A spectrophotometric method has been developed for the quantitative determination of antioxidant capacity. The assay is based on the reduction of Mo(VI)to Mo(V) by the sample analyte and the subsequent formation of a green phosphate/Mo(V) complex atacidic pH. The method has been optimized and characterized with respect to linearity interval, repetitivity and reproducibility, and molar absorption coefficients for the quantitation of several antioxidants, including vitamin E. The phosphomolybdenum method, in combination with hexane monophasic extraction, has also been adapted for the specific determination of vitamin E in seeds. The results obtained with the proposed method were validated by comparison with a standard HPLC method. The phosphomolybdenum method is routinely applied in our laboratory to evaluate the total antioxidant capacity of plant extracts and to determine vitamin E in a variety of grains and seeds, including corn and soybean.There is an increasing interest in the use and measurement of antioxidants in food, pharmaceutical, and cosmetic industries. This interest is rooted in the cumulative evidence that connects oxidative stress with numerous d egenerative d isorders ranging f rom p remature aging, pr ostaglandin-mediated i nflammatory processes, cancer, and a l ong series of diseases i n which free r adicals ar e implicated (1, 2). In addition, many states implement very rigorous regulations on the use of food preservatives, so that they only allow the use of natural antioxidants. Basically, these include vitamin C (ascorbic acid), vitamin E (tocopherols and tocotrienols), and relatively complex extracts from a number of plant species (Rosmarinus officinalis, Nerium oleander, and Myrtus comunis).The e arly works by Ch ipault et a l. (3-5) w ere t he precursors o f m any s tudies o n t he antioxidant capacity of a number of plant extracts with potential applications as preservatives in the food, cosmetics, and p harmaceutical industries (6 -12). The c urrent i nterest of our l aboratory i n t he b iosynthesis o f tocopherols in plants and in the identification of alternative sources of natural antioxidants prompted us to develop a m ethod for t he e valuation o f water-soluble a nd fa t-soluble a ntioxidant capacity. T he phosphomolybdenum method that we propose in this article is now systematically used by us in extensive screenings o f samples o f v ery different origins a nd composition i n our search f or natural sources of vitamin E and other powerful antioxidants. MATERIALS AND METHODSChemicals an d r eagents. Ammonium mo lybdate; ascorbic ac id; r educed g lutathione; b utyl hydroxytoluene (BHT);3 α-, γ-, an d δ-tocopherol; a nd t he internal standard α-tocopherol a cetate were obtained from Sigma (St. Louis, MO). HPLC gr ade methanol, h exane, ethanol, dimethyl sulfoxide, and analytical grade sodium phosphate and sulfuric a cid were from Merck (Darmstadt, Germany). All other chemicals were of analytical grade.Instrumentation. The m olecular a bsorption s pectra and absorbance a t s pecifi...

The antioxidant potential of roselle (Hibiscus sabdariffa L.) extracts was studied. Different plant organs, including seeds, stems, leaves, and sepals, were analyzed with respect to their water-soluble antioxidant capacity, lipid-soluble antioxidant capacity, and tocopherol content, revealing that roselle seeds are a good source of lipid-soluble antioxidants, particularly gamma-tocopherol. Roselle seed oil was extracted and characterized, and its physicochemical parameters are summarized: acidity, 2.24%; peroxide index, 8.63 meq/kg; extinction coefficients at 232 (k(232)) and 270 nm (k(270)), 3.19 and 1.46, respectively; oxidative stability, 15.53 h; refractive index, 1.477; density, 0.92 kg/L; and viscosity, 15.9 cP. Roselle seed oil belongs to the linoleic/oleic category, its most abundant fatty acids being C18:2 (40.1%), C18:1 (28%), C16:0 (20%), C18:0 (5.3%), and C19:1 (1.7%). Sterols include beta-sitosterol (71.9%), campesterol (13.6%), Delta-5-avenasterol (5.9%), cholesterol (1.35%), and clerosterol (0.6%). Total tocopherols were detected at an average concentration of 2000 mg/kg, including alpha-tocopherol (25%), gamma-tocopherol (74.5%), and delta-tocopherol (0.5%). The global characteristics of roselle seed oil suggest that it could have important industrial applications, adding to the traditional use of roselle sepals in the elaboration of karkade tea.

A ureidoglycolate-degrading activity was analyzed in different organs of chickpea (Cicer arietinum). Activity was detected in all the tissues analyzed, but highest levels of specific activity were found in pods, from which it has been purified and characterized. This is the first ureidoglycolate-degrading activity that has been purified to homogeneity from any photosynthetic organism. Only one ureidoglycolate-degrading activity was found during the purification. The enzyme was purified 1,500-fold, and specific activity for the pure enzyme was 8.6 units mg Ϫ1 , which corresponds with a turnover number of 1,600 min Ϫ1 . The native enzyme has a molecular mass of 180 kD and consists of six identical or similar-sized subunits of 31 kD each. The enzyme exhibited hyperbolic, Michaelian kinetics for (Ϫ) ureidoglycolate with K m values of 6 and 10 m in the presence or absence of Mn 2ϩ , respectively. Optimum pH was between 7 and 8 and maximum activity was found at temperatures above 70°C, the enzyme being extremely stable and resistant to heat denaturation. The activity was inhibited by EDTA and enhanced by several bivalent cations, thus suggesting that the enzyme is a metalloprotein. This enzyme has been characterized as a ureidoglycolate urea-lyase (EC 4.3.2.3), which catalyzes the degradation of (Ϫ) ureidoglycolate to glyoxylate and urea. This is the first time that such an activity is detected in plant tissues. A possible function for this activity and its implications in the context of nitrogen mobilization in legume plants is also discussed.

Little is known about the molecular basis of the influence of external carbon/nitrogen (C/N) ratio and other abiotic factors on phytohormones regulation during seed germination and plant developmental processes, and the identification of elements that participate in this response is essential to understand plant nutrient perception and signaling. Sugars (sucrose, glucose) and nitrate not only act as nutrients but also as signaling molecules in plant development. A connection between changes in auxin transport and nitrate signal transduction has been reported in Arabidopsis thaliana through the NRT1.1, a nitrate sensor and transporter that also functions as a repressor of lateral root growth under low concentrations of nitrate by promoting auxin transport. Nitrate inhibits the elongation of lateral roots, but this effect is significantly reduced in abscisic acid (ABA)-insensitive mutants, what suggests that ABA might mediate the inhibition of lateral root elongation by nitrate. Gibberellin (GA) biosynthesis has been also related to nitrate level in seed germination and its requirement is determined by embryonic ABA. These mechanisms connect nutrients and hormones signaling during seed germination and plant development. Thus, the genetic identification of the molecular components involved in nutrients-dependent pathways would help to elucidate the potential crosstalk between nutrients, nitric oxide (NO) and phytohormones (ABA, auxins and GAs) in seed germination and plant development. In this review we focus on changes in C and N levels and how they control seed germination and plant developmental processes through the interaction with other plant growth regulators, such as phytohormones.

This article presents a study of the influence of the sputtering parameters on the preferred orientation of polycrystalline aluminum nitride thin films. Aluminum nitride films were grown by rf reactive sputtering of an aluminum target in an N2/Ar gas mixture for different values of the deposition parameters: total pressure, nitrogen content in the discharge gas, and substrate bias voltage. The preferred orientation of the films was analyzed by x-ray diffraction. Films with different preferred orientations were obtained, ranging from c-axis oriented films to films with the c axis tilted by up to 61.6° from the substrate normal. The different mechanisms influencing the preferred orientation of the films have been considered, especially the transfer of energy to the adatoms on the substrate by particle bombardment. An analysis of the relation between the deposition parameters and the crystal orientation has allowed us to determine the relative importance of the different particles in the supply of energy to the substrate. We have found that Ar ion bombardment of the film during growth is the most influential mechanism on the preferred orientation of the films. As bombardment becomes more energetic, microcrystals in the film tend to grow with the c axis along the surface normal. The energy of Ar bombardment can be best controlled through the substrate bias voltage, a characteristic that we have employed to obtain AlN films exhibiting pure (00.2) preferred orientation and rocking curves with a full width at half maximum as low as 4.2°.

Summary  Asparagine metabolism in sunflower (Helianthus annuus) was investigated by cDNA cloning, sequence characterization and expression analysis of three genes encoding different isoforms of asparagine synthetase (AS, EC 6.3.5.4).  The AS‐coding sequences were searched for in leaves, roots and cotyledons by using a methodology based on the simultaneous amplification of different cDNAs. Three distinct AS‐coding genes, HAS1, HAS1.1 and HAS2, were identified.  HAS1 and HAS1.1 are twin genes with closely related sequences that share some regulatory features. By contrast, HAS2 is a singular sequence that encodes an incomplete AS polypeptide and shows an unusual regulation. The functionality of both the complete HAS1 and the truncated HAS2 proteins was demonstrated by complementation assays. Northern analysis revealed that HAS1, HAS1.1 and HAS2 were differentially regulated dependent on the organ, the physiological status, the developmental stage and the light conditions.  Asparagine synthetase from sunflower is encoded by a small gene family whose members have achieved a significant degree of specialization to cope with the major situations requiring asparagine synthesis.

A Chlamydomona~' reinhardtff molybdenum cofactor (MoCo).carrier protein (CP), capable of reconstituting nitrate redugtase activity with apoprotein from the Neurospora crassa mutant nit.l, was subjected to experiments of diffusion through a dialysis membrane and gel filtration. CP bonded firmly MoCo and did not release it efficiently unles-~ aponitrate reduetase was present in the incubation mixture, Stability of MoCo bound to CP against air and heat was very similar to that of fre¢-MoCo released from milk xanthine oxidase. Our data strongly suggest that MoCo is directly transferred from CP to aponitrate reductase to form an active enzyme, Molybdenum ¢ofactor; Molybdopterin; Nitrate reductase; Xanthine oxidase

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris L.). A 2-kb cDNA clone of this gene (PVAS1) encodes a protein of 579 amino acids with a predicted molecular mass of 65,265 Da, an isoelectric point of 6.3, and a net charge of -9.3 at pH 7.0. The PVAS1 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS1 complemented an Escherichia coli asparagine auxotroph, which demonstrates that it encodes a glutamine-dependent AS. The PVAS1 protein showed the highest similarity to soybean SAS1, and piled up with other legume ASs to form an independent dendritic group of type-I AS enzymes. Northern blot analyses revealed that the expression pattern of PVAS1 resembles that of PVAS2, another AS previously described in the common bean. Unlike PVAS2, however, PVAS1 was not expressed in the nodule and was not repressed by light, suggesting different functions for these two AS genes.