Jack Bean Seeds Research Articles

Global and targeted proteomics in developing jack bean (Canavalia ensiformis) seedlings: an investigation of urease isoforms mobilization in early stages of development

Jack bean (Canavalia ensiformis) seeds are toxic for insects and the toxicity is due in part to an entomotoxic peptide enzymatically released from ureases in the midgut of susceptible insects. To characterize expression of urease isoforms in jack bean seed, particularly the more abundant urease isoform (JBU), quantitative proteomics was performed. Quiescent through 5-day germinating seeds were analyzed at 1-day intervals using a total proteomics approach (TPA) and also after co-immunoprecipitation (co-IP) with anti-JBU monoclonal antibodies. Jack bean proteins for TPA and co-IP were pre-fractionated by SDS-PAGE, segmented for in-gel trypsin digestion, and analyzed by liquid chromatography coupled to nanospray ionization tandem mass spectrometry (LC-MS/MS). Acquired MS(2) data were searched against a comprehensive plant database and the MEROPS peptidase database, in the absence of a jack bean EST database. Proteins detected in TPA were quantified by label-free spectral counting. A total of 234 and 106 non-redundant proteins were detected in TPA and co-IP, respectively. Mobilization of JBU was observed beginning 3-days after imbibition indicating that the entomotoxic peptide was not formed before this stage. A predicted urease isoform, JBURE-IIb, was detected in the co-IP study. Additionally, 46 plastid proteins, including RuBisCO and plastid ATPase were pulled down with JBU antibodies. These data shed new light on the behavior of urease isoforms during the early stages of plant development.

COMPARATIVE EVALUATION OF RAW AND COOKED JACKBEAN (Canavalia ensiformis) ON THE PERFORMANCE OF WEANER RABBITS.

Jackbean (Canavalis ensiformis) is a tropical legume which has shown potential as source of forage and seed. The seed contains up to 32 % CP. But as with other tropical legumes, the raw seed contains toxic substances. A 42-day feeding trial was conducted to compare the effect of raw and cooked jackbean on the performance of weaner rabbits. Raw Jackbean seeds were dried and milled and incorporated at 10 and 20 % dietary levels respectively while another batch was cooked for 40 minutes, dried, ground and incorporated also at 10 and 20 % dietary level respectively and each diet fed to 8 rabbits. At dietary level of 20 %, raw jackbean significantly (P<0.05) depressed performance of rabbits (ADG 16.2g/d vs 20.5 for the control). Cooking for 40 minutes improved the nutritive value of jackbean for weaner rabbits up to 20 % dietary level (ADG of 26.7 and 21.9 g/day for 10% and 20% levels respectively).

Jack bean urease alters serotonin-induced effects on Rhodnius prolixus anterior midgut

Urease isoforms from jack bean seeds are toxic to insects, and this entomotoxic effect is mostly due to the release of a peptide by insect digestive enzymes. We previously demonstrated that jack bean urease (JBU) has antidiuretic effects on Rhodnius prolixus Malpighian tubules, decreasing the serotonin-stimulated secretion of fluid. Now, we evaluate the toxicity of the intact JBU and its effect on R. prolixus anterior midgut, to further elucidate the mechanism of action of JBU in insects. JBU decreases the serotonin-induced fluid transport by the anterior midgut in vitro when injected into the lumen. A decrease in the levels of cAMP is observed in tissues treated with JBU (in the presence of serotonin). JBU also causes a dose-dependent increase in the frequency of serotonin-induced contractions in the anterior midgut, but does not alter the frequency of spontaneous contractions. The cyclooxygenase inhibitor indomethacin and the prostaglandin antagonist AH6809 block JBU's potentiation of serotonin-induced contractions, indicating that prostaglandins might act as second messengers for JBU action. Prostaglandin E 2 (PGE 2) increases the frequency of serotonin-induced contractions, again supporting the role of prostaglandins as second messengers for JBU action. JBU and PGE 2 increase cGMP levels in the anterior midgut, indicating that this molecule might also be part of the JBU pathway.

Specific Ions Effect on Emulsions, Foams, and Gels of a Seed Protein

A protein concentrate was prepared from the seeds of jack bean (Canavalia ensiformis), and the influence of selected Hofmeister salts on functional properties of the protein concentrate was investigated. Results indicated that kosmotropic salts (Na2SO4, NaCl, NaBr) improved water absorption capacity and least gelation concentration of the proteins more than chaotropic salts (NaI, NaClO4, NaSCN) did. The reduction in water absorption capacity followed the Hofmeister trend (Na2SO4 > NaCl > NaBr > NaI > NaClO4 > NaSCN). However, a reverse trend was observed with the foaming and emulsifying properties. These findings are insightful in understanding the structure–property relations of the concentrate.

Proximate and Mineral Elements Composition of Water Soaked Carnavalia ensiformis Seeds

The study examines the proximate and mineral elements constituents of water soaked Jack Bean (Carnavalia ensiformis) seeds. The seeds were subjected to four treatments which comprised: 24 hrs, 48 hrs, 72 hrs and 96 hrs soaking in cold water with each treatment made up of three replicates. After the expiration of each soaking period, the water was discarded, sun-dried for 7 days and thereafter milled to pass through 1 mm sieve and subjected to laboratory analysis. The study revealed the following: dry matter increased after 96 hrs of soaking. Crude protein (CP) however decreased in value from 21% to 17%. Soaking for 48 hrs led to a reduction in the crude fibre (CF) content of seeds. Prolonged soaking period of 96 hrs increased the ether extract (EE) level. However, total ash content was reduced in seeds soaked for 96 hrs soaking. Soaking for 96 hrs increased Mg, Fe and Cu contents but Zn content recorded a decrease. Differences between treatments for all the parameters considered in the study were significant (P < 0.01).

Lectin of Concanavalin A as an anti-hepatoma therapeutic agent

Liver cancer is the predominant cause of cancer mortality in males of Southern China and Taiwan. The current therapy is not satisfactory, and more effective treatments are needed. In the search for new therapies for liver tumor, we found that Concanavalin A (Con A), a lectin from Jack bean seeds, can have a potent anti-hepatoma effect. Con A after binding to the mannose moiety on the cell membrane glycoprotein is internalized preferentially to the mitochondria. An autophagy is triggered which leads to cell death. Con A as a T cell mitogen subsequently activates the immune response in the liver and results in the eradication of the tumor in a murine in situ hepatoma model. The liver tumor nodule formation is inhibited by the CD8+ T cells, and a tumor antigen-specific immune memory is established during the hepatic inflammation. The dual properties (autophagic cytotoxicity and immunomodulation) via the specific carbohydrate binding let Con A exert a potent anti-hepatoma therapeutic effect. The novel mechanism of the Con A anti-hepatoma effect is discussed. The prototype of Con with an anti-hepatoma activity gives support to the search for other natural lectins as anti-cancer compounds.

Effect of simple cook and defatten processing method on the protein quality of jack bean (&lt;i&gt;Canavalia ensiformis&lt;/i&gt;) seeds

The protein quality of the raw and processed canavalia ensiformis seeds were studied, using 20 weanling wistar albino rats weighing between 55 to 65 g. In compounding of the test diets, the raw seed, cooked and cooked-defatted seeds were used differently as sources of proteins for raw (RD), cooked (CD) and cooked-defatted (CDD) diets respectively. Two controls, nitrogen-free diet (NFD) and casilan diet (CAD) were also prepared. These were assigned to different groups of test animals for 14 days treatment. The cooked-defatted diet (CDD) and casilan diet (CAD) supported growth while Nitrogen-free diet (NFD), raw diet (RD) and cooked diet (CD) did not support growth. The protein efficiency ratio (PER), net protein utilization (NPU), apparent and true digestibility of the raw diets were very low (-4.01 ± 1.66, 48.35 ± 7.64, 44.90 ± 5.22 and 61.01 ± 5.32 respectively), but improved significantly (p

Evaluation and Nutrient Quality of Detoxified Jackbean Seeds, Cooked in Distilled Water or Trona Solution, as a Substitute for Soybean Meal in Nile Tilapia, Oreochromis niloticus, Diets

ABSTRACT Jackbean, Canavalia ensiformis, seeds were detoxified using eight different wet thermal detoxification methods in either distilled water (5% w/v) or trona (NaCO2.NaHCO3.2H2O) solution. The effects of the detoxification methods on hemagglutinating, antitryptic and urease activities, and on the digestibility coefficient of crude protein and gross energy in the jackbean seeds were investigated. Cracked jackbean seeds cooked (100°C) in trona solution for one hour proved to be more effective as a method of inactivating the antinutritional factors in jackbean seeds. Dried detoxified jackbean seed meals were incorporated in dry practical diets providing 20% or 30% of total protein and fed to Nile tilapia, Oreochromis niloticus, fingerlings (mean weight, 6.7 g) twice daily to apparent satiation for 70 days. Mortality was low and no abnormal fish behavior was noted even when the detoxified jackbean meals provided 30% of total protein in diet. Growth and feed utilization indices, were similar (P > 0.05) between the diet in which cracked jackbean seeds cooked in water provided 20% of total protein and in diets in which cracked jackbean seeds cooked in trona solution provided 20% or 30% of total protein. Carcass composition and hepatosomatic index showed no definite trend among O. niloticus fingerlings fed the experimental diets.

Single step immobilized metal ion affinity precipitation/chromatography based procedures for purification of concanavalin A and Cajanus cajan mannose-specific lectin

Concanavalin A and a mannose-specific lectin could be precipitated specifically from extracts of jack bean and Cajanus cajan seeds, respectively, using metal charged EGTA. Single step purification of the lectins was also possible using iminodiacetic acid-Sepharose charged with metal ions. Nondenaturing electrophoresis in polyacrylamide gel and that performed in presence of SDS ascertained homogeneity of the isolated lectins. The migration behavior of the purified lectins was comparable with those of the lectins purified using alternative procedures.

Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori

Mulberry leaves are the sole diet of the silkworm, Bombyx mori. The host urease is incorporated into the larval hemolymph and involved in nitrogen metabolism in the insect. To investigate the selective absorption of the host urease to the larvae, crude urease was prepared from mulberry leaves and roots. Root urease was identical to leaf urease on the basis of electrophoretic analyses: (1) the urease activity appeared in the same migration position in a native gel; (2) There was no difference in molecular mass of the subunit. The root urease was orally injected to the fifth instar larvae of the silkworm. Just before spinning, the larvae absorbed intact urease from the midgut lumen to the hemolymph without the loss of activity. The capacity to absorb urease occurred only at the specific stage. Localization of host urease in midgut tissue was observed using confocal laser scanning microscopy and transmission electron microscopy. Based on spatial distribution of immunofluorescent signals and immunogold particles, host urease specifically attached to the surfaces of microvilli existing in the apical side of columnar cells and appeared in the cytoplasm of the cells for transport to the hemolymph. The incorporation efficiency of root urease into the hemolymph was significantly higher than for ureases from jack bean seeds and Bacillus pasteurii. The urease that was transported to the hemolymph was electrophoretically altered, compared with the host urease extracted.

Separation of jack bean (Canavalia ensiformis) urease isoforms by immobilized metal affinity chromatography and characterization of insecticidal properties unrelated to ureolytic activity

In this work we described the separation of two isoforms of urease from jack bean seeds, the “classical” jack bean urease (JBU) and canatoxin (CNTX), using immobilized metal affinity chromatography (IMAC). Jack bean urease isoforms presented differential behavior on a cobalt-loaded iminodiacetic acid (IDA)–Sepharose column and IMAC is proposed as an efficient method to isolate the isoenzymes. The metal content of the urease isoforms was determined by particle induced X-ray emission (PIXE). CNTX displays ca. 1 eq. of nickel per monomer, contrasting with 2 eq. of nickel found per monomer for JBU. Beside nickel, CNTX contains 1 eq. of zinc per monomer, while no zinc is found in JBU. The insecticidal property of these ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Both ureases were lethal to the insects, being CNTX more potent than JBU. This property was not affected by treatment with p-hydroxymercurybenzoate (pHMB), an irreversible inhibitor of ureolytic activity. Altogether the data show that IMAC is a suitable method for separating jack bean urease isoforms and that the isoenzymes display entomotoxic effects, independent of their ureolytic activity, suggestive of a role in plant defense against insect predators.

Separation of jack bean ( Canavalia ensiformis) urease isoforms by immobilized metal affinity chromatography and characterization of insecticidal properties unrelated to ureolytic activity

In this work we described the separation of two isoforms of urease from jack bean seeds, the “classical” jack bean urease (JBU) and canatoxin (CNTX), using immobilized metal affinity chromatography (IMAC). Jack bean urease isoforms presented differential behavior on a cobalt-loaded iminodiacetic acid (IDA)–Sepharose column and IMAC is proposed as an efficient method to isolate the isoenzymes. The metal content of the urease isoforms was determined by particle induced X-ray emission (PIXE). CNTX displays ca. 1 eq. of nickel per monomer, contrasting with 2 eq. of nickel found per monomer for JBU. Beside nickel, CNTX contains 1 eq. of zinc per monomer, while no zinc is found in JBU. The insecticidal property of these ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Both ureases were lethal to the insects, being CNTX more potent than JBU. This property was not affected by treatment with p-hydroxymercurybenzoate (pHMB), an irreversible inhibitor of ureolytic activity. Altogether the data show that IMAC is a suitable method for separating jack bean urease isoforms and that the isoenzymes display entomotoxic effects, independent of their ureolytic activity, suggestive of a role in plant defense against insect predators.

PIXE analysis of urease isoenzymes isolated from Canavalia ensiformis (jack bean) seeds

The main goal of this work was to perform a stoichiometric analysis of metals, in particular Ni and Zn, in two isoforms of urease isolated from jack bean seeds. The urease isoforms were separated by immobilized metal affinity chromatography. The samples were prepared for PIXE by acetone precipitation of the proteins, which were deposited onto regenerated cellulose acetate membranes by filtration. The resulting mass of the samples was of the order of 0.20 mg (dry weight). The results show that urease contains two equivalents of Ni per 90 kDa monomer, in agreement with the results reported in the literature. For canatoxin, the other urease isoform, the presence of one equivalent of Zn per monomer was observed, besides Ni, previously detected by atomic absorption spectrometry as one equivalent per 90 kDa. Problems and further improvements concerning the preparation of the samples are discussed.

Diversity in nutritional composition of wild jack bean (Canavalia ensiformis L. DC) seeds collected from south India

Nine accessions of the wild jack bean (Canavalia ensiformis L. DC) seeds collected from nine different locations of south India were analysed for proximate composition and mineral profiles. The major findings of the study were as follows: crude protein ranged from 28.9 to 35.0%, crude lipid 3.4–4.7%, crude fibre 7.0–10.7%, ash 3.0–5.8%, carbohydrates 46.1–54.5% and energy levels 1469–1574 kJ 100 g−1 DM. Significant (P<0.05) diversity was observed in crude protein and carbohydrate contents among the accessions collected from different locations based on analysis of variance (ANOVA) analysis. Potassium was the most abundant mineral, which ranged from 634 mg 100 g−1 in Dasukuppam accession to 1017 mg 100g−1 in Valacode accession. Sodium and magnesium levels are generally low with mean values of 63 and 250 mg 100 g−1, respectively. In the present study, in all minerals, significant diversity was observed among the accessions collected from different locations.

Deglycosylation is necessary but not sufficient for activation of proconcanavalin A.

Concanavalin A (ConA), one of the most studied plant lectins, is formed in jack bean (Canavalia ensiformis) seeds. ConA is synthesized as an inactive glycoprotein precursor proConA. Different processing events such as endoproteolytic cleavages, ligation of peptides and deglycosylation of the precursor are required to generate the different polypeptides constitutive of mature ConA. Among these events, deglycosylation of the prolectin appears as a key step in the lectin activation. The detection of deglycosylated proConA in immature jack bean seeds indicates that endoproteolytic cleavages are not prerequisite for its deglycosylation. Both the structure of the lectin precursor N-glycans Man8-9GlcNAc2 and the capacity of Endo H to cleave these oligosaccharide from native proConA in vitro favoured Endo H-type glycosidases as candidates for proConA deglycosylation in planta. Evidence for pH-dependent changes in the prolectin folding were obtained from analysis of the N-glycan accessibility and activation of the deglycosylated lectin precursor in acidic conditions. These data are consistent with the observation that both deglycosylation and acidification of the pH are the minimum requirements to convert the inactive precursor into an active lectin.

Purification and characterization of a basic amino acid-specific peptidase from seeds of jack bean (Canavalia ensiformis).

A peptidase was purified from seeds of Canavalia ensiformis by extraction with water, ammonium sulfate precipitation, and successive chromatographies on DEAE-Toyopearl 650M, butyl-Toyopearl 650M, and G-3000 SW columns. The enzyme has an apparent molecular weight of 41,000. Activity is maximal at pH 9 and 60 degrees C. The enzyme hydrolyzed synthetic substrates at Arg-X and Lys-X bonds more rapidly than bovine trypsin did, and did not cleave protein or ester substrates. The enzyme was inhibited by alkylamines and several serine protease inhibitors such as diisopropylfluorophosphate, chymostatin, leupeptin, and benzamidine. Cysteine protease-, metalloprotease-, and proteinous trypsin inhibitors were ineffective. Inhibition by alkylamines was dependent on length of the alkyl chains. From the substrate specificity and susceptibility to chemicals, the enzyme is a unique peptidase with trypsin-like specificity.

Jack Bean Seed Coat Contains a Protein with Complete Sequence Homology to Bovine Insulin

Abstract: The present study reports that a protein with complete sequence homology to bovine insulin is present in Jack bean (Canavalia ensiformis) seed coats. This protein was recognized by anti-human insulin antibodies and lowered blood glucose levels of alloxanized mice, indicating that it possesses biological potency. Our results suggest that insulin-like activities previously found in diverse plant tissues may be indeed due to insulin and indicate that insulin-dependent signaling pathways have been remarkably conserved during evolution.

Characterization of a class II chitinase from jack bean (Canavalia ensiformis) seeds

Characterization of a class II chitinase from jack bean (Canavalia ensiformis) seeds

Effects of Concanavalin A, fed as a constituent of Jack bean (Canavalia ensiformis L.) seeds, on the humoral immune response and performance of broiler chickens

An experiment was conducted to investigate the effects of the lectin, Concanavalin A (Con A), contained in raw Jack bean (JB) (Canavalia ensiformis, L.) seeds on the immunological response of broilers. A maize-soybean meal basal diet was prepared to which either 2.5, 5, or 10% of ground raw Jack bean (RJB) seeds was added. The RJB seeds contained 24 g Con A/kg on a dry matter basis, as measured by rocket immunoelectrophoresis. Similar diets were prepared by using the same levels of JB after toasting at 190 C for 16 min. In addition, the basal diet was pair-fed to groups of chicks at the level of feed intake of chicks fed the 10% RJB diet. Each diet was fed to six groups of six chicks for 6 wk. At 5 wk, 15 of chicks from each diet were immunized against Brucella abortus (BA) and the anti-BA antibody titers were determined 1 wk later by ELISA. Antibody production against Con A was also measured by the same method. Binding of Con A to intestinal villi and subsequent endocytosis were confirmed by microscopic examination using a specific peroxidase-antiperoxidase-staining technique. Performance was recorded weekly. Feed intake and weight gain were reduced (P < 0.05) only by the diet containing 10% RJB, indicating that broiler chicks can tolerate daily intakes of 100 mg of Con A over 6 wk without affecting growth. Toasted JB diets supported adequate chick performance. The antibody response to BA did not differ with dietary treatment. Serum from chicks fed raw JB also contained antibodies against Con A. The bursa of Fabricius, thymus, spleen, and pancreas dry weights, as a percentage of dry body weight, were not affected by the experimental diets. The data indicated that Con A binds to the cells of the gastrointestinal tract, passes into the general circulation and, eventually, elicits an immunological response without affecting the production of antibodies to BA.

Some physicochemical properties and anti-nutritional factors of raw, cooked and germinated Jack bean ( Canavalia ensiformis)

Physicochemical properties and anti-nutritional factors of raw, cooked and germinated Jack bean ( Canavalia ensiformis) were studied. Jack bean seeds were relatively large, measuring 1.88 cm in length, 1.32 cm in width and 1.09 cm in thickness. The 1000 seed weight was 1.78 kg. The seed coat formed about 13.3% of the whole seed, resulting in high fibre content of the meal. The Jack bean was high in protein (23.3%) and starch (24.7%). Hydrogen cyanide and phytate levels in the raw beans were 11.2 mg/100 g and 2.78 g/100 g dry sample, respectively. Haemagglutinin and trypsin inhibitor activities were detected in the bean. Cooking and germination had varied effects on bean constituents. Raw bean flour had a fairly good foam capacity which was reduced considerably by cooking and germination. The Brabender amylograph showed that the bean starch had poor cooked paste properties and this may, in part, explain why the bean is difficult to cook.