Action of natural and synthetic antioxidants on the electrical parameters and stability of natural and artificial membranes

The effect of antioxidants alpha-tocopherol and ionol on membranes of human red cells and bilayer lipid membrane (BLM) from azolektin has been studied. Ionol at concentration 4-10 mM induces the hemolysis of erythrocytes, the cells form changes are observed at concentration 2 mM alpha-tocopherol doesn't show the hemolytic properties at concentration 23 mM. The ionol concentration 1 mM doesn't change the form of the cells, but influence the passive electric parameters: the capacity (Cs) of erythrocytic membrane increases and the intracellular conductance (chi i) decreases. Tocopherol (3 mM) induces the decrease both Cs and chi i. The fast increase of membrane conductance is almost immediately registered on one side of BLM at addition of ionol (0,2-0,4 g/ml). Phosphatidylionol synthesized from ionol and contining the acyl chains C15H31 and C17H35 doesn't influence the electrical properties of BLM.     

Antioxidant protection of phospholipid bilayers by alpha-tocopherol. Control of alpha-tocopherol status and lipid peroxidation by ascorbic acid and glutathione

Factors affecting the balance between pro- and antioxidant effects of ascorbic acid and glutathione were studied in soybean phosphatidylcholine liposomes challenged with Fe2+/H2O2. Effective antioxidant protection by alpha-tocopherol appeared to be due to efficient reaction with lipid oxy-radicals in the bilayer rather than to interception of initiating oxygen radicals. At concentrations above a threshold level of approximately 0.2 mol % (based on phospholipid content), alpha-tocopherol completely suppressed lipid oxy-radical propagation, which was measured as malondialdehyde production. Both ascorbic acid and glutathione, alone or in combination, enhanced lipid oxy-radical propagation. Alpha-Tocopherol, incorporated into liposomes at concentrations above its threshold protective level, reversed the pro-oxidant effects of 0.1-1.0 mM ascorbic acid but not those of glutathione. Ascorbic acid also prevented alpha-tocopherol depletion. The combination of ascorbic acid and subthreshold levels of alpha-tocopherol only temporarily suppressed lipid oxy-radical propagation and did not maintain the alpha-tocopherol level. Glutathione antagonized the antioxidant action of the alpha-tocopherol/ascorbic acid combination regardless of alpha-tocopherol concentration. These observations indicate that membrane alpha-tocopherol status can control the balance between pro- and antioxidant effects of ascorbic acid. The data also provide the most direct evidence to date that ascorbic acid interacts directly with components of the phospholipid bilayer.     

An assay for the alpha-tocopherol binding protein mediated transfer of vitamin E between membranes

A model system consisting of donor membrane (egg lecithin liposomes) and acceptor membrane (human erythrocyte ghosts or rat liver mitochondria) were used to investigate the alpha-tocopherol binding protein (alpha TBP) mediated transfer of alpha-tocopherol. Liposomes containing RRR-[alpha-3H]tocopherol ([alpha-3H]T) were incubated with acceptor membrane at 37 degrees C for 0-45 min in the presence or absence of rat liver cytosol or a dialyzed 30-60% saturated ammonium sulfate precipitated fraction of rat liver cytosol (Fraction B). Erythrocyte ghosts and liver mitochondria were compared and found to behave similarly in the presence of Fraction B. alpha-Tocopherol transfer activity (alpha TTA) typically varied 0- to 27-fold greater than buffer blanks, depending upon type and concentration of protein preparation. Gel filtration of Fraction B yielded one alpha TTA peak (liver mitochondria as acceptor) with an estimated Mr of 39,000. [alpha-3H]T recovered from erythrocyte ghosts pellets by HPLC suggest that the [alpha-3H]T was transferred intact. alpha TTA of Fraction B in the presence of varying concentrations of erythrocyte ghosts and liposomal [alpha-3H]T followed saturation kinetics. Optimal concentrations gave alpha TTA responses directly proportional to rat liver cytosol concentration. alpha TTA was inhibited only 5% in the presence of a 32-fold excess of cold liposomal alpha-tocopheryl acetate suggesting that the free hydroxyl group on the chromanol ring of alpha-tocopherol is needed for transfer. Coefficient of variation of repeated measures of alpha TTA in rat liver cytosol was 2.9%. Thus, the intermembrane transfer phenomenon of alpha-tocopherol can be studied quantitatively and can be used to compare liver protein preparations exhibiting transfer activity.     

Circular dichroism study of membrane dynamics focused on effect of monosialogangliosides

Monosialogangliosides (GM) purified from bovine brain were incorporated into circular dichroism (CD)-active liposomes and the effects of GM on the membrane dynamics were studied by CD spectroscopy. In the presence of 7 mol% of GM, the phase transition temperature (Tc) of the membrane increased by ca. 10 degrees C compared with the membrane without GM and characteristic CD spectra were observed for CD-active liposomes incorporating GM at low temperature. Asialogangliosides had no effect on the CD spectra or Tc. We have also studied the role of GM in reducing leakage of [3H]sucrose from liposomes composed of egg phosphatidylcholine, dipalmitoyl phosphatidic acid, cholesterol and alpha-tocopherol with a molar ratio of 4 : 1 : 5 : 0.1 in the presence of human plasma at 25 degrees C. The half-life of [3H]-sucrose leakage was 173 h for liposomes incorporating 7 mol% of GM. On the other hand, the half-lives for liposomes incorporating 7 mol% of asialogangliosides and liposomes without glycolipids were 45 and 42 h, respectively. These results indicate that sialic acid on the membrane surface contributes to the increase of Tc, to the change of the aggregation state of phospholipids and to the stabilization of liposomes in plasma.     

Antioxidant and free radical scavenging properties of N-acetylcysteine amide (NACA) and comparison with N-acetylcysteine (NAC)

The antioxidant potential of N-acetylcysteine amide (NACA), also known as AD4, was assessed by employing different in vitro assays. These included reducing power, free radical scavenging capacities, peroxidation inhibiting activity through linoleic acid emulsion system and metal chelating capacity, as compared to NAC and three widely used antioxidants, alpha-tocopherol, ascorbic acid and butylated hydroxytoluene (BHT). Of the antioxidant properties that were investigated, NACA was shown to possess higher 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging ability and reducing power than NAC, at all the concentrations, whereas the scavenging ability of H(2)O(2) differed with concentration. While NACA had greater H(2)O(2) scavenging capacity at the highest concentration, NAC was better than NACA at lower concentrations. NAC and NACA had a 60% and 55% higher ability to prevent beta-carotene bleaching, respectively, as compared to control. The chelating activity of NACA was more than 50% that of the metal chelating capacity of EDTA and four and nine times that of BHT and alpha-tocopherol, respectively. When compared to NACA and NAC; alpha-tocopherol had higher DPPH scavenging abilities and BHT and alpha-tocopherol had better beta-carotene bleaching power. These findings provide evidence that the novel antioxidant, NACA, has indeed enhanced the antioxidant properties of NAC.     

Hemifluorinated surfactants: a non-dissociating environment for handling membrane proteins in aqueous solutions?

The instability of membrane proteins in detergent solution can generally be traced to the dissociating character of detergents and often correlates with delipidation. We examine here the possibility of substituting detergents, after membrane proteins have been solubilized, with non-detergent surfactants whose hydrophobic moiety contains a perfluorinated region that makes it lipophobic. In order to improve its affinity for the protein surface, the fluorinated chain is terminated by an ethyl group. Test proteins included bacteriorhodopsin, the cytochrome b(6)f complex, and the transmembrane region of the bacterial outer membrane protein OmpA. All three proteins were purified using classical detergents and transferred into solutions of C(2)H(5)C(6)F(12)C(2)H(4)-S-poly-Tris-(hydroxymethyl)aminomethane (HF-TAC). Transfer to HF-TAC maintained the native state of the proteins and prevented their precipitation. Provided the concentration of HF-TAC was high enough, HF-TAC/membrane protein complexes ran as single bands upon centrifugation in sucrose gradients. Bacteriorhodopsin and the cytochrome b(6)f complex, both of which are detergent-sensitive, exhibited increased biochemical stability upon extended storage in the presence of a high concentration of HF-TAC as compared to detergent micelles. The stabilization of cytochrome b(6)f is at least partly due to a better retention of protein-bound lipids.     

Deuterium NMR study of the interaction of alpha-tocopherol with a phospholipid model membrane

The effects of 5, 10, and 20 mol % incorporation of alpha-tocopherol (vitamin E) on 50 wt % aqueous multilamellar dispersions of sn-2-substituted [2H31]palmitoylphosphatidylcholine (PC-d31), a saturated, deuterated phospholipid prepared from egg lysophosphatidylcholine, have been studied by deuterium nuclear magnetic resonance (2H NMR) and differential scanning calorimetry (DSC). Moment analysis of the 2H NMR spectra as a function of temperature and DSC heating curves demonstrate that the main gel to liquid-crystalline phase transition is progressively broadened and its onset temperature lowered by increasing concentrations of alpha-tocopherol. Below the transition temperature (40 degrees C) for PC-d31 bilayers, the 2H NMR spectra indicate that acyl chain motion is increased by addition of alpha-tocopherol and that this effect extends to lower temperatures with higher alpha-tocopherol content. Above the transition, average carbon-deuterium bond order parameters calculated from the first spectral moment establish that alpha-tocopherol increases acyl chain ordering within the PC-d31 bilayer by as much as 17% at 20 mol % incorporation. Profiles of order parameter vs. chain position, constructed from 2H NMR spectra following application of the depaking technique, show that despite higher order the general form of the profile is not significantly altered by alpha-tocopherol.     

Sugar-induced increase in cytosolic Ca(2+) in Arabidopsis thaliana whole plants.

Using Ca(2+)-dependent photoprotein aequorin-transformed Arabidopsis thaliana, sugar-induced increase in cytosolic free Ca(2+ )concentration ([Ca(2+)](cyt))( )was investigated by luminescence imaging technique. When 0.1 M sucrose was fed to roots of autotrophically grown intact whole plants whose roots had been incubated overnight with coelenterazine to reconstitute aequorin systemically, strong and transient (within 20 s) luminescence was observed in the roots; that luminescence was followed by weak luminescence moving from the lower leaves to the upper leaves. The moving rate of luminescence was roughly comparable to that of [(14)C]sucrose. Application of 0.1 M glucose or fructose induced transient luminescence in excised leaves. No such luminescence was observed in heterotrophically grown (with sucrose) whole plants or in excised tissues. mRNA levels of sucrose-H(+) symporter genes AtSUC1 and AtSUC2 were higher in autotrophic plants than in heterotrophic plants. These results indicate that influx of transported sucrose together with H(+) into the mesophyll cells of autotrophic plants may depolarize the membrane potential, and subsequently activate a voltage-gated Ca(2+) channel on the plasma membrane, resulting in a [Ca(2+)](cyt) increase. The [Ca(2+)](cyt) increase might initiate Ca(2+ )signaling leading to the expression of genes related to biosynthesis of storage carbohydrates. Hexoses, when applied, might also be involved in the [Ca(2+)](cyt) increase mediated by monosaccharide-H(+) co-transporters.     

New aspects of the inhibition of soybean lipoxygenase by alpha-tocopherol. Evidence for the existence of a specific complex

The formation of alpha-tocopherol--lipoxygenase complex was elucidated using immobilized affinity purified soybean lipoxygenase and [D-3H]alpha-tocopherol. The alpha-tocopherol--lipoxygenase complex did not dissociate on addition of linoleic acid. Iodoacetate modified immobilized lipoxygenase did not form the complex with alpha-tocopherol. Lipoxygenase attached to an aminoethyl linoleyl Sepharose column was eluted by alpha-tocopherol. DL-alpha-Tocopherol acetate at a concentration of 3 X 10(-3) M inhibited 80% of linoleate oxidation by soybean lipoxygenase. The lipoxygenase--alpha-tocopherol complex did not give the usual soybean lipoxygenase antigenic pattern in immunodiffusion. Digestion of the [3H]alpha-tocopherol--lipoxygenase complex with proteolytic enzymes showed that most of the radioactivity is incorporated into one peptide.     

Antioxidant activity of quercetin and myricetin in liposomes

The antioxidant activity during storage at 30 degrees C of quercetin, myricetin and alpha-tocopherol in small unilamellar liposomes has been investigated. Myricetin was more effective than alpha-tocopherol as an antioxidant in liposomes under all conditions studied. At pH 5.4 with a concentration of 10(-2) mol/mol phospholipid, myricetin has been shown to be the strongest antioxidant followed by quercetin and alpha-tocopherol. Cupric chloride and ferric chloride strongly reduced the antioxidant activity of myricetin and quercetin with cupric chloride causing a stronger reduction in activity than ferric chloride. At a pH of 7.4, quercetin was less effective than alpha-tocopherol at a concentration of 10(-2) mol/mol phospholipid, but it's activity increased more strongly with concentration and it was very effective at a concentration of 5 x 10(-2) mol/mol phospholipid.     

The effects of alpha-tocopherol on site-specific lipid peroxidation induced by iron in charged micelles

alpha-Tocopherol inhibited H2O2-Fe2+-induced lipid peroxidation of linoleic acid (LA) by scavenging OH radicals in tetradecyltrimethylammonium bromide (TTAB) micelles. The inhibiting ability of alpha-tocopherol was much greater than that of OH-radical scavengers mannitol and t-butanol. In contrast, alpha-tocopherol enhanced linoleic acid hydroperoxide (LOOH)-Fe2+-induced lipid peroxidation through regeneration of Fe2+ in sodium dodecyl sulfate (SDS) micelles containing LA. alpha-Tocopherol was oxidized by Fenton's reagent (FeSO4 + H2O2) at a higher rate in SDS micelles than in TTAB micelles. The likely oxidants were OH radicals in the former and Fe3+ in the latter. Both reagents formed in the Fenton reaction. Ferrous ion catalyzed in a dose-dependent manner the decomposition of LOOH and conjugated diene compounds in SDS but not in TTAB micelles. alpha-Tocopherol and Fe3+ individually had no effect on the decomposition of LOOH, but together were quite effective. The rate of the decomposition was a function of the concentration of alpha-tocopherol. The mechanism of "site-specific" antioxidant action of alpha-tocopherol in charged micelles is discussed.     

Modification of the structure of plasmatic membranes of the liver by the action of alpha-tocopherol in vitro

The effect of the natural antioxidant alpha-tocopherol in a broad concentration range (10(-4) - 10(-25) M) on the viscosity characteristics and thermally induced structural transitions of a lipid bilayer of plasma membranes of murine hepatocytes in vitro has been studied. Changes in the rigidity of surface (approximately Abb) of the lipid bilayer were measured on a Bruker EMX EPR spectrometer (Germany) by the method of spin probes. Stable nitroxyl radicals of 5- and 16-doxylstearic acid, localized at different depth in the membrane served as spin probes. It was shown that the concentration dependence of the effect of alpha-tocopherol is linear and polymodal with three statistically significant increases in three ranges of its concentration: (1) in the range of traditional physiological concentrations 10(-4)-10(-9) M, (2) in the range of superlow doses 10(-9) - 10(-17) M, and (3) in the range of "imaginary" concentrations 10(-17) - 10(-25) M. The mechanisms of action of alpha-tocopherol in each of the three ranges are discussed. When studying the temperature dependences of viscous characteristics, a new thermally induced structural transition in the range of "physiological" temperatures 309-313 K for those alpha-tocopherol concentrations (including superlow ones) to which the maxima on the dose dependence curves at constant temperature of 293 K corresponded.     

The influx of calcium ions into human erythrocytes during cold storage

1. When human erythrocytes, suspended in iso-osmotic sucrose containing CaCl(2), are stored at 3 degrees C, Ca(2+) influx into the cells occurs. Simultaneously, efflux of K(+), Na(+), Cl(-) and water takes place and cell volume diminishes. 2. The extent of Ca(2+) influx increases with duration of cold storage and with increasing concentration of Ca(2+) in the suspending medium. 3. Erythrocytes that have been thus loaded with Ca(2+) exhibit Ca(2+) efflux against a concentration gradient when subsequently incubated at 37 degrees C. 4. Ca(2+) influx likewise occurs when the sucrose of the medium is replaced by iso-osmotic solutions of other non-ionized compounds. 5. Replacement of sucrose by iso-osmotic KCl or NaCl greatly diminishes the rate of Ca(2+) influx during cold storage; however, in iso-osmotic choline chloride, Ca(2+) influx is as rapid as in sucrose. 6. Preincubation of erythrocytes in iso-osmotic sucrose at 37 degrees C causes rapid efflux of K(+) and Na(+) and renders the cell membranes highly permeable to Ca(2+) during subsequent cold storage. 7. Preincubation of erythrocytes in iso-osmotic NaCl at 37 degrees C with trypsin or neuraminidase is without effect on the permeability of the membrane towards Ca(2+). 8. The experimental results lead to the conclusion that the main prerequisite for Ca(2+) influx into erythrocytes is the partial depletion of the cells of their univalent cations.     

Metabolic syndrome in the rat: females are protected against the pro-oxidant effect of a high sucrose diet

Metabolic syndrome is more prevalent in men than in women. In an experimental dietary model of metabolic syndrome, the high-fructose-fed rat, oxidative stress has been observed in males. Given that estradiol has been documented to exert an antioxidant effect, we investigated whether female rats were better protected than males against the adverse effects of a high-sucrose diet, and we studied the influence of hormonal status in female rats. Males and females were first fed a sucrose-based or starch-based diet for 2 weeks. In the males, the plasma triglyceride (TG)-raising effect of sucrose was accompanied by significantly lowered plasma alpha-tocopherol and a significantly lowered alpha-tocopherol/TG ratio (30%), suggesting that vitamin E depletion may predispose lipoproteins to subsequent oxidative stress. In males, after exposure of heart tissue homogenate to iron-induced lipid peroxidation, thiobarbituric reactive substances were significantly higher in the sucrose-fed than in the starch-fed rats. In contrast, in sucrose-fed females, neither a decrease in vitamin E/TG ratio nor an increased susceptibility of heart tissue to peroxidation was observed, despite both a significantly decreased heart superoxide dismutase activity (14%) and a significant 3-fold increase in plasma nitric oxide concentration compared with starch-fed females. The influence of hormonal status in female rats was then assessed using intact, ovariectomized, or estradiol-supplemented ovariectomized female rats fed the sucrose or starch diet for 2 weeks. After exposure of heart tissue to iron-induced lipid peroxidation, higher susceptibility to peroxidation was found only in ovariectomized females fed the sucrose diet compared with the starch group and not in intact females or ovariectomized females supplemented with estradiol. Thus, estrogens, by their effects on antioxidant capacity, might explain the sexual difference in the pro-oxidant effect of sucrose diet resulting in metabolic syndrome in rats.     

Degradation of transplanted rat liver mitochondrial-outer-membrane proteins in hepatoma cells.

Reductively [3H]methylated 3H mitochondrial-outer-membrane vesicles from rat liver and vesicles where monoamine oxidase has been derivatized irreversibly by [3H]-pargyline have been deliberately miscompartmentalized by heterologous transplantation into hepatoma (HTC) cells by poly(ethylene glycol)-mediated vesicle-cell fusion. Fluorescein-conjugated mitochondrial-outer-membrane vesicles have also been used to show that transplanted material is patched, capped and internalized. Reductively methylated outer-membrane proteins and monoamine oxidase are destroyed at the same rate (t1/2 24 h). Mitochondrial-outer-membrane proteins are not degraded at the same rate as HTC plasma-membrane proteins, endogenous cell protein, or endocytosed protein. Transplanted radiolabelled mitochondrial-outer-membrane proteins accumulate intracellularly in structures that are distinct from plasma membrane and lysosomes. However, when mitochondrial-outer-membrane vesicles derivatized with [14C]sucrose are transplanted, the acid-soluble degradation products accumulate in the lysosomal fraction. [14C]Sucrose-conjugated HTC cell plasma membrane accumulates in intracellular structures that are again distinct from plasma membrane and lysosomes. In contrast with the above observations, homologously transplanted mitochondrial-outer-membrane proteins from rat liver are destroyed in hepatocytes at rates that are remarkably similar (t1/2 60-70 h) to the rates in rat liver in vivo [Evans & Mayer (1982) Biochem. Biophys. Res. Commun. 107, 51-58].     

Sugarcane ShSUT1: analysis of sucrose transport activity and inhibition by sucralose

Plant sucrose transporters (SUTs) are members of the glycoside-pentoside-hexuronide (GPH) cation symporter family (TC2.A.2) that is part of the major facilitator superfamily (MFS). All plant SUTs characterized to date function as proton-coupled symporters and catalyze the cellular uptake of sucrose. SUTs are involved in loading sucrose into the phloem and sink tissues, such as seeds, roots and flowers. Because monocots are agriculturally important, SUTs from cereals have been the focus of recent research. Here we present a functional analysis of the SUT ShSUT1 from sugarcane, an important crop species grown for its ability to accumulate high amounts of sucrose in the stem. ShSUT1 was previously shown to be expressed in maturing stems and plays an important role in the accumulation of sucrose in this tissue. Using two-electrode voltage clamping in Xenopus oocytes expressing ShSUT1, we found that ShSUT1 is highly selective for sucrose, but has a relatively low affinity for sucrose (K(0.5) = 8.26 mM at pH 5.6 and a membrane potential of -137 mV). We also found that the sucrose analog sucralose (4,1',6'-trichloro-4,1',6'-trideoxy-galacto-sucrose) is a competitive inhibitor of ShSUT1 with an inhibition coefficient (K(i)) of 16.5 mM. The presented data contribute to our understanding of sucrose transport in plants in general and in monocots in particular.     

Some permeability properties of isolated rat liver cell mitochondria

The rates of penetration of various solutes into isolated rat liver mitochondria have been studied. Sodium, potassium, and sucrose were observed to enter the mitochondria until an equilibrium concentration was reached. The diffusion of these solutes, after the first few minutes, followed the predicted diffusion curve for solutes entering a particle with a rate-limiting membrane and instantaneous mixing in the interior. Reasons for deviations from the predicted equation during the first few minutes of diffusion are suggested. The data show that at pH 7.4 sodium and potassium enter more rapidly than sucrose. I¹³¹-labelled albumin was found to enter very slowly, if at all. Increasing the pH from 7.4 reduced the rate at which sodium ion penetrated the mitochondria. The rate of diffusion of sucrose into mitochondria was considerably slower than diffusion of sucrose into a sphere of water of the same size. Sodium ion was not found to be concentrated in vitro against an external concentration gradient as has been reported by other investigators. It is concluded that the rate of diffusion of solutes between the external medium and the interior of mitochondria is probably restricted and controlled by a mitochondrial membrane exhibiting passive permeability characteristics.     

Effects of replacement of the hydroxyl group of cholesterol and tocopherol on the thermotropic behavior of phospholipid membranes

The role of the hydroxyl groups of cholesterol and tocopherol in mediating their interaction with phospholipid bilayers has been a subject of considerable interest. We have examined this question by using derivatives of cholesterol and tocopherol in which the hydroxyl group is esterified to succinate. The hemisuccinate esters of cholesterol and alpha-tocopherol can be readily incorporated into phospholipid membranes and in fact can by themselves form closed membrane vesicles as demonstrated by the encapsulation of [3H]sucrose. The thermotropic behavior of mixtures containing each succinate ester and phospholipid was studied by differential scanning calorimetry. The effect of cholesteryl hemisuccinate on the thermotropic properties of dipalmitoylphosphatidylcholine and dimyristoylphosphatidylethanolamine is very similar to that of cholesterol. This indicates that the 3 beta-OH is not required for the formation of a cholesterol-phospholipid complex. In mixtures of tocopherol acid succinate and phospholipids the peak transition temperature is progressively shifted to lower temperatures as the mole fraction of alpha-tocopherol succinate is increased, while the enthalpy of the transition is only slightly affected. At a tocopherol succinate/phospholipid molar ratio of 9/1 a phase transition is still detectable. A comparison between tocopherol succinate and tocopherol indicates that the substitution of the hydroxyl group reduces the interaction of tocopherol with phospholipids to a small but measurable extent. Thus, the hydroxyl group of tocopherol is more important than the hydroxyl group of cholesterol in influencing their interactions with phospholipids.     

Analysis of the transport activity of barley sucrose transporter HvSUT1

Localization studies indicate that barley (Hordeum vulgare) sucrose transporter HvSUT1 functions in sucrose uptake into seeds during grain filling. To further understand the physiological function of HvSUT1, we have expressed the HvSUT1 cDNA in Xenopus laevis oocytes and analyzed the transport activity by two-electrode voltage clamping. Consistent with a H(+)-coupled transport mechanism, sucrose induced large inward currents in HvSUT1-expressing oocytes with a K (0.5) of 3.8 mM at pH 5.0 and a membrane potential of -157 mV. Of 21 other sugars tested, four glucosides were also transported by HvSUT1. These glucosides were maltose, salicin (2-(hydroxymethyl) phenyl beta-D-glucoside), alpha-phenylglucoside and alpha-paranitrophenylglucoside. Kinetic analysis of transport of these substrates by HvSUT1 was performed and K (0.5) values were measured. The apparent affinity for all substrates was dependent on membrane potential and pH with lower K (0.5) values at lower external pH and more negative membrane potentials. HvSUT1 was more selective for alpha-glucosides over beta-glucosides than the Arabidopsis sucrose transporter AtSUC2. Several substrates transported by AtSUC2 (beta-phenylglucoside, beta-paranitrophenylglucoside, alpha-methylglucoside, turanose, and arbutin (hydroquinone beta-D-glucoside)) showed low or undetectable transport by HvSUT1. Of these, beta-paranitrophenylglucoside inhibited sucrose transport by HvSUT1 indicating that it interacts with the transporter while arbutin and alpha-methyl glucoside did not inhibit. The results demonstrate significant differences in substrate specificity between HvSUT1 and AtSUC2.     

Fermentative hydrogen production and bacterial community structure in high-rate anaerobic bioreactors containing silicone-immobilized and self-flocculated sludge

A novel continuously stirred anaerobic bioreactor (CSABR) seeded with silicone-immobilized sludge was developed for high-rate fermentative H2 production using sucrose as the limiting substrate. The CSABR system was operated at a hydraulic retention time (HRT) of 0.5-6 h and an influent sucrose concentration of 10-40 g COD/L. With a high feeding sucrose concentration (i.e., 30-40 g COD/L) and a short HRT (0.5 h), the CSABR reactor produced H2 more efficiently with the highest volumetric rate (VH2) of 15 L/h/L (i.e., 14.7 mol/d/L) and an optimal yield of ca. 3.5 mol H2/mol sucrose. The maximum VH2 value obtained from this work is much higher than any other VH2 values ever documented. Formation of self-flocculated granular sludge occurred during operation at a short HRT. The granule formation is thought to play a pivotal role in the dramatic enhancement of H2 production rate, because it led to more efficient biomass retention. A high biomass concentration of up to 35.4 g VSS/L was achieved even though the reactor was operated at an extremely low HRT (i.e., 0.5 h). In addition to gaining high biomass concentrations, formation of granular sludge also triggered a transition in bacterial community structure, resulting in a nearly twofold increase in the specific H2 production rate. According to denatured-gradient-gel-electrophoresis analysis, operations at a progressively decreasing HRT resulted in a decrease in bacterial population diversity. The culture with the best H2 production performance (at HRT = 0.5 h and sucrose concentration = 30 g COD/L) was eventually dominated by a presumably excellent H2-producing bacterial species identified as Clostridium pasteurianum.