Elfrieda Fodor

The Effects of Cadmium on the Fluidity and H+-ATPase Activity of Plasma Membrane from Sunflower and Wheat Roots

Summary The effects of cadmium on the H + -ATPase activity and on the membrane fluidity were studied in plasma membrane prepared from roots of sunflower and wheat. Plasma membrane purified from the microsomal fraction by phase partitioning showed a decreased ATPase activity by in vivo Cd 2+ treatment (30 % and 90 % decrease in wheat and sunflower, respectively) and the in vitro application caused an inhibition, as well. The ordering state of the membrane at the C-5 and C-16 levels of the fatty acid chains and at the membrane surface was determined by electron spin resonance (ESR) and by fluorescence spectroscopy, respectively. The presence of Cd 2+ increased the ordering of the plasma membrane in both in vivo treatments and in vitro experiments. Under Cd 2+ treatment, a more rigid structure of the PM was found at different depths in the membrane; changes were larger in sunflower than in wheat. The alterations can be the results of direct effects on the membrane constituents (proteins, lipids) or of the modification in lipid composition. Preliminary studies suggest that there are changes caused by Cd 2+ in the fatty acid composition of the phospholipid fraction in PM, which were different in the case of wheat and sunflower.

Molecular architecture and biophysical properties of phospholipids during thermal adaptation in fish: An experimental and model study

Phospholipids from livers of carps (Cyprinus carpio L.) adapted to winter (5°C) and summer (25°C) temperatures were isolated, and the fatty acid composition of total phospholipids, as well as molecular species composition of diacyl phosphatidylcholines and ethanolamines, were determined. Order parameter of 5-doxyl stearic acid and steady-state fluorescence anisotropy of different anthroyloxy fatty acids—[2-, 12(N-9-anthroyloxy)stearic acid and 16(N-9-anthroyloxy)palmitic acid—embedded in native and synthetic (16∶0/16∶0, 16∶0/22∶6, 18∶0/22∶6, 18∶1/22∶6, 20∶4/20∶4, 22∶6/22∶6 phosphatidylcholines and 16∶0/18∶1, 18∶1/22∶6 phosphatidylethanolamines) phospholipid vesicles was also determined between −30 and 30°C and 5 and 30°C, respectively. There is an accumulation of 1-monoenoic, 2-polyenoic diacyl phosphatidylcholine and ethanolamine with a concomitant reduction of 1-stearoyl,2-docosahexaenoyl species in the cold-adapted state. Despite a 30% accumulation of long-chain polyunsaturated fatty acids in phospholipids in cold, there is only a 5°C downshift in the solid-gel to liquid-crystalline phase transition temperature (−8 vs. −13°C). Vesicles from total phospholipids of cold-adapted fish proved to be more disordered in all segments than from the warmadapted ones when assayed using 2,12-(N-9-anthroyloxy)stearic and 16-(N-9-anthroyloxy)palmitic acid. Vesicles made from purified phosphatidylcholines showed the same pattern, but they were more disordered than the corresponding total phospholipids. This could be modelled using mixed phospholipid vesicles made of synthetic 16∶0/22∶6 phosphatidylcholine (75%) and either 18∶1/22∶6 phosphatidylethanolamine (25%) vs. 16∶0/18∶1 phosphatidylethanolamine (25%) and comparison of the anisotropy parameters of 100% 16∶0/22∶6 and 100% 18∶1/22∶6 phosphatidylcholine vesicles. Mixing either 16∶0/18∶1 (25%) or 18∶1/22∶6 (25%) phosphatidylethanolamines to 18∶0/22∶6 (75%) phosphatidylcholine shifted down or up, respectively, the transition temperature of vesicles compared to 100% 18∶0/22∶6 vesicles assayed by electron spin resonance spectroscopy using 5-doxylstearic acid. It is concluded that it is not the gross amount of long-chain polyunsaturated fatty acids in phospholipids, but rather their specific combination withcis Δ9 monounsaturated fatty acids in the positionsn-1, especially in phosphatidylethanolamines, that is important in determining the physical properties of biomembranes in relation to adaptational temperature.

Heat stress causes spatially-distinct membrane re-modelling in K562 leukemia cells

Cellular membranes respond rapidly to various environmental perturbations. Previously we showed that modulations in membrane fluidity achieved by heat stress (HS) resulted in pronounced membrane organization alterations which could be intimately linked to the expression and cellular distribution of heat shock proteins. Here we examine heat-induced membrane changes using several visualisation methods. With Laurdan two-photon microscopy we demonstrate that, in contrast to the enhanced formation of ordered domains in surface membranes, the molecular disorder is significantly elevated within the internal membranes of cells preexposed to mild HS. These results were compared with those obtained by anisotropy, fluorescence lifetime and electron paramagnetic resonance measurements. All probes detected membrane changes upon HS. However, the structurally different probes revealed substantially distinct alterations in membrane heterogeneity. These data call attention to the careful interpretation of results obtained with only a single label. Subtle changes in membrane microstructure in the decision-making of thermal cell killing could have potential application in cancer therapy.

Adaptation of composition and biophysical properties of phospholipids to temperature by the crustacean, Gammarus spp.

The compositions of lipid classes as well as the molecular species composition of subclasses (diacyl, alkylacyl, and alkenylacyl forms) of choline and ethanolamine phosphoglycerides in marine amphipod crustaceans, Gammarus spp., collected in the Baltic Sea at 8 and 15°C, were studied in relation to environmental temperature. The structural order of phospholipid multibilayers was also determined. Environmental temperature had little effect on fatty acid composition. The level of some polyunsaturated fatty acids, such as 20:4, even increased in choline and ethanolamine phosphoglycerides at 15°C. Ethanolamine phosphoglycerides were rich in alkenylacyl forms, especially in crustaceans collected at 15°C. The accumulation of sn-1 monoenic, sn-2 polyenic diacyl, alkyl, and alkenylacyl phosphatidylethanolamines and diacyl phosphatidylcholines was observed at 8°C. The phospholipid vesicles of crustaceans collected at 8°C were more disordered than expected compared to those obtained from animals collected at 15°C. It was concluded that, in addition to variations in the levels of sn-1 monoenic and sn-2 polyenic phospholipid molecular species with temperature, ethanolamine plasmalogens may play a role in controlling membrane biophysical properties in marine amphipod crustaceans.

Involvement of phospholipid molecular species in controlling structural order of vertebrate brain synaptic membranes during thermal evolution

Fluorescence anisotropy parameter of [p-(6-phenyl)-1,3,5-hexatrienyl]phenyl-propionic acid (DPH-PA) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) embedded in synaptic plasma membranes prepared from brains of cold (5°C) and warm (22°C) adapted fish (Cyprinus carpio L.), rat (Rattus norvegicus) and bird (Branta canadensis), was studied. Fatty acid composition of total lipids as well as molecular species composition of diacyl phosphatidylcholines and phosphatidylethanolamines was also determined. The amount of long-chain polyunsaturated fatty acids decreased with increasing body temperature. There was a nearcomplete compensation of membrane structural order for environmental/body tempeature over the evolutionary scale as seen by DPH-PA. Using TMA-DPH, the compensation was partial with rat and bird. Since DPH-PA and TMA-DPH differ in their charges, it is proposed, that the former reported membrane regions rich in cationic or zwitterionic (neutral) phospholipids and the latter, membrane regions rich in negatively charged phospholipids in the synaptic plasma membranes. Many different molecular species (20–25) of diacyl phosphatidylcholines and diacyl phosphatidylethanolamines were identified. The level of 16:0/22:6 phosphatidylcholine decreased while disaturated phosphatidylcholines increased with increase of environmental/body temperature from the fish through the bird. Level of 1-monoenoic 2-polyenoic phosphatidylethanolamines also decreased with an increase in environmental/body temperature. Experiments using vesicles made of mixed synthetic phosphatidylcholine vesicles (16:0/16:0, 16:0/18:1, 16:0/22:6 in various proportions) showed that increase in disaturated phosphatidylcholine species does not explain the observed complete adjustment of membrane structural order in synaptic plasma membranes. Change in level of 1-monoenoic, 2-polyenoic phosphatidylethanolamines might be one of the factors involved in controlling the biophysical properties of the membrane according to the temperature.

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 degrees C, as does the kidney enzyme at 42 degrees C (but not at 20 degrees C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (Tm approximately 45 degrees C) than does the kidney enzyme (Tm approximately 55 degrees C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 degrees C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.

Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage

BackgroundThe propensity for off-target activity of Streptococcus pyogenes Cas9 (SpCas9) has been considerably decreased by rationally engineered variants with increased fidelity (eSpCas9; SpCas9-HF1). However, a subset of targets still generate considerable off-target effects. To deal specifically with these targets, we generated new “Highly enhanced Fidelity” nuclease variants (HeFSpCas9s) containing mutations from both eSpCas9 and SpCas9-HF1 and examined these improved nuclease variants side by side to decipher the factors that affect their specificities and to determine the optimal nuclease for applications sensitive to off-target effects.ResultsThese three increased-fidelity nucleases can routinely be used only with perfectly matching 20-nucleotide-long spacers, a matching 5′ G extension being more detrimental to their activities than a mismatching one. HeFSpCas9 exhibit substantially improved specificity for those targets for which eSpCas9 and SpCas9-HF1 have higher off-target propensity. The targets can also be ranked by their cleavability and off-target effects manifested by the increased fidelity nucleases. Furthermore, we show that the mutations in these variants may diminish the cleavage, but not the DNA-binding, of SpCas9s.ConclusionsNo single nuclease variant shows generally superior fidelity; instead, for highest specificity cleavage, each target needs to be matched with an appropriate high-fidelity nuclease. We provide here a framework for generating new nuclease variants for targets that currently have no matching optimal nuclease, and offer a simple means for identifying the optimal nuclease for targets in the absence of accurate target-ranking prediction tools.

The highly conserved, N-terminal (RXXX)8 motif of mouse Shadoo mediates nuclear accumulation.

The prion protein (PrP)-known for its central role in transmissible spongiform encephalopathies-has been reported to possess two nuclear localization signals and localize in the nuclei of certain cells in various forms. Although these data are superficially contradictory, it is apparent that nuclear forms of the prion protein can be found in cells in either the healthy or the diseased state. Here we report that Shadoo (Sho)-a member of the prion protein superfamily-is also found in the nucleus of several neural and non-neural cell lines as visualized by using an YFP-Sho construct. This nuclear localization is mediated by the (25-61) fragment of mouse Sho encompassing an (RXXX)8 motif. Bioinformatic analysis shows that the (RXXX)n motif (n=7-8) is a highly conserved and characteristic part of mammalian Shadoo proteins. Experiments to assess if Sho enters the nucleus by facilitated transport gave no decisive results: the inhibition of active processes that require energy in the cell, abolishes nuclear but not nucleolar accumulation. However, the (RXXX)8 motif is not able to mediate the nuclear transport of large fusion constructs exceeding the size limit of the nuclear pore for passive entry. Tracing the journey of various forms of Sho from translation to the nucleus and discerning the potential nuclear function of PrP and Sho requires further studies.

New partner proteins containing novel internal recognition motif for human glutaminase interacting protein (hGIP)

Regulation of gene expression in cells is mediated by protein-protein, DNA-protein and receptor-ligand interactions. PDZ (PSD-95/Discs-large/ZO-1) domains are protein-protein interaction modules. PDZ-containing proteins function in the organization of multi-protein complexes controlling spatial and temporal fidelity of intracellular signaling pathways. In general, PDZ proteins possess multiple domains facilitating distinct interactions. The human glutaminase interacting protein (hGIP) is an unusual PDZ protein comprising entirely of a single PDZ domain and plays pivotal roles in many cellular processes through its interaction with the C-terminus of partner proteins. Here, we report the identification by yeast two-hybrid screening of two new hGIP-interacting partners, DTX1 and STAU1. Both proteins lack the typical C-terminal PDZ recognition motif but contain a novel internal hGIP recognition motif recently identified in a phage display library screen. Fluorescence resonance energy transfer and confocal microscopy analysis confirmed the in vivo association of hGIP with DTX1 and STAU1 in mammalian cells validating the previous discovery of S/T-X-V/L-D as a consensus internal motif for hGIP recognition. Similar to hGIP, DTX1 and STAU1 have been implicated in neuronal function. Identification of these new interacting partners furthers our understanding of GIP-regulated signaling cascades and these interactions may represent potential new drug targets in humans.

A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a ‘self-cleaving’ GFP-expression plasmid

Abstract The efficacies of guide RNAs (gRNAs), the short RNA molecules that bind to and determine the sequence specificity of the Streptococcus pyogenes Cas9 nuclease, to mediate DNA cleavage vary dramatically. Thus, the selection of appropriate target sites, and hence spacer sequence, is critical for most applications. Here, we describe a simple, unparalleled method for experimentally pre-testing the efficiencies of various gRNAs targeting a gene. The method explores NHEJ-cloning, genomic integration of a GFP-expressing plasmid without homologous arms and linearized in-cell. The use of ‘self-cleaving’ GFP-plasmids containing universal gRNAs and corresponding targets alleviates cloning burdens when this method is applied. These universal gRNAs mediate efficient plasmid cleavage and are designed to avoid genomic targets in several model species. The method combines the advantages of the straightforward FACS detection provided by applying fluorescent reporter systems and of the PCR-based approaches being capable of testing targets in their genomic context, without necessitating any extra cloning steps. Additionally, we show that NHEJ-cloning can also be used in mammalian cells for targeted integration of donor plasmids up to 10 kb in size, with up to 30% efficiency, without any selection or enrichment.