Biochemical basis of insect resistance in inged bean (Psophocarpus tetragonolobus) seeds

Mature seeds of the inged bean (Psophocarpus tetragonolobus) are toxic to developing larvae of a range of cosmopolitan storage Bruchidae of economic importance, including the copea seed eevil, Callosobruchus maculatus. Insect feeding trials ere carried out in hich protein fractions from seeds of inged bean ere incorporated at a range of concentrations into artificial seeds, and their effects upon development of C maculatus determined. Both albumin and globulin fractions ere toxic to the developing larvae and their toxicity correlated ith their haemagglutinating activity. Assay of Psophocarpin A, B and C fractions demonstrated Psophocarpin B to be the most insecticidal and to contain the highest haemagglutinating activity. Purified basic seed lectin as highly insecticidal to C maculatus larvae, ith an LC₅₀ value of c. 3·5 g kg^?1. The physiological level of this protein in inged bean seeds is sufficient to account for their resistance to attack by C maculatus. inged bean trypsin inhibitor as also purified and tested in artificial seeds against C maculatus. Hoever, even at concentrations in excess of tice the physiological concentration it had no deleterious effects upon development.     

Mechanism of seed lectin tolerance by a major insect storage pest of Phaseolus vulgaris,acanthoscelides obtectus

The bean weevil Acanthoscelides obtectus (Say) is a major storage peat of Phaseolus vulgaris L (kidney, haricot bean). The seeds of P vulgaris contain high levels (up to 30 mg g^?1 DM) of lectin, which has been shown to be toxic towards larvae of the related bruchid storage pest of cowpea, Callosobruchus maculatus F. The lack of toxicity of this lectin towards larvae of A obtectus is demonstrated. Unlike the strong binding of lectin to the midgut epithelium observed in larvae of C maculatus, no binding of lectin molecules was found to occur in the gut epithelial cells of A obtectus. This observation provides the basis for a hypothesis explaining the lack of toxicity of P vulgaris lectin towards A obtectus. Assays of proteolytic activity in gut homogenates of C maculatus and A obtectus suggest that the difference in susceptibility of the two insects towards the toxic effects of the lectin is not due to differential inactivation by proteolysis. Besides its effects on larval development, the lectin has a further effect at pupation, causing disruption of adipose tissues in C maculatus but having no effect on A obtectus.     

The effect of heating on the haemagglutinating activity and nutritional properties of bean (Phaseolus vulgaris) seeds

The toxic lectins present in red, white and black kidney beans (Phaseolus vulgaris) are sensitive to heat treatment and the efficiency of that treatment is greatly improved by pre-soaking of the seeds. Heating of pre-soaked seeds at all temperatures above 75°C caused a continuous reduction in both their haemagglutinating activity and toxicity. However, the only safe method of eliminating toxicity was to heat the fully hydrated seeds to 100°C for a minimum of 10 min.     

Biochemical basis of insect resistance in Vigna unguiculata

The bruchid beetle Callosobruchus maculatus (F.) causes extensive damage to seeds of the cowpea, Vigna unguiculata (L.) (Walp.), when this important tropical foodstuff is stored. A variety of cowpea resistant to attack by this pest has been described. In the present work seeds of a number of cowpea varieties, including the resistant one, were tested for the presence of a physical resistance to C. maculatus, in terms of repulsion of oviposition or of failure of larvae to enter the seeds. No evidence to suggest the presence of a physical resistance was found. When seeds of cowpea varieties were tested for the presence of various antimetabolic secondary compounds, only inhibitory activity against trypsin and, to a much lesser extent, chymotrypsin, could be detected. The resistant variety of cowpea contained a significantly higher level of inhibitors, about twice as much as any other variety. A proteinase inhibitor active against trypsin was purified from cowpea varieties by affinity chromatography on trypsin-Sepharose. The purified inhibitor was shown to inhibit chyraotrypsin also, in such proportions as to account for chymotrypsin inhibition by seed extracts. The inhibitor was shown to consist of a number of isoinhibitors by gel electrophoresis and isoelectric focusing, but no qualitative differences in the inhibitor between varieties could be detected. The antimetabolic nature of the cowpea trypsin inhibitor was confirmed by insect feeding trials in which various protein fractions were added to a basic meal and the effect on larval survival noted. The albumin proteins of cowpea (containing the trypsin inhibitors) at a level of 10% were toxic to larvae of C. maculatus whereas the globulin fractions were not. Further, if cowpea trypsin inhibitor was removed from the albumin proteins they ceased to be toxic. When purified cowpea trypsin inhibitor was added to the basic meal it was shown that a level slightly less than that found in the resistant variety of cowpea caused complete mortality of larvae, whereas lower levels had lesser or no effect. It is concluded that this example of insect resistance in the cowpea is due to an elevated level of trypsin inhibitor.     

The toxicity of Phaseolus vulgaris lectins. Nitrogen balance and immunochemical studies

Inclusion of pure lectins isolated from the seeds of kidney bean (Phaseolus vulgaris L. cv. Processor) in diets for rats increased both faecal and urinary losses of N and resulted in a negative N balance for the animals. The relationship between total body N change [y (mg)] and lectin concentration [x (g kg^?1)] of the diet was: y=421.5-75.1 x, which was statistically significant (P=0.05). These rats developed circulating antibodies of low avidity to the dietary lectins, while no other proteins of the diet elicited a similar antibody response. Additionally, in several serum samples from rats which had been fed raw beans, the presence of small amounts of a protein reactive with rabbit anti-lectin antibodies was detected. This protein was isolated from the immune precipitate and was shown by sodium dodecylsulphate-gel electrophoresis to contain a protein subunit of 30 000, which was very similar to that of pure lectins. It is tentatively suggested that lectin toxicity results from the combined effects of the interference with normal intestinal digestion and/or absorption of N through the damaged enterocytes and of systemic immune (and other) responses of the rat to the internalised lectin.     

Deterrent and insecticidal properties of bean seed (Phaseolus vulgaris L.) whole meal or protein extract incorporated into the diet of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)

Callosobruchus maculatus, a pest that causes serious damage to chickpea Cicer arietinum, cannot develop in the seeds of Phaseolus or Vigna spp. which contain lectins. The insecticidal activity towards C. maculatus in these seeds is attributed both to lectins with specific affinity to N-acetylglucosamine, the major component of insect chitin, and to alpha-amylase inhibitors (lectin-like proteins). The insecticidal properties of bean meal or bean protein extracts from different sources towards different pest species are variable and need to be experimentally evaluated. The main objective of this study was to determine through a feeding trial on artificial chickpea seed the potential of bean seed meal from a wild bean Vigna caracalla, four varieties of Phaseolus vulgaris, and of a protein extract of P. vulgaris seed, to alter different life history traits of C. maculatus. The chickpea weevil was set up on artificial chickpea seeds containing different amounts of bean meal to observe the effects on female oviposition, percentage of development to adulthood and juvenile development time. These traits were combined in a composite index measuring the alteration of the multiplication rate of C. maculatus fed on artificial seed. The activity of lectin-like extracts was observed on chickpea artificial seed spiked with bean seed extract. Incorporation of bean flour at a rate of 10 and 20% in chickpea artificial seed significantly decreased C. maculatus female fecundity, percentage of adult emergence, and greatly increased the development time. Feeding trials with protein extracts of P. vulgaris reduced fecundity and survival of C. maculatus. Incorporation of 10% V. caracalla bean seed meal in chickpea artificial seed, reduced the multiplication potential of C. maculatus by over 90% showing that bean seed lectin extracts are worthy of further investigation for post-harvest infestation control.     

Nutritional evaluation of kidney beans (phaseolus vulgaris): The isolation and partial Characterisation of Toxic Constituents

Comprehensive fractionation studies on the contents of the seeds of two varieties of kidney bean (Phaseolus vulgaris) were carried out. The extent of toxicity of the fractions obtained was assessed from the depression of the growth of rats fed on a mixed diet containing 5% casein protein and 5% unfractionated bean proteins or proportional amounts of fractionated materials. The low molecular weight diffusible materials and the structural water-insoluble carbohydrate-protein complexes (10 and 73% of the seeds, respectively) were found to have had little deleterious effects on rat growth performance. However, both protein fractions, the albumins (6.3% of the seeds) and the globulins (11% of the seeds), were found to be toxic. Further fractionation with continuous high-voltage electrophoresis indicated that most of the toxicity of the albumin fraction might be related to the presence of a range of isolectins. A similar, but not identical, isolectin might also be implicated in the toxicity of the globulin fraction. In addition, these results also demonstrated that the three major storage proteins, glycoprotein I and II and the trypsin inhibitors, which altogether made up about three quarters of the total protein content, could have made a small contribution only to the toxicity of kidney beans.     

Nutritional evaluation of kidney beans (phaseolus vulgaris): The toxic principle

Kidney bean (Phaseolus vulgaris) lectins were purified by affinity chromatography on a fetuin-Sepharose-4B column. The net protein utilisation (NPU) of rats fed on a 5% casein-containing diet was strongly depressed by these pure lectin preparations. The extent of this nutritional toxicity was found to be related to the lectin content of the diet according to the following equation: NPU = NPU_{5% casein} ?280 x (where x is the lectin content in % w/w). Thus, because in addition lectin-free kidney bean protein preparations were found to be non-toxic for rats, it is now suggested that the toxic principle of kidney bean is identical with its constituent lectins.     

Inhibitory action of spray dried blood plasma and whole egg powder on lectins in extracts of several legume seeds: a qualitative approach

Samples of seeds from eight legume species and Triticum vulgaris grains were extracted with buffer and lectin activity in the extracts was determined in haemagglutination experiments using normal or Pronase-treated rabbit erythrocytes. The effect of the addition of spray dried porcine and bovine plasma powder, whole egg powder, galactosides, whey powder and specific inhibitors (eg mannose, galactose, N-acetyl galactosamine, fetuin) on haemagglutination activity (HA) was determined. Plasma powders were potent inhibitors of HA in extracts of Pisum sativum, Vicia faba, Vicia sativa, Lens culinaris and Phaseolus vulgaris. HA in extracts of Lupinus sp and Phaseolus vulgaris was efficiently decreased by whole egg powder, while the lectin of Glycine max could only be inhibited by addition of galactosides, whole and defatted milk powder and whey powder. Inhibitors (plasma and whole egg powder and fetuin) were subjected to SDS-PAGE and Western blotting and blots were incubated with biotinylated lectins, except for Lupinus lectin. Results of the HA experiments were confirmed: lectins which were not influenced by inhibitory compounds in HA experiments also showed no binding with proteins of the blotted inhibitor. There were strong indications that lectins were not bound to the albumin fraction of the plasma powders. Results are discussed in view of future in vivo experiments. © 1998 SCI.     

Thermal stability of black turtle soup bean (Phaseolus vulgaris) lectins

A method for determining the thermal stability of porcine thyroglobulin (PTG)-binding lectins in whole black turtle soup beans (Phaseolus vulgaris L) is described. The procedure utilises PTG-Sepharose affinity chromatography and the Folin-Ciocalteau protein assay. The majority of lectin activity in whole black turtle soup beans was destroyed by heating presoaked beans at 97.8°C for 10 min whereas unsoaked beans required 20 min of heat treatment at 97.8°C. Residual lectin activity was eliminated by thermally processing the presoaked and unsoaked beans for 25 and 50 min at 97.8°C, respectively. Thermal inactivation of the lectin in the whole seed is a biphasic, first-order reaction mechanism. Lectin-rat intestinal epithelial cell binding studies indicated the presence of a second lectin in the BTS albumin protein fraction. The lectin lacked an affinity for PTG and was inactivated by heating unsoaked whole beans for 50 min at 97.8°C.     

Nutritional evaluation of kidney beans (Phaseolus vulgaris): chemical composition, lectin content and nutritional value of selected cultivars

The chemical composition, lectin content and nutritional value of selected cultivars of kidney beans (Phaseolus vulgaris) were determined. Mainly due to different amounts of reserve globulins deposited, seed N content varied between 3.4 and 5.0%. The amino acid composition and the corresponding chemical score values of 40-60 however, were similar for all the beans, with the sulphur-containing amino acids limiting. Despite similarities in composition, 11 out of the 13 cultivars examined were highly toxic for rats in the raw state. These were all found to contain high concentrations of haemagglutinating lectins (over 10% of the total protein). On the other hand, two low-lectin bean varieties, Pinto III and Great Northern, had no appreciable disruptive effects on the intestines and were essentially non-toxic for rats.     

Hard-to-cook phenomenon in beans: Changes in antinutrient factors and nitrogenous compounds during storage

Five different bean varieties (Phaseolus vulgaris) from Kenya, which were either freshly collected or stored for 5 years in tropical conditions (30–40°C; >75% humidity), were compared for their cooking characteristics. The beans under storage develop an irreversible phenomenon classified as ‘hard-to-cook’ (HTC) which results in undesirable characteristics limiting their acceptability. Our aims were to determine the effects of the HTC phenomenon on the proteins and antinutrient factors in these beans. Both fresh and HTC beans contained nutritionally significant amounts of lectins, trypsin and α-amylase inhibitors. HTC samples had higher lectin and lower α-amylase inhibitor contents, while the amounts of trypsin and chymotrypsin inhibitors were the same. Storage appeared to reduce water-extractable nitrogen. Fractionation with sodium dodecyl sulphate also showed that less protein may be available in HTC than fresh beans. We established that the undesirable changes in beans, such as increased cooking time, which often accompany the storage of harvested seeds under tropical conditions produce an overall decrease in soluble-N fractions, particularly proteins. The results can provide guidelines on how to utilise these seed(s) and their products. Furthermore, appropriate processing is needed to reduce their antinutrients for human nutrition.     

Characterisation and fractionation studies on seed proteins of some of the leguminosae by starch-gel electrophoresis

This investigation supplements the previously reported characterisation of 32 cultivars of Phaseolus vulgaris and led to results for 5 cultivars of Vicia faba and 3 cultivars of Pisum sativum. Moreover, proteins of S1 fractions of commercial seeds of three Phaseolus vulgaris, two Vicia faba and three Pisum sativum cultivars were fractionated; electrophoretic patterns obtained for the albumin fractions (F1) are characteristic for the species used. By fractionation, three different globulin fractions (F2-F4) are obtained; after electrophoresis optimum cultivar differences appear in the legumin patterns (F2).     

Differentiating the Lectin Activity in Twenty-Four Cultivars of Dry Beans (Phaseolus vulgaris L.)

Hemagglutination activity of 24 cultivars of dry beans (Phaseolus vulgaris) was estimated with rabbit, rat, bovine, and human erythrocytes. Net protein utilization (NPU) evaluated the nutritional toxicity to weanling rats of the beans fed at 10% protein. Nonprotein and 10%-casein control diets were included. SDS-polyacrylamide gel electrophoresis indicated lectin-containing beans. Beans evaluated (in or-der of decreasing lectin activity) were: Aurora, Sanilac, Royal Red, Red Kloud, Roza, Rufus, Harris, Viva, Fiesta, Black Turtle Soup, Chief, Hyden, UI-59, Sutter; (nontoxic): Blue Mountain, GN-1140, Holberg, Nodak, Olathe, Pindak, JM-126, NW-410, NW-590, UI-114. This study shows that combined hemagglutination tests and SDSPAGE enable rapid screening of dry beans for toxic lectins.     

Antinutritional and/or toxic factors in soybean (Glycine max (L) Merril) seeds: comparison of different cultivars adapted to the southern region of Brazil

Soybean (Glycine max (L) Merril) seeds are known to contain different proteins displaying antinutritional and/or toxic effects, such as soybean agglutinin (an N‐acetylgalactosamine‐specific lectin), proteinase inhibitors (Kunitz‐ and Bowman–Birk‐type trypsin and chymotrypsin inhibitors) and urease (seed and tissue isoforms). Two other toxic proteins were previously isolated from soybeans, soyatoxin (21 kDa) and soybean toxin (18.4 kDa), which are immunologically related to canatoxin, a toxic protein from Canavalia ensiformis (jackbean) seeds. In this work we have screened crude extracts from seeds of six different soybean cultivars, which together represent most of the crop harvested in the southern region of Brazil, for the presence of urease, trypsin inhibitory and haemagglutination activities, intraperitoneal toxicity in mice and immunoreactivity against anti‐canatoxin antibodies. Significant differences were found in the contents of proteinase inhibitors, lectin, urease activity and lethality in mice. The relevance of these findings to the agronomic qualities and to the choice of soybean cultivars to be used as food or feed is discussed. Copyright © 2004 Society of Chemical Industry     

Characterization and insecticidal properties of globulins and albumins from Luetzelburgia auriculata (Allemao) Ducke seeds towards Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)

We evaluated the development of Callosobruchus maculatus growing in artificial seeds composed of Vigna unguiculata (cowpea) seed flour mixed with exogenous proteins from Luetzelburgia auriculata. Albumin and globulin fractions from Luetzelburgia auriculata were characterized in terms of protein content, amino acid composition and antimetabolic proteins (trypsin/chymotrypsin inhibitory, porcine pancreatic alpha-amylase inhibitory, lectin activity and presence of chitin-binding proteins). Both fractions were distinct in terms of protein content and diversity as determined by electrophoresis. Lectin activity was present only in the globulins. Neither fraction exhibited inhibitory activity towards porcine pancreatic alpha-amylase but trypsin inhibition was observed. Interestingly, chitin-binding proteins were detected in both protein classes. Albumins had a severe effect upon larval development and were detrimental to insect emergence (LD₅₀=0.4%) while globulins displayed slight toxicity upon larval development and no effect towards insect emergence. The presence of serine proteinase inhibitory activity and chitin binding proteins could explain, at least in part, the harmful effects on C. maculatus development while lectin activity and amino acid availability seem not to correlate with any deleterious effects. Luetzelburgia auriculata would be an interesting source of seed proteins to study behavior of C. maculatus upon infestation and genes coding for insecticidal proteins could become candidates for molecular biology programs devoted to producing transgenic seeds expressing resistance towards the beetle.     

The effects of α-amylase inhibitors on insect storage pests: Inhibition of α-amylase in vitro and effects on development in vivo

Protein α-amylase inhibitors were prepared from wheat and their effects tested against insect storage pests both in vitro against the insect α-amylases and in vivo in insect feeding trials. Inhibitor fraction A was found to inhibit porcine pancreatic α-amylase but not insect α-amylases, whereas fractions B, C and D (0.28) did not inhibit porcine pancreatic α-amylase but were strong inhibitors of digestive α-amylases from larvae of Tribolium confusum, a storage pest of wheat products, and Callosobruchus maculatus, a storage pest of legume seeds. Fraction D, which was a single polypeptide of M_r 13 000 was the most effective inhibitor in vitro. It would appear that the degree of inhibition by the wheat α-amylase inhibitor preparations can be correlated with the presence of the M_r 13 000 (0.28) polypeptide since the purer this polypeptide the stronger was the inhibition; fraction A which contained two polypeptides of M_r 60 000 and 58 000 caused no inhibition. The effects of fractions B and C on larval development were determined in insect feeding trials. With C. maculatus both fractions were toxic, their relative effectiveness being directly paralleled by their effectiveness observed in vitro. Only fraction C was tested against T. confusum in feeding trials. Despite this fraction being equally effective against both pests in vitro it had very little effect upon larval development of T. confusum in vivo, thus suggesting that this organism is able to detoxify the wheat α-amylase inhibitors. As far as the authors are aware, this is the first time that the effects of identified inhibitor fractions have been monitored both in vitro and in vivo. The results, in contrast to previous proposals, suggest that selecting wheat varieties for high α-amylase inhibitory activity may not be a very reliable criterion in selecting for insect resistance.     

Nutritional response of mature rats to kidney bean (Phaseolus vulgaris) lectins

In a series of 10-day pair feeding experiments it was found that the nutritional value of diets containing beans was essentially the same for rats aged between 30 and 123 days. Thus net protein utilisation (NPU) values of 25–39 on diets containing Processor bean (35 g protein kg^?1) + egg albumin (65g protein kg^?1) were obtained. As food intakes were considerably reduced when rats were fed diets containing more than 35g protein kg^?1 of Processor bean, the measurement of protein utilisation became increasingly more difficult. The severe disruption of the brush borders of duodenal and jejunal enterocytes, originally observed when bean-containing diets were fed to young (30-day-old) rats was also found with rats up to the age of 120 days on similar diets. Similarly, the development of circulating anti-lectin antibodies in the animals showed no age dependence within the age limits investigated. It was also shown that oral immunisation did not protect the rats from the effects of toxicity and that the immune response was a result of continuous absorption of lectin throughout the feeding period. Thus the extent and the mechanism of toxicity of Phaseolus vulgaris bean lectins were found not to be dependent on the age or maturity of the animal.     

Laboratory assessment of toxic activity of purified peptides extracted from chickpea seeds to two strains of the rice weevil Sitophilus oryzae (L.) (Coleoptera: Curculionidae)

Several peptides with entomotoxic properties were extracted from seeds of different chickpea varieties, by either aqueous or alcoholic phase extraction. The extracts were purified by anion-exchange chromatography followed by membrane dialysis. The toxicity of the purified extracts was tested on two different strains of the rice weevil, Sitophilus oryzae: a French strain feeding only on cereal grain and sensitive to pea phytotoxins (SS strain) and a Chinese strain naturally breeding on split pea (RS strain). The toxic activity of the different fractions isolated from the purified extracts was evaluated by the ‘artificial kernel method’, incorporating the fraction into a reconstituted (‘artificial’) kernel made from whole wheat flour. The toxicity of the fractions was assessed by determining the mortality of young adults of each S. oryzae strain after 7 and 14 d feeding. With the more toxic acidic fraction incorporated into the artificial kernel, the mortality rate of SS strain weevils was 83% and 100% after 7 and 14 d feeding, respectively. With all the alcoholic fractions, mortality of the SS strain remained very low. With RS strain weevils, mortality was negligible after 7 d or 14 d feeding on any peptide fraction of the extracts.     

Anti‐proliferative activity of bovine blood hydrolysates towards cancer cells in culture

Four proteins, α/β globulin, serum albumin, γ‐globulin and fibrinogen, were isolated from bovine blood and hydrolysed using papain. Hydrolysates were assessed for non‐cellular and cellular antioxidant activity. The anti‐proliferative activity of hydrolysed fractions was assessed in a number of cancer cell lines including U937 lymphoma cells, MCF‐7 breast cancer cells, HepG2 hepatocytes and Caco‐2 epithelial colorectal adenocarcinoma cells. Anti‐inflammatory activity of the hydrolysates was also assessed. Hydrolysates generated from γ‐globulin or fibrinogen had significant antioxidant activity in non‐cellular assays. Hydrolysates were also found to be highly toxic to different cancer cell lines, in particular U937 lymphoma cells when assessed using the MTT assay. The fibrinogen hydrolysate was the most toxic sample and toxicity appeared to correlate with its non‐cellular antioxidant activity. None of the hydrolysates had significant anti‐inflammatory activity. The high cytotoxicity of the γ‐globulin and the fibrinogen hydrolysates towards cancer cells may indicate a potential use as anti‐proliferative agents.