On the use of the quenching of luminol luminescence to evaluate sod activity

Addition of horseradish peroxidase to a luminol solution (pH = 9.4) produces a burst of light followed by a steady luminescence that lasts for several minutes. This steady-state luminescence is readily quenched by SOD, with a concentration (the additive concentration needed to decrease by one-half the emitted luminescence intensity) of c.a. 4 ng/ml (14 mU/ml). The luminescence intensity decrease can then be employed to evaluate SOD activity in SOD-containing samples. However, the light intensity can also be quenched by additives, such as Trolox, that are able to trap luminol-derived intermediates. It is proposed that double quenching experiments must be performed in order to be able to relate the observed effect of an additive to its SOD-like activity.     

Guanine nucleotide regulatory protein alterations in young Milan hypertensive strain rats

Vascular smooth muscle cell membranes from prehypertensive rats of the Milan hypertensive strain (MHS) were used to examine adenylyl cyclase activity and its regulation by guanine nucleotide regulatory proteins (G-proteins). Basal adenylyl cyclase activity was similar in MHS and Milan normontensive strain (MNS) membranes. Forsokolin (10^?4 M) produced a significantly greater stimulatory response in MHS membranes, but this was not observed with NaF (10^?2 M). Isoporterenol (10^?4 M) caused a significantly decreased stimulation of adenylyl cyclase activity in MHS membranes, while prostaglandin E₁ (10^?5 M) produced similar responses in the two strains. G_i function and GTP responses, as observed by biphasic effects of GTP on isoproterenol-stimulated membranes, were similar in both strains. The levels of G_i2α and G_qα/G₁₁α were similar in the two strains, while the levels of G_sα (44 and 42 kDa forms) and the β-subunit were significantly reduced by ～20% in MHS membranes. The α-subunit of G_i3 was dramatically reduced by ～80% in MHS membranes. The affinities of β-adrenergic receptors for the antagonist, cyanophindolol, were similar in the two strains; however, the number of β-adrenoceptors was substantially reduced in MHS membranes. These findings may be of relevance to altered vascular reactivity and transmembrane ion distribution observed in the MHS.     

Assessment of cytoplasmic differences of near-isonuclear male-sterile lines in pearl millet

Four near-isonuclear polycytoplasmic versions of 81A and two of Pb 402A male-sterile lines of pearl millet (Pennisetum typhoides) were used in factorial matings with five inbred male testers in different combinations in three sets. The cytoplasmic differences were studied for several agronomic traits using mean values and general combining effects (gca) of male-sterile lines, and specific combining ability effects of hybrids. The fertility/ sterility behaviour of different male-sterile lines in crosses with common male parents was also studied. Significant differences among near-isonuclear polycytoplasmic lines were observed in mean values for a few traits such as plant height, leaf length and peduncle length, but the differences for combining ability were more pronounced. The A₃ cytoplasm was a better general combiner than the A₂ cytoplasm for grain yield and both A₂ and A₃ cytoplasms were better general combiners for leaf length and peduncle length. In addition, superiority of A₃ cytoplasm for gca was observed for plant height and ear characters over the A₂ cytoplasm in set II. A differential behaviour of cytoplasms, both in combination with a common pollinator and across pollinators, was observed for several traits. The results provide evidence for the distinctiveness of different cytoplasmic sources in pearl millet and for the influence of cytoplasmic factors on the phenotypic expression of nuclear genes. A diversification of male sterility sources in the breeding of pearl millet hybrids is suggested.     

The effects of enhanced ozone and enhanced carbon dioxide concentrations on biomass, pigments and antioxidative enzymes in spruce needles (Picea abies L.)

During one growing period, 5-year-old spruce trees (Picea abies L., Karst.) were exposed in environmental chambers to elevated concentrations of carbon dioxide (750 cm³ m^?3) and ozone (008 cm³ m^?3) as single variables or in combination. Control concentrations of the gases were 350cm³ m^?3CO₂ and 0.02 cm³ m ^?3 ozone. To investigate whether an elevated CO₂ concentration can prevent adverse ozone effects by reducing oxidative stress, the activities of the protective enzymes superoxide dismutase, catalase and peroxidase were determined. Furthermore, shoot biomass, pigment and protein contents of two needle age classes were investigated. Ozone caused pigment reduction and visible injury in the previous year's needles and growth reduction in the current year's shoots. In the presence of elevated concentrations of ozone and CO₂, growth reduction in the current year's shoots was prevented, but emergence of visible damage in the previous year's needles was only delayed and pigment reduction was still found. Elevated concentrations of ozone or CO₂ as single variables caused a significant reduction in the activities of superoxide dismutase and catalase in the current year's needles. Minimum activities of superoxide dismutase and catalase and decreased peroxidase activities were found in both needle age classes from spruce trees grown at enhanced concentrations of both CO₂ and ozone. These results suggest a reduced tolerance to oxidative stress in spruce trees under conditions of elevated concentrations of both CO₂ and ozone.     

Reversal of selenium and zinc deficiencies in chronic hemodialysis patients by intravenous sodium selenite and zinc gluconate supplementation

In six chronic dialyzed uremic patients, an intravenous sodium selenite (Se 50 μg during 5 wk and then 100 μg) and zinc gluconate (Zn 5 mg) supplementation was performed during 20 wk at each dialysis session three times weekly. Before supplementation, plasma Se and Zn, plasma and erythrocytes (RBC) antioxidant metalloenzymes glutathione peroxidase (GPX), and superoxide dismutase (SOD) were significantly decreased, whereas lipid peroxidation (as thiobarbituric acid reactants TBARs) was increased. To obtain a significative change in plasma selenium, we had to use an Se dose of 100 μg/dialysis session. Then, treatment-increased plasma Se (from 0.58 ±0.09 to 0.89±0.16 μmol/L) led to a repletion of RBC-GPX (from 29.6±6 to 43±5.8 U/g Hb) and increased plasma GPX levels (from 62±13 to 151±43 U/L). Plasma Zn and RBC-SOD did not vary significantly. The change of TBARs was not observed between wk 1 and 4. They decreased significantly between wk 4 (4.80±0.21μmol/L) and wk 20 (4.16±0.26 μmol/L). We noted a low correlation between TBARs and plasma GPX. A strong correlation was observed between Se and plasma GPX. The reversal of Se deficiencies should reduce oxidative damage observed in these patients.     

Effect of selenium and protein deficiency on selenium and glutathione peroxidase in rats

Twenty-four weanling male Wistar rats were divided into four groups fed diets containing adequate or deficient levels of selenium (0.5 ppm [+ Se] or <0.02 ppm [−Se] and protein (15% [+Pro] or 5% [−Pro]), but adequate levels of all other nutrients for 4 wk to determine the effects of Se deficiency and protein deficiency on tissue Se and glutathione peroxidase (GSHPx) activity in rats. Plasma, heart, liver, and kidney Se and GSHPx were significantly lower in Se-deficient groups in relation to Se-sufficient groups. In Se-deficient groups, Se and GSHPx were significantly higher in −Se−Pro rats in heart, liver, and kidney. Data analysis showed that there were significant interaction effects between dietary Se and protein on Se and GSHPx of rats. It is assumed that under the condition of Se deficiency. a low level of protein may decrease Se and GSHPx utilization, increase GSHPx synthesis, and result in Se redistribution. This could account for high levels of Se and GSHPx in the −Se−Pro rats compared to −Se+Pro rats.     

Molecular characterization of salt-stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases

A gene encoding for a citrus salt-stress-associated protein (Cit-SAP) was cloned from Citrus sinensis salt-treated cell suspension. The gene, designated csa, was isolated from a cDNA expression library. The partial amino acid sequence of the protein, as well as that deduced from the nucleotide sequence of csa, revealed a considerable homology to mammalian glutathione peroxidase (GP), and to clone 6P229 from tobacco protoplasts. The increased expression of Cit-SAP in NaCl-treated cultured citrus cells and in citrus plants irrigated with saline water, and its similarity to GP, raise the possibility that one of the effects of salt stress in plants may be the increase of the level of free radicals.     

Characterization of peroxidases in luminol chemiluminescence coupled with copper-catalysed oxidation of cysteamine

Hydrogen peroxide formed during the course of the copper(II)-catalysed oxidation of cysteamine with oxygen was continuously determined by a peroxidase (POD)-catalysed luminol chemiluminescence (CL) method. Horseradish peroxidase (HRP), lactoperoxidase (LPO) and Arthromyces ramosus peroxidase (ARP) were used as a CL catalyst. The respective PODs gave specific CL intensity-time profiles. HRP caused a CL delay, and ARP gave a time-response curve which followed the production rate of H₂O₂. LPO gave only a weak CL flash which decayed promptly. These differences of CL response curves could be explained in terms of the different reactivities of PODs for superoxide anion and the different formation rate of luminol radicals in the peroxidation of luminol catalysed by POD.     

Effects of D2O on permeation and gating in the Ca2+-activated potassium channel fromChara

We studied the effects of H₂O/D₂O substitution on the permeation and gating of the large conductance Ca²⁺-activated K⁺ channels inChara gymnophylla droplet membrane using the patchclamp technique. The selectivity sequence of the channel was: K⁺>Rb⁺≫Li⁺, Na⁺, Cs⁺ and Cl⁻. The conductance of this channel in symmetric 100mm KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D₂O as compared to H₂O. The blockade of channel conductance by cytosolic Ca²⁺ weakened in D₂O as a result of a decrease in zero voltage Ca²⁺ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D₂O primarily due to the change in Ca²⁺ binding to the channel during the activation step. The Hill coefficient for Ca²⁺ binding was 3 in D₂O and around 1 in H₂O. The values of the Ca²⁺ binding constant in the open channel conformation were 0.6 and 6 μm in H₂O and D₂O, respectively, while the binding in the closed conformation was much less affected by D₂O. The H₂O/D₂O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1mm internal Ca²⁺.     

Catalytic and interfacial aspects of enzymatic polymer synthesis in reversed micellar systems

The enzyme horseradish peroxidase, when encapsulated in reversed micelles, is capable of catalyzing the synthesis of phenolic and aromatic amine polymers. The synthesis of polyethylphenol is specifically considered in this article and is found to be extremely feasible in the micellar system. Polymer chain growth can be controlled to some degree by manipulating the ability of the solvent to sustain chain solubility; this is effectively done by adjusting the surfactant concentration. This results in a degree of control of polymer molecular weight. The synthesized polymer drops out of solution and can be easily recovered. (c) 1993 John Wiley & Sons, Inc.