Facilitation of Mitochondrial Outer and Inner Membrane Permeabilization and Cell Death in Oxidative Stress by a Novel Bcl-2 Homology 3 Domain Protein

We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-x_L, which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.     

Hin4II, a new prototype restriction endonuclease from Haemophilus influenzae RFL4: discovery, cloning and expression in Escherichia coli

The genes encoding restriction-modification system of unknown specificity Hin4II from Haemophilus influenzae RFL4 were cloned in Escherichia coli and sequenced. The Hin4II system comprises three tandemly arranged genes coding for m6A DNA methyltransferase, m5C DNA methyltransferase and restriction endonuclease, respectively. Restriction endonuclease was expressed in E. coli and purified to apparent homogeneity. The DNA recognition sequence and cleavage positions were determined. R.Hin4II recognizes the novel non-palindromic sequence 5'-CCTTC-3' and cleaves the DNA 6 and 5 nt downstream in the top and bottom strand, respectively. The new prototype restriction endonuclease Hin4II was classified as a potential candidate of HNH nuclease family after comparison against SMART database. An amino acid sequence motif 297H-X14-N-X8-H of Hin4II was proposed as forming a putative catalytic center.     

A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites

Apicomplexan parasites constitute one of the most significant groups of pathogens infecting humans and animals. The liver stage sporozoites of Plasmodium spp. and tachyzoites of Toxoplasma gondii, the causative agents of malaria and toxoplasmosis, respectively, use a unique mode of locomotion termed gliding motility to invade host cells and cross cell substrates. This amoeboid-like movement uses a parasite adhesin from the thrombospondin-related anonymous protein (TRAP) family and a set of proteins linking the extracellular adhesin, via an actin-myosin motor, to the inner membrane complex. The Plasmodium blood stage merozoite, however, does not exhibit gliding motility. Here we show that homologues of the key proteins that make up the motor complex, including the recently identified glideosome-associated proteins 45 and 50 (GAP40 and GAP50), are present in P. falciparum merozoites and appear to function in erythrocyte invasion. Furthermore, we identify a merozoite TRAP homologue, termed MTRAP, a micronemal protein that shares key features with TRAP, including a thrombospondin repeat domain, a putative rhomboid-protease cleavage site, and a cytoplasmic tail that, in vitro, binds the actin-binding protein aldolase. Analysis of other parasite genomes shows that the components of this motor complex are conserved across diverse Apicomplexan genera. Conservation of the motor complex suggests that a common molecular mechanism underlies all Apicomplexan motility, which, given its unique properties, highlights a number of novel targets for drug intervention to treat major diseases of humans and livestock.     

Phytoremediation of selenium using subsurface-flow constructed wetland

The potential of two plant species, Phragmites australis (common reed) and Typha latifolia (cattail), in the phytoremediation process of selenium (Se) was studied in subsurface-flow constructed wetland (SSF). Se was supplemented continuously at a concentration of 100 microg Se L(-1) in the inlet of the cultivation beds of the SSF. Water samples collected from the outlet of the Phragmites bed after 1, 3, 6, 9, and 12 wk of treatments showed that Se content was under detectable limits. Water samples collected from the Typha bed at the same five periods showed that Se concentrations in the outlet were 55, 47, 65, 76, and 25 microg/L, respectively. The results of bioaccumulation in the biomass of both species after 12 wk of treatment indicated that Typha plants accumulated Se mainly in fine roots. Phragmites accumulated Se mainly in leaves and rhizomes, and moderate levels were found in stems and fine organic materials. The results indicate that common reed is a very good species for Se phytoextraction and phytostabilization (immobilization) and that cattail is only a phytostabilization species. The use of common reed and cattail for Se phytoremediation in a SSF system and in constructed wetland models are discussed.     

Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemia-reperfusion: Therapeutic potential of mitochondrially targeted antioxidants

Mitochondrial reactive oxygen species generation has been implicated in the pathophysiology of ischemia-reperfusion (I/R) injury; however, its exact role and its spatial-temporal relationship with inflammation are elusive. Herein we explore the spatial-temporal relationship of oxidative/nitrative stress and inflammatory response during the course of hepatic I/R and the possible therapeutic potential of mitochondrial-targeted antioxidants, using a mouse model of segmental hepatic ischemia-reperfusion injury. Hepatic I/R was characterized by early (at 2h of reperfusion) mitochondrial injury, decreased complex I activity, increased oxidant generation in the liver or liver mitochondria, and profound hepatocellular injury/dysfunction with acute proinflammatory response (TNF-α, MIP-1α/CCL3, MIP-2/CXCL2) without inflammatory cell infiltration, followed by marked neutrophil infiltration and a more pronounced secondary wave of oxidative/nitrative stress in the liver (starting from 6h of reperfusion and peaking at 24h). Mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently attenuated I/R-induced liver dysfunction, the early and delayed oxidative and nitrative stress response (HNE/carbonyl adducts, malondialdehyde, 8-OHdG, and 3-nitrotyrosine formation), and mitochondrial and histopathological injury/dysfunction, as well as delayed inflammatory cell infiltration and cell death. Mitochondrially generated oxidants play a central role in triggering the deleterious cascade of events associated with hepatic I/R, which may be targeted by novel antioxidants for therapeutic advantage.     

Size of the pores created by an electric pulse: Microsecond vs millisecond pulses

Here, the sizes of the pores created by square-wave electric pulses with the duration of 100μs and 2ms are compared for pulses with the amplitudes close to the threshold of electroporation. Experiments were carried out with three types of cells: mouse hepatoma MH-22A cells, Chinese hamster ovary (CHO) cells, and human erythrocytes. In the case of a short pulse (square-wave with the duration of 100μs or exponential with the time constant of 22μs), in the large portion (30-60%) of electroporated (permeable to potassium ions) cells, an electric pulse created only the pores, which were smaller than the molecule of bleomycin (molecular mass of 1450Da, r≈0.8nm) or sucrose (molecular mass of 342.3Da, radius-0.44-0.52nm). In the case of a long 2-ms duration pulse, in almost all cells, which were electroporated, there were the pores larger than the molecules of bleomycin and/or sucrose. Kinetics of pore resealing depended on the pulse duration and was faster after the shorter pulse. After a short 100-μs duration pulse, the disappearance of the pores permeable to bleomycin was completed after 6-7min at 24-26°C, while after a long 2-ms duration pulse, this process was slower and lasted 15-20min. Thus, it can be concluded that a short 100-μs duration pulse created smaller pores than the longer 2-ms duration pulse. This could be attributed to the time inadequacy for pores to grow and expand during the pulse, in the case of short pulses.     

Oxidative effects of nanosecond pulsed electric field exposure in cells and cell-free media

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 (1-13kV/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.     

The impacts of heavy metals on oxidative stress and growth of spring barley

Oxidative stress is accepted to play a significant role in stress symptoms, caused by different stressors in a variety of organisms. In this study seedlings of spring barley (Hordeum vulgare L.) were exposed to a wide range of copper, zinc, chromium, nickel, lead and cadmium concentrations in order to determine the relationships between heavy metals-induced oxidative stress and plant growth inhibition. All investigated heavy metals induced an essential increase in lipid peroxidation and a reduction of dry biomass along with an increase in metal concentration in the nutrient solution. A very close and statistically significant exponential relationship between lipid peroxidation and growth inhibition was detected in this study. According to the results of analysis of variance (ANOVA), the intensity of nonspecific oxidative stress is identified as the main factor of barley growth inhibition, explaining 75% of total variance. Almost 10% of growth inhibition is attributed to the specific impact of heavy metals. The most pronounced increase of malondialdehyde content and growth inhibition was observed in Cu and Cd treatments, whereas the lowest changes in observed indicators were detected after exposure to Zn and Pb.     

Are we protected? Imported measles - on the way to eradication

In accordance with the 2015 regional goal for measles and rubella elimination of the WHO European Region, only a few imported cases have been documented of both diseases in Hungary for years.This paper presents a case of a Hungarian woman, born in 1975, who received measles vaccination at age of 12 months and later at age of 11 years, according to her certificate of vaccination. In 2009, after arriving home from a vacation in Ireland, she developed acute measles infection with clinical symptoms. It was confirmed by the detection of measles specific IgM, IgA and IgG antibodies, and by detection of viral nucleic acid from throat swab in virus transport medium.Additionally, an outbreak occurred in December of 2011 among a family emigrated from Romania to Hungary. No new measles cases were diagnosed among the contact persons of neither the young Hungarian woman returning from Ireland, nor the family emigrated from Romania. This observation refers to the effectiveness of the Hungarian vaccination program.