The plasmalemma redox system of a fresh-water alga and membrane electrical parameters

In order to learn more details about the plasmalemma redox system in the alga Hydrodictyon reticulatum the electrophysiological characteristics of the plasma membrane as influenced by artificial electron acceptors were estimated. Ferricyanide anion as well as TTF+ depolarized the membrane potential, the effect being more marked in the dark than in the light. In the presence of ferricyanide the membrane resistance in the light was decreased by about 29% on the average, in the dark it remained unchanged. On the other hand, TTF+ brought about a large increase in membrane resistance, notwithstanding the light conditions. The results are discussed in view of the impairing influence of both electron acceptors on the active inflow of chloride ions (Rybová, R. et al. (1990) Bot. Acta 103, 404-407). The electrogenic outflow of electrons appears to make the largest contribution to the membrane depolarization. The inhibition of a pumping mechanism coupling the active uptake of Cl- in some way to the transmembrane electron flow is envisaged as a plausible explanation for membrane resistance increase by TTF+.     

Measurements of redox activity at the plasmalemma

Salt (NaCl) tolerance of 3 eucalypt species ( Eucalyptus alba Reinw. ex Bl., E. camaldulensis Dehnh and E. microtheca f.v. Muell.) was studied: three-month-old seedlings grown in a greenhouse were watered by a saline solution (up to 700 m M ) for 1 month. Mineral, water and sugar contents were highly affected by the saline stress. Sodium, K and Ca were accumulated in the leaves. No significant differences were found between E. camaldulensis and E. microtheca , the tolerant species, in water and mineral contents. Sugar contents were greater in E. microtheca . In E. microtheca Na was highly accumulated in roots [up to 910 μmol (g fresh weight)⁻¹], less in stems [up to 580 μmol (g fresh weight)-1] and leaves [up to 410 μmol (g fresh weight)^−r]. Chloride was also accumulated, its content was greater than the total content of Na and K, especially in the salt-tolerant provenance. Potassium and Ca contents were variously affected by the saline stress whereas soluble protein, amino acid and sugar contents were increased. Just after the saline stress, plants showed a large increase in the Na content of the leaf while the decrease in the K content of the stem and leaf continued. Plants were killed by the stress, probably because of too high accumulation of Na in leaves or roots according to the provenance. Osmoregulation and especially the participation of Na are discussed.     

Evidence for two separate Ca2+ pathways in smooth muscle plasmalemma

Summary The activation of rabbit aortic smooth muscle was studied by two most widely used vascular smooth muscle stimulants: -adrenoceptor activation by norepinephrine (NE) and high-K⁺ depolarization. This was studied by measurements of isometric contractions and net as well as unidirectional Ca²⁺ fluxes. These parameters showed markedly differential sensitivities towards two smooth muscle inhibitors used in this study: D 600 and amrinone. By choosing an appropriate concentration of D 600 or amrinone, Ca²⁺ uptake or Ca²⁺ influx induced by high K⁺ or NE could be selectively inhibited. Furthermore, by using unidirectional flux measurements it was demonstrated that Ca²⁺ influx stimulated by NE and high K⁺ were additive in nature. The data from the addivity experiment exclude the interpretation of a common Ca²⁺ pathway with two separate mechanisms for opening it. The data on three criteria employed in this study provide evidence for the existence of two independent Ca²⁺ pathways, one for each mode of activation, for Ca²⁺ influx known to be associated with these contractions.     

Evidence for a K+-stimulated Na+ efflux at the plasmalemma of barley root cells

Summary The influence of K⁺ on the Na⁺ fluxes of barley root cells was investigated. A increased K⁺ concentration (K⁺ influx) results in a transient increase of the plasmalemma efflux of Na⁺ followed by a decrease, and in a decrease of the cytoplasmic content and the tonoplast influx of Na⁺. These results are consistent with a Na-K-pump at the plasmalemma.     

Evidence for suppression of serum LH without elevation in serum estradiol or prolactin with a brain-enhanced redox delivery system for estradiol

We developed a redox system for brain-enhanced delivery of estradiol based on an interconvertible dihydropyridine in equilibrium pyridinium salt carrier. Estradiol (E2), when combined with the lipoidal carrier, readily crosses the blood-brain barrier. The carrier, when oxidized, reduces the rate of exit of the estradiol-carrier complex from the brain. Subsequent hydrolysis of the carrier provides sustained production of estradiol in the brain. The aim of the study was to evaluate the effects of single vs. multiple injections of the estradiol-chemical delivery system (E2-CDS) on both central and peripheral estrogen-responsive tissues. Ovariectomized Sprague-Dawley rats received an intravenous injection of E2-CDS at 10, 33, 100 or 333 micrograms/kg BW or the drug vehicle, dimethyl sulfoxide (DMSO; 0.5 ml/kg) every 2 days for 7 injections (2 weeks) or a single injection only at 2 days before sacrifice. With a single injection, E2-CDS did not affect serum luteinizing hormone (LH) levels at the 10 micrograms/kg dose but caused a dose-dependent reduction in serum LH of 39-52% at the dose range of 33 to 333 micrograms/kg. By contrast, multiple injections of E2-CDS caused a 32 to 76% reduction in serum LH levels at doses ranging from 10 micrograms/kg to 333 micrograms/kg. Additionally, multiple doses of E2-CDs caused a dose-dependent reduction in body weight at the 10 and 33 micrograms/kg doses with the higher doses causing no further weight reduction. For both single and multiple dosage groups, serum E2 levels remained unchanged after doses of E2-CDS of 10 and 33 micrograms/kg, then increased to 21 pg/ml for the single dosage group and to 23 pg/ml for the multiple dosage group at the 100 micrograms/kg dose, and to 59 pg/ml for singly-injected rats and 60 pg/ml for multiply-injected rats at the 333 micrograms/kg dose. Serum prolactin concentrations were closely correlated with serum E2 levels for both the single and multiple dose groups. These data reveal that a single or multiple doses of E2-CDS can reduce serum LH levels without elevating serum E2 or prolactin concentrations, supporting the concept of brain-enhanced delivery of estradiol with an estradiol chemical delivery system.     

Evidence for the extracellular reduction of alpha-lipoic acid by Leishmania donovani promastigotes: a transplasma membrane redox system

Leishmania donovani cells, capable of reducing certain electron acceptors with redox potentials at pH 7.0 down to -290 mV, outside the plasma membrane, can reduce the oxidised form of alpha-lipoic acid. alpha-Lipoic acid has been used as natural electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane alpha-lipoic acid reduction by Leishmania was not inhibited by mitochondrial inhibitors as azide, cyanide, rotenone or antimycin A, but responded to hemin, modifiers of sulphhydryl groups and inhibitor of glycolysis. The protonophores carbonyl cyanide chlorophenylhydrazone and 2,4-dinitrophenol showed inhibition of alpha-lipoic acid reduction. This transmembrane redox system differs from that of mammalian cells in respect to its sensitivity of UV irradiation and stimulation by diphenylamine. Thus a naphthoquinone coenzyme appears to be involved in alpha-lipoic acid reduction by Leishmania cells.     

Cation stimulation of the proton-translocating redox activity at the plasmalemma of Catharanthus roseus cells

Cultured Catharanthus roseus cells exhibit transmembrane ferricyanide reduction through a plasma membrane redox system which may be associated with proton translocation. Evidence shows that endogenous pyridine nucleotides serve as hydrogen donors for the reaction. The proton translocating function of the redox system is confirmed, in intact cells and isolated protoplasts, by the ability of Ca²⁺ and other cations to increase both the redox activity and the efflux of protons. The role of the cations is seen to be not a simple general charge screening phenomenon as already described. By using ionic surfactants (CP⁺, SDS^–) it was shown that the net surface charge of the membrane can interact in the activation process via a cation attraction effect. It is proposed that specific binding of cations to the plasma membrane could alter the conformation of the redox system facilitating its interaction with NADH.Abbreviations CP⁺ cetylpyridinium - EGTA ethylene glycol bis (-aminoethyl)-N,N-tetraacetic acid - FeCN potassium ferricyanide - SDS^– sodium dodecyl sulfate - SHAM salicylhydroxamic acid     

Regulated redox processes at the plasmalemma of plant root cells and their function in iron uptake

Plants take up iron as ferric chelates or, after reduction, as ferrous ions. Ferric reduction takes place at the plasma membrane of the root epidermis cells by a transmembrane redox system, which can be activated when iron is getting short. It is proposed that this inducible system, with NADPH as electron donor, is separate from a system, presumably present in all plant cells, which transports electrons from NADH or NADPH to ferricyanide, or,in vivo, oygen.     

Evidence for the uptake of sucrose intact into sugarcane internodes

Application of [¹⁴C]fructosyl sucrose was used to determine whether sucrose cleavage was necessary for sucrose uptake by sugarcane (Saccharum spp.) internode tissue. Although approximately 25% of ¹⁴C in the apoplast was present as fructose, indicating some sucrose cleavage, less than 15% of the label was randomized in the sucrose that remained in the tissue after a 30 minute osmoticum rinse. This is insufficient to support cleavage and resynthesis as the sole sucrose transport scheme. The lack of randomization of label between the glucose and fructose moieties of the sucrose molecule was taken as presumptive evidence that sucrose does not have to be cleaved prior to uptake by parenchyma cells in sugarcane internode tissue.     

Evidence for the extracellular reduction of ferricyanide by rat liver. A trans-plasma membrane redox system

1. Reduction of ferricyanide by the isolated perfused rat liver and by isolated rat hepatocytes was studied. 2. Ferricyanide was reduced to ferrocyanide by the perfused liver at a linear rate of 0.22mumol/min per g of liver. Ferricyanide was not taken up by the liver and the perfusate concentration of ferricyanide+ferrocyanide remained constant throughout the perfusion. Perfusate samples from livers perfused without ferricyanide did not reduce ferricyanide. 3. Isolated hepatocytes reduced ferricyanide in a biphasic manner. The initial rate of 2.3mumol/min per g of cells proceeded for approx. 3min and derived from low-affinity sites (apparent K(m)>1.3mm). The secondary rate of 0.29mumol/min per g of cells was maintained for the remainder of the incubation and derived from higher affinity sites (apparent K(m)0.13mm). Disruption of the cells resulted in an increase in the low-affinity rate and a decrease in the high-affinity rate. 4. Ferrocyanide was oxidized by isolated hepatocytes but not by perfused liver. The apparent K(m) for ferrocyanide oxidation by hepatocytes was 1.3mm. 5. Oxidized cytochrome c was reduced by isolated hepatocytes in the presence of 1mm-KCN but at a rate less than that of the reduction of ferricyanide. 6. Properties of the ferricyanide-reducing activities of intact hepatocytes and the perfused liver were examined. The low-affinity rate, present only in cell and broken cell preparations, was inhibited by 1mum-rotenone and 0.5mm-ferrocyanide, and stimulated by 0.1mm-KCN. The mitochondrial substrate, succinate, also stimulated this rate. The perfused liver showed only a high-affinity activity for ferricyanide reduction. This activity was also present in liver cells and was unaffected by rotenone, antimycin A, KCN, NaN(3), or p-hydroxymercuribenzoate but was inhibited by 2.6mm-CaCl(2), 2-heptyl-4-hydroxyquinoline-N-oxide and ferrocyanide. Overall, these results are consistent with the occurrence of a trans-plasma membrane redox system of liver that reduces extracellular ferricyanide to ferrocyanide. The reduction process shows properties which are similar to that of the NADH:ferricyanide oxidoreductase found in isolated liver plasma membranes but different from that of mitochondria.     

Evidence for a DNA inversion system in Bordetella pertussis

An isolation procedure was developed to provide within one day microcystin-LR, a cyclic heptapeptide toxin from Microcystis aeruginosa PCC 7806. After ODS (octadecylsilyl) solid phase extraction, the crude toxin fraction was chromatographed using a strong anion exchange column. The toxin was eluted with 0.02 M ammonium bicarbonate. An at least 95% purity was revealed on HPLC separation by monitoring at 214 nm. Application of the procedure to the cyclic pentapeptide toxin nodularin from Nodularia spumigena AV2 was examined.     

Evidence for a trypanothione-dependent peroxidase system in Trypanosoma cruzi

Hydroperoxide metabolism in Crithidia fasciculata has recently been shown to be catalyzed by a cascade of three oxidoreductases comprising trypanothione reductase (TR), tryparedoxin (TXN1), and tryparedoxin peroxidase (TXNPx) (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). The existence of this metabolic system in the human pathogen Trypanosoma cruzi is supported here by immunohistochemistry. Epimastigotes of T. cruzi display strong immunoreactivity with antibodies raised against TXN1 and TXNPx of C. fasciculata. In addition, a full-length open reading frame presumed to encode a peroxiredoxin-type protein in T. cruzi (Acc. Nr. AJ 012101) was heterologously expressed in Escherichia coli and shown to exhibit tryparedoxin peroxidase activity. With TXN, TXNPx, trypanothione and TR, T. cruzi possesses all components constituting the crithidial peroxidase system. It is concluded that the antioxidant defense of T. cruzi also depends on the NADPH-fuelled, trypanothione-mediated enzymatic hydroperoxide metabolism.     

Evidence for a mobile semiquinone in the redox cycle of the mammalian cytochrome bc1 complex

Experimental evidence is presented to demonstrate that cytochromes b of the mammalian cytochrome bc1 complex may be rapidly oxidised by a pulse of oxidising equivalents which react with cytochrome c1, even when all cytochrome b is fully reduced before the pulse. The oxidation is sensitive both to antimycin and to myxothiazol. Such behaviour is inconsistent with models in which only the fully oxidised ubiquinone may move between the centres 'o' and 'i' of the complex. It is proposed that the charged semiquinone (Q-) may move between these centres, which may constitute separate reaction domains of a single ubiquinone-binding site. The bearing of this on the mechanism of electron, proton and charge transfer in the complex is discussed.     

Evidence for a DNA inversion system in Bordetella pertussis

The expression of virulence-associated genes in Bordetella pertussis can be lost in three ways: phase variation, antigenic modulation, or serotype conversion. The mechanism(s) of these alterations in gene expression is unclear. B. pertussis chromosomal DNA was probed with cloned pin genes from Escherichia coli and cloned hin genes from Salmonella typhimurium. DNA duplex melting temperature experiments indicated significant homology between B. Pertussis chromosomal DNA and both DNA inversion genes. Southern blots using the hin gene probe showed homology with a 15 kb EcoRI fragment of B. pertussis chromosomal DNA. We postulate here that B. pertussis contains a DNA inversion system which may be responsible for serotype conversion or virulence phase change in this organism.     

Evidence for rapid microscale bacterial redox cycling of iron in circumneutral environments

The potential for microscale bacterial Fe redox cycling was investigated in microcosms containing ferrihydrite-coated sand and a coculture of a lithotrophic Fe(II)-oxidizing bacterium (strain TW2) and a dissimilatory Fe(III)-reducing bacterium (Shewanella alga strain BrY). The Fe(II)-oxidizing organism was isolated from freshwater wetland surface sediments which are characterized by steep gradients of dissolved O₂ and high concentrations of dissolved and solid-phase Fe(II) within mm of the sediment–water interface, and which support comparable numbers (10⁵–10⁶ mL⁻¹) of culturable Fe(II)-oxidizing and Fe(III)-reducing reducing. The coculture systems showed minimal Fe(III) oxide accumulation at the sand-water interface, despite intensive O₂ input from the atmosphere and measurable dissolved O₂ to a depth of 2 mm below the sand–water interface. In contrast, a distinct layer of oxide precipitates formed in systems containing Fe(III)-reducing bacteria alone. Examination of materials from the cocultures by fluorescence in situ hybridization indicated close physical juxtapositioning of Fe(II)-oxidizing and Fe(III)-reducing bacteria in the upper few mm of sand. Our results indicate that Fe(II)-oxidizing bacteria have the potential to enhance the coupling of Fe(II) oxidation and Fe(III) reduction at redox interfaces, thereby promoting rapid microscale cycling of Fe. This revised version was published online in August 2006 with corrections to the Cover Date.     

Targeting the redox system for cardiovascular regeneration in aging

Cardiovascular aging presents a formidable challenge, as the aging process can lead to reduced cardiac function and heightened susceptibility to cardiovascular diseases. Consequently, there is an escalating, unmet medical need for innovative and effective cardiovascular regeneration strategies aimed at restoring and rejuvenating aging cardiovascular tissues. Altered redox homeostasis and the accumulation of oxidative damage play a pivotal role in detrimental changes to stem cell function and cellular senescence, hampering regenerative capacity in aged cardiovascular system. A mounting body of evidence underscores the significance of targeting redox machinery to restore stem cell self-renewal and enhance their differentiation potential into youthful cardiovascular lineages. Hence, the redox machinery holds promise as a target for optimizing cardiovascular regenerative therapies. In this context, we delve into the current understanding of redox homeostasis in regulating stem cell function and reprogramming processes that impact the regenerative potential of the cardiovascular system. Furthermore, we offer insights into the recent translational and clinical implications of redox-targeting compounds aimed at enhancing current regenerative therapies for aging cardiovascular tissues.     

Evidence that the redox state has a role in muscular contraction and relaxation

The present work reports that simple oxidizing agents are capable of inducing isotonic contraction of rat aorta in vitro, and that the concentration of agent required depends on its oxidizing potential. Conversely a reducing agent will reverse a muscular contraction induced by oxidizing agents.     

Reduction of vanadate by a microsomal redox system

The reduction of vanadate catalyzed by rat liver microsomes is demonstrated. This reaction is SOD-insensitive. It is specific for NADH and polyvanadate and is not obtained with metavanadate and NADPH.