Britta N. Krath

A short-term intervention trial with selenate, selenium-enriched yeast and selenium-enriched milk: effects on oxidative defence regulation.

Increased Se intakes have been associated with decreased risk of cancer and CVD. Several mechanisms have been proposed, including antioxidant effects through selenoproteins, induction of carcinogen metabolism and effects on the blood lipid profile. In a 4 x 1 week randomised, double-blind cross-over study, healthy young men supplemented their usual diet with selenate, Se-enriched yeast, Se-enriched milk or placebo (Se dose was 300 microg/d for selenate and Se-enriched yeast, and about 480 microg/d for Se-enriched milk) followed by 8-week washout periods. All Se sources increased serum Se levels after supplementation for 1 week. The effect of the organic forms did not differ significantly and both increased serum Se more than selenate. Conversely, thrombocyte glutathione peroxidase (GPX) was increased in the periods where subjects were supplemented with selenate but not in those where they were given Se-enriched yeast or Se-enriched milk. We found no effect on plasma lipid resistance to oxidation, total cholesterol, TAG, HDL- and LDL-cholesterol, GPX, glutathione reductase (GR) and glutathione S-transferase (GST) activities measured in erythrocytes, GPX and GR activities determined in plasma, or GR and GST activities in thrombocytes. Leucocyte expression of genes encoding selenoproteins (GPX1, TrR1 and SelP), and of electrophile response element-regulated genes (GCLC, Fra1 and NQO1) were likewise unaffected at all time points following intervention. We conclude that thrombocyte GPX is specifically increased by short-term selenate supplementation, but not by short-term supplementation with organic Se. Short-term Se supplementation does not seem to affect blood lipid markers or expression and activity of selected enzymes and a transcription factor involved in glutathione-mediated detoxification and antioxidation.

Effects of apples and specific apple components on the cecal environment of conventional rats: role of apple pectin

BackgroundOur study was part of the large European project ISAFRUIT aiming to reveal the biological explanations for the epidemiologically well-established health effects of fruits. The objective was to identify effects of apple and apple product consumption on the composition of the cecal microbial community in rats, as well as on a number of cecal parameters, which may be influenced by a changed microbiota.ResultsPrincipal Component Analysis (PCA) of cecal microbiota profiles obtained by PCR-DGGE targeting bacterial 16S rRNA genes showed an effect of whole apples in a long-term feeding study (14 weeks), while no effects of apple juice, purée or pomace on microbial composition in cecum were observed. Administration of either 0.33 or 3.3% apple pectin in the diet resulted in considerable changes in the DGGE profiles.A 2-fold increase in the activity of beta-glucuronidase was observed in animals fed with pectin (7% in the diet) for four weeks, as compared to control animals (P < 0.01). Additionally, the level of butyrate measured in these pectin-fed animal was more than double of the corresponding level in control animals (P < 0.01). Sequencing revealed that DGGE bands, which were suppressed in pectin-fed rats, represented Gram-negative anaerobic rods belonging to the phylum Bacteroidetes, whereas bands that became more prominent represented mainly Gram-positive anaerobic rods belonging to the phylum Firmicutes, and specific species belonging to the Clostridium Cluster XIVa.Quantitative real-time PCR confirmed a lower amount of given Bacteroidetes species in the pectin-fed rats as well as in the apple-fed rats in the four-week study (P < 0.05). Additionally, a more than four-fold increase in the amount of Clostridium coccoides (belonging to Cluster XIVa), as well as of genes encoding butyryl-coenzyme A CoA transferase, which is involved in butyrate production, was detected by quantitative PCR in fecal samples from the pectin-fed animals.ConclusionsOur findings show that consumption of apple pectin (7% in the diet) increases the population of butyrate- and β-glucuronidase producing Clostridiales, and decreases the population of specific species within the Bacteroidetes group in the rat gut. Similar changes were not caused by consumption of whole apples, apple juice, purée or pomace.

Biological effects of fruit and vegetables

A strong and persistent effect of plant-derived foods on the prevention of lifestyle diseases has emerged from observational studies. Several groups of constituents in plants have been identified as potentially health promoting in animal studies, including cholesterol-lowering factors, antioxidants, enzyme inducers, apoptosis inducers etc. In human intervention studies the dose levels achieved tend to be lower than the levels found to be effective in animals and sampling from target organs is often not possible. A controlled dietary human intervention study was performed with forty-three volunteers, providing 600 g fruit and vegetables/d or in the controls a carbohydrate-rich drink to balance energy intake. Surrogate markers of oxidative damage to DNA, protein and lipids, enzymic defence and lipid metabolism were determined in blood and urine. It was found that a high intake of fruit and vegetables tends to increase the stability of lipids towards oxidative damage. Markers of oxidative enzymes indicate a steady increase in glutathione peroxidase (GPX1) activity in erythrocytes during intervention with fruit and vegetables but there is no effect on GPX1 transcription levels in leucocytes. No change occurs in glutathione-conjugating or -reducing enzyme activities in erythrocytes or plasma, and there are no effects on the transcription of genes involved in phase 2 enzyme induction or DNA repair in leucocytes. Fruit and vegetable intake decreases the level of total cholesterol and LDL-cholesterol, but does not affect sex hormones. In conclusion, it has been shown that total cholesterol and LDL-cholesterol, markers of peripheral lipid oxidation, and erythrocyte GPX1 activity are affected by high intakes of fruit and vegetables. This finding provides support for a protective role of dietary fruit and vegetables against CVD.

Effect of long-term selenium yeast intervention on activity and gene expression of antioxidant and xenobiotic metabolising enzymes in healthy elderly volunteers from the Danish Prevention of Cancer by Intervention by Selenium (PRECISE) Pilot Study

Numerous mechanisms have been proposed to explain the anti-carcinogenic effects of Se, among them altered carcinogen metabolism. We investigated the effect of Se supplementation on activities of glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione S-transferase (GST) in different blood compartments, and expression of selected phase 1 and phase 2 genes in leucocytes (GPX1, gamma-glutamylcysteine ligase catalytic subunit (GCLC), AP-1 transcription factor Fos-related antigen 1 (Fra1), NAD(P)H:quinone oxidoreductase (NQO1), and aryl hydrocarbon receptor repressor (AhRR)). Healthy elderly Danes (n 105; age 71.3 (SD 4.26) years; 36% reporting use of multivitamin/mineral supplements) participated and were supplemented daily for 5 years with placebo, 100 microg, 200 microg or 300 microg Se as Se-enriched yeast (SelenoPrecise). Blood samples were collected after 5 years of intervention. When all four groups were compared we found no effect of Se supplementation on plasma GPX or GR, on erythrocyte GPX, GR or GST, or on thrombocyte GR or GST. We found increased thrombocyte GPX activity at the two highest dosage levels in women only, but not in men. No effects on GPX1, NQO1 or AhRR gene expression were found. When all Se-supplemented groups were pooled we found significant down regulation of the expression of some phase 2 genes (GCLC, Fra1). A significant increase in AhRR gene expression with smoking was found but was independent of Se supplementation. Down regulation of phase 2 genes could increase the risk of cancer. However, further studies are needed to establish whether the observed effect in leucocytes reflects a similar expression pattern in target tissues.

Cloning and sequencing of cDNAs specifying a novel class of phosphoribosyl diphosphate synthase in Arabidopsis thaliana.

cDNAs specifying four active phosphoribosyl diphosphate synthase isozymes were isolated from an Arabidopsis thaliana cDNA library. In contrast to other phosphoribosyl diphosphate synthases the activity of two of the A. thaliana isozymes are independent of Pi. Amino acid sequence comparison and phylogenetic analysis indicate that these two isozymes belong to a novel class of phosphoribosyl diphosphate synthases.

Effects of an onion by-product on bioactivity and safety markers in healthy rats

Onions are excellent sources of bioactive compounds including fructo-oligosaccharides (FOS) and polyphenols. An onion by-product was characterised in order to be developed as a potentially bioactive food ingredient. Our main aim was to investigate whether the potential health and safety effects of this onion by-product were shared by either of two derived fractions, an extract containing the onion FOS and polyphenols and a residue fraction containing mainly cell wall materials. We report here on the effects of feeding these products on markers of potential toxicity, protective enzymes and gut environment in healthy rats. Rats were fed during 4 weeks with a diet containing the products or a control feed balanced in carbohydrate. The onion by-product and the extract caused anaemia as expected in rodents for Allium products. No other toxicity was observed, including genotoxicity. Glutathione reductase (GR) and glutathione peroxidase (GPx1) activities in erythrocytes increased when rats were fed with the onion extract. Hepatic gene expression of Gr, Gpx1, catalase, 5-aminolevulinate synthase and NAD(P)H:quinone oxidoreductase was not altered in any group of the onion fed rats. By contrast, gamma-glutamate cysteine ligase catalytic subunit gene expression was upregulated but only in rats given the onion residue. The onion by-products as well as the soluble and insoluble fractions had prebiotic effects as evidenced by decreased pH, increased butyrate production and altered gut microbiota enzyme activities. In conclusion, the onion by-products have no in vivo genotoxicity, may support in vivo antioxidative defence and alter the functionality of the rat gut microbiota.

Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure

Spinach 5‐phospho‐d‐ribosyl α‐1‐diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of Pi; it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts ATP, dATP, GTP, CTP, and UTP as diphosphoryl donors. All of these properties are characteristic for class II PRPP synthases. Km values for ATP and ribose 5‐phosphate are 77 and 48 μM, respectively. Gel filtration reveals a molecular mass of the native enzyme of ∼110 kD, which is consistent with a homotrimer. Secondary structure prediction shows that spinach PRPP synthase isozyme 4 has a general folding similar to that of Bacillus subtilis class I PRPP synthase, for which the three‐dimensional structure has been solved, as the position and extent of helices and β‐sheets of the two enzymes are essentially conserved. Amino acid sequence comparison reveals that residues of class I PRPP synthases interacting with allosteric inhibitors are not conserved in class II PRPP synthases. Similarly, residues important for oligomerization of the B. subtilis enzyme show little conservation in the spinach enzyme. In contrast, residues of the active site of B. subtilis PRPP synthase show extensive conservation in spinach PRPP synthase isozyme 4.

Class II Recombinant Phosphoribosyl Diphosphate Synthase from Spinach PHOSPHATE INDEPENDENCE AND DIPHOSPHORYL DONOR SPECIFICITY

A recombinant form of spinach (Spinacia oleracea) phosphoribosyl diphosphate (PRPP) synthase isozyme 3 resembling the presumed mature enzyme has been synthesized in an Escherichia coli strain in which the endogenous PRPP synthase gene was deleted, and has been purified to near homogeneity. Contrary to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of Pi, and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows an unusual low specificity toward diphosphoryl donors by accepting dATP, GTP, CTP, and UTP in addition to ATP. The kinetic mechanism of the enzyme is an ordered steady state Bi Bi mechanism with K ATPand K Rib-5-P values of 170 and 110 μm, respectively, and a V maxvalue of 13.1 μmol (min × mg of protein)−1. The enzyme has an absolute requirement for magnesium ions, and maximal activity is obtained at 40 °C at pH 7.6.

Short-term effects of dietary advanced glycation end products in rats

Dietary advanced glycation end products (AGE) formed during heating of food have gained interest as potential nutritional toxins with adverse effects on inflammation and glucose metabolism. In the present study, we investigated the short-term effects of high and low molecular weight (HMW and LMW) dietary AGE on insulin sensitivity, expression of the receptor for AGE (RAGE), the AGE receptor 1 (AGER1) and TNF-α, F2-isoprostaglandins, body composition and food intake. For 2 weeks, thirty-six Sprague-Dawley rats were fed a diet containing 20% milk powder with different proportions of this being given as heated milk powder (0, 40 or 100%), either native (HMW) or hydrolysed (LMW). Gene expression of RAGE and AGER1 in whole blood increased in the group receiving a high AGE LMW diet, which also had the highest urinary excretion of the AGE, methylglyoxal-derived hydroimidazolone 1 (MG-H1). Urinary excretion of N ε-carboxymethyl-lysine increased with increasing proportion of heat-treated milk powder in the HMW and LMW diets but was unrelated to gene expression. There was no difference in insulin sensitivity, F2-isoprostaglandins, food intake, water intake, body weight or body composition between the groups. In conclusion, RAGE and AGER1 expression can be influenced by a high AGE diet after only 2 weeks in proportion to MG-H1 excretion. No other short-term effects were observed.

BACILLUS CALDOLYTICUS PRS GENE ENCODING PHOSPHORIBOSYLDIPHOSPHATE SYNTHASE

The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gcaD) encoding N-acetylglucosamine-1-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus PRPP synthase was resistant to heat treatment at 70 degrees C to a much higher extent than PRPP synthase from B. subtilis.