Molecular and FISH analyses of a 53-kbp intact DNA fragment inserted by biolistics in wheat (Triticum aestivum L.) genome.

KEY MESSAGE: A large, 53-kbp, intact DNA fragment was inserted into the wheat ( Triticum aestivum L.) genome. FISH analyses of individual transgenic events revealed multiple insertions of intact fragments. Transferring large intact DNA fragments containing clusters of resistance genes or complete metabolic pathways into the wheat genome remains a challenge. In a previous work, we showed that the use of dephosphorylated cassettes for wheat transformation enabled the production of simple integration patterns. Here, we used the same technology to produce a cassette containing a 44-kb Arabidopsis thaliana BAC, flanked by one selection gene and one reporter gene. This 53-kb linear cassette was integrated in the bread wheat (Triticum aestivum L.) genome by biolistic transformation. Our results showed that transgenic plants harboring the entire cassette were generated. The inheritability of the cassette was demonstrated in the T1 and T2 generation. Surprisingly, FISH analysis performed on T1 progeny of independent events identified double genomic insertions of intact fragments in non-homoeologous positions. Inheritability of these double insertions was demonstrated by FISH analysis of the T1 generation. Relative conclusions that can be drawn from molecular or FISH analysis are discussed along with future prospects of the engineering of large fragments for wheat transformation or genome editing.

Identification of glycosyltransferases involved in cell wall synthesis of wheat endosperm.

Plant cell walls are complex structures critical for plant fitness and valuable for human nutrition as dietary fiber and for industrial uses such as biofuel production. The cell wall polysaccharides in wheat endosperm consist of two major polymers, arabinoxylans and beta-glucans, as well as other minor components. Most of these polysaccharides are synthesized in the Golgi apparatus but the mechanisms underlying their synthesis have yet to be fully elucidated and only a few of the enzymes involved have been characterized. To identify actors involved in the wheat endosperm cell wall formation, we used a subcellular fractionation strategy to isolate Golgi-enriched fractions from endosperm harvested during active cell wall deposition. The proteins extracted from these Golgi-enriched fractions were analyzed by LC-MS/MS. We report the identification of 1135 proteins among which 64 glycosyltransferases distributed in 17 families. Their potential function in cell wall synthesis is discussed. In addition, we identified 63 glycosylhydrolases, some of which may be involved in cell wall remodeling. Several glycosyltransferases were validated by showing that when expressed as fusion proteins with a fluorescent reporter, they indeed accumulate in the Golgi apparatus. Our results provide new candidates potentially involved in cell wall biogenesis in wheat endosperm.

Retinol mobilization from cultured rat hepatic stellate cells does not require retinol binding protein synthesis and secretion.

Retinol mobilization from retinyl esters stores of hepatic stellate cells (HSCs) is a key step in the regulation of mammalian retinol homeostasis, but the precise mechanisms of such a mobilization are still poorly understood. Using primary cultures of HSCs, we first demonstrated that HSCs expressed immunoreactivity against retinol-binding-protein (RBP) when cultured in a medium containing RBP but were unable to synthesize RBP transcripts and proteins. Using pulse and chase-type experiments, we demonstrated that radioactive retinol was released in culture medium without binding proteins. Inhibition of protein secretion by brefeldin A did not modify quantitatively retinol release. This data ruled out, for the first time, the direct involvement of RBP in retinol mobilization from HSCs. Moreover, HSCs co-cultured with primary isolated hepatocytes displayed an increase of retinol transfer from HSCs to hepatocytes when they established direct physical contacts, as compared with co-cultures without contact. Based on this latter data, a mechanism of retinol mobilization from HSCs via the hepatocytes using retinol transfer through cellular membranes is proposed.

Comparison of the postprandial plasma vitamin A response in young and older adults.

To assess the influence of age on vitamin A intestinal and liver metabolism in humans, the postprandial plasma concentrations of intestinal-originated vitamin A, i.e., retinyl esters, and liver-originated vitamin A, i.e., retinol, were compared in eight young (20-30 years old) and eight elderly (64-72 years old) healthy men. Plasma and chylomicron retinyl esters and retinol concentrations were measured for up to 24 h following the intake of a test meal that contained 23,300 RE retinyl palmitate. The chylomicron retinyl palmitate response (area under the curve) was not significantly different between the two groups, but its peak was slightly delayed (1 h) in the elderly men. The proportion of the different retinyl esters secreted in the chylomicrons was not significantly different between the two groups. The postprandial plasma retinol concentration did not change in the young participants, whereas it significantly increased in the elderly. These results suggest that vitamin A intestinal absorption and retinol intestinal esterification processes are not markedly modified in the elderly, whereas the chylomicron clearance and the regulation of postprandial plasma retinol concentration are apparently altered in these subjects.

Postprandial chylomicron and plasma vitamin E responses in healthy older subjects compared with younger ones.

The effect of ageing on vitamin E bioavailability in humans was assessed by comparing chylomicron and plasma alpha-tocopherol postprandial concentrations after a dose of vitamin E (432 or 937 IU as d1-alpha-tocopherol acetate), in eight young (20-30 years old) and eight healthy elderly men (64-72 years old). The fasting plasma alpha-tocopherol concentration was significantly higher in the elderly (33 +/- 2 mumol L-1) than in the young (22 +/- 2 mumol L-1). In both groups, the plasma and chylomicron alpha-tocopherol postprandial concentrations were significantly, approximately twofold, higher after the 937-IU meal than after the 432-IU meal. For both test meals, the chylomicron alpha-tocopherol areas under the curve were significantly lower in the elderly than in the young subjects: 98.9 +/- 16.5 (young group) vs. 55.3 +/- 7.8 (elderly group) mumol L-1 h for the 937-IU test meal and 60.4 +/- 14.1 (young group) vs. 26.0 +/- 7.6 (elderly group) mumol L-1 h for the 432-IU test meal, whereas the plasma alpha-tocopherol area under the curve was significantly higher in elderly than in young subjects: 337.56 +/- 16.11 (937-IU test meal) vs. 159.81 +/- 35.55 (432-IU test meal) mumol L-1 h in the young group and 709.55 +/- 69.33 (937-IU test meal) vs. 436.39 +/- 41.08 (432-IU test meal) mumol L-1 h in the elderly group. We concluded that (a) the amount of vitamin E appearing in plasma is proportional to the dose ingested (up to 937 IU); (b) the intestinal absorption of vitamin E is not increased, even possibly decreased, in the elderly; and (c) the amount of vitamin E transported by non-chylomicron lipoproteins is apparently higher in the elderly. This suggests that vitamin E postprandial transport is affected by ageing, mainly as the consequence of age-related modifications of lipoprotein metabolism.

Dietary triglycerides, up to 40 g/meal, do not affect preformed vitamin A bioavailability in humans.

OBJECTIVE: Assessing the effect of the dose of dietary triglycerides on preformed vitamin A (retinyl esters) bioavailability in humans. DESIGN: Four test meals containing 15,000 RE retinyl-palmitate and either 0, 15, 30 or 40 g added triglycerides were ingested by eight healthy volunteers, at different days and in a randomized order. SETTING: The study was done in the Hospital Sainte Marguerite, Marseille, France. SUBJECTS: Eight healthy male volunteers were recruited by advertisement. INTERVENTION: Blood samples were collected every hour for seven hours after the test meals intake. Serum and chylomicron (Svedberg flotation unit > 1000) were prepared by centrifugation and retinyl esters were measured by HPLC. RESULTS: The serum retinyl ester response was not significantly lower after the intake of the meal without added triglycerides (7944 +/- 3262 nmol/L h) than after the intake of the fat meals (10012 +/- 2182, 7869 +/- 3157 and 10777 +/- 2067 nmol/L h for the 15, 30 and 40 g-fat meal, respectively), indicating that the serum retinyl ester response was not related to the amount of meal triglycerides. Chylomicron retinyl linoleate response stepwise increased when the amount of meal triglycerides increased while retinyl palmitate and retinyl stearate responses reached a maximum since 15 g triglycerides. Postprandial serum retinol concentration did not change whatever the meal ingested. CONCLUSIONS: (i) a significant amount of preformed vitamin A is apparently absorbed when ingested with trace amount of meal triglycerides only; (ii) meal triglycerides, up to 40 g/meal, do not increase preformed vitamin A bioavailability; (iii) the retinyl ester pattern recovered in the chylomicrons, and probably the esterification process of retinol, is affected by the amount of meal triglycerides; (iv) postprandial retinol homeostasis is not affected by dietary triglycerides.

Carotenoids in biological emulsions: solubility, surface-to-core distribution, and release from lipid droplets.

Data on the physico-chemical properties of carotenoids in biological emulsions are essential to our knowledge of carotenoid metabolism. Therefore, we determined the behavior of carotenoids in phospholipid-stabilized triglyceride emulsions, a model for biological emulsions such as dietary emulsions, triglyceride-rich lipoproteins, and intracellular storage droplets. The solubility of beta-carotene (a model for apolar carotenoids, carotenes) in pure bulk triglycerides (0.112 to 0.141 wt % according to triglycerides) was significantly higher than zeaxanthin (a model for polar carotenoids, xanthophylls) (0.022 to 0.088 wt %). The solubility of both carotenoids increased when the chain-length of the triglycerides' fatty acids decreased. The amount of zeaxanthin associated with lipid droplet dramatically increased in phospholipid-triglyceride droplets as compared to the pure corresponding triglyceride droplets, whereas the amount of beta-carotene associated with lipid droplets increased only slightly beta-Carotene distributed almost exclusively in the core of triolein-lecithin-carotenoid droplets, while zeaxanthin distributed preferentially at the droplet's surface. A significant percentage (8.3%) of zeaxanthin was spontaneously transferred from lipid droplets to aqueous phase and the remaining part was transferred during triglyceride hydrolysis catalysed by pancreatic lipase, while beta-carotene absolutely required triglyceride lipolysis to be transferred to the aqueous phase. Our results show that polar and apolar carotenoids behave differently in biological emulsions. They further our understanding of the bioavailability of polar and apolar carotenoids and of their distribution between lipoprotein particles.

Vitamin A contained in the lipid droplets of rat liver stellate cells is substrate for acid retinyl ester hydrolase.

Vitamin A is stored in the lipid droplets of liver stellate cells (LSCs), as retinyl esters whose hydrolysis is necessary for the secretion of retinol into the blood. Here, we isolated these retinyl esters under their physiological form, i.e., in LSC lipid droplets, which had retained their morphological and biochemical characteristics. These retinyl esters are substrate for an hydrolytic enzyme, whose optimum pH is 4.1, and which is kinetically similar to the acidic retinyl ester hydrolase (aREH) we had previously described (Mercier et al., Biochim. Biophys. Acta (1994) 1212, 176-182). The cellular and subcellular localizations of aREH activity in rat liver suggest that this enzyme could be involved in the hydrolysis of the esterified vitamin A stores.

Vitamin A status in the institutionalized elderly. Critical analysis of four evaluation criteria: dietary vitamin A intake, serum retinol, relative dose-response test (RDR) and impression cytology with transfer (ICT).

Infraclinical vitamin A deficiencies may be health-threatening for elderly people, yet they are difficult to assess unequivocally in this population. In this study, we evaluate the vitamin A status of an elderly institutionalized population (49 subjects, 83.6 +/- 6.1 years of age), by examining four different criteria: the dietary vitamin A intake, the retinol concentration in serum, the relative dose-response test and the impression cytology with transfer. The incidence of infra-clinical deficiencies was estimated to be 55% by examining dietary vitamin A intake, 21% by using the RDR test, 6% by the ICT and 2% from serum retinol values. These variations are not due to the choice of threshold values for each of the methods, yet rather to poor correlations between the results given by these methods. Canonical correlation analyses indicate that some parameters related to retinol secretion from the liver, including Zn, prealbumin and retinol-binding protein, can affect individual patient response towards the different methods. Validation of the RDR test in this elderly population was not successful because of poor reproducibility and moderate correction of RDR-detected vitamin A deficiencies by vitamin A supplementation. The method chosen for the determination of vitamin A status in elderly people must be carefully evaluated to account for possible age-related changes in the patient response to the method employed. In the institutionalized elderly population examined in this study, we observed a low vitamin A intake, whereas serum retinol and ICT are within normal ranges and while RDR test's responses appear too variable to draw any conclusion.