Potential of Energy Saving as a Tool for Increasing the Stability of Electrical Supply of the Kaliningrad Region

The Kaliningrad region remains to be energetically dependent on neighboring states, since the reliability and continuity of its power supply depends on transit flows of capacity and energy, as well as international relations with transit countries. In this regard, the sustainability of electricity supply is becoming a priority task for ensuring the livelihood of the Kaliningrad region. The basis for the sustainability of electricity supply for the region is the planum implementation of a set of technical and technological measures for optimal control of power consumption. One of the key procedures for optimal control of power consumption is the potentization procedure. It consists in determining the energy-saving potential, the amount of which can be reduced in the given time interval by the energy consumption of the region without affecting its normal functioning. Energy Saving Potential is the absolute difference between the energy consumption of a region's objects obtained without the implementation of energy-saving procedures, on the one hand, and the power consumption corresponding to the lower boundary of the variable confidence interval, on the other. The purpose of the article is to substantiate the need to implement a scientifically based approach to determine the energy saving potential of the Kaliningrad region to ensure the sustainability of the region's electricity supply. This approach will allow to reduce the power consumption and to cut costs on the necessary time interval without affecting the normal functioning of the region. Keywords: stability of electrical supply, energy saving, optimal power management, power system JEL Classifications: O25; Q43; Q4

Barnase as a New Therapeutic Agent Triggering Apoptosis in Human Cancer Cells

RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells.In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC(50)) ranging from 0.2 to 13 microM and to leukemia cell lines with IC(50) values ranging from 2.4 to 82 microM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC(50) = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3.These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells

Binding and internalization of scFv 4D5-dibarnase in BT-474 cells visualized by confocal microscopy.

<p>(A) Cells were incubated with scFv 4D5-dibarnase at 4°C or (B) at 37°C. The scFv 4D5-dibarnase was detected with rabbit anti-barnase antiserum followed by GAR-PE. Fluorescence was observed predominantly on the surface of cells incubated at 4°C and inside the cells incubated at 37°C. This difference in the localization of the fluorescent label suggests internalization of scFv 4D5-dibarnase at 37°C in BT-474 cells.</p

Effects of recombinant proteins on cell viability as determined by MTT assay.

<p>(A) The effects of barnase and scFv 4D5-dibarnase on the viability of human cancer and normal cells. SKOV-3 cells were treated for 72 h with barnase (long dashed line) or scFv 4D5-dibarnase (solid line), and hPBMCs were treated with barnase (short dashed line) or scFv 4D5-dibarnase (dashed-dotted line). (B) The competitive inhibition of scFv 4D5-dibarnase cytotoxicity by scFv 4D5. SKOV-3 cells were treated for 72 h with scFv 4D5-dibarnase in the absence (black circles) or presence (white triangles) of 300 nM scFv 4D5 or with scFv 4D5 alone (white squares). (C) The inhibition of barnase cytotoxicity and scFv 4D5-dibarnase cytotoxicity by barstar. SKOV-3 cells were treated for 72 h with barnase (white circles), barnase and equimolar amounts of barstar (white triangles), scFv 4D5-dibarnase (black circles), scFv 4D5-dibarnase with three-fold molar excess of barstar (black triangles), or barstar alone (black squares). (D) The effects of hRI on the cytotoxicity of scFv 4D5-dibarnase. SKOV-3 cells were treated for 72 h with either scFv 4D5-dibarnase in the absence of hRI (black circles), scFv 4D5-dibarnase in the presence of hRI (white diamonds), or hRI alone (black diamonds). Cell viability is expressed as the percentage of the metabolic activity of treated cells with respect to untreated cells (crosshair). Each regression curve in panel A (with 95% confidence intervals indicated by dotted lines) represents at least three independent experiments. Sigmoid regression was performed with SigmaPlot software. Curves in B–D represent typical experiments. Error bars (B–D) were obtained from triplicate measurements.</p

Cellular RNA undergoes degradation in SKOV-3 cells treated with barnase.

<p>SKOV-3 cells were exposed to 50 µM barnase for 24 h (lane 3) or 48 h (lane 4). Total RNA was isolated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002434#s4" target="_blank">Materials and Methods</a> and analyzed on a 9% polyacrylamide gel containing 7.5 M urea. Each sample lane was loaded with RNA from 2×10⁵ treated (+) or untreated (−) cells. Lane 2 corresponds to mock-treated control. The positions of the RNA molecular weight standards (lane 1) are shown as the number of bases to the left of panel. Asterisks indicate the most prominent bands that appear as a result of enzymatic cleavage of high molecular weight rRNA by barnase (lane 3).</p

Barnase and scFv 4D5-dibarnase cause DNA fragmentation in SKOV-3 cells.

<p>(A) Flow cytometric analysis of the cell cycle distribution was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002434#s4" target="_blank">Materials and Methods</a>. Histograms represent the differences in the percentages of cells between barnase- or scFv 4D5-dibarnase-treated and untreated cells for each cell cycle stage (sub-G1, G1, S, and G2/M) measured after 24 h (black bars), 48 h (blue bars), and 72 h (green bars) of treatment. Error bars show the standard deviation. Positive controls for DNA fragmentation were SKOV-3 cells cultured for 7 days in serum-free medium (orange bars). (B) DNA electrophoresis assay. Cells were treated with either 50 µM barnase or 50 nM scFv 4D5-dibarnase. Seventy-two hours later, genomic DNA of both treated (+) and untreated (−) cells was isolated and DNA from equal numbers of cells was resolved in non-denaturing 1.5% agarose gels. The DNA was visualized by ethidium bromide staining. Chromatin fragments resulting from internucleosomal cleavage were present in samples of DNA from cells treated with barnase (lane 2) and scFv 4D5-dibarnase (lane 6). DNA of serum-starved (ss) cells were cleaved irregularly (lane 7). Lanes 3 and 5 represent untreated controls. Lanes 1 and 4 are molecular weight markers ((M) HyperLadder I, Bioline). (C) Cells were exposed to 50 nM scFv 4D5-dibarnase for 72 h and then stained with acridine orange, analyzed by fluorescence microscopy, and photographed. A representative case of nuclear pyknosis and fragmentation (karyorrhexis) is shown (inset). Magnification, 400× (1200×, inset).</p

Ribonuclease activity assay.

<p>(A) The ribonuclease activities of barnase (dashed line and diamonds) and scFv 4D5-dibarnase (dotted line and circles) were determined according to the method of Rushizky et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002434#pone.0002434-Rushizky1" target="_blank">[58]</a>. The x-axis represents the concentration of barnase alone or the half-concentration of scFv 4D5-dibarnase. The absorbance of 0.5 AU₂₆₀ corresponds to the activity of 2 nM native barnase as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002434#pone.0002434-Hartley2" target="_blank">[27]</a>. (B) Susceptibility of barnase to hRI (solid line and circles) and of scFv 4D5-dibarnase to barstar (dashed line and triangles). Data are means±SD of triplicate determinations; the curves are the results of sigmoid regression performed with SigmaPlot software.</p

Binding of barnase and scFv 4D5-dibarnase to cells.

<p>The cell-binding ability of the recombinant proteins demonstrated by fluorescent microscopy. Cells were incubated at 4°C for 1 h with either 20 nM scFv 4D5-dibarnase (A, B and D), or a mixture of 20 nM scFv 4D5-dibarnase and 20 nM scFv 4D5 (C), or 20 µM barnase (E). Unbound proteins were removed, and then living (A–D) or fixed (E) cells were stained with rabbit anti-barnase antiserum and GAR-TR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002434#s4" target="_blank">Materials and Methods</a>. The scFv 4D5-dibarnase bound to HER2-positive SKOV-3 cells (A and B), this specific binding was inhibited by scFv 4D5 (C). The scFv 4D5-dibarnase did not bind to HER2-negative CTLL-2 cells (D). Cytoplasmic staining of SKOV-3 cells with 20 µM barnase was observed (E). Magnification, 400×.</p

Barnase and scFv 4D5-dibarnase induced apoptosis accompanied by phosphatidylserine externalization and caspase-3 activation.

<p>(A–C) SKOV-3 cells were mock-treated (A) or treated with either 50 µM barnase (B) or 50 nM scFv 4D5-dibarnase (C) for 72 h. Cells were analyzed for early apoptosis by Annexin-V-FITC/PI staining. The lower left quadrants of each panel show the viable cells, which exclude PI and are negative for Annexin-V-FITC binding. The upper right quadrants contain the non-viable, necrotic cells, which are positive for both Annexin-V-FITC binding and PI uptake. The lower right quadrants represent apoptotic cells, Annexin-V-FITC positive and PI negative. One representative experiment out of three is shown. (D and E) Caspase-3-like enzymatic activities of cells treated with either 50 µM barnase (D, unfilled peak) or 50 nM scFv 4D5-dibarnase (E, unfilled peak) for 72 h were assessed by the cleavage of the fluorogenic substrate PhiPhiLux-G₁D₂ and compared with that of untreated cells (filled peaks). M1 and M2 markers correspond to levels of caspase-3 activation in untreated and treated cells, respectively.</p