Heat-inactivated peroxidases and the role of calcium abstraction as a cause of their enhanced lipid oxidation activity: potential effects on the flavour quality of heat-processed vegetables

Cationic swede and anionic turnip peroxidases were partially purified by ion-exchange and gel-filtration chromatography, respectively. Heat treatment of these enzymes and of a commercial high purity horseradish peroxidase (HRP) caused a loss of enzyme activity and a corresponding increase in linoleic acid hydroperoxide formation activity. The hydroperoxide levels in model systems increased only in the early stages of the oxidation reaction and then declined as degradation became more significant. The presence of a dialysed blend of cooked swede markedly lowered the hydroperoxide level formed. Analysis of volatile compounds formed showed that hexanal predominated in a buffer system and in a blend of cooked turnip. In dialysed blends of cooked swede, hexanol was the primary volatile compound generated. After inactivation under mild conditions in the presence of EDTA, the peroxidases showed hydroperoxide formation activity and patterns of volatile compounds from linoleic acid that were similar to those found on heat-inactivation. This suggested that calcium abstraction from the peroxidases was critical for the enhancement of lipid oxidation activity. (C) 2002 Elsevier Science Ltd. All rights reserved.

Feline calicivirus p32, p39 and p30 proteins localize to the endoplasmic reticulum to initiate replication complex formation

In common with other positive-strand RNA viruses, replication of feline calicivirus (FCV) results in rearrangement of intracellular membranes and production of numerous membrane-bound vesicular structures on which viral genome replication is thought to occur. In this study, bioinformatics approaches have identified three of the FCV non-structural proteins, namely p32, p39 and p30, as potential transmembrane proteins. These proteins were able to target enhanced cyan fluorescent protein to membrane fractions where they behaved as integral membrane proteins. Immunofluorescence microscopy of these proteins expressed in cells showed co-localization with endoplasmic reticulum (ER) markers. Further electron microscopy analysis of cells co-expressing FCV p39 or p30 with a horseradish peroxidase protein containing the KDEL ER retention motif demonstrated gross morphological changes to the ER. Similar reorganization patterns, especially for those produced by p30, were observed in naturally infected Crandel-Rees feline kidney cells. Together, the data demonstrate that the p32, p39 and p30 proteins of FCV locate to the ER and lead to reorganization of ER membranes. This suggests that they may play a role in the generation of FCV replication complexes and that the endoplasmic reticulum may represent the potential source of the membrane vesicles induced during FCV infection.

Restricted expression of NADPH oxidase/peroxidase gene (Duox) in zone VII of the ascidian endostyle

The ascidian Ciona intestinalis, a marine invertebrate chordate, is an emerging model system for developmental and evolutionary studies. The endostyle, one of the characteristic organs of ascidians, is a pharyngeal structure with iodine-concentrating and peroxidase activities and is therefore considered to be homologous to the follicular thyroid of higher vertebrates. We have previously reported that a limited part of the endostyle (zone VII) is marked by the expression of orthologs of the thyroid peroxidase (TPO) and thyroid transcription factor-2 (TTF-2/FoxE) genes. In this study, we have identified the Ciona homolog of NADPH oxidase/peroxidase (Duox), which provides hydrogen peroxide (H2O2) for iodine metabolism by TPO in the vertebrate thyroid. Expression patterns assessed by in situ hybridization have revealed that Ciona Duox (Ci-Duox) is predominantly expressed in the dorsal part of zone VII of the endostyle. Furthermore, two-color fluorescent in situ hybridization with Ci-Duox and Ciona TPO (CiTPO) has revealed that the ventral boundary of the Ci-Duox domain of expression is more dorsal than that of CiTPO. We have also characterized several genes, such as Ci-Fgf8/17/18, 5HT7, and Ci-NK4, which are predominantly expressed in the ventral part of zone VII, in a region complementary to the Ci-Duox expression domain. These observations suggest that, at the molecular level, zone VII has a complex organization that might have some impact on the specification of cell types and functions in this thyroid-equivalent element of the ascidian endostyle.

Haem peroxidase activity in Daphnia magna: A biomarker for sub-lethal toxicity assessments of kerosene-contaminated groundwater

A novel biomarker was developed in Daphnia magna to detect organic pollution in groundwater. The haem peroxidase assay, which is an indirect means of measuring oxidase activity, was particularly sensitive to kerosene contamination. Exposure to sub-lethal concentrations of kerosene-contaminated groundwater resulted in a haem peroxidase activity increase by dose with a two-fold activity peak at 25%. Reproduction in D. magna remained unimpaired when exposed to concentrations below 25% for 21 days, and a decline in fecundity was only observed at concentrations above the peak in enzyme activity. The measurement of haem peroxidase activity in D. magna detected sublethal effects of kerosene in just 24 h, whilst offering information on the health status of the organisms. The biomarker may be useful in determining concentrations above which detrimental effects would occur from long-term exposure for fuel hydrocarbons. Moreover, this novel assay detects exposure to chemicals in samples that would normally be classified as non-toxic by acute toxicity tests.

Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury

Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of gamma-glutamylcysteine synthetase-heavy subunit (gamma-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis. (c) 2006 Elsevier Inc. All rights reserved.

Effects of dietary selenium supplementation on tissue selenium distribution and glutathione peroxidase activity in Chinese Ring Necked Pheasants

The objective of this study was to determine the concentration of total selenium (Se) and the proportions of total Se comprised as selenomethionine (SeMet) and selenocysteine (SeCys) in the post mortem tissues of female pheasants (Phasianus Colchicus Torquator) offered diets containing graded additions of selenized enriched yeast (SY) or sodium selenite (SS). Thiobarbituric acid reactive substances (TBARS) and tissue glutathione peroxidase (GSH-Px) activity of breast (Pectoralis Major) were assessed at 0 and 5 d post-mortem. A total of 216 female pheasant chicks were enrolled onto the study. 24 birds were euthanased at the start of the study and samples of blood, breast muscle, leg muscle (Peroneus Longus and M. Gastrocnemius), heart, liver, kidney and gizzard collected for determination of total Se. Remaining birds were blocked by live weight and randomly allocated to one of four dietary treatments (n=48 birds/treatment) that either differed in Se source (SY vs. SS) or dose (Con [0.2 mg total Se/kg], SY-L and SS-L [0.3 mg/kg total Se as SY and SS, respectively], and SY-H [0.45 mg total Se/kg]). Following 42 and 91 days of treatment 24 birds/treatment were euthanased and samples of blood, breast muscle, leg muscle, heart, liver, kidney and gizzard retained for determination of total Se and the proportion of total Se comprised as SeMet or SeCys. Whole blood GSH-Px activity was determined at each time point. Tissue GSH-Px activity and TBARS were determined in breast tissue at the end of the study. There were positive responses (P<0.001) in both blood and tissues to the graded addition of SY to the diet but the same responses were not apparent in the blood and tissues of selenite supplemented birds receiving comparable doses. Although there were differences between tissue types in the distribution of SeMet and SeCys there were few differences between treatments. There were effects of treatment on erythrocyte GSH-Px activity (P = 0.012) with values being higher in treatments SY-H and SS-L when compared to the negative control and treatment SY-L. There were no effects of treatment on tissue GSH-Px activity which is reflected in the overall lack of any treatment effects on TBARS.

Tolerance of ruminant animals to high dose in-feed administration of a selenium-enriched yeast

The objective of the study was to determine if there were adverse effects on animal health and performance when a range of ruminant animals species were fed at least 10 times the maximum permitted European Union (EU) selenium (Se) dietary inclusion rate (0.568 mg Se/kg DM) in the form of selenium enriched yeast (SY) derived from a specific strain of Saccharomyces cerevisiae CNCM I-3060. In a series of studies, dairy cows, beef cattle, calves and lambs were offered either a control diet which contained no Se supplement or a treatment diet which contained the same basal feed ingredients plus a SY supplement which increased total dietary Se from 0.15 to 6.25, 0.20 to 6.74, 0.15 to 5.86 and 0.14 to 6.63 mg Se/kg DM, respectively. The inclusion of the SY supplement (P < 0.001) increased whole blood Se concentrations, reaching maximum mean values of 716, 1,505, 1,377, and 724 ng Se/mL for dairy cattle, beef cattle, calves and lambs, respectively. Selenomethionine accounted for 10% of total whole blood Se in control animals whereas the proportion in SY animals ranged between 40 and 75%. Glutathione peroxidase (EC 1.11.1.9) activity was higher (P < 0.05) in SY animals when compared with controls. A range of other biochemical and hematological parameters were assessed, but few differences of biological significance were established between treatments groups. There were no differences between treatment groups within each species with regard to animal physical performance or overall animal health. It was concluded that there were no adverse effects on animal health, performance and voluntary feed intake to the administration of at least ten times the EU maximum, or approximately twenty times the US FDA permitted concentration of dietary Se in the form of SY derived from a specific strain of Saccharomyces cerevisiae CNCM I-3060.

Effect of dietary supplementation with selenium-enriched yeast or sodium selenite on selenium tissue distribution and meat quality in beef cattle

The objective was to determine the concentration of total selenium (Se) and the proportion of total Se comprised as selenomethionine (SeMet) and selenocysteine (SeCys) in post mortem tissues of beef cattle offered diets containing graded additions of selenized enriched yeast (SY) [Saccharomyces cerevisae CNCM I-3060]), or sodium selenite (SS). Oxidative stability and tissue glutathione peroxidase (GSH-Px) activity of edible muscle tissue were assessed 10 d post-mortem. Thirty two beef cattle were offered, for a period of 112 d, a total mixed ration which had either been supplemented with SY (0, 0.15 or 0.35 mg Se/kg DM) or SS (0.15 mg Se/kg DM). At enrollment (0 d) and at 28, 56, 84 and 112 d following enrollment, blood samples were taken for Se and Se species determination, as well as whole blood GSH-Px activity. At the end of the study beef cattle were euthanized and samples of heart, liver, kidney, and skeletal muscle (LM and psoas major) were retained for Se and Se species determination. Tissue GSH-Px activity and thiobarbituric acid reactive substances (TBARS) were determined in skeletal muscle tissue (LM only). The incorporation into the diet of ascending concentrations of Se as SY increased whole blood total Se and the proportion of total Se comprised as SeMet, as well as GSH-Px activity. There was also a dose dependant response to the graded addition of SY on total Se and proportion of total Se as SeMet in all tissues and GSH-Px activity in skeletal muscle tissue. Furthermore, total Se concentration of whole blood and tissues was greater in those animals offered SY when compared with those receiving a comparable dose of SS, indicating an improvement in Se availability and tissue Se retention. Likewise, GSH-Px activity in whole blood and LM was greater in those animals offered SY when compared with those receiving a comparable dose of SS. However, these increases in tissue total Se and GSH-Px activity appeared to have little or no effect in meat oxidative stability.

Effects of dietary supplementation with selenium enriched yeast or sodium selenite on selenium tissue distribution and meat quality in lambs

The objective was to determine the concentration of total selenium (Se) and the proportion of total Se comprised as selenomethionine (SeMet) and selenocysteine (SeCys), as well as meat quality in terms of oxidative stability in post mortem tissues of lambs offered diets with an increasing dose rate of selenized enriched yeast (SY), or sodium selenite (SS). Fifty lambs were offered, for a period of 112 d, a total mixed ration which had either been supplemented with SY (0, 0.11, 0.21 or 0.31 mg/kg DM to give total Se contents of 0.19, 0.3, 0.4 and 0.5 mg Se/kg DM for treatments T1, T2, T3 and T4, respectively) or SS (0.11 mg/kg DM to give 0.3 mg Se/kg DM total Se [T5]). At enrolment and at 28, 56, 84 and 112 d following enrolment, blood samples were taken for Se and Se species determination, as well as glutathione peroxidase (GSH-Px) activity. At the end of the study lambs were euthanased and samples of heart, liver, kidney, and skeletal muscle were retained for Se and Se species determination. Tissue GSH-Px activity and thiobarbituric acid reactive substances (TBARS) were determined in Longissimus Thoracis. The incorporation into the diet of ascending concentrations of Se as SY increased whole blood total Se and the proportion of total Se comprised as SeMet, and erythrocyte GSH-Px activity. Comparable doses of SS supplementation did not result in significant differences between these parameters. With the exception of kidney tissue, all other tissues showed a dose dependant response to increasing concentrations of dietary SY, such that total Se and SeMet increased. Selenium content of Psoas Major was higher in animals fed SY when compared to a similar dose of SS, indicating improvements in Se availability and retention. There were no significant treatment effects on meat quality assessments GHS-Px and TBARS, reflecting the lack of difference in the proportion of total Se that was comprised as SeCys. However, oxidative stability improved marginally with ascending tissue Se content, providing an indication of a linear dose response whereby TBARS improved with ascending SY inclusion.

Enhancement of germination of spores of Verticillium dahliae and Fusarium oxysporum esp vasinfectum in vascular fluid from cotton plants infected with the root-knot nematode

Root-knot nematode [RKN] (Meloidogyne incognita) can increase the severity of Verticillium (V dahliae) and Fusarium (F oxysporum f.sp. vasinfectum) wilt diseases in cotton (Gossypium hirsutum). This study was conducted to determine some of the physiological responses caused by nematode invasion that might decrease resistance to vascular wilt diseases. The effect of RKN was investigated on spore germination and protein, carbohydrate and peroxidase content in the xylem fluids extracted from nematode-infected plants. Two cotton cultivars were used with different levels of resistance to both of the wilt pathogens. Spore germination was greater in the xylem fluids from nematode-infected plants than from nematode-free plants. The effect on spore germination was greater in the Fusarium-resistant cultivar (51%). Analysis of these fluids showed a decrease in total protein and carbohydrate levels for both wilt-resistant cultivars, and an increase in peroxidase concentration. Fluids from nematode-free plants of the Verticillium-resistant cultivar contained 46% more peroxidase than the Fusarium-resistant cultivar. The results provide further evidence that the effect of RKN on vascular wilt resistance is systemic and not only local. Changes in metabolites in the xylem pass from the root to the stem, accelerating disease development.

Zea mays annexins modulate cytosolic free Ca2+ and generate a Ca2+-permeable conductance

Regulation of reactive oxygen species and cytosolic free calcium ([Ca2+](cyt)) is central to plant function. Annexins are small proteins capable of Ca2+-dependent membrane binding or membrane insertion. They possess structural motifs that could support both peroxidase activity and calcium transport. Here, a Zea mays annexin preparation caused increases in [Ca2+] cyt when added to protoplasts of Arabidopsis thaliana roots expressing aequorin. The pharmacological profile was consistent with annexin activation (at the extracellular plasma membrane face) of Arabidopsis Ca2+-permeable nonselective cation channels. Secreted annexins could therefore modulate Ca2+ influx. As maize annexins occur in the cytosol and plasma membrane, they were incorporated at the intracellular face of lipid bilayers designed to mimic the plasma membrane. Here, they generated an instantaneously activating Ca2+-permeable conductance at mildly acidic pH that was sensitive to verapamil and Gd3+ and had a Ca2+-to-K+ permeability ratio of 0.36. These results suggest that cytosolic annexins create a Ca2+ influx pathway directly, particularly during stress responses involving acidosis. A maize annexin preparation also demonstrated in vitro peroxidase activity that appeared independent of heme association. In conclusion, this study has demonstrated that plant annexins create Ca2+-permeable transport pathways, regulate [Ca2+] cyt, and may function as peroxidases in vitro.

The cell wall and secretory proteome of a tobacco cell line synthesising secondary wall

The utility of plant secondary cell wall biomass for industrial and biofuel purposes depends upon improving cellulose amount, availability and extractability. The possibility of engineering such biomass requires much more knowledge of the genes and proteins involved in the synthesis, modification and assembly of cellulose, lignin and xylans. Proteomic data are essential to aid gene annotation and understanding of polymer biosynthesis. Comparative proteomes were determined for secondary walls of stem xylem and transgenic xylogenic cells of tobacco and detected peroxidase, cellulase, chitinase, pectinesterase and a number of defence/cell death related proteins, but not marker proteins of primary walls such as xyloglucan endotransglycosidase and expansins. Only the corresponding detergent soluble proteome of secretory microsomes from the xylogenic cultured cells, subjected to ion-exchange chromatography, could be determined accurately since, xylem-specific membrane yields were of poor quality from stem tissue. Among the 109 proteins analysed, many of the protein markers of the ER such as BiP, HSP70, calreticulin and calnexin were identified, together with some of the biosynthetic enzymes and associated polypeptides involved in polymer synthesis. However 53% of these endomembrane proteins failed identification despite the use of two different MS methods, leaving considerable possibilities for future identification of novel proteins involved in secondary wall polymer synthesis once full genomic data are available.

EfeUOB (YcdNOB) is a tripartite, acid-induced and CpxAR-regulated, low-pH Fe2+ transporter that is cryptic in Escherichia coli K-12 but functional in E-coli O157 : H7

Escherichia coli possesses iron transporters specific for either Fe2+ or Fe3+. Although Fe2+ is far more soluble than Fe3+, it rapidly oxidizes aerobically at pH >= 7. Thus, FeoAB, the major Fe2+ transporter of E. coli, operates anaerobically. However, Fe2+ remains stable aerobically under acidic conditions, although a low-pH Fe2+ importer has not been previously identified. Here we show that ycdNOB (efeUOB) specifies the first such transporter. efeUOB is repressed at high pH by CpxAR, and is Fe2+-Fur repressed. EfeU is homologous to the high-affinity iron permease, Ftr1p, of Saccharomyces cerevisiae and other fungi. EfeO is periplasmic with a cupredoxin N-terminal domain; EfeB is also periplasmic and is haem peroxidase-like. All three Efe proteins are required for Efe function. The efeU gene of E. coli K-12 is cryptic due to a frameshift mutation - repair of the single-base-pair deletion generates a functional EfeUOB system. In contrast, the efeUOB operon of the enterohaemorrhagic strain, O157:1147, lacks any frameshift and is functional. A 'wild-type' K-12 strain bearing a functional EfeUOB displays a major growth advantage under aerobic, low-pH, low-iron conditions when a competing metal is provided. Fe-55 transport assays confirm the ferrous iron specificity of EfeUOB.

Preliminary X-ray diffraction analysis of YcdB from Escherichia coli: a novel haem-containing and Tat-secreted periplasmic protein with a potential role in iron transport

YcdB is a periplasmic haem-containing protein from Escherichia coli that has a potential role in iron transport. It is currently the only reported haem-containing Tat-secreted substrate. Here, the overexpression, purification, crystallization and structure determination at 2.0 angstrom resolution are reported for the apo form of the protein. The apo-YcdB structure resembles those of members of the haem-dependent peroxidase family and thus confirms that YcdB is also a member of this family. Haem-soaking experiments with preformed apo-YcdB crystals have been optimized to successfully generate haem-containing YcdB crystals that diffract to 2.9 angstrom. Completion of model building and structure refinement are under way.

Comparative expression analysis of transcription factor genes in the endostyle of invertebrate chordates

The endostyle of invertebrate chordates is a pharyngeal organ that is thought to be homologous with the follicular thyroid of vertebrates. Although thyroid-like features such as iodine-concentrating and peroxidase activities are located in the dorsolateral part of both ascidian and amphioxus endostyles, the structural organization and numbers of functional units are different. To estimate phylogenetic relationships of each functional zone with special reference to the evolution of the thyroid, we have investigated, in ascidian and amphioxus, the expression patterns of thyroid-related transcription factors such as TTF-2/MoxE4 and Pax2/5/8, as well as the forkhead transcription factors FoxQ1 and FoxA. Comparative gene expression analyses depicted an overall similarity between ascidians and amphioxus endostyles, while differences in expression patterns of these genes might be specifically related to the addition or elimination of a pair of glandular zones. Expressions of Ci-FoxE and BbFoxE4 suggest that the ancestral FoxE class might have been recruited for the formation of thyroid-like region in a possible common ancestor of chordates. Furthermore, coexpression of FoxE4, Pax2/5/8, and TPO in the dorsolateral part of both ascidian and amphioxus endostyles suggests that genetic basis of the thyroid function was already in place before the vertebrate lineage. (c) 2005 Wiley-Liss, Inc.