Disruptive effects of glucocorticoids on glutathione peroxidase biochemistry in hippocampal cultures

Glucocorticoids (GCs), the adrenal steroids secreted during stress, compromise the ability of hippocampal neurons to survive various necrotic insults. We have previously observed that GCs enhance the hippocampal neurotoxicity of reactive oxygen species and, as a potential contributor to this, decrease the activity of the antioxidant enzyme, glutathione peroxidase (GSPx). In this report, we have studied the possible mechanisms underlying this GC effect upon GSPx in primary hippocampal cultures and have observed several results. (i) Corticosterone (the GC of rats) decreased glutathione levels; this was predominately a result of a decrease in levels of reduced glutathione (GSH), the form of glutathione which facilitates GSPx activity. (ii) Corticosterone also decreased levels of NADPH; this may help explain the effect on GSH as NADPH is required for regeneration of GSH from oxidized glutathione. (iii) However, the corticosterone effect on total glutathione levels could not just be caused by the NADPH effect, as there were also reduced levels of oxidized glutathione. (iv) Corticosterone caused a small but significant decrease in GSPx activity over a range of glucose concentrations; this occurred under circumstances of an excess of glutathione as a substrate, suggesting a direct effect of corticosterone on GSPx activity. (v) This corticosterone effect was likely to have functional implications, in that enhancement of GSPx activity (to the same magnitude as activity was inhibited by corticosterone) by GSPx overexpression protected against an excitotoxin. Thus, GCs have various effects, both energetic and non-energetic in nature, upon steps in GSPx biochemistry that, collectively, may impair hippocampal antioxidant capacity.     

Methods for preparation of recombinant cytokine proteins V. mutant analogues of human interferon-gamma with higher stability and activity

Mutant analogues of recombinant human immune interferon (IFN-gamma) with higher stability and biological activity were prepared. Depending on the analogue, protein structure modification might involve introduction of an intramonomer disulfide bond (through replacements of Glu7Cys and Ser69Cys), C-terminal shortening by 10 amino acid residues, as well as Gln133Leu substitution in truncated variant. Isolation, purification, and renaturation of the IFN-gamma analogues expressed in Escherichia coli as inclusion bodies were performed according to the scheme developed earlier for wild-type protein. The main idea of this scheme is to remove cellular impurities before recombinant protein renaturation. Folding kinetics of IFN-gamma was studied by reversed-phase HPLC. IFN-gamma and mutant proteins were characterized by their thermal stability and biological activity. Introduction of the intramolecular disulfide bond together with C-terminal shortening and replacement of C-terminal residue was shown to result in increasing the thermal stability by 19 degrees C and four times enhancement of biological activity compared with intact IFN-gamma molecule.     

Optical and magneto-optical activity of cytochrome bd from Geobacillus thermodenitrificans

Cytochromes bd are terminal oxidases in the respiratory chains of many prokaryotic organisms. They reduce O? to 2H?O at the expense of electrons extracted from quinol. The oxidases can be divided into two subfamilies, L and S, based on the presence of either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I designated as 'Q-loop'. The L-subfamily members, e.g. the enzyme from Escherichia coli, are relatively well-studied and were shown to generate proton-motive force. The S-subfamily comprises the majority of cytochromes bd including the enzyme from Geobacillus thermodenitrificans but is very poor studied. We compared the properties of cytochromes bd from G. thermodenitrificans and E. coli at room temperature using a combination of absorption, CD and MCD spectroscopy. The G. thermodenitrificans enzyme does contain the high-spin heme b(HS) ("b(595)") despite the fact that its characteristic Q(00)-band ("α"-band) at 595nm is not seen in the absorption spectra; stoichiometry of hemes b(LS), b(HS) and d per the enzyme complex is suggested to be 1:1:1. At 1mM CO, 20-25% of ferrous heme b(HS) in the G. thermodenitrificans oxidase binds the ligand, while in case of the E. coli enzyme such a reaction is minor. In the G. thermodenitrificans oxidase, the excitonic interaction between ferrous hemes b(HS) and d decreased as compared to that in the E. coli bd. The latter may suggest that the two enzymes differ in the distance between heme d and heme b(HS) and/or in the angle between their porphyrin planes.     

Suppression of basal autophagy reduces lung cancer cell proliferation and enhances caspase-dependent and -independent apoptosis by stimulating ROS formation

Autophagy is a catabolic process involved in the turnover of organelles and macromolecules which, depending on conditions, may lead to cell death or preserve cell survival. We found that some lung cancer cell lines and tumor samples are characterized by increased levels of lipidated LC3. Inhibition of autophagy sensitized non-small cell lung carcinoma (NSCLC) cells to cisplatin-induced apoptosis; however, such response was attenuated in cells treated with etoposide. Inhibition of autophagy stimulated ROS formation and treatment with cisplatin had a synergistic effect on ROS accumulation. Using genetically encoded hydrogen peroxide probes directed to intracellular compartments we found that autophagy inhibition facilitated formation of hydrogen peroxide in the cytosol and mitochondria of cisplatin-treated cells. The enhancement of cell death under conditions of inhibited autophagy was partially dependent on caspases, however, antioxidant NAC or hydroxyl radical scavengers, but not the scavengers of superoxide or a MnSOD mimetic, reduced the release of cytochrome c and abolished the sensitization of the cells to cisplatin-induced apoptosis. Such inhibition of ROS prevented the processing and release of AIF (apoptosis-inducing factor) and HTRA2 from mitochondria. Furthermore, suppression of autophagy in NSCLC cells with active basal autophagy reduced their proliferation without significant effect on the cell-cycle distribution. Inhibition of cell proliferation delayed accumulation of cells in the S phase upon treatment with etoposide that could attenuate the execution stage of etoposide-induced apoptosis. These findings suggest that autophagy suppression leads to inhibition of NSCLC cell proliferation and sensitizes them to cisplatin-induced caspase-dependent and -independent apoptosis by stimulation of ROS formation.     

Long-term ecology and conservation of the Kungur forest-steppe (pre-Urals,Russia): case study Spasskaya Gora

Biodiversity and Conservation - The Kungur forest-steppe is the northernmost outpost of European forest-steppe, located in the western pre-Urals within the boreal climatic zone. The co-existence of...     

The fusion of synaptic vesicle membranes studied by lipid mixing: the R18 fluorescence assay validity

The various experimental approaches and octadecyl rhodamine B chloride (R18) assay's capability to meet the criteria for examining the Ca²⁺dependent synaptic vesicles (SVs) fusion with target membranes have been investigated. The existence of at least two simultaneous processes one of which attributed to real Ca²⁺-dependent membrane fusion, while another is considered to be non-specific probe transfer has been shown. The differences in response to temperature changes were found for R18 fluorescence dequenching upon stimulation of membrane fusion or nonspecific probe transfer. The temperature dependences of the probe dequenching rate were the same for heterotypic and homotypic membrane systems and increased with the temperature growth. The combination of R18 fluorescence studies with the data obtained by dynamic light scattering (DLS) offers a unique opportunity for the determination of SVs aggregation and the membrane fusion. The cholesterol content of the synaptosomal plasma membrane was modulated by methyl-β-cyclodextrin (MCD). The MCD molecule has proven to bind directly the membrane cholesterol and interact with lipophilic probe R18 that affects its fluorescence. The obvious distinctions in probe dequenching due to the membrane mixing or the MCD effect were observed. The cholesterol depletion from the synaptosomal plasma membranes was found to inhibit the process of Ca²⁺-induced membrane fusion with SVs. Thus, the manipulations with conditions of R18 probe dequenching at the model conditions, specific for the Ca²⁺-triggered fusion steps of regulated exocytosis, allowed us to determine the relative contribution of probe transfer and genuine membrane fusion to the overall fluorescence signal.     

Oxygenated complex of cytochrome bd from Escherichia coli: stability and photolability

Cytochrome bd is one of the two terminal ubiquinol oxidases in the respiratory chain of Escherichia coli catalyzing reduction of O2 to H2O. The enzyme is expressed under low oxygen tension; due to high affinity for O2 it is isolated mainly as a stable oxygenated complex. Direct measurement of O2 binding to heme d in the one-electron reduced isolated enzyme gives K(d(O2)) of approximately 280 nM. It is possible to photolyse the heme d oxy-complex by illumination of the enzyme for several minutes under microaerobic conditions; the light-induced difference absorption spectrum is virtually identical to the inverted spectrum of O2 binding to heme d.     

In vivo expression and characteristics of novel alpha-D-mannose-rich glycoprotein markers of apoptotic cells

We recently established that an increased expression of alpha-D-mannose (Man)- and beta-D-galactose-rich plasma membrane glycoproteins (GPs) is characteristic for apoptotic cells in vitro [Bilyy, R.O., Stoika, R.S., 2003. Lectinocytochemical detection of apoptotic murine leukemia L1210 cells. Cytometry 56A, 89-95]. It was independent of cell line or apoptosis-inducing agent, and can therefore be considered as a selective marker for identification and isolation of apoptotic cells [Bilyy, R.O., Antonyuk, V.O., Stoika, R.S., 2004. Cytochemical study of role of alpha-D-mannose- and beta-D-galactose-containing glycoproteins in apoptosis. J. Mol. Histol. 35, 829-838]. The main goals of the present study were: (1) to determine whether an increased expression of specific GPs also takes place after apoptosis induction in vivo; and (2) to identify additional characteristics of the membrane GP markers of the apoptotic cells. To reach these goals, we studied the expression of alpha-Man-rich membrane GPs in murine leukemia L1210 cells inoculated into abdominal cavities of mice which were then subjected to the action of apoptosis inducer doxorubicin. Another experimental model used in the present work was splenocytes obtained from mice treated with dexamethasone. Lectin-affinity chromatography and PAGE electrophoresis, or PAGE electrophoresis and lectinoblot analysis were applied for isolation of plasma membrane GPs (34 kDa, and high M(W) of approximately 600 and 800 kDa) whose expressions were increased during apoptosis. Triton X-114 treatment of cell membrane samples showed that the apoptotic cell-specific GPs were localized in the peripheral and integral compartments of plasma membrane. Apoptosis in vitro and in vivo was accompanied by an increased expression of the same GP, identified by MALDI-TOF MS analysis as the microtubule-actin cross-linking factor 1. Other GPs, whose expressions were also increased at apoptosis, were similarly identified as G-protein beta-subunit like (Acc# BAA06185.1) and dystonin isoform beta.     

Correlation of the cytotoxic activity of four different alkaloids, from Chelidonium majus (greater celandine), with their DNA intercalating properties and ability to induce breaks in the DNA of NK/Ly murine lymphoma cells

The purpose of this study was to examine the relationship between the DNA intercalating characteristics and the DNA damaging capacity of four alkaloids extracted from Chelidonium majus L, as well as their toxicity towards murine NK/Ly lymphoma cells. Chelerythrine, sanguinarine and coptisine were found to be intercalated into the DNA isolated from NK/Ly cells, meanwhile, chelidonine exhibited no affinity to DNA. Sanguinarine exhibited the greatest toxicity toward NK/Ly cells, and the toxicity of the other three decreased in descending order: chelerythrine, coptisine and chelidonine. Chelerythrine and sanguinarine caused DNA damage, illustrated by the formation of comets of the third class. Coptisine was less toxic than chelerythrine and sanguinarine, and affected the formation the same class of comets in higher concentration. The quantity of comets induced by chelidonine were negligible, a finding consistent with its inability to intercalate into DNA structure. The ability of four main alkaloids of Chelidonium majus L., to intercalate into DNA isolated from murine NK/Ly lymphoma cells, correlated with their ability to induce breaks in cellular DNA and with their toxic effect towards those cells.