Mechanism of haemolysis by Vibrio vulnificus haemolysin

The haemolytic action of Vibrio vulnificus haemolysin (VVH) was compared to that of streptolysin O (SLO). Both were cholesterol-binding haemolysins, but differed in the release of haemoglobin (Hb). In the first step of haemolysis, the haemolysins were temperature-independently bound to the cholesterol site on the target erythrocyte membrane. This was followed by the rapid release of K+, which is an intra-erythrocyte marker. Hb was then released, in different ways. In the case of VVH, Hb was released slowly after a relatively long lag, whereas with SLO, Hb was released as rapidly as K+. Haemolysis by VVH was inhibited by the addition of 30 mM-dextran 4 (mean Mr 4000), which is considered to be an effective colloid-osmotic protectant. The results therefore indicated that haemolysis by VVH (like that by Escherichia coli alpha-haemolysin and Staphylococcus aureus alpha-toxin) was caused by a colloid-osmotic mechanism. Both K+ and Hb release caused by VVH proceeded temperature-dependently, and the membrane fluidity of liposomes prepared with lipids extracted from sheep red blood cell membranes increased above 20 degrees C. These results suggest that the temperature-dependence of the haemolysis by VVH is due to the requirement for an increase in the membrane fluidity during the formation of a transmembrane pore.     

Characterization of mitochondrial glutamate dehydrogenase from dark‐grown soybean seedlings

Purified preparations of NAD(H)‐glutamate dehydrogenase (GDH, EC 1.4.1.2.) were assayed to determine the effects of mono‐ and divalent cations, nucleotides and select carbon compounds on NAD(H)‐dependent GDH activity. The amination reaction was stimulated 2‐ to 17‐fold by divalent cations (Ca²⁺ > Cd²⁺ > Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ between 1 and 1000 µ M ), but the reaction was unaffected by monovalent cations (Na ⁺ and K ⁺). The amination reaction was most responsive to changes in Ca²⁺ in a NADH‐dependent manner. The addition of EDTA or EGTA nullified the stimulatory effects of Ca²⁺. Calmodulin alone or in combination with calmodulin antagonists did not affect the amination reaction. Divalent cations (at 1 m M ) inhibited the rate of the deamination reaction by 15 to 25%, while monovalent cations had no effect. ATP inhibited the amination reaction by 10 to 60%, while ADP had little or no effect. ATP or ADP decreased the rate of the deamination reaction 23 to 60 or 20 to 38%, respectively. Many tricarboxylic acid cycle intermediates inhibited the amination reaction, 20 to 50% of the inhibition could be attributed to the chelating capacity of intermediates. Conversely, most of the carbon sources tested did not affect the deamination reaction, the only appreciable differences were increases in activity with sucrose (21%) and glucose (41%) and a decrease in activity with pyruvate (34%). Inhibitors of sulfhydryl groups were used to examine the importance of reduced thiol groups in the amination or deamination reactions. The amination was not dependent on reduced thiol groups, whereas the deamination reaction was dependent on reduced thiol groups.     

Purification and properties of l-lactate dehydrogenase from Acinetobacter calcoaceticus

Abstract Membrane-bound l -lactate dehydrogenase has been purified almost to homogeneity from Acinetobacter calcoaceticus . The enzyme is an oligomeric protein of sub-unit M _r 40 000 containing non-covalently bound FMN as a prosthetic group. Purified l -lactate dehydrogenase has an apparent K _m of 83 μM for l -lactate but has no activity with, and is not inhibited by, d -lactate. The enzyme is strongly inhibited by HgCl₂, but other thiol reagents and metal-chelating compounds have little or no effect upon its activity.     

Effect of SO2, on the activity of adenosine 5'-phosphosulfate sulfotransferase from spruce trees (Picea abies) in fumigation chambers and under field conditions

The effect of SO₂ on adenosine 5'-phosphosulfate sulfotransferase activity and various other parameters of needles from spruce ( Picea abies L.) was studied using potted grafts in outdoor fumigation chambers and trees growing near a factory. In summer and autumn fumigation of grafted spruce, SO₂, decreased the extractable activity of adenosine 5'-phosphosulfate sulfotransferase to 12–50% of the controls, and reduced the amount of ³⁵S from sulphate incorporated into protein by excised branches to a comparable degree. SO₂ treatment in January and February inhibited the increase in adenosine 5'phosphosulfate sulfotransferase activity measured in the controls during this time. ATP-sulfurylase activity was less affected by SO₂. fumigation. In trees growing near a factory with high SO₂. emission, the activity of adenosine 5'-phosphosulfate sulfotransferase was about 35% of that of trees from a control area. The low enzyme activity was correlated with a high content of sulfate and compounds containing thiol groups.     

Some properties of Vibrio vulnificus hemolysin

Some properties of hemolysin produced by Vibrio vulnificus were investigated. The hemolysin was heat labile, and the hemolytic activity was inhibited by adding cholesterol or divalent cations. Cholesterol inhibited the temperature-independent hemolysin-binding step, suggesting that cholesterol made up the binding site of the cell membrane, whereas the divalent cations inhibited the temperature-dependent membrane-degradation step. However, the V. vulnificus hemolysin was stable to oxygen and sulfhydryl reagents and was not inactivated by antiserum against streptolysin O, suggesting that the V. vulnificus hemolysin differs from oxygen-labile hemolysins which bind to cholesterol. The V. vulnificus hemolysin seems to be one of the exceptional cholesterol-binding hemolysins.     

HEAVY-METAL INDUCED CHANGES IN NONPROTEINACEOUS THIOL LEVELS AND HEAVY-METAL BINDING PEPTIDE IN TETRASELMIS TETRATHELE (PRASINOPHYCEAE)

The effects of mercury and cadmium on the intracellular level of nonproteinaceous thiols in a unicellular green alga Tetraselmis tetrathele (West) Butcher (Prasinophyceae) were investigated by using a fluorescent dye, 5-chloromethylfluorescein (5CMF), as a probe for nonproteinaceous thiols. The 5CMF fluorescence was observed in cytoplasm, and the intensity of the fluorescence was decreased by exposure of the cells to HgCl₂. Analysis of the fluorescent intensity of 5CMF by flow cytometry made it possible to distinguish cells in three states during the dying process caused by HgCl₂: a normal state, a thiol-depleted state, and a dead state. Depletion of nonproteinaceous thiols began within 30 min, and they were completely depleted at 2 h. Most cells died after 24 h of exposure to more than 3.0 μM HgCl₂, whereas exposure up to 1.0 mM CdCl₂ did not cause depletion of nonproteinaceous thiols or cell death within 48 h.   HPLC analyses revealed that glutathione was a major nonprotein thiol in T. tetrathele and that it was oxidized by exposing the cells to HgCl₂. Phytochelatins, which play a great role in the tolerance to heavy metals of higher plants and many algae, could not be found in T. tetrathele. However, a tripeptide, Arg-Arg-Glu, was found to be abundant, and it showed ability to bind Hg²⁺, suggesting that it functions to scavenge heavy metals as well as thiol molecules.     

Senescence in French-bean nodules: Occurrence of different proteolytic activities

A decline in nitrogenase activity (C₂H₂ reduction) of nodules of Phaseolus vulgaris L. cv. Contander was correlated with a decrease in their soluble protein including leghe-moglobin. Concomitantly, two distinct proteolytic activities against leghemoglobin with acidic and alkaline pH optima were detected. The corresponding proteases were purified about 30-fold by ammonium sulfate precipitation, gel filtration and hydroxy-apatite chromatography. Both the acidic (pH optimum 3.5) and the alkaline (pH optimum 8.0) proteases were thiol enzymes. They were characteristic of senescing nodules, whereas only an acidic serine protease was present in functional nodules.     

Proteolytic activity of a rumen anaerobic fungus

Abstract A strain of the anaerobic phycomycetous fungus Neocallimastix frontalis isolated from the rumen of a sheep had a high proteolytic activity which became predominantly extracellular during growth. Proteolytic activity appeared to be due to a metalloprotease, as it was inhibited by 1,10-phenanthroline, EDTA and other chelators but not by phenylmethylsulphonyl fluoride (PMSF). Inhibition by EDTA was fully reversed by the addition of Zn²⁺, Ca²⁺ or Co²⁺, whereas addition of metal ions in the presence of 1,10-phenanthroline restored only a little activity. p -Chloromercuribenzoate (PCMB) was also inhibitory in dialysed supernatant fluid. N-α-p-Tosyl- l -lysine chloromethylketone (TLCK) inhibited proteolysis, suggesting that the protease(s) has a trypsin-like specificity, but benzoylarginine p -nitroanilide was not hydrolysed. Protease activity has a broad pH profile with a maximum at pH 7.5. Gel fractionation indicated that most of the activity was in a high- M _r form.     

The Aromatic Ring of Phenylalanine 334 Is Essential for Oligomerization of Vibrio vulnificus Hemolysin

Vibrio vulnificus hemolysin (VVH) is thought to be a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins. To date, the structure-function relationships of CDCs produced by Gram-negative bacteria remain largely unknown. We show here that the aromatic ring of phenylalanine residue conserved in Vibrionaceae hemolysins is essential for oligomerization of VVH. We generated the VVH mutants; substituted Phe 334 for Ile (F334I), Ala (F334A), Tyr (F334Y), or Trp (F334W); and tested their binding and oligomerizing activity on Chinese hamster ovary cells. Binding in all mutants fell by approximately 50% compared with that in the wild type. Oligomerizing activities were completely eliminated in F334I and F334A mutants, whereas this ability was partially retained in F334Y and F334W mutants. These findings indicate that both hydrophobicity and an aromatic ring residue at the 334th position were needed for full binding activity and that the oligomerizing activity of this toxin was dependent on the existence of an aromatic ring residue at the 334th position. Our findings might help further understanding of the structure-and-function relationships in Vibrionaceae hemolysins.Vibrio vulnificus hemolysin (VVH) is a pore-forming toxin produced by the Gram-negative bacterium Vibrio vulnificus (6, 11). VVH binds directly to cholesterol and is oligomerized in vitro. Once VVH forms the VVH-cholesterol complex, it can no longer bind to susceptible cells (10). Therefore, VVH could be considered a member of the cholesterol-dependent cytolysin (CDC) toxin family (35).A wide variety of Gram-positive and some Gram-negative bacteria produce CDCs, which require cellular cholesterol to exert their cytotoxicity (22, 38). Structure-function relationships between CDCs produced by Gram-positive bacteria (gpCDCs) have been studied intensively for over a decade, whereas CDCs produced by Gram-negative bacteria remain largely unknown. On the other hand, it is well known that some Vibrionaceae bacteria, such as Vibrio vulnificus, Vibrio cholerae, Aeromonas hydrophila, and Aeromonas sobria, produce pore-forming toxins/hemolysins. Among them, it was reported that VVH and Vibrio cholerae cytolysin (VCC) required cholesterol to exert their activity (12, 35). Thus, Vibrionaceae hemolysins are thought to be members of the CDC family. The generalized toxic steps are thought to be similar for both gpCDCs and Vibrionaceae hemolysins (22); i.e., monomers interact with a susceptible cell membrane, these monomers are assembled to form oligomers by membrane fluidity, and transmembrane pore formation follows (5, 22, 27, 30, 37). Although gpCDCs and Vibrionaceae hemolysins have common toxic steps, the following differences exist between them. (i) There is no similarity in amino acid sequences. (ii) gpCDCs have a highly conserved tryptophan-rich motif, which is involved in membrane recognition (3, 9, 27), whereas this motif does not exist in Vibrionaceae hemolysins. (iii) gpCDCs, such as perfringolysin and intermedilicine, are composed of four domains, whereas Vibrionaceae hemolysins are composed of two or three domains (21, 24, 25). (iv) Vibrionaceae hemolysins form pores that are smaller (2 to 3 nm in diameter) (33, 36) than those formed by gpCDCs (approximately 30 nm) (1, 2, 19).Recently, the crystal structure of VCC was determined (21). VCC is composed of three domains, namely the cytolysin domain, the β-trefoil lectin domain, and the β-prism lectin domain (21). The proposed mechanisms of action of VCC are as follows: (i) monomer binding to cell surfaces via interactions with the cytolysin domain, (ii) binding to carbohydrate receptors by the β-prism lectin domain, (iii) oligomerization via the cytolysin domain, and (iv) pore formation by insertion of a stem-loop from the cytolysin domain into the cellular membrane (21). On the other hand, from the analysis of the VVH amino acid sequence, it has been predicted that VVH is composed of two domains (21) and is missing the β-prism lectin domain, which binds to carbohydrate receptors on the cellular membrane (21). Therefore, the structure and functions of VVH are thought to be slightly different from those of VCC. Thus, analysis of the structure-function relationship of VVH will aid in the understanding of the evolutionary process of CDCs as well as of the toxic mechanism of VVH.In this study, we show that phenylalanine in the 334th position (F334) is required for the binding and oligomerizing ability of VVH. In particular, the benzene ring of this phenylalanine is a prerequisite for its oligomerizing ability. Because of the high conservation of this phenylalanine in other Vibrionaceae hemolysins, our results will contribute to a better understanding of the structure-function relationships of Vibrionaceae hemolysins.     

Buoyancy regulation and aggregate formation in Amoebobacter purpureus from Mahoney Lake

Abstract The meromictic Mahoney Lake (British Columbia, Canada) contains an extremely dense layer of purple sulfur bacteria ( Amoebobacter purpureus ). The buoyant density of Amoebobacter cells grown in pure culture at saturating light intensity was significantly higher (1027–1034 kg m⁻³) than the density of lake water (1015 kg m⁻³). When stationary cultures were shifted to the dark, the gas-vesicle content increased by a factor of 9 and buoyant density decreased to 1002 kg m⁻³ within three days.
A novel mechanism of cell aggregation was detected for the Mahoney Lake strain. Dense cell aggregates were formed after depletion of sulfide. Formation of aggregates was correlated with an increase in cell surface hydrophobicity. Cell aggregates could be disintegrated within less than 1 s by addition of sulfide or various thiol compounds. Mercaptanes with a branched structure in the vicinity of the terminal thiol group, compounds with esterified thiol groups (methylmercaptanes), reducing compounds lacking thiol groups and detergents did not influence aggregate stability. Cell aggregates disintegrated upon addition of urea or of proteinase K. Addition of various sugars had no effect on aggregation; this points to the absence of lectins. The results indicate that cell-to-cell adhesion in A, purpureus ML1 is mainly caused by a hydrophobic effect and includes a specific mechanism possibly mediated by a surface protein.
Extrapolation of laboratory results to field conditions demonstrated that both regulation of buoyant density and formation of cell aggregates result in passive accumulation of cells at the chemocline and contribute to the narrow stratification of A. purpureus in Mahoney Lake.     

Bouyancy regulation and aggregate formation in Amoebobacter purpureus from Mahoney Lake

Abstract The meromictic Mahoney Lake (British Columbia, Canada) contains an extremely dense layer of purple sulfur bacteria ( Amoebobacter purpureus ). The buoyant density of Amoebobacter cells grown in pure culture at saturating light intensity was significantly higher (1027–1034 kg m⁻³) than the density of lake water (1015 kg m⁻³). When stationary cultures were shifted to the dark, the gas-vesicle content increased by a factor of 9 and buoyant density decreased to 1002 kg m⁻³ within three days.
A novel mechanism of cell aggregation was detected for the Mahoney Lake strain. Dense cell aggregates were formed after depletion of sulfide. Formation of aggregates was correlated with an increase in cell surface hydrophobicity. Cell aggregates could be disintegrated within less than 1 s by addition of sulfied or various thiol compounds. Mercaptanes with a branched structure in the vicinity of the terminal thiol group, compounds with esterified thiol groups (methyl-mercaptanes), reducing compounds lacking thiol groups and detergents did not influence aggregate stability. Cell aggregates disintegrated upon addition of urea or of proteinase K. Addition of various sugars had no effect on aggregation; this points to the absence of lectins. The results indicate that cell-to-cell adhesion in A. purpureus ML1 is mainly caused by a hydrophobic effect and includes a specific mechanism possibly mediated by a surface protein.
Extrapolation of laboratory results to field conditions demonstrated that both regulation of buoyant density and formation of cell aggregates result in passive accumulation of cells at the chemocline and contribute to the narrow stratification of A. purpureus in Mahoney Lake.     

Inhibition of Adenylyl Cyclase Activity by a Homogeneous Population of Dopamine Receptors: Selective Blockade by Antisera Directed Against Gi1 and/or Gi2

Abstract: The 7315c pituitary tumor cell expresses a homogeneous population of dopamine receptors that are functionally similar to brain dopamine D₂ receptors. [³H]-Sulpiride binding to 7315c cell homogenates was specific and saturable, and K _i values for compounds to compete for these sites were highly correlated with values for the same compounds at D₂ receptors in brain. Dopamine maximally inhibited ∼65% of forskolin-stimulated cyclase activity in cell membranes. Some D₂ agonists had lower efficacies, suggesting that some compounds are partial agonists at this receptor. Removal of GTP from the assay buffer or pretreatment of the tissue with pertussis toxin abolished the inhibition of adenylyl cyclase by dopamine. Immunodetection of most of the known Gα subunits revealed that G_i1, G_i2, G_i3, G_o, G_q, and G_s are present in the 7315c membrane. Pretreatment with the AS antibody (which recognizes the C-terminal regions of Gα_i1 and Gα_i2) significantly attenuated the inhibition of adenylyl cyclase activity by dopamine, whereas antibodies to C-terminal regions of the other Gα subunits had no effect. These findings suggest that the dopamine D₂ receptor regulates cyclase inhibition predominantly via G_i1 and/or G_i2 and that the 7315c tumor cells provide a useful model for studying naturally expressed dopamine D₂ receptors in the absence of other dopamine receptor subtypes.     

Different effects of supplied ammonium on glutamine synthetase activity in mustard (Sinapis alba) and pine (Pinus sylvestris) seedlings

The activity of glutamine synthetase (GS) in mustard ( Sinapis alba L.) and Scots pine ( Pinus sylvestris L.) seedlings was used as an index to evaluate the capacity to cope with excessive ammonium supply. In these 2 species GS activity was differently affected by the application of nitrogen compounds (NH₄⁺ or NO₃⁻). Mustard seedlings older than 5 days showed a considerable increase in GS activity after NH₄⁺ or NO₃⁻ application. This response was independent of the energy flux, but GS activity in general was positively affected by light. Endogenous NH₄⁺ did not accumulate greatly after nitrogen supply. In contrast, seedlings of Scots pine accumulated NH₄⁺ in cotyledons and roots and showed no stimulation of GS activity after the application of ammonium. In addition, root growth was drastically reduced. Thus, the pine seedlings seem to have insufficient capacity to assimilate exogenously supplied ammonium. NO₃⁻, however, did not lead to any harmful effects.     

Sulfhydryl Groups in Receptor Binding of Thyrotropin-Releasing Hormone to Rat Amygdala

Abstract: Binding of [³H]-[3-Me-His²]thyrotropin-releasing hormone ([³H]MeTRH) to TRH receptors in rat amygdala was decreased by sulfhydryl reagents in a time-, temperature-, and concentration-dependent manner. A pronounced reduction in receptor density, with little or no change in binding affinity, was apparent following disulfide bond reduction by dithiothreitol (DTT), alkylation of thiol groups by N -ethylmaleimide (NEM), and their oxidation by 5,5'-dithiobis (2-nitrobenzoic acid). Heavy metals (Cd²⁺, Hg²⁺), which complex with reactive -SH residues, also potently inhibited binding. The pharmacological specificity of residual [³H]MeTRH binding in chemically modified amygdala membranes was the same as that in control preparations. Sequential exposure to thiol reagents, in the presence or absence of cations, revealed possible additive effects. Pretreatment of membranes with TRH (10^--⁸-^-10^--⁶ M ), and its continued presence during modification, afforded protection against DTT and NEM. These results indicate the possible importance of thiol groups in the maintenance of TRH receptor conformation.     

Endothelin-1 Increases Arachidonic Acid Release in C6 Glioma Cells Through a Potassium-Modulated Influx of Calcium

Abstract: Endothelin-1 (Et-1) but not a range of other receptor agonists stimulated the release of arachidonic acid (AA) in C6 glioma. Et-1 activation was concentration dependent and was inhibited by chelation of extracellular calcium. The calcium ionophores A23187 and ionomycin could also stimulate release of AA. Et-1 caused an early increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) followed by a sustained but lower plateau level. The sensitivity of the response to quinacrine, its dependence on Ca²⁺, and the demonstration of an increase in phospholipase A₂ (PLA₂) activity that was insensitive to dithiothreitol suggested that the release of AA was due to activation of cytosolic PLA₂ in the cells. Staurosporine, a protein kinase C (PKC) inhibitor, had no effect on Et-1-induced AA release but abolished that by phorbol 12-myristate 13-acetate, demonstrating that the Et-1 response was PKC independent. Raised levels of extracellular KCI inhibited both AA release and the increase in [Ca²⁺]_i triggered by Et-1, whereas valinomycin, which causes K⁺ efflux, not only caused a rapid rise in [Ca²⁺]_i but also caused AA mobilisation. The results therefore suggest that Et-1 activation of PLA₂ in this cell type requires calcium influx dependent on K⁺ efflux.     

Role of oxygen and metal ions in the instability of streptolysin O.

Thiol-containing preparations of streptolysin O (SLO) and pure cysteine generate superoxide radicals in alkaline buffer on autoxidation of the thiol groups. Autoxidation is stimulated by cupric ions. Reconstituted SLO preparations accumulate hydrogen peroxide with a concomitant loss of activity on storage at room temperature. Short-term protection of hemolytic activity was achieved by inclusion of catalase in the preparation; no apparent protection was observed by superoxide dismutase, whereas 1,10-O-phenanthroline offered long-term protection of the hemolysin.     

Inhibition of anthocyanin synthesis and phenylalanine ammonialyase activity by Co2+ in leaf disks of Terminalia catappa

Treatment with sucrose induced anthocyanin synthesis and phenylalanine ammonialyase (PAL, EC 4. 3. 1. 5) activity in leaf disks of Indian almond ( Terminalia catappa L. Duthie). Co²⁺, an inhibitor of ethylene biosynthesis, inhibited anthocyanin synthesis and PAL activity when given together with sucrose. Ethephon (an exogenous source of ethylene) given along with sucrose, promoted anthocyanin synthesis and PAL activity, but in the presence of Co²⁺ its effectiveness decreased. In an attempt to understand the inhibitory action of Co²⁺ in the presence of ethephon, the effect of Co²⁺ on PAL activity was studied in vitro. A kinetic study showed an uncompetitive type of inhibition of PAL by Co²⁺, which was not time dependent. Addition of 2-mercaptoethanol, cysteine or glutathione overcame the in vitro effect of Co²⁺, and 2-mercaptoethanol also restored the activity of PAL extracted from Co²⁺-treated leaf disks. It is suggested that sulfhydryl group(s) might be involved in the inactivation of PAL by Co²⁺. The effects of N-ethylmaleimide (NEM) and HgCl₂ (other sulfhydryl reagents) were also studied. Both NEM and Hg²⁺ competitively inhibited PAL activity in vitro, and the inhibition could be reversed by sulfhydryl compounds.     

K+ATP channel opening prevents succinate-dependent H2O2 generation by plant mitochondria

The role of a recently identified K⁺_ATP channel in preventing H₂O₂ formation was examined in isolated pea stem mitochondria. The succinate-dependent H₂O₂ formation was progressively inhibited, when mitochondria were resuspended in media containing increasing concentration of KCl (from 0.05 to 0.15  M ). This inhibition was linked to a partial dissipation of the transmembrane electrical potential (ΔΨ) induced by KCl. Conversely, the malate plus glutamate-dependent H₂O₂ formation was not influenced. The succinate-sustained H₂O₂ generation was also unaffected by nigericin (a H⁺/K⁺ exchanger), but completely prevented by valinomycin (a K⁺ ionophore). In addition, cyclosporin A (a K⁺_ATP channel opener) inhibited this H₂O₂ formation, while ATP (an inhibitor of the channel opening) slightly increased it. The inhibitory effect of ATP was strongly stimulated in the presence of atractylate (an inhibitor of the adenine nucleotide translocase), thus suggesting that the receptor for ATP on the K⁺ channel faces the intermembrane space. Finally, the succinate-dependent H₂O₂ formation was partially prevented by phenylarsine oxide (a thiol oxidant).     

Effect of Peroxynitrite on the Mitochondrial Respiratory Chain: Differential Susceptibility of Neurones and Astrocytes in Primary Culture

Abstract: The effect of the neurotoxic nitric oxide derivative, the peroxynitrite anion (ONOO⁻), on the activity of the mitochondrial respiratory chain complexes in cultured neurones and astrocytes was studied. A single exposure of the neurones to ONOO⁻ (initial concentrations of 0.01–2.0 m M ) caused, after a subsequent 24-h incubation, a dose-dependent decrease in succinate-cytochrome c reductase (60% at 0.5 m M ) and in cytochrome c oxidase (52% at 0.5 m M ) activities. NADH-ubiquinone-1 reductase was unaffected. In astrocytes, the activity of the mitochondrial complexes was not affected up to 2 m M ONOO⁻. Citrate synthase was unaffected in both cell types under all conditions studied. However, lactate dehydrogenase activity released to the culture medium was increased by ONOO⁻ in a dose-dependent manner (40% at 0.5 m M ONOO⁻) from the neurones but not from the astrocytes. Neuronal glutathione concentration decreased by 39% at 0.1 m M ONOO⁻, but astrocytic glutathione was not affected up to 2 m M ONOO⁻. In isolated brain mitochondria, only succinate-cytochrome c reductase activity was affected (22% decrease at 1 m M ONOO⁻). We conclude that the acute exposure of ONOO⁻ selectively damages neurones, whereas astrocytes remain unaffected. Intracellular glutathione appears to be an important factor for ameliorating ONOO⁻-mediated mitochondrial damage. This study supports the hypothesis that the neurotoxicity of nitric oxide is mediated through mitochondrial dysfunction.     

Relationship between localization on cellular membranes and cytotoxicity of Vibrio vulnificus hemolysin

Vibrio vulnificus secretes a hemolysin/cytolysin (VVH) that induces cytolysis in target cells. A detergent resistant membrane domain (DRM) fraction of the cells after sucrose gradient centrifugation includes cholesterol-rich membrane microdomains which have been called "lipid rafts". It was reported that some pore-forming toxins require association with DRM and/or lipid rafts to exert their cytotoxicity. It has also been thought that cellular cholesterol is involved in VVH cytotoxicity because VVH cytotoxicity was inhibited by pre-incubation with cholesterol. However, both cellular localization and mode of action of VVH cytotoxicity remain unclear. In this study, we investigated the relationship between VVH localization on the cellular membrane and its cytotoxicity. Oligomers of VVH were detected from DRM fractions by sucrose gradient ultracentrifugation but all of these oligomers shifted from DRM fractions to non-DRM fractions after treatment with methyl-beta-cyclodextrin (MβCD), a cholesterol sequestering agent. On the other hand, immunofluorescence analysis showed that VVH did not co-localize with major lipid raft markers on cellular membrane of CHO cells. These data suggested that VVH localized at membrane regions which are relatively abundant in cholesterol but which are not identical with lipid rafts. To determine the linkage between localization and cytotoxicity of VVH, cytotoxicity was evaluated in MβCD-treated CHO cells. The cytotoxicity of VVH was not decreased by the MβCD treatment. In addition, the amount of VVH oligomer did not decrease in MβCD-treated CHO cells. Thus, we found that the amount of oligomer on cellular membrane is important for induction of cytotoxicity, whereas localization to lipid rafts on the cellular membrane was not essential to cytotoxicity.