Modification Of Membrane Structure Research Articles

THE CONCEPTUAL APPROACH TO THE USE OF POSTBIOTICS BASED ON BACTERIAL MEMBRANE NANOVESICLES FOR PROPHYLAXIS OF ASTRONAUTS’ HEALTH DISORDERS

The functional fermented foods containing live microorganisms and their components are necessary for the normal functioning of the human body as normal gut microbiota needs fuel from external microbial organisms and their nanostructures — membrane vesicles (MVs), excreting outside. The сoncept that MVs may contribute to astronauts’ health probably to the same extent as their parental microbial cells do and be a temporary substitute for living microbial cells until we know more about the behavior of microbes in the space environment. The advantage of MVs is that they are not alive and cannot be changed under unfavorable conditions as microbial organisms may be. As the model, we selected MVs of a robust to environmental factors kombucha multimicrobial culture (KMC), known for its health-promoting characteristics for humans. We exposed KMC on the International Space Station in a hybrid space/Mars-like environment for an initial proof-of-concept stage. In the exposure study, KMC has survived a long-term period in harsh conditions, and the MVs generated by post-flight kombucha community members did not acquire toxicity, despite the changed membrane composition in the environment imitated conditions on the Mars surface. This observation, together with our KMC metagenomic and comparative genomic analyses of the dominant KMC bacterium Komagataeibacter oboediens, showed that the ground reference sample and spaceexposed ones were similar in topology and maintained their stability. In the next stage, we assessed the fitness, safety, and biodistribution of MVs of post-flight K. oboediens and showed that they were altered, but the modifications in membrane structure did not result in toxicity acquisition. Our proof-of-concept strategy is discussed in this review in line with the literature.

Tailoring microstructure of polysulfone membranes via novel hexagonal ZnO particles to achieve improved filtration performance

Incorporation of nanofillers into polymeric membranes has been the trend in the realm of membrane research owing to the improvements in many filtration characteristics with the modifications of membrane structure, morphology and bulk properties. The major drawback of the nanoparticle embedded membrane is the uncontrollable agglomeration, which could be mitigated by tailoring microstructure of the matrix by using designed nanoparticles. In the present study, polysulfone (PSf) based ultrafiltration membranes incorporated with designed MicNo® platelets composed of strongly bonded ZnO nanoparticles were prepared as single- and double-layer membranes using immersion precipitation technique. Filtration performance, morphological and bulk properties of the novel composite membranes containing MicNo® powders of varying amounts (0.01–1%, wt.) and particle sizes were determined in comparison to those of pristine PSf membrane. Single-layer composite membrane containing 0.5% MicNo® exhibited the greatest permeance improvement of 74% and a marked enhancement of 116% could be achieved with double-layer membrane system containing 0.5% and 0.1% MicNo® in the upper and bottom layers, respectively. The results demonstrated that MicNo® powders were successfully distributed throughout the membrane matrix without any surface imperfections owing to the well alignment of ZnO particles on their hexagonal platelet surfaces, which prevented the agglomeration of primary ZnO particles that ensured a more efficient hydrophilic surface generation in the membrane.

Synaptic Function and Dysfunction in Lysosomal Storage Diseases.

Lysosomal storage diseases (LSDs) with neurological involvement are inherited genetic diseases of the metabolism characterized by lysosomal dysfunction and the accumulation of undegraded substrates altering glial and neuronal function. Often, patients with neurological manifestations present with damage to the gray and white matter and irreversible neuronal decline. The use of animal models of LSDs has greatly facilitated studying and identifying potential mechanisms of neuronal dysfunction, including alterations in availability and function of synaptic proteins, modifications of membrane structure, deficits in docking, exocytosis, recycling of synaptic vesicles, and inflammation-mediated remodeling of synapses. Although some extrapolations from findings in adult-onset conditions such as Alzheimer’s disease or Parkinson’s disease have been reported, the pathogenetic mechanisms underpinning cognitive deficits in LSDs are still largely unclear. Without being fully inclusive, the goal of this mini-review is to present a discussion on possible mechanisms leading to synaptic dysfunction in LSDs.

CO2 capture by modified hollow fiber membrane contactor: Numerical study on membrane structure and membrane wettability

This work aims at capturing CO2 from natural gas using a verified finite element mathematical model by improving the membrane wettability, membrane structure, the type of absorbents used in the system, and the physical parameters like membrane porosity and the number of fibers used in hollow fiber membrane contactor (HFMC). The model is developed to simulate the CO2 removal using membranes by considering mass transfer equations in the presence of chemical reactions. Simulations were performed using a membrane system including 10 fibers with 175 mm length, 830 μm outer fiber radius and 450 μm inner fiber radius. The effect of adding montmorillonite nanoparticles (MMT) in weight percentages of 1%, 3%, and 5% to the PVDF membrane was perused to demonstrate the influence of membrane structure modification on system performance. The results show that the modification of membrane structure with montmorillonite nanoparticles could increase the efficiency of the system in the removal of CO2 particles by 8%. Four absorbents including MEA (monoethanol amine), PZ (piperazine), TEA (triethanolamine), and EDA (ethylenediamine) are used to identify the most effective absorbent where PZ absorbent shows the highest and EDA absorbent has the lowest carbon capture efficiency. Wettability is found to have a considerably negative impact on membrane carbon capture capacity. For instance, the modeling results show that even a 10% wetting of the membrane reduces the efficiency of the CO2 removal process by more than 47%. In addition, the results illustrate that the increase in membrane tortuosity and gas velocity have negative impacts on the separation process, while increase in the absorbent concentration, packing density and porosity enhance the separation of CO2.

A Virulence-Associated Glycolipid with Distinct Conformational Attributes: Impact on Lateral Organization of Host Plasma Membrane, Autophagy, and Signaling.

Mycobacterium tuberculosis (Mtb) serves as the epitome of how lipids-next to proteins-are utilized as central effectors in pathogenesis. It synthesizes an arsenal of structurally atypical lipids (C60-C90) to impact various membrane-dependent steps involved in host interactions. There is a growing precedent to support insertion of these exposed lipids into the host membrane as part of their mode of action. However, the vital role of specific virulence-associated lipids in modulating cellular functions by altering the host membrane organization and associated signaling pathways remain unanswered questions. Here, we combined chemical synthesis, biophysics, cell biology, and molecular dynamics simulations to elucidate host membrane structure modifications and modulation of membrane-associated signaling using synthetic Mycobacterium tuberculosis sulfoglycolipids (Mtb SL). We reveal that Mtb SL reorganizes the host cell plasma membrane domains while showing higher preference for fluid membrane regions. This rearrangement is governed by the distinct conformational states sampled by SL acyl chains. Physicochemical assays with SL analogues reveal insights into their structure-function relationships, highlighting specific roles of lipid acyl chains and headgroup, along with effects on autophagy and cytokine profiles. Our findings uncover a mechanism whereby Mtb uses specific chemical moieties on its lipids to fine-tune host lipid interactions and confer control of the downstream functions by modifying the cell membrane structure and function. These findings will inspire development of chemotherapeutics against Mtb by counteracting their effects on the host-cell membrane.

Lectin-induced oxidative stress in human platelets

Previously we have shown that wheat germ agglutinin (WGA) and, with minor potency, Phaseolus vulgaris agglutinin (PHA), but not lens culinarian agglutinin (LCA), induce platelet aggregation, through the PLCƴ2 activation by the concerted action of src/syk and PI3K/BTK pathways. In this study, we have investigated platelet oxidative stress induced by lectins. Several parameters indicative of oxidative stress, such as reactive oxygen species (ROS), superoxide anion, lipid peroxidation and the efficiency of the aerobic metabolism, have been measured. It was found that ROS, superoxide anion formation and lipid peroxidation are significantly increased upon platelet treatment with WGA and PHA while LCA is ineffective. WGA is always more effective than PHA in all experimental conditions tested. In addition, the involvement of NADPH oxidase 1, syk and PI3K in oxidative stress induced by WGA and PHA has been shown. Concerning the lectins effect on aerobic metabolism, WGA and PHA, but not LCA, act as uncoupling agents, determining an increase of oxygen consumption and a decrease of ATP synthesis, with a consequent decrease of P/O value. These results are confirmed by the impairment of platelets proton gradient formation, evaluated by membrane potential, in platelets treated with WGA and PHA. In conclusion lectins, especially WGA, induce oxidative stress in platelets and decrease energy availability through modifications of membrane structure leading to the inefficiency of the aerobic machinery that steers platelets toward death as suggested by the decreased metabolic activity of platelets and the increased lactic dehydrogenase release.

Antibiotic resistance and host immune evasion in Staphylococcus aureus mediated by a metabolic adaptation

Staphylococcus aureus is a notorious human bacterial pathogen with considerable capacity to develop antibiotic resistance. We have observed that human infections caused by highly drug-resistant S. aureus are more prolonged, complicated, and difficult to eradicate. Here we describe a metabolic adaptation strategy used by clinical S. aureus strains that leads to resistance to the last-line antibiotic, daptomycin, and simultaneously affects host innate immunity. This response was characterized by a change in anionic membrane phospholipid composition induced by point mutations in the phospholipid biosynthesis gene, cls2, encoding cardiolipin synthase. Single cls2 point mutations were sufficient for daptomycin resistance, antibiotic treatment failure, and persistent infection. These phenotypes were mediated by enhanced cardiolipin biosynthesis, leading to increased bacterial membrane cardiolipin and reduced phosphatidylglycerol. The changes in membrane phospholipid profile led to modifications in membrane structure that impaired daptomycin penetration and membrane disruption. The cls2 point mutations also allowed S. aureus to evade neutrophil chemotaxis, mediated by the reduction in bacterial membrane phosphatidylglycerol, a previously undescribed bacterial-driven chemoattractant. Together, these data illustrate a metabolic strategy used by S. aureus to circumvent antibiotic and immune attack and provide crucial insights into membrane-based therapeutic targeting of this troublesome pathogen.

Polyimide-TiO2 nanocomposites and their corresponding membranes: Synthesis, characterization, and gas separation applications

In this work at firstly a new class of polyimide/TiO2 (PI/TiO2) composites containing amino acid (Methionine) and benzimidazole fractions in the structure with chemical bonds between the polyimide and TiO2 network has been synthesized by the sol-gel reaction and characterized and then secondly carbon (C) membranes were prepared by carbonization of PI/TiO2 at different temperatures under vacuum and nitrogen flow. Carbonization temperature was critical in the modification of membrane structure. At the same temperature, the carbon membranes fabricated under nitrogen atmosphere had higher gas permeations than those fabricated under vacuum. With the introduction of the TiO2, there was no significant enhancement of the gas separation in the resulting PI/TiO2 composite membranes over the PI membrane, which still suffered a ‘‘trade-off’’ relationship between permeability and selectivity. However, the derived C/PI/TiO2 composite membranes exhibited better gas separation properties. The C/PI/TiO2 15% composite membrane produced the highest permeations of 650.47, 520.56, 187.92 and 115.22 GPU for He, CO2, O2 and N2, respectively. The bionanocomposites were analyzed for antibacterial activity against Gram-positive and Gram-negative bacteria. The results indicated that the synthesized bionanocomposites is effective against all of the studied bacteria, and its effectiveness is higher for Pseudomonas aeruginosa.

The relationship between self-organization and membrane effects of aqueous dispersion systems of the thyroliberin oligopeptide

The relationship between rearrangement of the dispersed phase inducing considerable changes in the pH and nonmonotonic concentration dependences of membrane effects in aqueous systems of the endogenous regulatory peptide, thyroliberin (thyrotropin-releasing hormone), in 10–3–10–16 mol/L concentration range was demonstrated for the first time. The membrane structure modification in the 10–13–10–16 mol/L range was found to be due to accumulation of nanoassociates, while the oppositely directed pronounced structural changes in the 10–6–10–12 mol/L range may be associated with the coexistence and rearrangement of dispersed phases of various nature (domains and associates) whose action on membrane lipid components is regulated in this concentration range by the correlated changes in the dispersed phase parameters and pH.

Unique Modification of Membrane Structure by Lithium: A Molecular Dynamics Study

Lithium is profoundly different from other ions in the body in multiple ways. Extracellular concentrations of calcium, sodium, potassium, and hydrogen ions must be closely regulated for well-being. On the other hand, the body does not closely regulate lithium but rather adapts reasonably well to lithium concentration variation of over a thousandfold, in spite of the fact that lithium interacts with many biological molecules. This stability coupled with interactiveness implies the existence of myriad mutually compensating lithium-modulated processes, which are almost completely not understood. In this study we explore the interaction of lithium, sodium, potassium, and rubidium with bilayer membranes comprised of POPC, using molecular dynamics simulations. Lithium, but not any of the others, forms a long-lasting noncovalent bond simultaneously with the headgroup phosphate and the CO group of the Sn2 chain. This interaction significantly alters the structure and the surface area of the membrane.

Novel regulation targets of the metal-response BasS–BasR two-component system of Escherichia coli

The BasS-BasR two-component system is known as an iron- and zinc-sensing transcription regulator in Escherichia coli, but so far only a few genes have been identified to be under the direct control of phosphorylated BasR. Using Genomic SELEX (systematic evolution of ligands by exponential enrichment) screening, we have identified a total of at least 38 binding sites of phosphorylated BasR on the E. coli genome, and based on the BasR-binding sites, have predicted more than 20 novel targets of regulation. By DNase I footprint analysis for high-affinity BasR-binding sites, a direct repeat of a TTAAnnTT sequence was identified as the BasR box. Transcription regulation in vivo of the target genes was confirmed after Northern blot analysis of target gene mRNAs from both wild-type E. coli and an otherwise isogenic basR deletion mutant. The BasR regulon can be classified into three groups of genes: group 1 includes the genes for the formation and modification of membrane structure; group 2 includes genes for modulation of membrane functions; and group 3 includes genes for stress-response cell functions, including csgD, the master regulator of biofilm formation.

Cell shape and plasma membrane alterations after static magnetic fields exposure

The biological effects of static magnetic fields (MFs) with intensity of 6 mT were investigated in lymphocytes and U937 cells in the presence or absence of apoptosis-inducing drugs by transmission (TEM) and scanning (SEM) electron microscopy. Lectin cytochemistry of ConA-FITC conjugates was used to analyze plasma membrane structural modifications. Static MFs modified cell shape, plasma membrane and increased the level of intracellular [Ca++] which plays an antiapoptotic role in both cell types. Modifications induced by the exposure to static MFs were irrespective of the presence or absence of apoptotic drugs or the cell type. Abundant lamellar-shaped microvilli were observed upon 24 hrs of continuous exposure to static MFs in contrast to the normally rough surface of U937 cells having numerous short microvilli. Conversely, lymphocytes lost their round shape and became irregularly elongated; lamellar shaped microvilli were found when cells were simultaneously exposed to static MFs and apoptosis-inducing drugs. In our experiments, static MFs reduced the smoothness of the cell surface and partially impeded changes in distribution of cell surface glycans, both features being typical of apoptotic cells. Cell shape and plasma membrane structure modifications upon static MFs exposure were time-dependent. Lamellar microvilli were clearly observed before the distortion of cell shape, which was found at long times of exposure. MFs exposure promoted the rearrangement of F-actin filaments which, in turn, could be responsible for the cell surface modifications. Here we report data that support biological effects of static MFs on U937 cells and human lymphocytes. However, the involvement of these modifications in the onset of diseases needs to be further elucidated.

Effect of the Nature of the 3β-Substitution in Manoyl Oxides on the Thermotropic Behavior of DPPC Lipid Bilayer and on DPPC Liposomes

Functionalized manoyl oxide derivatives have been proved over the years to evoke several biological responses. Among them, 3β-hydroxy-manoyl oxide (1) and 3β-acetoxy-manoyl oxide (2) have been shown to exhibit in vitro antimicrobial and cytotoxic activity, while N-imidazole-3 β-thiocarbonyl ester of manoyl oxide (3) was found to exhibit potent cytotoxic effect. Their partitioning into phospholipid bilayers may lead to membrane structure modifications that are crucial in liposome development as they may influence their maintenance and integrity. DSC was used to study the modifications induced in DPPC bilayers by incorporating increasing concentrations of the three manoyl oxide derivatives. All derivatives were found to strongly affect the bilayer structural organization in terms of a decrease of the cooperativity, the fluidization and partially destabilization of the gel phase and the induction of a lateral phase separation in clustering domains. Derivatives 1 and 3 were incorporated into DPPC liposomes and their physicochemical stability was monitored at 4°C. The stability of liposomes was strongly influenced by the presence of 1 and 3 at any molar ratio studied. DPPC/1 liposomes were found to retain its stability for 48 h at low concentration of 10% mol, while at higher concentrations up to 30% mol they collapsed into aggregated material. In all cases DPPC/3 liposomes were found unstable and sticky aggregated structures precipitated from the bulk suspension.

Calorimetric study on the induction of interdigitated phase in hydrated DPPC bilayers by bioactive labdanes and correlation to their liposome stability: The role of chemical structure

Labd-7,13-dien-15-ol (1), labd-13-ene-8α,15-diol (2), and labd-14-ene-8,13-diol (sclareol) have been found to exhibit cytotoxic and cytostatic effects. Their partitioning into phospholipid bilayers may induce membrane structure modifications, crucial in the development of liposomes. DSC was used to elucidate the profile of modifications induced in DPPC bilayers by incorporating increasing concentrations of the labdanes. Labdanes 1, 2 and sclareol were incorporated into SUV liposomes composed of DPPC their physicochemical stability was monitored (4 °C) and was compared to liposomes incorporating cholesterol. All labdanes strongly affect the bilayer organization in a concentration dependent manner in terms of a decrease of the cooperativity, the fluidization and partially destabilization of the gel phase, the induction of a lateral phase separation and the possible existence of interdigitated domains in the bilayer. The physicochemical stability of liposomes was strongly influenced by the chemical features of the labdanes. The liposomal preparations were found to retain their stability at low labdane concentration (10 mol%), while at higher concentrations up to 30 mol% a profound decrease in intact liposomes occurred, and a possible existence of interdigitated sheets was concluded.

Effects of carbonisation on pore evolution and gas permeation properties of carbon membranes from Kapton ® polyimide

Carbon membranes were prepared by carbonisation of Kapton ® polyimide at different temperatures under vacuum and nitrogen flow. Pore structure development of the membranes during carbonisation was studied. Carbonisation temperature was critical in the modification of membrane structure. At the same temperature, the carbon membranes fabricated under nitrogen atmosphere had higher gas permeances than those fabricated under vacuum. During heat treatment, the value of d-spacing for the carbon membranes decreased with increasing temperature, however, vacuum and nitrogen atmosphere had different influences on the changes in the d-spacing. CO 2 adsorption showed that the carbon membranes prepared at 1273 K under vacuum had the highest micropore volume whilst the membranes prepared at 1073 K under vacuum had the highest characteristic adsorption energy. N 2 adsorption showed that the samples obtained at 873 K under vacuum had the highest nitrogen uptake. Mesopores were deemed to be connected through micropores and narrow channels between meso- and/or micropores were supposedly present. The micropores predominantly controlled the transport properties of the carbon membranes. The membrane samples obtained at 1173 K under vacuum yielded ideal separation factors of 558.27, 60.87, 19.69 and 138.53 for He/N 2, CO 2/N 2, O 2/N 2 and CO 2/CH 4, respectively, with permeances of 7.26, 0.79, 0.26, 0.13 and 0.006 mol/(m 2 s Pa) for He, CO 2, O 2, N 2 and CH 4, respectively.

Gene expression profile of Campylobacter jejuni in response to growth temperature variation.

The foodborne pathogen Campylobacter jejuni is the primary causative agent of gastroenteritis in humans. In the present study a whole genome microarray of C. jejuni was constructed and validated. These DNA microarrays were used to measure changes in transcription levels over time, as C. jejuni cells responded to a temperature increase from 37 to 42 degrees C. Approximately 20% of the C. jejuni genes were significantly up- or downregulated over a 50-min period after the temperature increase. The global change in C. jejuni transcriptome was found to be essentially transient, with only a small subset of genes still differentially expressed after 50 min. A substantial number of genes with a downregulated coexpression pattern were found to encode for ribosomal proteins. This suggests a short growth arrest upon temperature stress, allowing the bacteria to reshuffle their energy toward survival and adaptation to the new growth temperature. Genes encoding chaperones, chaperonins, and heat shock proteins displayed the most dramatic and rapid upregulation immediately after the temperature change. Interestingly, genes encoding proteins involved in membrane structure modification were differentially expressed, either up- or downregulated, suggesting a different protein membrane makeup at the two different growth temperatures. Overall, these data provide new insights into the primary response of C. jejuni to surmount a sudden temperature upshift, allowing the bacterium to survive and adapt its transcriptome to a new steady state.

Positive effects of growth at suboptimal temperature and high salt concentration on long-term survival of Lactobacillus sakei

Lactobacillus sakei is a lactic acid bacterium commonly found on fresh meat and represents the predominant flora of vacuum-packed meat. In the present article, we studied the behavior of L. sakei in a chemically defined medium under various growth conditions relative to temperature or NaCl concentration. Growth occurred at each temperature, but growth rate and final cell density decreased at low temperature and survival was enhanced. In the presence of NaCl, we also observed a longer doubling time and a lower final cell density together with an enhanced long-term survival. When both conditions were combined, the long-term survival was greatly increased to about 28 weeks. Differences in cell morphology were observed under electron microscopy when cells were grown at low temperature in the presence of salt. We speculate that these are due to modifications in membrane structure. These results show that L. sakei is able to adapt to these environmental conditions and that slow growth is clearly associated with enhanced long-term survival.

Sodium chloride stimulus-response experiments in spiral wound reverse osmosis membranes: a new method to detect fouling

Fouling is generally described in terms of salt rejection and permeation flow, but these data seem to evolve very slowly, and when they reach a dramatic level, fouling is irreversible. We propose a method to determine the state of the wear of membranes by analyzing sodium chloride stimulus-response experiments. Indeed, it turns out that the shape of the distribution (RTD) of sodium chloride in the permeate flow of the membrane reveals the solute permeation mechanisms for used membranes. For new membranes the distribution of sodium chloride collected in the permeate side as well in the rejection side is unimodal. For fouled membranes we note a singular distribution form with the presence of several modes. The existence of a salt leakage peak, as well as an earlier detection of salt for all the fouled membranes, give evidence of the membrane structure modification. The intensive use of the membranes might have created an enlargement of the pore sizes; salt and solvent permeabilities are improved as well. It turns out that each permeate side sodium chloride distribution can be fitted by a weighted average of Gaussian distributions, the new membrane RTD and the shifted new membrane RTD. The coefficients of this average give us the fraction of the membrane which is enlarged, hampered by the deposited layer and not modified.

Alterations in Mitochondrial Function During Acute Hypoglycemia in Newborn Piglets • 1496

Decreased substrate availability may lead to increased generation of oxygen free radicals in mitochondria due to partial reduction of oxygen. The present study tests the hypothesis that acute hypoglycemia alters mitochondrial function, resulting in oxygen free radical-induced mitochondrial damage. Studies were performed in newborn piglets exposed to normoglycemia (n=7) or 2 hr of hypoglycemia (n=6). Hypoglycemia (blood glucose 1 mmol/L) was induced with a bolus of regular insulin, 100 U/kg IV, followed by a continuous IV infusion of insulin, 2 U/kg/min. Mitochondria were isolated from cerebral cortex using a discontinuous Ficoll gradient and mitochondrial DNA was extracted. Cerebral oxidative phosphorylation was assessed biochemically by measuring tissue levels of ATP and phosphocreatine (PCr). Lipid peroxidation products (fluorescent compounds and conjugated dienes) were measured in mitochondrial membranes as an index of free radical-induced modification of membrane structure. Mitochondrial DNA analysis was performed using 1% agarose gel electrophoresis as an additional marker of mitochondrial damage. During hypoglycemia, blood glucose concentration decreased from the baseline value(mean ± SD) of 5.8 ± 1.2 mmol/l to 1.2 ± 0.4 mmol/L (p<0.005). ATP concentration (mmoles/g brain) was 4.7 ± 1.0 and 3.8± 0.7 in control and hypoglycemia respectively (p=ns); PCr (mmoles/g brain) decreased by 50% during hypoglycemia to 1.4 ± 0.8 from the control value of 2.7 ± 0.4 (p<0.05). Conjugated dienes were significantly increased in mitochondrial membranes during hypoglycemia to 4.14± 1.72 mmoles/g protein (p<0.05 compared to the reference value of 0 in controls). Fluorescent compounds (mg quinine sulfate/g protein) were also increased during hypoglycemia from the control value of 15.2 ± 9.5 to 62.2 ± 15.8 (p<0.001). Mitochondrial DNA from hypoglycemic brain showed marked fragmentation with a random distribution of fragment sizes ranging from 100-1500 bp, while control DNA showed no significant fragmentation. Thus we conclude that 2 hr of acute hypoglycemia in newborn piglets increased mitochondrial free radical generation as demonstrated by peroxidation of mitochondrial membranes. The observed pattern of mitochondrial DNA fragmentation could also be due to modification of DNA by oxygen free radicals. We speculate that free radical-induced changes in mitochondrial function during acute hypoglycemia may lead to progressive mitochondrial failure and subsequent cell death.

Apoptosis dependent decrease of the intramembrane ion traffic in cultured mouse fibroblasts shown by conductivity dispersion.

We have investigated the intramembranal ion traffic in apoptotic 3T6 cells in culture. Apoptosis was induced by various treatments, such as serum deprivation, high density growth and hydrogen peroxide at subnecrotic doses. Cell death was assessed by nucleosomal DNA fragmentation, single cell electrophoresis, immunofluorescence and histological staining. To study the modifications of membrane structure and function, we adopted a well established biophysical strategy based on the measurement of the electrical conductivity of cell suspensions, as a function of the frequency of the electrical field applied to the sample. A comparison between the conductivity of normal and apoptotic cell suspensions shows that programmed cell death causes a decrease of membrane conductivity which indicates a diminished intramembranal ion traffic. Our results strongly suggest that one of the early events in the triggering of apoptosis is represented by an overall reduction of plasma membrane function. Finally, our results are in agreement with the idea that the nucleus is not the sole target of the apoptotic process.