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

Spectrally Selective Detection of Short Spin Waves in Magnetoplasmonic Nanostructures via the Magneto-Optical Intensity Effect

A method of spectrally selective detection of short spin waves (or magnons) by means of the transverse magneto-optical (MO) intensity effect in transmission in the magnetoplasmonic nanostructure is proposed. We considered the spin waves with a wavelength equal to or less than (by an integer number of times) the period of the plasmonic structure, that is, of the order of hundreds of nanometers or 1–2 μm. The method is based on the analysis of the MO effect spectrum versus the modulation of the sample magnetization (created by the spin wave) and related spatial symmetry breaking in the magnetic layer. The spatial symmetry breaking leads to the appearance of the MO effect modulation at the normal incidence of light in the spectral range of the optical states (the SPP and the waveguide modes) and the breaking of the antisymmetry of the effect with respect to the sign of the incidence angle of light. We reveal that the magnitude of the MO effect varies periodically depending on the spatial shift of the spin wave with respect to the plasmonic grating. The period of this modulation is equal to the period of the spin wave. All these facts allow for the detection of spin waves of a certain wavelength propagating in a nanostructure by measuring the MO response

Evaluating characteristics of turbulent flames by using IR thermography and PIV

The paper describes experimental results of determining scales of turbulent eddies in diffusion flames. The results are obtained by analyzing a set of 2D cross-sections cut from 3D semi-transparent flames. The comparison of data obtained by using the particle image velocimetry (PIV) method and infrared (IR) thermography is presented to demonstrate similarity between hydro- and thermodynamic parameters

Особенности законодательной регламентации построения и функционирования систем национальных баз данных ДНК (на примере Великобритании, США, Китая и России)

The study considers the history of establishment, current condition and perspective of the legislative regulation in establishing and functioning of national DNA database systems in some European and Asian countries. The authors study the peculiarities in establishing systems of DNA registration regarding a case study of Great Britain, China and the USA as the countries having a considerable experience in this field and the biggest national DNA databases. Thus, the main developmental characteristics in the forensic registration of these countries are identified. The aim of the study is to examine the theoretical aspects of the legislative regulation in establishing and functioning of national DNA database systems including the purposes to create the best model of the legislative regulation to meet the requirements of a modern society. The gaps in the state regulation of genomic registration are noted. It is determined that the existing laws and regulations in Russia do not completely satisfy the demands of the times for they considerably limit the sphere of application of DNA registration systems. As a result, the real potential of the DNA registration system in Russia is identified. Consequently, it will assist in establishment of a more effective model of DNA registration system taking into account the world’s experience. Regulatory developments in legal activities as ways to improve legislative regulation of the genomic registration in Russia are set.В статье рассмотрена история создания, современное состояние и перспективы правового регулирования формирования и функционирования национальных баз данных ДНК в странах Европы и Азии. Авторы исследуют особенности построения систем ДНК-регистрации на примере стран, имеющих значительный опыт в данной области, администрирующих самые крупные в мире базы данных ДНК, — Великобритании, США, Китая. При этом выявлены основные особенности развития этого направления криминалистической регистрации в указанных странах. Цель настоящей работы состоит в том, чтобы исследовать теоретические аспекты правовой регламентации построения и функционирования систем национальных баз данных ДНК, в том числе в целях формирования оптимальной модели законодательного регулирования, учитывающей потребности современного общества. В работе также отмечены пробелы в отечественном законодательном регулировании геномной регистрации. Установлено, что действующие нормативные правовые акты в России не в полной мере отвечают требованиям времени, поскольку существенно ограничивают сферу применения систем ДНК-регистрации. При этом выявлен действительный потенциал системы ДНК-регистрации в Российской Федерации, позволяющий с учетом мирового опыта создать наиболее эффективную модель ее построения. Определены направления нормотворческой деятельности на пути совершенствования правового обеспечения геномной регистрации населения в Росси

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

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

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