Intracellular Na+ and K+ contents of Zygosaccharomyces rouxii mutants defective in salt tolerance

Two of five Zygosaccharomyces rouxii mutants defective in salt tolerance, 152S (sat1) and 1717S (SAT3), were inviable in a nutrient medium (YPD) containing more than 1% NaCl. These two mutant cells contained significantly higher amounts of Na⁺ (298 μmol and 285 μmol per g cells of 152S and 1717S, respectively) but lower amounts of K⁺ (242 μmol and 176 μmol per g cells of 152S and 1717S, respectively) than three other mutants, 41S (sat2-1 [98 μmol Na⁺ and 326 μmol K⁺/g cells]), 197S (sat2-2 [103μmol Na⁺ and 336 μmol K⁺/g cells]), 1611S (SAT4 [139 μmol Na⁺ and 294 μmol K⁺/g cells]), as well as a wild-type strain, AN39 (61 μmol Na⁺ and 349 μmol K⁺/g cells), when cultured in YPD medium containing 0.8% NaCl. A KCl supplement, optimally 0.6 M, added to the medium somewhat restored the NaCl-hypersensitivity of 152S and 1717S with a concomitant decrease of intracellular Na⁺. This finding suggests that the NaCl-hypersensitive mutations are due to a defect in the Na⁺-regulating mechanism. The other three mutants showed weak responses to KCl in high NaCl-YPD. These five salt sensitive mutants and the wild-type strain retained the same levels of intracellular glycerol and arabitol when transferred into NaCl (5%)-YPD from YDP medium. This suggests that polyol accumulation is not the only mechanism of salt tolerance in Z. rouxii.     

Evaluation of the participation of ferredoxin in oxygen reduction in the photosynthetic electron transport chain of isolated pea thylakoids

The contribution to reduction of oxygen by ferredoxin (Fd) to the overall reduction of oxygen in isolated pea thylakoids was studied in the presence of Fd versus Fd + NADP⁺. The overall rate of electron transport was measured using a determination of Photosystem II quantum yield from chlorophyll fluorescence parameters, and the rate of oxidation of Fd was measured from the light-induced redox changes of Fd. At low light intensity, increasing Fd concentration from 5 to 30 μM in the absence of NADP⁺ increased the proportion of oxygen reduction by Fd from 25–35 to 40–60% in different experiments. This proportion decreased with increasing light intensity. When NADP⁺ was added in the presence of 15 μM Fd, which was optimal for the NADP⁺ reduction rate, the participation of Fd in the reduction of oxygen was low, no more than 10%, and it also decreased with increasing light intensity. At high light intensity, the overall oxygen reduction rates in the presence of Fd + NADP⁺ and in the presence of Fd alone were comparable. The significance of reduction of dioxygen either by water-soluble Fd or by the membrane-bound carriers of the photosynthetic electron transport chain for redox signaling under different light intensities is discussed.     

Stimulation-evoked release of purines from the rabbit retina

The evoked release of purines from rabbit retinae preloaded with [³H]adenosine was studied in vitro. Potassium (8.6–43.6 mM) and ouabain (1 or 10 μM) increased the release of radioactivity in a concentration-dependent manner. The K⁺-evoked release was significantly reduced when the superfusion was carried out at 2–4°C. The effect of K⁺ (8.6, 13.6 and 23.6 mM) and of ouabain (1 μM) were completely abolished when the retinae were superfused with a Ca²⁺-free medium containing 0.1 mM EGTA. Calcium removal only partially reduced the effect of higher K⁺ and ouabain concentrations (43.6 mM and 10 μM, respectively). Further, the effect of K⁺ was found to be independent of extracellular Ca²⁺ when retinae were pretreated with ouabain for 30 min. Stimulation of the retina with light flashes induced a small, persistent increase in the release of radioactivity observable for several minutes after the end of stimulation.The superfusate contained mainly hypoxanthine and inosine. There were no significant changes in the relative proportions of the different purine compounds released before or in response to either K⁺ (23.6 mM) or ouabain (10 μM) stimulation. Potassium stimulation significantly increased the release of adenosine, inosine and hypoxanthine. Addition of the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), significantly increased the relative proportions of released endogenous adenosine and inosine.The results indicate that K⁺ stimulation induces the release of purines from the rabbit retina by a Ca²⁺- and energy-dependent process. Light flashes also induce a purine release. The results suggest an active role for adenosine in retinal neurotransmission.     

Sodium and chloride ions contribute synergistically to salt toxicity in wheat

The effects of supplying excess mineral salts, involving sodium as a cation and a range of counteranions, including chloride, on the growth and photosynthetic capacity of a salt susceptible bread wheat were studied. Plant performance was much more affected by the NaCl treatment than by the same concentration of either of the two component ions. With the exception of K⁺, other alkali metal chlorides also greatly inhibit plant growth and the electron flow through photosystem 2. The ranking of toxicity of these cations is Li⁺>Na⁺>K⁺. The synergistic effect of sodium (and other alkali and alkaline earth metals) and chloride shows that neither of these ions alone is responsible for salt stress induced damage.     

Endothelial cell oxidant generation during K+-induced membrane depolarization

We tested the hypothesis that membrane depolarization may initiate oxidant generation in the endothelial cell. Depolarization was produced in bovine pulmonary arterial endothelial cells (BPAEC) in monolayer culture with varying external K⁺, or with glyburide (10 μM), tetraethylammonium (TEA, 10 mM), gramicidin (1 μM), or nigericin (2 μM). Evaluation of bisoxonol fluorescence of BPAEC indicated concentration-dependent depolarization by high K⁺ (2% change in fluorescence/mV change in membrane potential in the 5.9–48 mM range of K⁺) and essentially complete depolarization with glyburide. Generation of oxidants was assessed with o-phenylenediamine dihydrochloride (o-PD) oxidation in the presence of horseradish peroxidase (HRP). There was a time-dependent increase in o-PD oxidation with 24 mM K⁺, nigericin, and gramicidin over 2 hours compared with control. In 1 hour o-PD oxidation increased 2.8-fold for 24 mM and 3.7-fold for 48 mM K⁺ compared with control. Catalase reduced 24 mM K⁺-induced o-PD oxidation by 50%, while Cu/Zn-superoxide dismutase (SOD) abolished the increase. Oxidation of o-PD was reduced by 57% in the absence of HRP in the system. With K⁺ channel blockade, o-PD oxidation increased 3.8-fold with glyburide and 4.6-fold with TEA compared with control. These data indicate formation of H₂O₂ and possibly other oxidants with depolarization and suggest involvement of K⁺-channels in this process. © 1996 Wiley-Liss, Inc.     

In vitro study of the antioxidant properties of non steroidal anti-inflammatory drugs by chemiluminescence and electron spin resonance (ESR)

Objectives. To determine the antioxidant activities of nonsteroidal anti-inflammatory drugs (NSAIDS), we examined by chemiluminescence (CL) and electron spin resonance (ESR) their scavenging properties towards lipid peroxides, hypochlorous acid and peroxynitrite.

Methods. The antioxidant properties of nimesulide (NIM), 4-hydroxynimesulide (4-HONIM), aceclofenac (ACLO), 4-hydroxyaceclofenac (4-HOA-CLO), diclofenac (DICLO) and indomethacin (INDO) were tested on four different reactive oxygen species (ROS) generating systems: (I) phorbol-myristate acetate (PMA)-activated neutrophils, (II) Fe²⁺/ascorbate-induced lipid peroxidation, (III) HOCl-induced light emission, (IV) the kinetics of ONOO^- decomposition followed by spectrophotometry. ROS production was monitored by luminol-enhanced CL or by ESR using two different spin traps.

Results. At 10 μM, ACLO, NIM, 4-HONIM, 4-HOA-CLO, and DICLO decreased luminol-enhanced CL generated by PMA-activated neutrophils. Inversely, INDO increased the luminol enhanced CL. Interestingly, hydroxylated metabolites were more potent antioxidants than the parent drugs. Furthermore, all drugs tested, excepted ACLO, lowered lipid peroxidation induced by Fe²⁺/ascorbate system. ACLO and DICLO, even at the highest concentration tested (100 μM), did not significantly lower HOCl induced CL, whereas the other drugs were potent scavengers. Finally, all the NSAIDS accelerated decomposition of ONOO^-, suggesting a potential capacity of the molecules to scavenge peroxynitrite.

Conclusion. The NSAIDs possess variable degrees of antioxidant activities, linked to their ability to react with HOCl, lipid peroxides or ONOO^-. These antioxidant activities could offer interesting targeted side-effects in the treatment of joint inflammatory diseases.     

Interaction of hemoglobin with salts: Effects on the functional properties of human hemoglobin

Oxygen isotherms of human hemoglobin measured in distilled water and in solutions of different inorganic salts in the concentration range from below 10^?3 m to above 1·5 m at neutral pH indicate that the oxygen affinity decreases with increasing salt concentration in the lower range of ionic strength; above the physiological range, there is in most cases a further decrease in oxygen affinity, but this varies with the nature of the salt and, in some instances, the affinity goes through a maximum.The effect of cations, which is opposite to that of anions, operates primarily in the higher concentration range; i.e. above 0·1 m. This effect is especially large for Li⁺, Ca²⁺ and Mg²⁺.The alkaline Bohr effect depends strongly on anion concentration, being displaced towards higher pH values and being reduced in magnitude as chloride concentration is increased. On the other hand, the acid Bohr effect, observed below pH 6, appears to be independent of chloride concentration from 6 × 10^?2 m to 2 m.The overall heat of oxygenation has been determined for the isoionic protein as well as at different concentrations of chloride and phosphate. The average intrinsic heat of reaction of hemoglobin with oxygen in solution is found to be ?14·6 kcal/mol of O₂.     

Design of a cytochrome P450BM3 reaction system linked by two‐step cofactor regeneration catalyzed by a soluble transhydrogenase and glycerol dehydrogenase

A cytochrome P450BM3‐catalyzed reaction system linked by a two‐step cofactor regeneration was investigated in a cell‐free system. The two‐step cofactor regeneration of redox cofactors, NADH and NADPH, was constructed by NAD⁺‐dependent bacterial glycerol dehydrogenase (GLD) and bacterial soluble transhydrogenase (STH) both from Escherichia coli. In the present system, the reduced cofactor (NADH) was regenerated by GLD from the oxidized cofactor (NAD⁺) using glycerol as a sacrificial cosubstrate. The reducing equivalents were subsequently transferred to NADP⁺ by STH as a cycling catalyst. The resultant regenerated NADPH was used for the substrate oxidation catalyzed by cytochrome P450BM3. The initial rate of the P450BM3‐catalyzed reaction linked by the two‐step cofactor regeneration showed a slight increase (approximately twice) when increasing the GLD units 10‐fold under initial reaction conditions. In contrast, a 10‐fold increase in STH units resulted in about a 9‐fold increase in the initial reaction rate, implying that transhydrogenation catalyzed by STH was the rate‐determining step. In the system lacking the two‐step cofactor regeneration, 34% conversion of 50 μM of a model substrate (p‐nitrophenoxydecanoic acid) was attained using 50 μM NADPH. In contrast, with the two‐step cofactor regeneration, the same amount of substrate was completely converted using 5 μM of oxidized cofactors (NAD⁺ and NADP⁺) within 1 h. Furthermore, a 10‐fold dilution of the oxidized cofactors still led to approximately 20% conversion in 1 h. These results indicate the potential of the combination of GLD and STH for use in redox cofactor recycling with catalytic quantities of NAD⁺ and NADP⁺. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Purification and characterization of malate dehydrogenase from the phototrophic bacterium,Rhodopseudomonas capsulata

Malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37) has been purified about 480-fold from crude extract of the facultative phototrophic bacterium, Rhodopseudomonas capsulata by only two purification steps, involving Red-Sepharose affinity chromatography. The enzyme has a molecular mass of about 80 kDa and consists of two subunits with identical molecular mass (35 kDa). The enzyme is susceptible to heat inactivation and loses its activity completely upon incubation at 40°C for 10 min. Addition of NAD⁺, NADH and oxaloacetate, but not l-malate, to the enzyme solution stabilized the enzyme. The enzyme catalyzes exclusively the oxidation of l-malate, and the reduction of oxaloacetate and ketomalonate in the presence of NAD⁺ and NADH, respectively, as the coenzyme. The pH optima are around 9.5 for the l-malate oxidation, and 7.75–8.5 and 4.3–7.0 for the reduction of oxaloacetate and ketomalonate, respectively. The K_m values were determined to be 2.1 mM for l-malate, 48 μM for NAD⁺, 85 μM for oxaloacetate, 25 μM for NADH and 2.2 mM for ketomalonate. Initial velocity and product inhibition patterns of the enzyme reactions indicate a random binding of the substrates, NAD⁺ and l-malate, to the enzyme and a sequential release of the products: NADH is the last product released from the enzyme in the l-malate oxidation.     

Carotenoid oxidative degradation products inhibit Na+-K+-ATPase

This study investigates the biological significance of carotenoid oxidation products using inhibition of Na⁺-K⁺-ATPase activity as an index. β-Carotene was completely oxidized by hypochlorous acid and the oxidation products were analyzed by capillary gasliquid chromatography and high performance liquid chromatography. The Na⁺-K⁺-ATPase activity was assayed in the presence of these oxidized carotenoids and was rapidly and potently inhibited. This was demonstrated for a mixture of β-carotene oxidative breakdown products, β-Apo-10′-carotenal and retinal. Most of the β-carotene oxidation products were identified as aldehydic. The concentration of the oxidized carotenoid mixture that inhibited Na⁺-K⁺-ATPase activity by 50% (IC₅₀) was equivalent to 10μM non-degraded β-carotene, whereas the IC₅₀ for 4-hydroxy-2-nonenal, a major lipid peroxidation product, was 120 μM. Carotenoid oxidation products are more potent inhibitors of Na⁺-K⁺-ATPase than 4-hydroxy-2-nonenal. Enzyme activity was only partially restored with hydroxylamine and/or β-mercaptoethanol. Thus, in vitro binding of carotenoid oxidation products results in strong enzyme inhibition. These data indicate the potential toxicity of oxidative carotenoid metabolites and their activity on key enzyme regulators and signal modulators.     

Resveratrol improves ex vivo expansion of CB-CD34+ cells via downregulating intracellular reactive oxygen species level

Intracellular reactive oxygen species (ROS) play important roles in the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). In this study, the effects of resveratrol (RES), on the ex vivo expansion of HSPCs were investigated by analyzing CD34⁺ cells expansion and biological functions, with the objective to optimize ex vivo culture conditions for CD34 ⁺ cells. Among the five tested doses (0, 0.1, 1, 10, 20, and 50 μM), 10 μM RES was demonstrated to be the most favorable for ex vivo CD34 ⁺ cells expansion. In the primary cultures, 10 μM RES favored higher expansion folds of CD34 ⁺ cells, CD34 ⁺CD38 ⁻ cells, and colony-forming units (CFUs) ( P < 0.05). It was found that the percentages of primitive HSPCs (CD34 ⁺CD38 ⁻CD45R ⁻CD49f ⁺CD90 ⁺ cells) in 10 μM RES cultures were higher than those without RES. Further, in the secondary cultures, expanded CD34 ⁺ cells derived from primary cultures with 10 μM RES exhibited significantly higher total cells and CD34 ⁺ cells expansion ( P < 0.05). In the semisolid cultures, the frequency of CFU-GM and total CFUs of 10 μM RES group were both higher than those of without RES group, demonstrating that CD34 ⁺ cells expanded with 10 μM RES possessed better biological function. Furthermore, the addition of 10 μM RES downregulated the intracellular ROS level via strengthening the scavenging capability of ROS, and meanwhile reducing the percentages of apoptotic cells in cultures. Collectively, RES could stimulate the ex vivo expansion of CD34 ⁺ cells, preserved more primitive HSPCs and maintain better biological function by alleviating intracellular ROS level and cell apoptosis in cultures.     

Molecular dynamic simulations study of the effect of salt valency on structure and thermodynamic solvation behaviour of anionic polyacrylate PAA in aqueous solutions

The effect of salt concentration and valency on intermolecular structure and solvation thermodynamic properties of aqueous solution containing polyacrylicacid (PAA) chains and multi-valent salts calcium chloride (CaCl₂) and aluminium chloride (AlCl₃) as a function of charge density was investigated using atomistic molecular dynamic simulations with explicit solvent. Salt-free solution favours the self-association of uncharged (acidic form) PAA chains facilitated by inter-chain hydrogen bonds. The ionised (charged) PAA chains are not associated in salt-free aqueous solutions and undergo self-association in the salt solutions due to bridging effect induced by condensed salt ions in agreement with scattering investigations available in literature. The collapse behaviour of PAA in presence of CaCl₂ and re-expansion behaviour of PAA chains in case of AlCl₃ salt solutions are observed. The rigidity of PAA chains decrease with increase in salt concentration, in agreement with experimental results available in literature. The trivalent salt favours relatively the greater extent of shrinking of PAA chains as well as inter-chain interactions as compared to divalent salts as evident from radius-of-gyration, H-bond and pair-wise solvation enthalpy data. The conformation and hydration behaviour of the acid form of PAA chains are not significantly altered by added salt ions. The hydration behaviour of ionised PAA chains is significantly reduced by added salts due to screening effect of the condensed salt ions. The pair correlation functions of solutions species such as Ca²⁺, Al³⁺, Na⁺ and Cl^? with respect to PAA oxygen show the greater affinity of PAA units with the higher valency Al³⁺ ions over Ca²⁺ and Na⁺ in solution. With increase in concentration of AlCl₃ and CaCl₂ salts, a decrease in effective charge density of ionised PAA chains is observed from the existence of unfavourable PAA–water, PAA–Ca²⁺ and PAA–Al³⁺ interactions.     

Enhancement of chemiluminescence of the KIO4–luminol system by gallic acid,acetaldehyde and Mn2+: application for the determination of catecholamines

Chemiluminescence (CL) from the oxidation of luminol with potassium periodate in strong alkaline solutions was greatly enhanced by the combined effect of gallic acid, acetaldehyde and Mn²⁺. The CL spectra exhibited only one emission band at 425 nm, indicating 3‐aminophthalate as the emitting species. Various scavengers for superoxide anion, hydroxyl radical and singlet oxygen quenched the CL emission very efficiently (74–100%), suggesting the possible involvement of these reactive oxygen species (ROS) in the CL reactions. It is postulated that oxidation of gallic acid and acetaldehyde by periodate catalyzed by Mn²⁺ generates these ROS, which then react with luminol to enhance the CL emission. We also found that the enhanced CL emission was strongly inhibited by catecholamines, probably because of their effective scavenging of ROS. Based on this observation, a simple, rapid and sensitive new CL method was developed for the determination of catecholamines. The detection limits (3σ) for dopamine, l‐ dopa, norepinephrine and epinephrine were 0.63, 1.37, 0.56 and 14.3 nmol/L, respectively. The linear range was 1–10 nmol/L; relative standard deviations were 0.71–1.34% for 0.1 µmol/mL catecholamines. This CL method was applied to the determination of catecholamines in pharmaceutical injections with satisfactory results. Copyright © 2009 John Wiley & Sons, Ltd.     

The conformational transition of κ-carrageenan using microcalorimetry

The interactions between inorganic cations and κ-carrageenan in aqueous solutions have been studied using microcalorimetry. The enthalpy of dilution and the enthalpy of mixing of κ-carrageenan in the coil conformation with an alkaline metal chloride have been investigated and are in agreement with theoretical predictions. The formation of helical dimer in a K⁺ salt form in the absence of a gel formation is confirmed.     

Light-induced turnover of chloroplast cytochrome b-559 in the presence of N-methylphenazonium methosulphate

In the presence of 0.1–5 μM N-methylphenazonium methosulphate approx. 50–70% oxidation of cytochrome b-559 can be induced by far-red light. The oxidation is best observed with long wavelength far-red light (732 nm) of moderate intensities (approx. 10⁴ ergs/cm² per s) and is reversed by subsequent illumination with red light. Concentrations of N-methylphenazonium methosulphate above 5 μM are inhibitory probably due to cyclic electron flow. The far-red oxidation is inhibited by low concentrations of the plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, while 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibits red light reduction and increases the amplitude of far-red oxidation. The effect of N-methylphenazonium methosulphate is mimicked by N-methyl-phenazonium ethosulphate, but not by pyocyanine or diaminodurene. Low concentrations (2–3 μM) of N-methylphenazonium methosulphate also stimulate a 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-inhibitable red light reduction of cytochrome f.     

Effects of four types of sodium salt stress on plant growth and photosynthetic apparatus in sorghum leaves

Sorghum variety Longza 17 was used as the experimental organism in a study of the effects of different types of sodium salt (two neutral salts, NaCl and Na₂SO₄; and two alkaline salts, NaHCO₃ and Na₂CO₃), at an equivalent Na⁺ concentration (100?mmol·L^?1) on leaf growth parameters and PSII and PSI function by using the Fast Chlorophyll Fluorescence Induction Dynamics technique and 820?nm light reflectance curves. The results showed that at Na⁺ concentration of 100?mmol·L^?1, different types of sodium salt stress significantly inhibited the growth of sorghum plants. Different types of sodium salt stress showed significant inhibition on the activities of PSII and PSI in sorghum leaves, the impact of different types of sodium salt on the activities of PSII and PSI in sorghum leaves was consistent, listed from greatest to least impact as Na₂CO₃ > NaHCO₃ > Na₂SO₄ > NaCl. The effects of alkaline salt stress on the growth and photosynthetic properties of sorghum were greater than those under the neutral salt stress, therefore, in addition to considering the impact of Na⁺ concentration in the sorghum planting area, emphasis should also be given to the influence of the degree of alkalization, especially the higher alkalinity of Na₂CO₃.     

Effect of CdS preparation on the photo-catalyzed decomposition of hydrogen sulfide in alkaline aqueous media

Band-gap irradiation of CdS dispersions in alkaline aqueous media (pH 14) containing 0.1 M Na₂S produces hydrogen and sulfur. The reaction is photo-decomposition of hydrogen sulfide by two quanta of visible light (λ > 400 nm). Various batches of commercially available cadmium sulfide, as well as CdS precipitated from nitrate, sulfate, and chloride solutions at neutral pH, produce different amounts of hydrogen. Electronically pure CdS (puratronic grade) generates almost no hydrogen. By contrast, CdS precipitates prepared in the presence of excess cadmium yield forty times more hydrogen than CdS prepared in the presence of excess sodium sulfide. Differences are rationalized in terms of possible surface modification and/or changes in the active sites by anions present as ‘impurities’ which could affect separation and recombination of the charge carries, e_CB⁻ and h_VB⁺, in CdS.     

The effect of silver and other metal ions on the in vitro growth of root-rotting Phytophthora and other fungal species

A range of metal ions and the oxoanion WO₄^2-were toxic to zoospores of Phytophthora nicotianae parasitica in the order: Ag⁺ > Cu⁺⁺ > WO₄^2-> Ni⁺ > Co⁺⁺ > Zn⁺. The LD₅₀ for Ag⁺, 0.11 μM (11.4 ppb), compared with 1.84 μm (117 ppb) for Cu⁺⁺. Silver was similarly toxic to a range of pathogens including Pythium aphanidermatum, Thielaviopsis basicola and Fusarium oxy-sporum f.spp. Most zoospores of Phytophthora spp. were killed by Ag⁺ in the range 46–460 nM (5–50 ppb), bursting at the higher concentrations. A small sub-population of most propagules exhibited greater tolerance to silver than the whole. In 0.93 μM (100 ppb) Ag⁺ 1.4% of P. nicotianae parasitica zoospores survived but were all killed at 500 ppb. A population of P. cryptogea (1.9%) surviving 0.47 μm (50 ppb) were killed at 0.93 μM (100 ppb). Zoospore cysts and germlings showed the same sensitivity to silver. Oospores were mostly killed over the range 0.23–0.93 μm (25–100 ppb) Ag⁺, some surviving up to the lethal concentration of 9.26 μM (1000 ppb). Mycelium of P. cryptogea was generally less sensitive, with some growth occurring at 9.26 μm (100 ppb). Zoosporangiogenesis was unaffected over the range 0.47–4.65 μm (50–500 ppb). Toxicity increased with increased pH over the range 5.0–6.5. Ionic silver was lost from solution during a microscope slide bioassay by binding to the glass surface. In the presence of chloride ions, colloidal AgCl formed which was equally toxic to P. cryptogea. Silver and AgCl were further lost from solution by colloidal agglomeration - Ostwald ripening - and by AgCl adsorption to glass. Silver, < 90 nM (10 ppb) Ag⁺ as AgNO₃ and particles of silver chloride were both strongly attractive to zoospores of P. cryptogea. Spores burst or failed to germinate on entering lethal concentrations. The results are discussed in the context of the use of silver salts to control Phytophthora root-rot pathogens and the importance of ion availability in in vitro toxicity assays.     

Mechanism of alcohol‐enhanced lucigenin chemiluminescence in alkaline solution

The chemiluminescence (CL) of lucigenin (Luc²⁺) can be enhanced by different alcohols in alkaline solution. The effect of different fatty alcohols on the CL of lucigenin was related to the carbon chain length and the number of hydroxyl groups. Glycerol provides the greatest enhancement. UV/Vis absorption spectra and fluorescence spectra showed that N‐methylacridone (NMA) was produced in the CL reaction in the presence of different alcohols. The peak of the CL spectrum was located at 470 nm in all cases, indicating that the luminophore was always the excited‐state NMA. The quenching of lucigenin CL by superoxide dismutase (SOD) and the electron spin resonance (ESR) results with the spin trap of 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO) demonstrated that superoxide anions (O₂^?–) were generated from dissolved oxygen in the CL reaction and that glycerol and dihydroxyacetone (DHA) can promote O₂^?? production by the reduction of dissolved oxygen in alkaline solution. It was assumed that the enhancement provided by different alcohols was related to the solvent effect and reducing capacity. Glycerol and DHA can also reduce Luc²⁺ into lucigenin cation radicals (Luc^?+), which react with O₂^?? to produce CL, and glycerol can slowly transform into DHA, which is oxidized quickly in alkaline solution. Copyright © 2015 John Wiley & Sons, Ltd.     

Control of respiration in proteoliposomes containing cytochrome aa3. I. Stimulation by valinomycin and uncoupler

1. Both valinomycin and p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) are required for full release of respiration by cytochrome c oxidase-containing proteoliposomes (prepared by sonicating beef heart cytochrome aa₃ in salt solution with 4 parts phosphatidylcholine, 4 parts phosphatidylethanolamine and 2 parts cardiolipin) in the presence of external ascorbate and cytochrome c. In the absence of valinomycin the response to FCCP is rather sluggish, as reported by Wrigglesworth et al. (1976) (Abstracts, 10th Int. Congr. Biochem., No. 06-6-230).2. The K_m for cytochrome c in 67 mM, pH 7.4, phosphate buffer with ascorbate as substrate, was 9 μM in both absence and presence of valinomycin and FCCP. Energization thus acts non-competitively towards cytochrome c oxidation.3. The apparent K_m for oxygen is greater in the energized than in the deenergized state; double reciprocal plots of respiration rate versus oxygen concentration are concave downward in the absence of uncouplers, as found with intact mitochondria. Energization thus acts “competitively” towards oxygen.4. Despite the lack of a functional ATPase system, all the kinetic features of energization found in intact mitochondria can be mimicked in the reconstituted liposomes. This supports the chemiosmotic idea that electrical and perhaps H⁺ gradients modify the oxidase activity in reconstituted vesicles.