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Exposure of living cells to intense nanosecond pulsed electric field (nsPEF) increases membrane permeability to small solutes, presumably by the formation of nanometer-size membrane lesions. Mechanisms responsible for the restoration of membrane integrity over the course of minutes after nsPEF have not been identified. This study explored if ESCRT-III and Annexin V calcium-dependent repair mechanisms, which play critical role in resealing large membrane lesions, are also activated by electroporation and contribute to the membrane resealing. The extent of membrane damage and the time course of resealing were monitored by the time-lapse imaging of propidium (Pr) uptake in human cervical carcinoma (HeLa) cells exposed to trains of 300-ns PEF. The removal of the extracellular Ca2+ slowed down the resealing, although did not prevent it. Recruitment of CHMP4B protein, a component of ESCRT-III complex, to the electroporated plasma membrane was not observed, thus providing no evidence for possible contribution of the macro-vesicle shedding mechanism. In contrast, silencing the AnxA5 gene impaired resealing and reduced the viability of nsPEF-treated cells. We conclude that Annexin V but not ESCRT-III was involved in the repair of HeLa cells permeabilized by 300-ns stimuli, but it was not the only and perhaps not the main repair mechanism.

Transforming growth factor (TGF) β1, β2, and β3 (TGF-β1–TGF-β3, respectively) are small secreted signaling proteins that each signal through the TGF-β type I and type II receptors (TβRI and TβRII, respectively). However, TGF-β2, which is well-known to bind TβRII several hundred-fold more weakly than TGF-β1 and TGF-β3, has an additional requirement for betaglycan, a membrane-anchored nonsignaling receptor. Betaglycan has two domains that bind TGF-β2 at independent sites, but how it binds TGF-β2 to potentiate TβRII binding and how the complex with TGF-β, TβRII, and betaglycan undergoes the transition to the signaling complex with TGF-β, TβRII, and TβRI are not understood. To investigate the mechanism, the binding of the TGF-βs to the betaglycan extracellular domain, as well as its two independent binding domains, either directly or in combination with the TβRI and TβRII ectodomains, was studied using surface plasmon resonance, isothermal titration calorimetry, and size-exclusion chromatography. These studies show that betaglycan binds TGF-β homodimers with a 1:1 stoichiometry in a manner that allows one molecule of TβRII to bind. These studies further show that betaglycan modestly potentiates the binding of TβRII and must be displaced to allow TβRI to bind. These findings suggest that betaglycan functions to bind and concentrate TGF-β2 on the cell surface and thus promote the binding of TβRII by both membrane-localization effects and allostery. These studies further suggest that the transition to the signaling complex is mediated by the recruitment of TβRI, which simultaneously displaces betaglycan and stabilizes the bound TβRII by direct receptor–receptor contact.

Depending on the initiating stimulus, cancer cell death can be immunogenic or non-immunogenic. Inducers of immunogenic cell death (ICD) rely on endoplasmic reticulum (ER) stress for the trafficking of danger signals such as calreticulin (CRT) and ATP. We found that nanosecond pulsed electric fields (nsPEF), an emerging new modality for tumor ablation, cause the activation of the ER-resident stress sensor PERK in both CT-26 colon carcinoma and EL-4 lymphoma cells. PERK activation correlates with sustained CRT exposure on the cell plasma membrane and apoptosis induction in both nsPEF-treated cell lines. Our results show that, in CT-26 cells, the activity of caspase-3/7 was increased fourteen-fold as compared with four-fold in EL-4 cells. Moreover, while nsPEF treatments induced the release of the ICD hallmark HMGB1 in both cell lines, extracellular ATP was detected only in CT-26. Finally, in vaccination assays, CT-26 cells treated with nsPEF or doxorubicin equally impaired the growth of tumors at challenge sites eliciting a protective anticancer immune response in 78% and 80% of the animals, respectively. As compared to CT-26, both nsPEF- and mitoxantrone-treated EL-4 cells had a less pronounced effect and protected 50% and 20% of the animals, respectively. These results support our conclusion that nsPEF induce ER stress, accompanied by bona fide ICD.

Disruption of the actin cytoskeleton structures was reported as one of the characteristic effects of nanosecond-duration pulsed electric field (nsPEF) in both mammalian and plant cells. We utilized CHO cells that expressed the monomeric fluorescent protein (mApple) tagged to actin to test if nsPEF modifies the cell actin directly or as a consequence of cell membrane permeabilization. A train of four 600-ns pulses at 19.2 kV/cm (2 Hz) caused immediate cell membrane poration manifested by YO-PRO-1 dye uptake, gradual cell rounding and swelling. Concurrently, bright actin features were replaced by dimmer and uniform fluorescence of diffuse actin. To block the nsPEF-induced swelling, the bath buffer was isoosmotically supplemented with an electropore-impermeable solute (sucrose). A similar addition of a smaller, electropore-permeable solute (adonitol) served as a control. We demonstrated that sucrose efficiently blocked disassembly of actin features by nsPEF, whereas adonitol did not. Sucrose also attenuated bleaching of mApple-tagged actin in nsPEF-treated cells (as integrated over the cell volume), although did not fully prevent it. We conclude that disintegration of the actin cytoskeleton was a result of cell swelling, which, in turn, was caused by cell permeabilization by nsPEF and transmembrane diffusion of solutes which led to the osmotic imbalance.

Opening of long-lived pores in the cell membrane is the principal primary effect of intense, nanosecond pulsed electric field (nsPEF). Here we demonstrate that the evolution of pores, cell survival, the time and the mode of cell death (necrotic or apoptotic) are determined by the level of external Ca(2+) after nsPEF. We also introduce a novel, minimally disruptive technique for nsEP exposure of adherent cells on indium tin oxide (ITO)-coated glass coverslips, which does not require cell detachment and enables fast exchanges of bath media. Increasing the Ca(2+) level from the nominal 2-5μM to 2mM for the first 60-90min after permeabilization by 300-nsPEF increased the early (necrotic) death in U937, CHO, and BPAE cells. With nominal Ca(2+), the inhibition of osmotic swelling rescued cells from the early necrosis and increased caspase 3/7 activation later on. However, the inhibition of swelling had a modest or no protective effect with 2mM Ca(2+) in the medium. With the nominal Ca(2+), most cells displayed gradual increase in YO-PRO-1 and propidium (Pr) uptake. With 2mM Ca(2+), the initially lower Pr uptake was eventually replaced by a massive and abrupt Pr entry (necrotic death). It was accompanied by a transient acceleration of the growth of membrane blebs due to the increase of the intracellular osmotic pressure. We conclude that the high-Ca(2+)-dependent necrotic death in nsPEF-treated cells is effected by a delayed, sudden, and osmotically-independent pore expansion (or de novo formation of larger pores), but not by the membrane rupture.

Recent papers showed that when dealing with nanosecond pulsed electric fields (nsPEFs), bipolar pulses proved far less effective at membrane permeabilization and at cell killing than monopolar ones, contrary to what happens in the microsecond range. The mechanism at the basis of such selective response of cells is not fully elucidated. One hypothesis worthwhile to test could be a direct action of nsPEFs on intracellular enzyme reactions. The goal of this study is to investigate possible enzyme response due to the application of nsPEF monopolar and bipolar of intensity of 108 V/m and duration of 100 ns on the superoxide dismutase (SOD, Cu-Zn) enzyme, by means of a molecular dynamics (MD) modelling approach.

Summary Community-acquired respiratory distress syndrome (CARDS) toxin from Mycoplasma pneumoniae is a 591-amino-acid virulence factor with ADP-ribosyltransferase (ADPRT) and vacuolating activities. It is expressed at low levels during in vitro growth and at high levels during colonization of the lung. Exposure of experimental animals to purified recombinant CARDS toxin alone is sufficient to recapitulate the cytopathology and inflammatory responses associated with M. pneumoniae infection in humans and animals. Here, by molecular modelling, serial truncations and site-directed mutagenesis, we show that the N-terminal region is essential for ADP-ribosylating activity. Also, by systematic truncation and limited proteolysis experiments we identified a portion of the C-terminal region that mediates toxin binding to mammalian cell surfaces and subsequent internalization. In addition, the C-terminal region alone induces vacuolization in a manner similar to full-length toxin. Together, these data suggest that CARDS toxin has a unique architecture with functionally separable N-terminal and C-terminal domains.

Electroporation by nanosecond electric pulses (nsEP) is an emerging modality for tumor ablation. Here we show the efficient induction of apoptosis even by a non-toxic nsEP exposure when it is followed by a 30-min chilling on ice. This chilling itself had no impact on the survival of U-937 or HPAF-II cells, but caused more than 75% lethality in nsEP-treated cells (300 ns, 1.8-7 kV/cm, 50-700 pulses). The cell death was largely delayed by 5-23 hr and was accompanied by a 5-fold activation of caspase 3/7 (compared to nsEP without chilling) and more than 60% cleavage of poly-ADP ribose polymerase (compared to less than 5% in controls or after nsEP or chilling applied separately). When nsEP caused a transient permeabilization of 83% of cells to propidium iodide, cells placed at 37 °C resealed in 10 min, whereas 60% of cells placed on ice remained propidium-permeable even in 30 min. The delayed membrane resealing caused cell swelling, which could be blocked by an isosmotic addition of a pore-impermeable solute (sucrose). However, the block of swelling did not prevent the delayed cell death by apoptosis. The potent enhancement of nsEP cytotoxicity by subsequent non-damaging chilling may find applications in tumor ablation therapies.

Nanosecond-duration electric stimuli are distinguished by the ability to permeabilize intracellular membranes and recruit Ca2+ from intracellular stores. We quantified this effect in non-excitable cells (CHO) using ratiometric Ca2+ imaging with Fura-2. In a Ca(2+)-free medium, 10-, 60-, and 300-ns stimuli evoked Ca2+ transients by mobilization of Ca2+ from the endoplasmic reticulum. With 2 mM external Ca2+, the transients included both extra- and intracellular components. The recruitment of intracellular Ca2+ increased as the stimulus duration decreased. At the threshold of 200-300 nM, the transients were amplified by calcium-induced calcium release. We conclude that nanosecond stimuli mimic Ca2+ signaling while bypassing the usual receptor- and channels-mediated cascades. The recruitment of the intracellular Ca2+ can be controlled by the duration of the stimulus.

Cell permeabilization by electric pulses (EP), or electroporation, is widely used for intracellular delivery of drugs and plasmids, as well as for tumour and tissue ablation. We found that cells pre-treated with 100-μs EP develop delayed hypersensitivity to subsequent EP applications. Sensitizing B16 and CHO cells by splitting a single train of eight 100-μs EP into two trains of four EP each (with 5-min. interval) decreased the LD(50) 1.5-2 times. Sensitization profoundly enhanced the electroporation-assisted uptake of bleomycin, a cell-impermeable cytotoxic agent accepted for killing tumours by electrochemotherapy. EP exposures that were not lethal per se caused cell death in the presence of bleomycin and proportionally to its concentration. Sensitizing cells by a split-dose EP exposure increased bleomycin-mediated lethality to the same extent as a 10-fold increase in bleomycin concentration when using a single EP dose. Likewise, sensitization by a split-dose EP exposure (without changing the overall dose, pulse number, or amplitude) enhanced the electroporative uptake of propidium up to fivefold. Enhancement of the electroporative uptake appears a key mechanism of electrosensitization and may benefit electrochemotherapy and numerous applications that employ EP for cell permeabilization.

Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of membranous structures and intracellular delivery of xenobiotics. We hypothesized that oxidative effects of nsPEF could be a separate primary mechanism responsible for bioeffects. ROS production in cultured cells and media exposed to 300-ns PEF (1-13 kV/cm) was assessed by oxidation of 2',7'-dichlorodihydrofluoresein (H(2)DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H(2)DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 2',7'-dichlorofluorescein (DCF) increased proportionally to the pulse number and cell density. DCF emission increased with time after exposure in nsPEF-sensitive Jurkat cells, but remained stable in nsPEF-resistant U937 cells. In cell-free media, nsPEF facilitated the conversion of H(2)DCF into DCF. This effect was not related to heating and was reduced by catalase, but not by mannitol or superoxide dismutase. Formation of H(2)O(2) in nsPEF-treated media was confirmed by increased oxidation of Amplex Red. ROS increase within individual cells exposed to nsPEF was visualized by oxidation of DHE. We conclude that nsPEF can generate both extracellular (electrochemical) and intracellular ROS, including H(2)O(2) and possibly other species. Therefore, bioeffects of nsPEF are not limited to electropermeabilization; concurrent ROS formation may lead to cell stimulation and/or oxidative cell damage.

Acinetobacter baumannii is an environmentally resilient healthcare-associated opportunistic pathogen responsible for infections at many body sites. In the last 10 years, clinical strains resistant to many or all commonly used antibiotics have emerged globally. With few antimicrobial agents in the pharmaceutical pipeline, new and alternative agents are essential. Platelets secrete a large number of proteins, including proteins with antimicrobial activity. In a previous study, we demonstrated that donor platelet supernatants and plasma significantly inhibited the growth of a reference strain of A. baumannii in broth and on skin. This inhibition appeared to be unrelated to the platelet activation state. In this study, we demonstrate that this growth inhibition extends to clinical multidrug resistant isolates. We also demonstrate that there is no relationship between this activity and selected platelet-derived antimicrobial proteins. Instead, the donor plasma components complement and alpha-2 macroglobulin are implicated.

Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8- and 9-μs EP.Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry.10-ns EP caused apoptotic or necrotic death within 2-20 h. Survival (S, %) followed the absorbed dose (D, J/g) as: S=alphaD((-K)), where coefficients K and alpha determined the slope and the "shoulder" of the survival curve. K was similar in all groups, whereas alpha was cell type- and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only.1.8- and 9-μs EP cause cell death efficiently and indiscriminately (LD₅₀ 1-3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD₅₀ 50-80 J/g for Jurkat and 400-500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores ("nanopores"), triggering different cell death mechanisms.Nanosecond EP can selectively target certain cells in medical applications like tumor ablation.

Significance The airway pathogen Mycoplasma pneumoniae ( Mp ) produces a virulence factor with ADP-ribosyltransferase and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). Mammalian cells exposed to recombinant CARDS TX exhibit distinct patterns of ADP-ribosylated proteins and unusual vacuolation phenotypes with disruption of monolayer integrity, leading to cell death. Recombinant CARDS TX administered to naive mice induces an allergic-type inflammatory response and airway hyperreactivity, suggesting that an analogous response in humans may play a causal role in Mp -associated asthma. Here we present the structure of CARDS TX, the newest member of the well-recognized group of ADP-ribosylating toxins. The structure enhances understanding of Mp pathogenicity and provides a platform for the development of therapies to treat Mp -associated acute and chronic airway diseases.

Transforming growth factor-beta (TGFbeta) isoforms initiate signaling by assembling a heterotetrameric complex of paired type I (TbetaRI) and type II (TbetaRII) receptors on the cell surface. Because two of the ligand isoforms (TGFbetas 1, 3) must first bind TbetaRII to recruit TbetaRI into the complex, and a third (TGFbeta2) requires a co-receptor, assembly is known to be sequential, cooperative and isoform-dependent. However the source of the cooperativity leading to recruitment of TbetaRI and the universality of the assembly mechanism with respect to isoforms remain unclear. Here, we show that the extracellular domain of TbetaRI (TbetaRI-ED) binds in vitro with high affinity to complexes of the extracellular domain of TbetaRII (TbetaRII-ED) and TGFbetas 1 or 3, but not to either ligand or receptor alone. Thus, recruitment of TbetaRI requires combined interactions with TbetaRII-ED and ligand, but not membrane attachment of the receptors. Cell-based assays show that TbetaRI-ED, like TbetaRII-ED, acts as an antagonist of TGFbeta signaling, indicating that receptor-receptor interaction is sufficient to compete against endogenous, membrane-localized receptors. On the other hand, neither TbetaRII-ED, nor TbetaRII-ED and TbetaRI-ED combined, form a complex with TGFbeta2, showing that receptor-receptor interaction is insufficient to compensate for weak ligand-receptor interaction. However, TbetaRII-ED does bind with high affinity to TGFbeta2-TM, a TGFbeta2 variant substituted at three positions to mimic TGFbetas 1 and 3 at the TbetaRII binding interface. This proves both necessary and sufficient for recruitment of TbetaRI-ED, suggesting that the three different TGFbeta isoforms induce assembly of the heterotetrameric receptor complex in the same general manner.

Protein SRP19 is an important component of the signal recognition particle (SRP) as it promotes assembly of protein SRP54 with SRP RNA and recognizes a tetranucleotide loop. Structural features and RNA binding activities of SRP19 of the hyperthermophilic archaeon Archaeoglobus fulgidus were investigated. An updated alignment of SRP19 sequences predicted three conserved regions and two alpha-helices. With Af-SRP RNA the Af-SRP54 protein assembled into an A. fulgidus SRP which remained intact for many hours. Stable complexes were formed between Af-SRP19 and truncated SRP RNAs, including a 36-residue fragment representing helix 6 of A. fulgidus SRP RNA.

Community-acquired respiratory distress syndrome toxin (CARDS TX) is a 591-amino-acid protein with ADP-ribosyltransferase and vacuolating activities that damages the cells lining the respiratory tracts of patients infected with the bacterial pathogen Mycoplasma pneumoniae. Crystals of CARDS TX were grown in space group C2, with unit-cell parameters a = 191.4, b = 107.4, c = 222.1 A, beta = 90.6 degrees. A complete 2.2 A data set was obtained from a single CARDS TX crystal.

Binding of yeast ribosomal protein L5 with 5S rRNA has long been considered a promising model for studying molecular mechanisms of protein-RNA interactions. However, in vitro assembly of a ribonucleoprotein (RNP) complex from purified yeast ribosomal protein L5 (also known as L1, L1a, or YL3) and 5S rRNA proved to be difficult, thus limiting the utility of this model. In the present report, we present data on the successful in vitro assembly of a RNP complex using a fusion (MBP-L5) protein consisting of the yeast ribosomal protein L5 fused to the carboxyl terminus of the E. coli maltose-binding protein (MBP). We demonstrated that: 1) the MBP-L5 protein binds yeast 5S rRNA but not 5.8S rRNA in vitro; 2) the MBP protein itself does not bind yeast 5S rRNA; 3) formation of the RNP complex is proportional to the concentration of MBP-L5 protein and 5S rRNA; and 4) the MBP moiety of the fusion protein in the RNP complex can be removed with factor Xa. The electrophoretic mobility of the resultant RNP complex is indistinguishable from that of L5-5S rRNA complex isolated from the ribosome. Using this new experimental approach, we further showed that the RNA binding capability of a mutant L5 protein is decreased by 60% compared to the wild-type protein. Additionally, the mutant RNP complex migrates slower than the wild-type RNP complex suggesting that the mutant RNP complex has a less compact conformation. The finding provides a probable explanation for an earlier observation that the 60S ribosomal subunit containing the mutant protein is unstable.

Effects of millimeter waves (MMW) on cell survival and ultraviolet-induced reciprocal and non-reciprocal recombination and mutagenesis were studied in the diploid D7 strain of the yeast Saccharomyces cerevisiae. MMW exposures lasted for 30 min (0.13 mW cm−2, 61.02–61.42 GHz) and were followed in 60 min by a 100 J m−2 dose of 254 nm ultraviolet (UV) radiation. The effect of the exposures was evaluated from the colony-forming ability of the cells on complete and selective media, and from the number of aberrant colonies formed. The MMW pretreatment did not alter cell survival or the frequency of reverse mutations. The incidence of conversions was higher in most cases in the MMW-treated cells (p < 0.05). MMW also increased the scores of crossover and colored aberrants in isolated experiments, though the average increse for all experiments (performed with different MMW frequencies) was not statistically significant. The results suggested that the MMW irradiation did not alter the UV-induced mutagenesis, but could facilitate recombinagenic processes.

Genetic effects of ultrawide-band (UWB) radiation pulses were studied in the D7 strain of yeast Saccharomyces cerevisiae. Yeast cells were exposed in suspension for 30 min at the UWB pulse repetition rates of 0 Hz (sham treatment), 16 Hz, and 600 Hz. Measured parameters of UWB pulses were as following: 101-104 kV/m peak voltage, 164-166 ps rise time, 1.01-1.02 ns pulse width, and 0 Hz-2 GHz spectral bandwidth. Cells were plated immediately after the exposure and incubated for 7 days at 30°C; then normal and aberrant colonies (mitotic crossovers, segregants, revertants, and convertants) were scored. The experiments established no statistically significant effect of UWB exposure on the colony-forming ability of yeast cells or the occurrence of mutations and chromosome recombinations.

High-amplitude electric pulses of nanosecond duration, also known as nanosecond pulsed electric field (nsPEF), are a novel modality with promising applications for cell stimulation and tissue ablation. However, key mechanisms responsible for the cytotoxicity of nsPEF have not been established. We show that the principal cause of cell death induced by 60- or 300-ns pulses in U937 cells is the loss of the plasma membrane integrity (“nanoelectroporation”), leading to water uptake, cell swelling, and eventual membrane rupture. Most of this early necrotic death occurs within 1–2 hr after nsPEF exposure. The uptake of water is driven by the presence of pore-impermeable solutes inside the cell, and can be counterbalanced by the presence of a pore-impermeable solute such as sucrose in the medium. Sucrose blocks swelling and prevents the early necrotic death; however the long-term cell survival (24 and 48 hr) does not significantly change. Cells protected with sucrose demonstrate higher incidence of the delayed death (6–24 hr post nsPEF). These cells are more often positive for the uptake of an early apoptotic marker dye YO-PRO-1 while remaining impermeable to propidium iodide. Instead of swelling, these cells often develop apoptotic fragmentation of the cytoplasm. Caspase 3/7 activity increases already in 1 hr after nsPEF and poly-ADP ribose polymerase (PARP) cleavage is detected in 2 hr. Staurosporin-treated positive control cells develop these apoptotic signs only in 3 and 4 hr, respectively. We conclude that nsPEF exposure triggers both necrotic and apoptotic pathways. The early necrotic death prevails under standard cell culture conditions, but cells rescued from the necrosis nonetheless die later on by apoptosis. The balance between the two modes of cell death can be controlled by enabling or blocking cell swelling.

In this study, we determined the LD(50) (50% lethal dose) for cell death, and the ED(50) (50% of cell population staining positive) for propidium (Pr) iodide uptake, and phosphatidylserine (PS) externalization for several commonly studied cell lines (HeLa, Jurkat, U937, CHO-K1, and GH3) exposed to 10-ns electric pulses (EP). We found that the LD(50) varied substantially across the cell lines studied, increasing from 51 J/g for Jurkat to 1861 J/g for HeLa. PS externalized at doses equal or lower than that required for death in all cell lines ranging from 51 J/g in Jurkat, to 199 J/g in CHO-K1. Pr uptake occurred at doses lower than required for death in three of the cell lines: 656 J/g for CHO-K1, 634 J/g for HeLa, and 142 J/g for GH3. Both Jurkat and U937 had a LD(50) lower than the ED(50) for Pr uptake at 780 J/g and 1274 J/g, respectively. The mechanism responsible for these differences was explored by evaluating cell size, calcium concentration in the exposure medium, and effect of trypsin treatment prior to exposure. None of the studied parameters correlated with the observed results suggesting that cellular susceptibility to injury and death by 10-ns EP was largely determined by cell physiology. In contrast to previous studies, our findings suggest that permeabilization of internal membranes may not necessarily be responsible for cell death by 10-ns EP. Additionally, a mixture of Jurkat and HeLa cells was exposed to 10-ns EP at a dose of 280 J/g. Death was observed only in Jurkat cells suggesting that 10-ns EP may selectively kill cells within a heterogeneous tissue.

Cell samples of the yeast Saccharomyces cerevisiae were exposed to 100 J/m2 of 254 nm ultraviolet (UV) radiation followed by a 30 min treatment with ultra-wide band (UWB) electromagnetic pulses. The UWB pulses (101-104 kV/m, 1.0 ns width, 165 ps rise time) were applied at the repetition rates of 0 Hz (sham), 16 Hz, or 600 Hz. The effect of exposures was evaluated from the colony-forming ability of the cells on complete and selective media and the number of aberrant colonies. The experiments established no effect of UWB exposure on the UV-induced reciprocal and non-reciprocal recombination, mutagenesis, or cell survival.

The signal recognition particle (SRP) RNA helix 6 of archaea and eukaryotes is essential for the binding of protein SRP19 and the assembly of a functional complex. The conserved adenosine at the third position of the tetraloop of helix 6 (A149) is crucial for the binding of protein SRP19 in the mammalian SRP. Here we investigated the significance of the equivalent adenosine residue at position 159 (A159) of Archaeoglobus fulgidus SRP RNA. The A159 of A. fulgidus and A149 of human SRP RNA were changed to C, G or U, and fragments containing helix 6 or helices 6 and 8 were synthesized by run-off transcription with T7 RNA polymerase. The ability of recombinant A. fulgidus and human SRP19 to form ribonucleoprotein complexes was measured in vitro. The simultaneous presence of A149 and helix 8 is required for the high-affinity binding of SRP19 to the human SRP RNA. In contrast, A. fulgidus SRP19 binds to the SRP RNA fragments with high affinity irrespective of the nature of the nucleotide, demonstrating that A159 does not directly participate in protein binding. Instead, as indicated by the resistance of the wild-type A. fulgidus RNA towards digestion by RNase A, this residue allows the formation of a tightly folded RNA molecule. The high affinity between A. fulgidus SRP19 and RNA molecules that contain both helices 6 and 8 suggests that A159 is likely to initiate archaeal SRP assembly by forming a conserved tertiary RNA– RNA interaction.

Protein SRP19 is an essential RNA-binding component of the signal recognition particle (SRP) in Archaea and Eucarya. A three-dimensional solution structure of the 104 residue SRP19 from the hyperthermophilic archaeon Archaeoglobus fulgidus, designated as Af19, was determined by NMR spectroscopy. Af19 contains three beta-strands, two alpha-helical regions, arranged in a betaalphabetabetaalpha topology, a 3(10) helix, and a disordered C-terminal tail. This fold is similar to the betaalphabetabetaalphabeta RNP motif present in numerous other RNA-binding proteins, which engage their cognate RNAs using conserved sequence motifs present within beta-strands 1 and 3. Mutagenesis studies of human SRP19, however, reveal the major contact sites with SRP RNA reside within loops 1, 3, and 4. These contacts were verified by the crystal structure of human SRP19 complexed to SRP RNA helix 6 reported subsequent to the submission of the manuscript. The crystal structure also reveals that, unlike canonical RNP motifs, SRP19 does not engage specific RNA bases through conserved sequence motifs present within beta-strands 1 and 3. Instead, SRP19 uses residues both within and flanking beta-strand 1 to stabilize the complex through direct and indirect contacts to the phosphate backbone of the tetraloop, leaving the bases of the tetraloop exposed. This, coupled with the fact that SRP19 appears relatively rigid and undergoes only minor changes in structure upon RNA binding, may underlie the molecular basis by which SRP19 functions to initiate SRP assembly.

Background Electroporation is a method of disrupting the integrity of cell membrane by electric pulses (EPs). Electrical modeling is widely employed to explain and study electroporation, but even most advanced models show limited predictive power. No studies have accounted for the biological consequences of electroporation as a factor that alters the cell's susceptibility to forthcoming EPs. Methodology/Principal Findings We focused first on the role of EP rate for membrane permeabilization and lethal effects in mammalian cells. The rate was varied from 0.001 to 2,000 Hz while keeping other parameters constant (2 to 3,750 pulses of 60-ns to 9-µs duration, 1.8 to 13.3 kV/cm). The efficiency of all EP treatments was minimal at high rates and started to increase gradually when the rate decreased below a certain value. Although this value ranged widely (0.1–500 Hz), it always corresponded to the overall treatment duration near 10 s. We further found that longer exposures were more efficient irrespective of the EP rate, and that splitting a high-rate EP train in two fractions with 1–5 min delay enhanced the effects severalfold. Conclusions/Significance For varied experimental conditions, EPs triggered a delayed and gradual sensitization to EPs. When a portion of a multi-pulse exposure was delivered to already sensitized cells, the overall effect markedly increased. Because of the sensitization, the lethality in EP-treated cells could be increased from 0 to 90% simply by increasing the exposure duration, or the exposure dose could be reduced twofold without reducing the effect. Many applications of electroporation can benefit from accounting for sensitization, by organizing the exposure either to maximize sensitization (e.g., for sterilization) or, for other applications, to completely or partially avoid it. In particular, harmful side effects of electroporation-based therapies (electrochemotherapy, gene therapies, tumor ablation) include convulsions, pain, heart fibrillation, and thermal damage. Sensitization can potentially be employed to reduce these side effects while preserving or increasing therapeutic efficiency.

SummaryIt is shown that a fraction of damage induced by high energy electrons (25 MeV) in certain rad mutants of the yeast Saccharomyces cerevisiae can be photoreactivated. The photoreactivable damage contributes to the lethal effect of this type of irradiation and modifies the oxygen effect. Using photoreactivating light or nigrosin, the amount of photoreactivable damage is reduced and the oxygen enhancement ratio (OER) for yeast mutants increases approximately to the OER found in wild-type cells.