malonate addition. The increase in respiratory intensity and re-entry of K+ from the incubation medium into the cells were observed during following hours of incubation. We assume that reparation and adaptation processes took place in this case. The observed contacts of endoplasmic reticulum lumens with mitochondria are indicative of possible synthesis of an enzyme able to metabolize malonate to acetyl-CoA and CO2. We propose that torus shape of mitochondria is due to the increase in their outer surfaces, that, in turn, is a result of activation of external NAD(P)H-dehydrogenase. These findings may be evidence of possible adaptation of the root cells to the joint effect of the inhibitors.

[Selective labeling of barstar in a T7 polymerase system by 15N-isotopes].

It has been shown that the use of a special growth medium enriched with amino acids and an inhibitor of aminotransferases alpha-aminooxyacetic acid makes possible the selectivity of labeling of barstar with 15N-leucine and 15N-tryptophan. The system of selective labeling, which was previously optimized with respect to the time of introducing the label relative to the time of introducing the inductor IPTG and the inhibitor of cell polymerase rifampicin, was substantially refined by the use of the transamination inhibitor. The inhibition of aminotransferases enables one to completely eliminate the redistribution of the isotope, which is a necessary step in NMR studies even if the strongly metabolizable 15N-leucine is used. The suppression of the redistribution of the isotope by alpha-aminooxyacetic acid is a successful approach to preparation of any selectively labeled proteins in the T7 polymerase system.

[The influence of protonophore high concentration on the structure and functions of wheat root cells].

Protonophore induced structural and functional changes in cells of excised roots of wheat seedlings have been investigated. The vector transfer of H+ inside the cells was accompanied by a decrease in energy supply of these cells (suppression of oxygen consumption and heat release), an output of K+ ions to the incubation medium, and by an increase in its pH value. The initial increase in heat release by roots (1 h) apparently reflects the process of dissipation of deltamicro H+ in plasma membrane. Within the first 5-10 min of exposure of 50 microM CCCP, changes in cell ultrastructure were observed that involved activation of Golgi apparatus, secretion of vesicle contents to the vacuole, and swelling of endoplasmic reticulum canals. Following a 2 h treatment with CCCP, structural and functional changes acquired a destructive character, and after 5-6 h of treatment with protonophore a complete desintegration of cell structure occurred demonstrating formations of myelin-like bodies, fragmentation of plasma membrane, and destruction of the nucleus. Thus, the protonophore induced proton excessive transport inside cells is fast and may cause an irreversible cell de-energization followed by serious disruption of ultrastructural organization of cells leading eventually to their death.

[Dynamics of structural and functional changes in wheat root cells under the action of protonophore]. / Dinamika strukturno-funktsional'nykh izmenenii v kletkakh kornei pshenitsy pri deistvii protonofora.

Structural and functional changes in wheat root cells during long-term action of a protonophore--carbonyl cyanide 3-chlorophenylhydrazone (CCCP)--were studied. It was demonstrated that CCCP affected the electrical potential and inward resistance of cells, increased K+ ions release to the incubation medium, inhibits oxygen uptake for 1-4 h, which was followed by oxygen uptake stimulation for 6 h of treatment. These changes of physiological processes were accompanied with a variety of ultrastructural changes in cell organization, namely in the structure of mitochondria, endoplasmic reticulum canals, and the nucleus. The role of protons is discussed, in particular, in the regulation of metabolic state of mitochondria, and in general regulation of structural and functional conditions of cells.