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PubMed1980

Biosynthesis of Ureides from Purines in a Cell-free System from Nodule Extracts of Cowpea [Vigna unguiculata (L) Walp.].

The synthesis of (14)C-labeled xanthine/hypoxanthine, uric acid, allantoin, allantoic acid, and urea from [8-(14)C]guanine or [8-(14)C]hypoxanthine, but not from [8-(14)C]adenine, was demonstrated in a cell-free extract from N(2)-fixing nodules of cowpea (Walp.). The (14)C recovered in the acid/neutral fraction was present predominantly in uric acid and allantoin (88-97%), with less than 10% of the (14)C in allantoic acid and urea. Time courses of labeling in the cell-free system suggested the sequence of synthesis from guanine to be uric acid, allantoin, and allantoic acid. Ureide synthesis was confined to soluble extracts from the bacteroid-containing tissue, was stimulated by pyridine nucleotides and intermediates of the pathways of aerobic oxidation of ureides, but was completely inhibited by allopurinol, a potent inhibitor of xanthine dehydrogenase (EC 1.2.1.37). The data indicated a purine-based pathway for ureide synthesis by cowpea nodules, and this suggestion is discussed.

Journal Article↗
PubMed1981

Ureide Synthesis in a Cell-Free System from Cowpea (Vigna unguiculata [L.] Walp.) Nodules : STUDIES WITH O(2), pH, AND PURINE METABOLITES.

The effect of O(2) and pH on the in vitro synthesis of (14)C-labeled ureides from [8-(14)C]hypoxanthine in a cell-free system from cowpea nodules was investigated. Under conditions which suppressed uricase (EC 1.7.3.3) activity, namely low O(2) concentrations and low pH, ureide synthesis was inhibited and the (14)C label incorporated into uric acid was increased. Conversely, conditions which increased uricase activity, namely high O(2) concentrations and high pH, also stimulated ureide synthesis, and the (14)C label was incorporated principally into allantoin. The overall response of the system to O(2) concentration and pH indicated that the per cent distribution of total (14)C label incorporated into uric acid was inversely related to that into allantoin. In the present study there was evidence that uricase (EC 1.7.3.3) controlled the in vitro rate of ureide synthesis in the cell-free system. Adenine and guanine inhibited xanthine dehydrogenase (EC 1.2.1.37) and as a consequence ureide synthesis from [8-(14)C]hypoxanthine was also inhibited.

Journal Article↗
PubMed1981

The Appearance of New Active Forms of Trypsin Inhibitor in Germinating Mung Bean (Vigna radiata) Seeds.

Ungerminated seeds of mung bean contain a single major species (F) of trypsin inhibitor with five minor species (A-E) separable on diethylaminoethyl-cellulose. During germination the level of trypsin inhibitory activity decreases from 1.8 units/grams dry weight in ungerminated cotyledons to 1.2 units/grams in cotyledons from seeds germinated 5 days. This decrease is accompanied by major changes in the distribution of inhibitory activity among the inhibitor species. By 48 hours of germination, inhibitor F has largely disappeared with an accompanying rapid increase in inhibitor C. Similarly, though less rapidly, inhibitor E decreases while inhibitor A increases. A similar sequence of changes is found in vitro when purified inhibitor F is incubated with extracts from seeds germinated 96 hours. The combined in vivo and in vitro data suggest a conversion sequence of: F --> E --> C --> A. The in vitro conversion is inhibited by phenylmethyl sulfonyl fluoride but not by iodoacetamide, indicating that at least the initial phases of inhibitor conversion are not catalyzed by the mung bean vicilin peptidohydrolase.

Journal Article↗
PubMed1985

Effects of Ni Deficiency on Some Nitrogen Metabolites in Cowpeas (Vigna unguiculata L. Walp).

Cowpeas grown in nutrient solutions, from which Ni had been removed by a ligand exchange technique, accumulated urea in most tissues. Urea levels were highest (up to 3.1 percent dry weight) in necrotic leaf tips. Urea accumulation in Ni-deficient cowpea tissues amounted to about 1 percent of the total N. The accumulation of urea was presumably associated with the catabolism of N compounds in older tissues and the redistribution of N catabolites within the plant during the reproductive growth. The exclusion of N salts from the nutrient media at a late stage of growth, either with or without added Ni, led to a general amelioration of urea accumulation and a lower level of the related amino acid, arginine, in root and stem tissue. Plant leaves that contained toxic levels of urea and displayed necrotic symptoms had tissue Ni levels ranging from less than 0.01 to 0.15 mug Ni per gram dry weight. Nickel concentrations in tissue from plants not treated with Ni, were initially very low, but increased as the cowpeas matured. Apparently, there was a source of Ni contamination in the Ni-deficient growth media which provided a source of Ni for uptake by the plants during growth. Ureide levels were low and unaffected by Ni deprivation. No evidence for free purines or uric acid accumulation in plant tissues could be found. It is hypothesized that Ni (and urease) participates in the normal N metabolism of these plants during the reproductive phase of growth.

Journal Article↗
PubMed1986

Lipid Composition of Plasma Membranes and Tonoplasts Isolated from Etiolated Seedlings of Mung Bean (Vigna radiata L.).

The lipid composition of plasma membranes and tonoplasts from etiolated mung bean hypocotyls was examined in detail. Phospholipids, sterols, and ceramide monohexoside(s) were the major lipid classes in both membranes. The content of phospholipids on a protein basis was higher in the tonoplast, but the content of total sterols was similar in both membranes. Accordingly, the sterol to phospholipid molar ratio in the plasma membrane was higher than that of the tonoplast. Phosphatidylethanolamine and phosphatidylcholine comprised the major phospholipids in both membranes. Phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol were identified as minor phospholipid components. The content of phosphatidylinositol and phosphatidylglycerol was relatively high in the tonoplast, comprising 11 and 5% of the total phospholipids, respectively. Although special care was taken against the degradative action of phospholipase D and phosphatidic acid phosphatase during the isolation of these membranes, by adding EDTA, EGTA, KF, choline, and ethanolamine to the homogenizing medium, significant amounts of phosphatidic acid, about 15% of the total phospholipids, were detected in the plasma membrane. On the other hand, the content of phosphatidic acid in tonoplasts and other membrane fractions was very low. This fact may indicate that high levels of phosphatidic acid occur naturally in plasma membranes. Phosphatidylglycerol in both membranes and phosphatidylinositol in the tonoplast contained high levels of palmitic acid, which comprised more than 50% of the total fatty acids. Significant differences were observed in the sterol compositions of plasma membranes and tonoplasts. More than 90% of the sterols in the plasma membrane were unesterified, while the tonoplast was enriched in glycosylated sterols, especially acylated sterylglycosides. Ceramide monohexoside was found to be specifically located in these membranes, in particular, in the tonoplast, in which it comprised nearly 17% of the total lipids.

Journal Article↗
PubMed1999

Immunogold labeling of rosette terminal cellulose-synthesizing complexes in the vascular plant vigna angularis

The catalytic subunit of cellulose synthase is shown to be associated with the putative cellulose-synthesizing complex (rosette terminal complex [TC]) in vascular plants. The catalytic subunit domain of cotton cellulose synthase was cloned using a primer based on a rice expressed sequence tag (D41261) from which a specific primer was constructed to run a polymerase chain reaction that used a cDNA library from 24 days postanthesis cotton fibers as a template. The catalytic region of cotton cellulose synthase was expressed in Escherichia coli, and polyclonal antisera were produced. Colloidal gold coupled to goat anti-rabbit secondary antibodies provided a tag for visualization of the catalytic region of cellulose synthase during transmission electron microscopy. With a freeze-fracture replica labeling technique, the antibodies specifically localized to rosette TCs in the plasma membrane on the P-fracture face. Antibodies did not specifically label any structures on the E-fracture face. Significantly, a greater number of immune probes labeled the rosette TCs (i.e., gold particles were 20 nm or closer to the edge of the rosette TC) than did preimmune probes. These experiments confirm the long-held hypothesis that cellulose synthase is a component of the rosette TC in vascular plants, proving that the enzyme complex resides within the structure first described by freeze fracture in 1980. In addition, this study provides independent proof that the CelA gene is in fact one of the genes for cellulose synthase in vascular plants.

Journal Article↗
PubMed2003

Crystallization and preliminary X-ray diffraction studies of the eukaryotic iron superoxide dismutase (FeSOD) from Vigna unguiculata.

Eukaryotic iron superoxide dismutases (FeSODs) are homodimeric proteins that constitute a fundamental protection against free radicals, which can damage essential cellular mechanisms. The protein was cloned and overexpressed in Escherichia coli with an N-terminal His tag. Crystallization experiments of the protein resulted, after several refined screenings, in crystals suitable for X-ray diffraction analysis. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 82.54, b = 48.41, c = 64.28 A, alpha = gamma = 90, beta = 119.66 degrees, and contain one molecule per asymmetric unit. At cryogenic temperatures, the crystals diffracted to a resolution limit of 1.80 A using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF).

Crystallization↗
PubMed2005

Birch pollen-related food allergy to legumes: identification and characterization of the Bet v 1 homologue in mungbean (Vigna radiata), Vig r 1.

BACKGROUND: Recently allergic reactions to legumes mediated by Bet v 1-homologous food allergens were described for soy and peanut. In this study we assessed allergic reactions to another legume, to mungbean seedlings, and identified its Bet v 1-homologous allergen Vig r 1. METHODS: Ten patients were selected who had a history of allergic reactions to mungbean seedlings and a respiratory allergy to birch pollen. The Bet v 1 homologue in mungbean seedlings, Vig r 1, was cloned by a PCR strategy, expressed in Escherichia coli, and purified by preparative SDS-PAGE. In all sera, specific IgE against birch pollen, Bet v 1, Bet v 2, Vig r 1, and the Bet v 1 homologues in soy (Gly m 4) and cherry (Pru av 1) was determined by CAP-FEIA. Cross-reactivity of specific IgE with Vig r 1, Bet v 1, Gly m 4, and Pru av 1 was assessed by immunoblot inhibition. Expression of Vig r 1 during development of mungbean seedlings and under wounding stress was analysed by immunoblotting. The Vig r 1 double band was analysed by matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography/tandem mass spectrometry (LC/MS/MS). RESULTS: All patients were sensitized to birch pollen and Bet v 1, 20% to Bet v 2, and 90% to Gly m 4. Seventy percent of the patients showed IgE binding to a double band at 15 kDa in mungbean extract that was inhibited after pre-incubation of sera with rBet v 1. PCR cloning revealed that the mungbean homologue of Bet v 1 had a molecular weight of 16.2 kDa, a calculated pI of 4.6% and 42.8% amino acid sequence identity with Bet v 1. MS analysis confirmed similarity of the double band with the deduced Vig r 1 sequence, but also indicated the existence of other Vig r 1 isoforms. ImmunoCAP analysis detected IgE against Vig r 1 in 80% of the sera. IgE binding to Vig r 1 was inhibited with Gly m 4 in six of six and with rPru av 1 in four of six patients. Vig r 1 expression occurred during development of seedlings and was increased by wounding stress. CONCLUSIONS: Food allergy to mungbean seedlings can be caused by primary sensitization to birch pollen and is mediated by Vig r 1 in the majority of the patients with birch pollen-related allergy to mungbean seedlings.

Allergens↗
PubMed2006

Effect of plant growth promoting rhizobacteria containing ACC-deaminase on maize (Zea mays L.) growth under axenic conditions and on nodulation in mung bean (Vigna radiata L.).

AIMS: This study was conducted to test the hypothesis that the bacterial strains possessing 1-aminocyclopropane-1-carboxylic acid (ACC)-deaminase activity may also promote growth of inoculated plants and could increase nodulation in legumes upon co-inoculation with rhizobia. METHODS AND RESULTS: Several rhizobacteria were isolated from maize rhizosphere through enrichment on ACC as a sole N source. Purified isolates were screened for growth promotion in maize under axenic conditions and for in vitro ACC-deaminase activity. A significant positive correlation was observed between in vitro ACC-deaminase activity of bacterial cells and root elongation. None of the isolates produced auxins. Bradyrhizobium japonicum produced less amount of auxins but did not carry ACC-deaminase activity. Results of pot experiment revealed that co-inoculation with Bradyrhizobium and plant growth promoting rhizobacteria (PGPR) isolates enhanced the nodulation in mung bean compared with inoculation with Bradyrhizobium alone. CONCLUSIONS: It is highly expected that inoculation with rhizobacteria containing ACC-deaminase hydrolysed endogenous ACC into ammonia and alpha-ketobutyrate instead of ethylene. Consequently, root and shoot growth as well as nodulation were promoted. SIGNIFICANCE AND IMPACT OF THE STUDY: The ACC-deaminase trait could be employed as an efficient tool to screen effective PGPR, which could be successfully used as biofertilizers to increase the growth of inoculated plants as well as nodulation in legumes.

Carbon-Carbon Lyases↗
PubMed2005

Similarities and differences between the characteristics of gibberellin-binding protein and gibberellin 2-oxidases in adzuki bean (Vigna angularis) seedlings.

Gibberellin-binding proteins (GBPs) were purified ca. 230,000 fold. The characteristics of adzuki GBP were examined and compared with those of a recombinant gibberellin 2-oxidase (rVaGA2oxA1) that was fused with glutathione S-transferase (GST). VaGA2oxA1 was most abundantly expressed in etiolated adzuki bean seedlings, and VaGA2oxA1 and GBPs from adzuki bean seedlings showed gibberellin-binding activity when incubated with 2-oxoglutarate and Co2+. Both rVaGA2oxA1 and partially purified GBPs from adzuki bean seedlings showed very similar selectivity to gibberellins in binding assays, where biologically active gibberellins such as GA4, GA3, GA7, and GA1 showed higher binding affinity than biologically inactive gibberellins such as GA8, GA34, and 3-epi-GA4. The polyclonal antibody raised against rVaGA2oxA1 cross-reacted with all rVaGA2oxs (rVaGA2oxA1, rVaGA2oxA2, rVaGA2oxB1, rVaGA2oxB2, and rVaGA2oxB3) whose cDNAs were cloned from adzuki bean seedlings. Treated with the antibody, the recombinants that originally showed gibberellin-binding activity lost both binding activity and enzymatic activity. In contrast to the recombinants, the gibberellin-binding activity of GBPs from adzuki bean seedlings was hardly affected by the antibody treatment. The GBPs showed very weak gibberellin 2-oxidase-like activity, and it was not affected by the antibody treatment either. These observations suggest that a major component that showed GA-binding activity was apparently different from any gibberellin 2-oxidase cloned from the seedlings.

Carrier Proteins↗
PubMed2005

Potential ability of hot water adzuki (Vigna angularis) extracts to inhibit the adhesion, invasion, and metastasis of murine B16 melanoma cells.

The 40% ethanol eluent of the fraction of hot-water extract from adzuki beans (EtEx.40) adsorbed onto DIAION HP-20 resin has many biological activities, for example, antioxidant, antitumorigenesis, and intestinal alpha-glucosidase suppressing activities. This study examined the inhibitory effect of EtEx.40 on experimental lung metastasis and the invasion of B16-BL6 melanoma cells. EtEx.40 was found significantly to reduce the number of tumor colonies. It also inhibited the adhesion and migration of B16-BL6 melanoma cells into extracellular matrix components and their invasion into reconstituted basement membrane (matrigel) without affecting cell proliferation in vitro. These in vivo data suggest that EtEx.40 possesses a strong antimetastatic ability, which might be a lead compound in functional food development.

Animals↗
PubMed1997

Some physicochemical and functional properties of cowpea (Vigna unguiculata) isoelectric protein isolate as a function of pH and salt concentration.

Cowpea isoelectric protein isolate was prepared by alkali extraction followed by acid precipitation at pH 4.5. Physicochemical and functional properties of the isolate were determined at various combinations of pH (3-8) and NaCl concentrations (0.5-2.0 M). Results showed that at all NaCl concentrations, emulsifying activity index, heat coagulability, aromatic hydrophobicity (ARH) and fluorescence intensity (FI) were high at low pH and gradually decreased with increasing pH. Protein solubility and foam stability were low at low pH and high NaCl concentrations, but increased with increase in pH. The relationships of ARH, FI and UV-absorption maxima (Amax) with the functional properties were determined using multiple regression analyses. Significant (P < or = 0.05) equations describing the functional properties on the basis of ARH, FI and Amax were generated; however, the contribution of these parameters was low as indicated by the low correlation coefficients. Regression results suggest that other physicochemical properties (e.g. electrostatic interactions) were also important contributors to the functional properties examined.

Fabaceae↗
PubMed2004

Hot-water extracts from adzuki beans (Vigna angularis) suppress not only the proliferation of KATO III cells in culture but also benzo(a)pyrene-induced tumorigenesis in mouse forestomatch.

Treatment of human stomach cancer KATO III cells with hot-water extracts from adzuki beans led to their growth inhibition as well as apoptosis induction. There are morphological changes in the cultured cells treated with the extracts, by which DNA fragmentation characteristic of apoptosis was actualized both concentration- and time-dependently. In contrast, N-acetyl-L-cysteine suppressed such DNA fragmentation, implying that the extracts from adzuki beans might exert antitumorigenicity via active oxygen-induced apoptosis. In order to verify this hypothesis in animal experiment, the 40% ethanol fraction of hot-water extracts was examined for its preventive effect against benzo(a)pyrene-induced tumorigenesis in the forestomach of A/J mice, given as drinking water containing the above fraction at 0.5-2.0% levels. Consequently, forestomach cancer has turned out to be reduced by 36-62% in tumor weight relative to the control. These results suggest that the fraction of hot-water adzuki extracts may serve as a nutrapharmaceutical or functional food available for cancer prevention.

Animals↗
PubMed2026

Bacillus subtilis isolated from medicinal plants rhizosphere effectively controls Cercospora leaf spot and improves plant growth in mung bean (Vigna radiata).

BACKGROUND: Mung bean is an important leguminous crop, which is reported to face devastating yield losses of up to 70% due to Cercospora leaf spot (CLS) disease. Traditional methods, such as the application of agrochemicals and fungicides, have been used to control CLS, but their intensive use has toxic effects on edible crops. METHODS: To find out a sustainable alternative, this study characterizes a strain, Bacillus subtilis Medicinal_04, isolated from Cannabis sativa rhizosphere and explores its role as an eco-friendly biofungicide and biostimulant. The species level identification of the isolate was confirmed by Average Nucleotide Identity (ANIb) and a digital DNA-DNA hybridization (dDDH). The antagonistic efficacy of B. subtilis Medicinal_04 against Cercospora canescens was evaluated in vitro as well as in planta assays. RESULTS: ANIb of 97.80% and a dDDH score of 85.90% against the reference B. subtilis str. 168. confirmed this isolate as B. subtilis. The in-vitro results showed that B. subtilis robustly inhibited C. canescens growth by 81.5%, strongly correlated with positive chitinolytic activity and a diverse genomic array of secondary metabolite biosynthetic gene clusters. The in planta results demonstrated that B. subtilis seed priming reduced disease incidence by 80 and 71.4%, while foliar application resulted in reductions of 90 and 85.7% for NM-51 and NM-20-21 varieties, respectively. Furthermore, fungicide application successfully reduced disease, however it caused noticeable phytotoxic reductions in root-shoot architecture and chlorophyll content. In contrast, biological interventions completely bypassed these trade-offs as B. subtilis application displayed improved root-shoot length, pod number, and chlorophyll content, while simultaneously enhancing antioxidative enzyme activities (SOD, POD, and CAT) and PR-1 gene expression. CONCLUSION: These findings demonstrate that B. subtilis Medicinal_04 has the potential to serve as a multifunctional biocontrol agent and is capable of securing high-level disease suppression and optimizing plant productivity, offering a valuable toolkit for climate-smart, sustainable agriculture.

Bacillus subtilis↗
PubMed2000

A complex containing both trypsin inhibitor and dehydroascorbate reductase activities isolated from mitochondria of etiolated mung bean (Vigna radiata L. (Wilczek) cv. Tainan no. 5) seedlings.

A complex containing trypsin inhibitor (TI) activity was extracted with 0.1 M TRIS buffer (pH 7.9) from trypsin-treated mitochondria of etiolated mung bean seedlings, and further purified with a Superdex 200 FPLC column. This partially purified complex with an M(r) about 820 kDa exhibited additional dehydroascorbate (DHA) reductase activity with specific activities of 0.21, 1.53 and 1.54 mumol ascorbate formed min-1 mg-1 protein at pH 6.0, 6.5 and 7.0, respectively, when glutathione was added. Much lower DHA reductase activity (0.013 and 0.026 mumol ascorbate formed min-1 mg-1 protein at pH 6.5 and 7.0, respectively) was found when glutathione was omitted. The isolated complex gave positive results when it was tested by TI activity staining after SDS-PAGE, and could be recognized by a polyclonal antibody which was raised against 38 kDa sweet potato Kunitz-type TI, one of the root storage proteins of sweet potato. The possible physiological functions of this complex with both TI and DHA reductase activities were discussed.

Chromatography↗