The formation of homogentisate in the biosynthesis of tocopherol and plastoquinone in spinach chloroplasts

Homogentisate is the precursor in the biosynthesis of -tocopherol and plastoquinone-9 in chloroplasts. It is formed of 4-hydroxyphenylpyruvate of the shikimate pathway by the 4-hydroxyphenylpyruvate dioxygenase. In experiments with spinach the dioxygenase was shown to be localized predominatedly in the chloroplasts. Envelope membranes exhibit the highest specific activity, however, because of the high stromal portion of chloroplasts, 60–80% of the total activity is housed in the stroma. The incorporation of 4-hydroxyphenylpyruvate into 2-methyl-6-phytylquinol as the first intermediate in the tocopherol synthesis by the two-step-reaction: 4-Hydroxyphenylpyruvate Homogentisate 2-Methyl-6-phytylquinol was demonstrated by using envelope membranes. Homogentisate originates directly from 4-hydroxyphenylpyruvate of the shikimate pathway. Additionally, a bypass exists in chloroplasts which forms 4-hydroxyphenylpyruvate from tyrosine by an L-amino-acid oxidase of the thylakoids and in peroxisomes by a transaminase reaction. Former results about the dioxygenase in peroxisomes were verified.     

Effect of Age on Vitamin E Concentrations in Various Regions of the Brain and a Few Selected Peripheral Tissues of the Rat, and on the Uptake of Radioactive Vitamin E by Various Regions of Rat Brain

Abstract: The concentrations of tocopherols in selected areas of the brains and a few peripheral tissues of 3-, 14-, and 30-month-old male Fischer 344 rats were determined by a high-performance liquid chromatographic method. Throughout the time period studied, α-tocopherol was the only tocopherol detected in the brain. Concentrations of α-tocopherol increased significantly with age in medulla and spinal cord whereas no such change was seen in other brain areas. Among the peripheral tisues, total tocopherol concentrations increased with age in the liver and adipose tissue while no significant changes were observed in the heart. The pattern of uptake of radioactive α-tocopherol from the serum by the various areas of the brain was similar for the 3-and 14-month-old animals even though the brains from the 14-month-old animals took up less of the radioactive compound. Measurable amounts of tocopherol esters were not present in the tissues of the 30-month-old animals.     

Effect of High Doses of Dietary Vitamin E on the Concentrations of Vitamin E in Several Brain Regions, Plasma, Liver, and Adipose Tissue of Rats

The object of this study was to assess the influence of high levels of dietary vitamin E on vitamin E concentrations in specific areas of the brain. Four-week-old male rats were fed vitamin E-deficient, control, and high-vitamin E (1,000 IU/kg) diets for 4 months. Concentrations of alpha-tocopherol in serum, adipose tissue, liver, cerebrum, cerebellum, and striatum were determined by liquid chromatography with fluorescence detection. In the high-vitamin E group, alpha-tocopherol concentrations in cerebrum, cerebellum, and striatum increased uniformly to 1.4-fold of values in controls; serum, adipose tissue, and liver attained even higher concentrations: 2.2-, 2.2-, and 4.6-fold, respectively, of control values. As observed before, brain levels of alpha-tocopherol were somewhat resistant to vitamin E deficiency, in contrast to the peripheral tissues.     

Increase of the tocochromanol content in transgenic Brassica napus seeds by overexpression of key enzymes involved in prenylquinone biosynthesis

Lipid soluble tocochromanols, only synthesised in photosynthetic organisms, are industrially interesting compounds because of their antioxidative properties and their essential function in nutrition. In order to increase the tocochromanol content in the seed oil of transgenic plants, approaches were undertaken to engineer the flux of substrates and intermediates through the pathway. To this end, we overexpressed genes encoding hydroxyphenylpyruvate dioxygenases, alone or in combination with chimeric homogentisate phytyltransferase and tocopherol cyclase genes, in seeds of transgenic Brassica napus plants and analysed total tocochromanol content and composition. Overexpression of chimeric hydroxyphenylpyruvate dioxygenase genes, both in the cytosol or in the plastids of developing seeds, yielded a slight although significant increase in total tocochromanol level. Coexpression of a hydroxyphenylpyruvate dioxygenase gene with both a homogentisate phytyltransferase gene and a tocopherol cyclase gene elevated this effect with maximum increases of up to two-fold in individual lines and this phenotype was found to be stably inherited. These data showed that the three enzymes are critical in determining the total tocochromanol content in the seed oil of Brassica napus plants, while the tocopherol cyclase, unlike hydroxyphenylpyruvate dioxygenase and homogentisate phytyltransferase, had additionally an effect on the relative abundance of individual tocochromanol species and resulted in an increase of δ-tocopherol and plastochromanol-8 in the seeds.Mirela Raclaru and Jens Gruber contributed equally     

Analysis of the pattern of gene expression during human adipogenesis by DNA microarray

High density DNA microarrays containing over 5000 cDNA clones were used to carry out a comprehensive investigation of gene expression during adipogenesis. Complex probes synthesized from total RNA were hybridized to the arrays to determine the level of mRNA expression of each arrayed gene. Thirty three genes (29 known and 4 ESTs with no identified homologies) have been found to alter their level of expression more than 2.5-fold after differentiation. The quantitative measurement by DNA array was in good agreement with conventional Northern blot analysis of selected genes. Our results demonstrate that utilization of a DNA array is a speedy, efficient and quantitative approach to profile the expression of a large number of genes.     

Vitamin E sensitive genes in the developing rat fetal brain: a high-density oligonucleotide microarray analysis

Vitamin E (tocopherols and tocotrienols) is essential for normal neurological function. Recently we have reported that the neuroprotective properties of tocotrienols are much more potent than that of the widely studied tocopherols (Sen, C.K., Khanna, S., Roy, S. and Parker, L. (2000) J. Biol. Chem. 275, 13049–13055). The objective of this study was to evaluate whether (i) oral supplementation of tocotrienols during pregnancy is bioavailable to fetal and mother brains; (ii) short-term change in dietary vitamin E levels of pregnant rats influences gene expression profile of developing fetal brains. We report that dietary tocotrienol is bioavailable to both mother and fetal brains. The enrichment is more in fetal brain tissue. Using a GeneChip microarray expression profiling approach we have identified a specific set of vitamin E sensitive genes in the developing rat fetal brain.     

Genetic dissection of tocopherol content and composition in maize grain using quantitative trait loci analysis and the candidate gene approach

Tocopherols are essential micronutrients for humans and animals, with several beneficial effects in plants. Among cereals, only maize grains contain high concentrations of tocopherols. In this investigation we analyzed, during 2004 and 2005, by high-performance liquid chromatography (HPLC), a population of 233 recombinant inbred lines (RIL) which were derived from two diverse parents and had extremely variable tocopherol content and composition. A genetic map was constructed using 208 polymorphic molecular markers including gene-targeted markers based on six candidate genes of the tocopherol biosynthesis pathway (HPPD, VTE1, VTE3, VTE4, P3VTE5, and P4VTE5). Thirty-one quantitative trait loci (QTL) associated with quantitative variation of tocopherol content and composition were identified by composite interval mapping (CIM); these were located on sixteen genomic regions covering all the chromosomes except chromosome 4. Most (65%) QTL were co-located, suggesting that in some cases the same QTL predominantly affected the amounts of more than one tocopherol. Two candidate genes, HPPD and VTE4 showed co-localization with major QTL for tocopherol content and composition whereas only one interval (umc1075–umc1304) on chromosome eight exhibited a QTL for α, δ, γ, and total tocopherols with high LOD and PVE values. The candidate genes associated with tocopherol content and with composition, especially VTE4 and HPPD, could be precisely used for alteration of the tocopherol content and composition of maize grains by development of functional markers. Other identified major QTL especially those on chromosomes 8, 1, and 2 (near candidate gene VTE5) can also be used for improvement of maize grain quality by marker-assisted selection.     

Effect of aging on the ability of preconditioning to protect rat hearts from ischemia-reperfusion injury

Ischemic preconditioning (IP) reduces infarct size in young animals; however, its impact on aging is underinvestigated. The effect of variations in IP stimuli was studied in young, middle-aged, and aged rat hearts. Isolated hearts underwent 35 min of regional ischemia and 120 min of reperfusion. Hearts with IP were subjected to either one ischemia-reperfusion cycle (5 min of ischemia and 5 min of reperfusion per cycle) or three successive cycles before 35 min of regional ischemia. Additional studies investigated the effects of pharmacological preconditioning in aged hearts using the adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine, the protein kinase C analog 1,2-dioctanoyl-sn-glycerol, and the mitochondrial ATP-sensitive potassium (K(ATP))-channel opener diazoxide. Infarct sizes indicated that the aged rat heart could not be preconditioned via ischemic or pharmacological means. The middle-aged rat heart had a blunted IP response compared with the young adult (only an increased IP stimulus caused a significant reduction in infarct size). These results suggest that there are defects within the IP signaling cascade of the aged heart. Clinical relevance is important if we are to use any IP-like mimetics to the benefit of an aging population.     

The effect of gamma-tocopherol on proliferation, integrin expression, adhesion, and migration of human glioma cells

The effect of vitamin E on proliferation, integrin expression, adhesion, and migration in human glioma cells has been studied. gamma-tocopherol at 50 microM concentration exerted more inhibitory effect than alpha-tocopherol at the same concentration on glioma cell proliferation. Integrin alpha5 and beta1 protein levels were increased upon both alpha- and gamma-tocopherol treatments. In parallel, an increase in the alpha5beta1 heterodimer cell surface expression was observed. The tocopherols inhibited glioma cell-binding to fibronectin where gamma-tocopherol treatment induced glioma cell migration. Taken together, the data reported here are consistent with the notion that the inhibition of glioma cell proliferation induced by tocopherols may be mediated, at least in part, by an increase in integrin alpha5 and beta1 expression. Cell adhesion is also negatively affected by tocopherols, despite a small increase in the surface appearance of the alpha5beta1 heterodimer. Cell migration is stimulated by gamma-tocopherol. It is concluded that alpha5 and beta1 integrin expression and surface appearance are not sufficient to explain all the observations and that other integrins or in general other factors may be associated with these events.     

Analysis of the variable effect of dietary vitamin E supplements on experimental atherosclerosis

Vitamin E inhibits processes thought to be important in the development of atherosclerosis but clinical trials to determine its effect on cardiovascular disease have given variable results, the majority being negative. The reasons for this are unclear. Animal trials can be better controlled and use more rigorous measures of lesion progression than human trials. The present study reviewed trials using rabbits and mice to determine whether they also are variable and, if so, to uncover methodological differences that may account for the different outcomes. A large number of trials examining the effect of vitamin E supplements on experimental atherosclerosis were identified. Using rigorous selection criteria, a well-defined group was selected for further investigation. Almost all the mice trials showed a significant effect of vitamin E, but only around one-third of the rabbit trials did so. When the rabbit trials were divided into those that did and those that did not observe significant effects, no single factor was found that could account for the dichotomy. However, when the percentage reduction in disease was considered, rather than the within-trial significance level, there were clear dose-dependent effects of vitamin E on disease severity in heritable hyperlipidaemic rabbits, and in genetically normal rabbits made hyperlipidaemic with cholesterol alone; the dose dependence was different in the two groups, the heritable hyperlipidaemic rabbits showing a near ten-fold lower sensitivity. The high doses required to affect experimental atherosclerosis may, if applicable to other species, help explain the absence of effects in many human trials.     

NTGLO: a tobacco homologue of the GLOBOSA floral homeotic gene of Antirrhinum majus: cDNA sequence and expression pattern

We report the cloning and DNA sequence of a cDNA from Nicotiana tabacum, NTGLO, as well as the pattern of expression of the NTGLO gene in wild-type tobacco plants. The NTGLO cDNA encodes a protein of 209 amino acids, which shows 73% identity with the GLO protein encoded by the GLO gene of Antirrhinum majus, a homeotic gene involved in the genetic control of flower development. Northern blot analysis shows that the NTGLO gene is expressed mainly in floral organs and, within the flower, expression is restricted to petals and stamens. The NTGLO gene most probably represents a true homologue of the GLO gene because: i) the MADS boxes, of the two genes are highly homologous (56 out of 58 amino acids are identical): ii) at the carboxyterminal a block of 19 amino acids is perfectly conserved between the NTGLO and GLO proteins and iii) their expression patterns in floral organs are identical.     

Characterizing the expression of the human olfactory receptor gene family using a novel DNA microarray

Background  

Olfactory receptor (OR) genes were discovered more than a decade ago, when Buck and Axel observed that, in rats, certain G-protein coupled receptors are expressed exclusively in the olfactory epithelium. Subsequently, protein sequence similarity was used to identify entire OR gene repertoires of a number of mammalian species, but only in mouse were these predictions followed up by expression studies in olfactory epithelium. To rectify this, we have developed a DNA microarray that contains probes for most predicted human OR loci and used that array to examine OR gene expression profiles in olfactory epithelium tissues from three individuals.     

Effect of serial passage on gene expression in MC3T3-E1 preosteoblastic cells: a microarray study

The osteoblastic function of mouse preosteoblastic MC3T3-E1 cells, as measured by alkaline phosphatase activity and osteocalcin secretion, decreases after serial passage. To uncover genes responsible for decreased osteoblastic function in high-passage cells, we have studied passage-dependent change of gene expression in MC3T3-E1 cells. Changes in the expression pattern of 2000 selected genes were examined simultaneously by comparing mRNA levels between MC3T3-E1 cells at passage 20 and passage 60 using the cDNA microarray analysis. Significant changes in the steady-state abundance of 27 mRNAs were observed in response to different passage numbers, including 17 known genes, 4 ESTs with homology to known genes, and 6 genes with no previously described function or homology. Northern blot analysis was used to verify and quantify the expression of selected genes, and revealed a significant higher level of up- and down-regulation compared to microarray data. These results indicate the existence of a significant change in gene expression in osteoblastic cells undergoing serial passages. Such changes might be responsible for a reduction in bone regeneration in older osteoblasts. Potential roles of selected genes in bone aging are discussed.