Induced Membrane Changes Research Articles

Flavonol clustering in model lipid membranes: DSC, AFM, force spectroscopy and MD simulations study

We here report on flavonols (myricetin (MCE) and its glycoside myricitrin (MCI)) – 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membrane interactions focusing on the effects of flavonol clustering on the membrane thermotropic and nanomechanical properties.Atomic force microscopy (AFM), force spectroscopy (FS) and differential scanning calorimetry (DSC) together with molecular dynamics (MD) simulations provided a consistent picture of flavonol - DMPC membrane interactions. DMPC membrane as a supported lipid bilayer preserved its integrity even at higher flavonol molar fraction x. When present at x = 0.1 – 0.3, MCE and MCI both slightly improve DMPC bilayer fluidity which is evidenced by the decrease in the main phase transition temperature Tm. MCE is found within the interior of the bilayer, while MCI incorporates in the head group-water interface region. AFM and FS confirmed clusters as protrusions with an average height of 0.012 μm and average diameters of 0.60 and 0.24 μm for MCE and MCI clusters, respectively. The average membrane thickness in DMPC fluid phase decreases for 7% at xMCE = 0.30, while only 4% at xMCI = 0.27.The induced membrane changes are dependent on the chemical and physical properties of inserted flavonols. The hypothesis regarding the tendency of flavonol to clustering in membranes by increasing flavonol molar fraction has been confirmed.

Methylmercury inhibits growth and induces membrane changes in Pseudomonas putida

A bacterial model system (Pseudomonas putida DSM 50026) was used in this research to assess toxicity of the environmentally relevant concentrations of mercury species (MeHg and Hg(II)) that represent important pollutants of aquatic ecosystems at sites of industrial or mining activities. In addition to direct monitoring of bacterial growth, we also analyzed fatty acid profiles of exposed and non-exposed cultures to determine possible toxic effects manifested on membrane level. The results showed that exposure of P. putida to Hg(II) in concentrations of 0.2–200 µg/L did not have any significant effects on growth nor fatty acid composition of exposed bacterial culture. On the other hand, when bacteria were exposed to up to 1600-times lower concentrations of MeHg (0.12–12.5 µg/L), growth inhibition as well as significant changes in fatty acid composition were detected. Observed adaptive membrane changes due to MeHg exposure were similar to those associated with responses to organic solvents and some other membrane-disrupting compounds.

Whole-body heat exposure induces membrane changes in spermatozoa from the cauda epididymidis of laboratory mice

This study was carried out to determine if exposure to hot environmental temperatures had a direct, detrimental effect on sperm quality. For this the effect of whole-body heat exposure on epididymal spermatozoa of laboratory mice was investigated. C57BL/6 mice (n = 7) were housed in a microclimate chamber at 37 degrees C-38 degrees C for 8 h per day for three consecutive days, while control mice (n = 7) were kept at 23 degrees C-24 degrees C. Cauda epididymal spermatozoa were obtained 16 h after the last heat treatment. The results showed that sperm numbers were similar in the two groups (P = 0.23), but after heat treatment, a significant reduction in the percentage of motile sperm was present (P < 0.0001). Membrane changes of the spermatozoa were investigated by staining with phycoerythrin (PE)-conjugated Annexin V, which detects exteriorization of phosphotidylserine from the inner to the outer leaflet of the sperm plasma membrane, and 7-aminoactinomycin D (7-AAD), which binds to the sperm nucleus when the plasma membrane is damaged. The percentage of spermatozoa showing positive staining with Annexin V-PE or 7-AAD or both, was significantly higher (P < 0.05) in heat-exposed mice compared with controls. These results show that whole-body heat exposure to 37 degrees C-38 degrees C induces membrane changes in the epididymal spermatozoa of mice, which may lead to apoptosis.

Methylmercury inhibits growth and induces membrane changes in Pseudomonas putida

1 univ. of Ljubljana, Biotechnical Fac., dept. of Animal Science, groblje 3, SI-1230 domžale, Slovenia, e-mail: masa.vodovnik@bf.uni-lj.si 2 Laboratory for environmental science and engineering, national institute of chemistry, hajdrihova 19, SI-1000 Ljubljana, Slovenia 3 Same address as 1, Ph.d. 4 Same address as 1, Ph.d., e-mail: romana.marinsek@bf.uni-lj.si most toxic metals is certainly mercury, which represents a significant concern especially in aquatic ecosystems at sites with industrial or mining activities. Mining operations in areas rich in cinnabar ore may represent strong sources of hg for many years even after mining has been discontinued (Benoit et al., 1994). One of mercury (hg) affected sites also lies in western part of Slovenia where Methylmercury inhibits growth and induces membrane changes in Pseudomonas putida A bacterial model system (Pseudomonas putida dSM 50026) was used in this research to assess toxicity of the environmentally relevant concentrations of mercury species (Mehg and hg(II)) that represent important pollutants of aquatic ecosystems at sites of industrial or mining activities. In addition to direct monitoring of bacterial growth, we also analyzed fatty acid profiles of exposed and non-exposed cultures to determine possible toxic effects manifested on membrane level. The results showed that exposure of P. putida to hg(II) in concentrations of 0.2–200 μg/L did not have any significant effects on growth nor fatty acid composition of exposed bacterial culture. On the other hand, when bacteria were exposed to up to 1600-times lower concentrations of Mehg (0.12–12.5 μg/L), growth inhibition as well as significant changes in fatty acid composition were detected. Observed adaptive membrane changes due to Mehg exposure were similar to those associated with responses to organic solvents and some other membrane-disrupting compounds.

Dual topology of the processed hepatitis C virus protein NS4B is influenced by the NS5A protein

Among the least-known hepatitis C virus proteins is the non-structural protein 4B (NS4B). It localizes to the endoplasmic reticulum (ER) membrane and induces membrane changes, resulting in a membranous web that is reported to be the locale for virus replication. A model was presented previously for the topology of recombinant HCV NS4B of the 1a genotype based on in vitro data. In this model, the N-terminal tail of a considerable fraction of the NS4B molecules was translocated into the ER lumen via a post-translational process, giving the protein a dual transmembrane topology. It is now reported that translocation of the N terminus also occurs for processed NS4B expressed in cells in the context of the polyprotein. In the presence of NS5A, however, a lower degree of translocation was observed, which may indicate that NS5A influences the topology of NS4B. In vitro expression studies of NS4B from all major genotypes demonstrated that translocation of the N terminus to the ER lumen is conserved across genotypes. This clearly suggests an important function for this feature. Furthermore, when disrupting a previously reported amphipathic helix (AH) in the N terminus of NS4B, translocation was inhibited. As a disrupted AH also abolished the ability of NS4B to rearrange membranes, these data indicate for the first time an association between translocation of the N terminus and membrane rearrangement. Finally, the present experiments also confirm the predicted location of the first luminal loop to be around aa 112.

In vitro studies on radiation induced membrane oxidative damage in apoptotic death of mouse thymocytes

Radiation oxidative damage to the plasma membrane of cells and its consequences in the mechanism of apoptotic death have been receiving growing attention of radiation scientists in recent years. We have employed a fluorescence probe method to determine changes in the permeability and fluidity of the plasma membrane of mouse thymocytes following &#947; irradiation mwithin a low to moderate radiation dose range (a few cGy to 10 Gy;·0.71 Gy/min). In vitro studies have shown that radiation induced membrane changes were correlated with the induction of apoptotic cell death. Studies have also been carried out to evaluate the generation of intracellular reactive oxygen species (ROS) in response to radio-oxidative damage to thymocytes using a cytosolic fluorescence probe, 2'-7'- dichlorodihydrofluorescien diacetate (DCH-FDA). The apoptosis of the thymocytes was determined by measuring nuclear condensation using propidium iodide (PI) and DNA fragmentation by gel electrophoresis. The viability of the thymocytes was determined by the trypan blue exclusion method which was found to gradually decrease after incubation of irradiated cells for 7 h. Thymocytes labeled with DCH-FDA in PBS at 25°C showed remarkably increased fluorescence intensity, measured after 30 min of irradiation (10 Gy), indicating an enhanced formation of intracellular reactive oxygen species. The DCH-FDA probe was able to sensitively detect irradiation effects on cells at lower doses. The population of thymocytes with reduced nuclear diameter was found to progressively increase with the postirradiation incubation time, following exposure to a particular radiation dose. Moreover, fragmentation of nuclear DNA in irradiated thymocytes, as observed by the pattern of ladder formation on agarose gel, was found to significantly increase on incubations beyond 3 h at 37°C. Results indicate that the enhanced amount of intracellular reactive oxygen species generated in response to radiooxidative stress were linked with the induction of &#947; irradiation of apoptotic death in thymocytes. These observations and a highlight of emerging scenario in the area of low dose radiation effects at cellular and membrane levels are described.

Inhibition of tert-butyl hydroperoxide-induced cell membrane bleb formation by α-tocopherol and glutathione

Multiple bleb formation on cell membrane is common during cell death. The effects of α-tocopherol and glutathione (GSH) on tert-butyl hydroperoxide (TBH)-induced membrane changes in rat hepatocytes were studied. Over 60 min of exposure, TBH (0.5–2.0 m m) caused a dose-dependent membrane blebbing. Cells pretreated with buthionine sulfoximine, a GSH synthesis inhibitor, had significantly greater blebbing and lactate dehydrogenase (LDH) leakage under 0.5 m m TBH treatment as compared to cells without pretreatment. However, the protective effect of GSH disappeared when the TBH concentration was increased to 2.0 m m. In the presence of α-tocopheryl succinate (TS) pretreatment, it was noted that bleb formation, expressed as the percentage of cells bearing blebs, the average bleb size, or the onset of blebbing, was partially suppressed even when TBH concentration was 2.0 m m. TBH-induced thiobarbituric acid reactive substances and LDH leakage were completely abolished by TS pretreatment. Accompanying bleb formation, membrane-insoluble actin was noted to decrease by immunoblot assay . The decrease in actin was also suppressed by TS. These results indicated that intracellular GSH and α-tocopherol status are important to the TBH-induced cell membrane abnormality. Furthermore, TS plays a defensive role against blebbing when GSH is exhausted by TBH.

Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen.

The sensitivity of dog sperm cells for extracellular Ca(2+)/Ca(2+)-ionophore challenge was compared to the detrimental effects of an optimized freeze/thawing protocol. Three sperm-rich fractions of ejaculates from 9 dogs were obtained, and one aliquot of each ejaculate was washed in a modified Tyrode's medium (HBT containing 0.1 mM Ca(2+)), without (control sample) and with 2.5 microM Ca(2+)-ionophore (induced sample) and incubated for 60 min at 38 degrees C in humidified atmosphere. Another aliquot from the same semen fractions was diluted, washed in a Tris buffer, and packed into 0.5-ml straws with a Tris buffer containing 7.5 vol % glycerol. The samples were stored for 1 week in liquid nitrogen after a computer-driven three-step freeze protocol and subsequently thawed for 50 sec in a 37 degrees C water bath and reconstituted into HBT. The acrosome integrity was determined using fluorescein-conjugated peanut agglutinin (PNA-FITC) as an acrosomal marker, while the vitality of the sperm cells was simultaneously assessed with the membrane impermeable DNA supravital stain ethidium homodimer 1 (EthD-1) using fluorescence microscopy and flow cytometry. The motility of frozen/thawed sperm samples was evaluated by microscopic as well as computerized motility analyses. Remarkably, the percentage sperm cells that underwent acrosome reactions induced by Ca(2+)-ionophore correlated very positively (r = 0.93) with the amount of acrosome damage observed in cryopreserved sperm samples. Furthermore, the degree of cellular damage induced by Ca(2+)-ionophore treatment correlated very negatively (r = -0.99) with the relative amount of sperm cells that remained motile after cryopreservation. Such clear correlations between Ca(2+)-ionophore induced acrosome reaction and motility parameters for frozen/thawed dog sperm cells were not found, suggesting that the generation of acrosome leakage and sperm immotility are two independent detrimental processes occurring during cryopreservation. From these results it can be concluded that Ca(2+)-ionophore treatment followed by simultaneous determination PNA-FITC and EthD-1 staining can be used to predict the cryopreservability of ejaculates from individual dogs used as donors.

Environmental stresses affect tomato microsomal membrane function differently than natural ripening and senescence

Abstract On the premise that environmental stresses may induce changes in microsomal membrane function characteristic of accelerated senescence, the effects of physical damage (PD), chilling injury (CI), heat shock (HS) and controlled atmosphere (CA) on tomato fruit ( Lycopersicon esculentum cv. Trust) were compared to changes occurring during normal ripening and senescence. A physiological pattern for ripening and senescence of tomato fruit was established based on weight loss patterns, colour development, ion leakage, lipid fatty acid profiles of microsomal membrane lipids, microsomal K + stimulated ATPase activity and electrophoretically separated protein patterns of microsomal membranes. Control fruit displayed increases in redness, weight loss, ion leakage and saturation index (SI) of membrane lipids, as well as the appearance of membrane-associated polygalacturonase (polygalacturonase isozyme II—PGII) and its β-subunit and decreased microsomal membrane K + stimulated ATPase activity during a three-week storage period. Relative to controls, PD and CI fruit displayed increased ion leakage and a decreased rate of red colour development, increased ATPase activity and SI. Membrane-associated PGII appeared sooner in PD fruit, but its appearance was delayed in CA and CI fruit, relative to controls. Tomatoes exposed to HS and CA treatments, however, displayed ripening and senescence physiological symptoms similar to control fruit. Thus, environmental stresses induced membrane changes that were expressed differently than seen during senescence.

The effects of prolactin and prostaglandin F2 alpha on plasma membrane changes during luteolysis in the rat.

The ability of PRL to modify prostaglandin F2 alpha (PGF2 alpha)-induced membrane changes during functional corpus luteum regression was examined in the pseudopregnant rat. Fluorescence polarization studies conducted 24 h after sc injection of PGF2 alpha revealed a marked and significant increase in the polarization parameter, which is suggestive of reduced plasma membrane fluidity. At the same time, there was a decrease in hCG binding and a significant increase in apparent phospholipase-A2 activity during incubation of dispersed rat luteal cells. Each of these changes was attenuated when the animals were pretreated with PRL 30 min before PGF2 alpha. The decrease in plasma progesterone caused by PGF2 alpha treatment was also inhibited by PRL. PGF2 alpha also stimulated a significant polarization increase in dispersed cells prepared from ovaries removed 1 h after injection of this luteolytic agent, although this effect could not be demonstrated in plasma membrane samples. These results indicate that PRL and PGF2 alpha affect the same membrane pathway in the rat corpus luteum and that this pathway appears to be closely coupled to luteal cell function.

Expression and electrophysiological identification of the receptor for bombesin and gastrin-releasing peptide in Xenopus laevis oocytes injected with polyA + RNA from rat brain

The receptor for bombesin and the related peptide, gastrin-releasing peptide (GRP) has been induced in frog oocytes by injection of polyA + RNA from rat brain. The primed oocytes responded to peptides of the bombesin family (GRP, neuromedin C of bombesin) by showing dose-dependent oscillations in membrane currents as recorded by the voltage-clamp method. The induced membrane changes were suppressed when oocytes were pretreated with a bombesin-receptor antagonist.

A comparative study of permeabilization induced by extracellular ATP and by Sendai virus

Both Sendai virus and extracellular ATP induce membrane changes in 3T6 cells which allow passage of phosphorylated metabolites and normally impermeant aqueous solutes. The two processes share many features in common, including their kinetics and the effects of temperature, Ca 2+, and various metabolic inhibitors. Furthermore, in each case permeabilization is preceded by net changes in intracellular cations. However, there are significant differences in that only ATP-dependent permeabilization is influenced by changes in the ionic strength of the medium, by inhibition of the Na +, K +, Cl − cotransporter and by preincubation of 3T6 cells with dithiothreitol.

L929 cells infected with temperature sensitive mutants of vesicular stomatitis virus: virus replication is necessary for induction of changes in membrane permeability.

Infection of L929 murine cells with vesicular stomatitis virus (VSV) results in inhibition of host protein synthesis and appearance of membrane alterations at a time when cells are still actively engaged in viral protein synthesis. VSV temperature-sensitive (ts) mutants have been used to explore the role(s) played by the virus-coded proteins in the genesis of these effects. Cells were infected with each of five ts mutants representing the known complementation groups of VSV Indiana serotype, and incubated at permissive (32 degrees C) and non-permissive temperatures (39 degrees C). Protein synthesis in the presence and absence of Hygromycin B (Hyg. B) was analyzed during virus infection via incorporation of 35S-methionine in acid-precipitable material and SDS-polyacrylamide gel electrophoresis. Data indicate that mutants belonging to groups I (L protein), II (NS protein) and IV (N protein) do not inhibit host protein synthesis and do not induce any membrane changes when grown at the non-permissive temperature. Mutants of group III (M protein) and V (G protein), instead, do inhibit cell protein synthesis and induce membrane changes also when grown at the non-permissive temperature; this suggests that these effects do not correlate with the biological activity of these proteins and their interaction with the cellular membrane. On the other hand, mutants exhibiting defective steps of nucleocapsid replication are apparently unable to induce these effects once more suggesting that virus replication per se is essential, as also indirectly shown by experiments employing cycloheximide to mimic shut-off.

Phorbol-induced recruitment of lymphocytes for spontaneous cytolysis of natural killer-insensitive tumor targets

Natural killer (NK) cells lyse a variety of tumor cells in vitro whereas NK-depleted unsensitized lymphocytes do not have this effect. In studies designed to elucidate the NK phenomenon, a series of experiments was carried out to identify properties of NK-sensitive targets and compare these with those of NK-insensitive targets and with targets rendered sensitive by treatment with phorbol esters. Following brief exposure to phorbol-12-myristate-13-acetate (PMA), the targets were thoroughly washed, and then incubated with lymphocyte preparations which were either enriched for or depleted of NK cells. PMA treatment increased the susceptibility of sensitive targets to NK-enriched fractions by only 20–30%, but made the NK-cell-insensitive targets markedly vulnerable to these effectors (80% lysis). Unexpectedly, brief PMA exposure also rendered cells susceptible to lysis by NK-cell-depleted lymphocytes. Yet, such targets were not killed by monocytes or B lymphocytes. Elimination of T8 lymphocytes from the NK-depleted fractions abolished lysis. To explore whether PMA had induced membrane changes not detectable on electron microscopy of thin sections, freeze-fracture studies were carried out on target cells before and after treatment with PMA. Freeze-fracture replicas of target cells which had been exposed to PMA exhibited a 50% reduction of the intramembranous particles (IMP) on the external leaflet of the plasma membrane but no changes in the number or size of the IMP associated with the protoplasmic leaflet face. The exact relationship of the structural changes and enhanced susceptibility to cytolysis has not yet been established. However, the observation that normal and tumor cells can be rendered vulnerable to lysis by lymphocytes which have not been sensitized immunologically may have practical applications.

Sequential onset of permeability changes in mouse ascites cells induced by sendai virus

(1) The addition of haemolytic Sendai virus to cells induces membrane changes in the following sequence: (i) Increased permeability to ions, (ii) increased permeability to low molecular weight metabolites, (iii) increased permeability to proteins. (2) The consequences of an increased permeability to ions are: (a) alteration of membrane potential, (b) net changes in intracellular cations and (c) cell swelling, in that order. (3) Depending on virus: cell ratio, Ca 2+ concentration and temperature, it is possible to observe ion leakage without metabolite or protein leakage, and ion and metabolite leakage without protein leakage. (4) A model for the induction of permeability changes is presented.

Interaction of inactivity and nerve breakdown products in the origin of acute denervation changes in rat skeletal muscle.

The action of nerve breakdown products on innervated fibres of soleus and extensor digitorum longus muscles was investigated with the following procedures: partial denervation, sensory or sympathetic denervation, section of a previously transplanted foreign nerve. Each procedure was performed either in isolation or combined with chronic muscle inactivity obtained by blocking impulse conduction along the sciatic nerve. Silastic cuffs containing tetrodotoxin (TTX) and sodium chloride were utilized for the block. Partial denervation induced extrajunctional sensitivity to acetylcholine (ACh) and resistance to tetrodotoxin not only in the denervated but also in the innervated fibres. The effects in the innervated fibres were equal in magnitude to those in the denervated fibres, provided they were paralysed. The onset of the membrane changes was synchronous in the two classes of fibres and their amount correlated with the extent of partial denervation. If the innervated fibres were normally active, the membrane changes were still detectable, but considerably smaller than in the denervated fibres. Sensory denervation (removal of dorsal root ganglia L4 and L5) was followed by the development of moderate ACh supersensitivity and TTX resistance in chronically paralysed muscles. Furthermore, section of radicular nerves (total denervation, i.e. efferent plus afferent) induced larger membrane changes than those observed following section of ventral roots alone (efferent denervation). Sympathetic denervation was ineffective even when associated with chronic muscle paralysis. Section of a previously transplanted mixed nerve (superficial fibular) was ineffective if the soleus muscle was normally active, while it induced marked extrajunctional ACh sensitivity and TTX resistance when combined with chronic paralysis of the muscle. Section of a transplanted sensory nerve (sural) also induced extrajunctional membrane changes in paralysed soleus muscles, but their magnitude was much smaller than after section of mixed nerves. We conclude that products of nerve destruction, especially those of motor axons, induce membrane changes of striking magnitude when potentiated by muscle inactivity. Such an action may also explain the greater efficacy of denervation vs. pure inactivity, at least at early times after their onset.

Vesicular stomatitis virus induced membrane changes: A spin label study

Synchronized entry of Vesicular Stomatitis Virus (VSV) into spin labeled cultured human cells resulted in an increase in the rigidity of cell membranes as measured by Electron Spin Resonance Spectroscopy. Treatment of spin labeled cells with homologous interferon alpha did not influence the membrane fluidity, neither did it significantly prevent the VSV induced membrane changes despite its anti-viral protection.

Target level blocking of T-cell cytotoxicity for human malignant melanoma by monoclonal antibodies

Cytotoxic T lymphocytes (CTL) for autologous malignant melanoma in culture of a patient AV were induced by restimulation of PBL (peripheral blood leukocytes) with AV melanoma cells in vitro and subcultured in interleukin 2 (IL-2) conditioned media. Monoclonal antibodies detecting six antigenic systems on melanoma cell surfaces were tested for blocking activity on the effector function of subcultured cytolytic T lymphocytes for autologous melanoma cells. The monoclonal antibodies R 24 (γ3), specific for the G D3 disialoganglioside on melanoma cell surfaces and I 24 (γM), detecting a similar antigenic determinant, blocked autologous T lymphocytotoxicity for malignant melanoma cells on the target level. The effector function of alloantigen activated cytolytic T lymphocytes generated by coculture of allogeneic PBL with Epstein-Barr virus (EBV) transformed AV B lymphocytes, was blocked by monoclonal antibody R 24 when tested against AV melanoma targets, but not when tested against AV B lymphocyte targets. It is concluded that blocking by mAb R 24 occurs in this system as a nonspecific effect, unrelated to the specific target antigen recognition by cytotoxic T lymphocytes. Steric hindrance or antibody induced membrane changes may account for the blocking effect of monoclonal antibody R 24.

Glucose induces membrane changes detected by fluorescence polarization in endocrine pancreatic cells

Pancreatic endocrine cells prepared from rat islets were labelled with 1,6-diphenyl-1,3,5-hexatriene and examined in a microviscosimeter. Glucose caused a dose-related decrease in fluorescence polarization. This decrease was observed within 1 min after increasing the concentration of glucose. These findings suggest that glucose affects membrane viscosity in pancreatic endocrine cells. At a glucose concentration of 16.7 mM, the estimated viscosity was 15 ± 3 per cent lower than basal value (2.01 ± 0.12 P).

Tumor promoters induce membrane changes detected by fluorescence polarization

The probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to determine if tumor promoting agents alter cell membranes. The active tumor promoters TPA (12-0-tetra-decanoyl-phorbol-13-acetate), PDD (phorbol-12,13-didecanoate) and PDB (phorbol-12,13-dibenzoate) were found to decrease fluorescence polarization of DPH in rat embryo cells, whereas the inactive tumor promoting compounds phorbol and 4α-PDD failed to induce this change.