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

ArticleinJournal of the Science of Food and Agriculture 84(3) · February 2004with 2,549 Reads
Abstract
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

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    Ureases catalyze the hydrolysis of urea into ammonia and carbon dioxide and, thus, are involved in the metabolism and bioavailability of nitrogen. Ureases occur in plants, fungi, and bacteria. In plants, besides their enzymatic activity, ureases as proteins play a role in defense against insect and phytopathogenic fungi. Little is known about the regulation of urease in plants under stress and whether or not phytohormones may be involved. In this study, we addressed the regulation of ubiquitous urease (ubSBU) gene expression after phytohormone applications, insect herbivory, and mechanical damage in soybean (Glycine max cv. Williams 82). Stress-related phytohormones were applied. In addition, Spodoptera littoralis feeding and mechanical damage by MecWorm were performed. Ureolytic activity and transcripts for ubSBU and UreG were quantified. Roots and leaves showed the highest levels of ubSBU transcripts. The results show a significant increase of ubSBU transcripts upon jasmonic acid application and after herbivory, but downregulation after MecWorm treatment. UreG transcripts were downregulated after MecWorm, S. littoralis, and application of gibberellic acid, but upregulated by jasmonic acid. However, the ureolytic activities in leaves were influenced neither by phytohormones nor by herbivory and MecWorm. We conclude that the enzymatic activity of ureases is constitutive and basal levels of the enzyme are sufficient to perform the ureolytic activities in defense against insects and fungi. The defense role of ureases, which does not require the ureolytic activity, may underlie their differential regulation in response to different stress stimuli.
  • Article
    Compared to other processing methods used in soybeans, roasting has received limited attention in soymilk processing despite its known potential role in improving soymilk quality. In this study, the effect of soybean roasting at 110 °C for 20 to 100 min and at 120 °C for 20 min on the distribution of soymilk volatile compounds, antinutritional factors and storage parameters: pH, total titratable acidity, emulsion stability (ES) and rheology were investigated and comparison was made with the control sample prepared from soybeans which were not roasted. Results have shown that roasting soybeans improved soymilk properties by significantly decreasing the levels of each of the following objectionable volatile compounds: hexanal, 1-hexanol and Furan, 2-pentyl respectively from 43.58% to a minimum level of 17.95, 11.67–2.28 and 4.09–0.82%. In addition, the levels of trypsin inhibitors were considerably decreased from 5.86 to 1.91 mg/gm thus reducing the overall heat treatment time of the soymilk from 19.4 to 9.9 min. ES increased significantly from 23.97 to 102.84 min while viscosity at the shear rates of 0.1 and 1.0/s decreased respectively from 3.00 and 0.73 to 0.27 and 0.03 Pa s. Furthermore, all the samples were found with the behavior flow indices values of less than unity (0.16–0.38 respectively) suggesting that they were pseudo plastics. From the results, it can therefore be concluded that careful selection and application of soybean roasting parameters in terms of temperature and time can be a useful strategy in improving the quality of soymilk.
  • Article
    Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea to produce ammonia and carbon dioxide These enzymes, which are found in fungi, bacteria, and plants, show very similar structures. Despite an abundance of urease in vegetal tissues, the physiological role of this enzyme in plants is still poorly understood. It has been previously described that ureases from the legumes jackbean ( Canavalia ensiformis) and soybean ( Glycine max) have insecticidal activity and antifungal properties. This work presents the physicochemical purification and characterization of a urease from cotton ( Gossypium hirsutum) seeds, the first description of this enzyme in Malvaceae. The urease content varied among different cotton cultivars. Cotton seed urease (98.3 kDa) displayed low ureolytic activity but exhibited potent antifungal properties at sub-micromolar concentrations against different phytopathogenic fungi. As described for other ureases, the antifungal effect of cotton urease persisted after treatment with an irreversible inhibitor of its enzyme activity. The data suggest an important role of these proteins in plant defense.
  • Article
    A toxic protein present in the crude extract of Canavalia ensiformis seeds induces within 24 hr dyspnoea, ataxia, hypothermia, coma, tonic convulsions and death in mice injected i.p. with 100–200 mg of protein per kg. The toxin was separated from concanavalin A by affinity adsorption of the latter on Sephadex G-100, and further purified by sequential fractionation by (a) removal of polysaccharides with 30% v/v ethanol; (b) ammonium sulfate precipitation at 0.35–0.55 saturation and (c) DEAE-cellulose chromatography. The purified toxin, with an ld50 of 2–5 mg of protein/kg mouse, is unstable. Gel-filtration of the purified toxin on Bio-Gel P-200 showed a single protein peak with a molecular weight corresponding to 88,000 daltons. Polyacrylamide gel electrophoresis of this material indicated the presence of two small contaminants. A central convulsive effect of the toxin can be proposed since no effects were found on isolated pharmacological preparations. The name CANATOXIN is suggested for this new toxic protein.
  • Book
    Among the numerous approaches to expanding food supplies in the developing world in environmentally benign ways is the bioengineering of crops. Bioengineering has much to contribute, but it is a novel system and possible risks need to be evaluated carefully. Opposition to bioengineering research and its application has already arisen, not all of it carefully thought out. As the World Bank has recognized, a considered and technically competent understanding of both the potential and the perceived risks of bioengineered crops is a requisite to their successful development and use. Public perceptions that genetically engineered crops and animal products pose specific dangers must be carefully considered and addressed if such products are to reach widespread use. In 1996 Ismail Serageldin, the World Bank's vice president for Environmentally and Socially Sustainable Development and chairman of the CGIAR, initiated a study panel to assess the potential of crop bioengineering as well as the inherent risks. The panel was to provide the Bank with guidance in its activities, including its support to the CGIAR. This is the panel's report. In what follows we review the status of world food supplies and the prospects and needs for the future with emphasis on the developing world. We then describe bioengineering technology and the potential contributions that transgenic crops might make to the alleviation of problems of food security. After that we deal with possible risks from the widespread deployment of genetically altered crops. Finally, we offer some conclusions and recommendations.
  • Article
    Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.
  • Article
    A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
  • Chapter
    The turbidity produced when protein is mixed with low concentrations of any of the common protein precipitants can be used as an index of protein concentration. The resulting turbidity is maximum after about 10 minutes and may be measured spectrophotometrically in the wavelength region of 600 m. Standardization may be effected by comparison with the turbidity produced by a suspension of a dried protein precipitate, or reference may be had to the methyl acrylate-styrene polymer. Turbidimetric techniques are rapid and convenient, but they yield different values with different proteins. They do not permit differentiation between protein and acid-insoluble compounds such as nucleic acids. Protein estimation with the Folin-Ciocalteu reagent include (1) biuret reaction of protein with copper ion in alkali, and (2) reduction of the phosphomolybdic-phosphotungstic reagent by the tyrosine and tryptophan present in the treated protein. Protein estimation by ultraviolet absorption takes advantage of the fact that nucleic acid, however, absorbs much more strongly at 260 mμ than at 280 mμ, whereas with protein the reverse is true. This advantage is used to eliminate, by calculation, the interference of nucleic acids in the estimation of protein.
  • Article
    Tannin in plant extracts can be determined by reacting the tannin with a protein and quantitating the precipitated complex. In the new assay described here, a tannin-containing solution is placed in a well in a protein-containing agar slab. As the tannin diffuses into the gel and complexes with protein, a visible ring of precipitation develops. The area of the ring is proportional to the amount of tannin in the extract. The detection limit of the method is 0.025 mg tannic acid or condensed tannin and the precision is 6% (relative standard deviation). Tests with extracts of a variety of plants show that the new method gives results comparable to other precipitation methods and that the new method is superior for samples of unusual composition, such as aspen buds. The method has several advantages over other methods for determining tannin: The new method is very simple and requires neither complex reagents nor instruments. Components of the plant extract such as non-tannin phenolics or water-insoluble compounds do not interfere with the method. The assay is not subject to interference from the organic and aqueous solutions which are commonly used to extract tannin from plants.
  • Article
    The distribution of three cross-reactive materials (CRMs), a toxic protein analogous to canatoxin, CNTX-CRM, a lectin analogous to concanavalin A, Con A-CRM, and a major storage protein, canavalin-CRM, was investigated during successive stages of maturation of Canavalia brasiliensis Mart. seeds. The data obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunological analyses indicated that these proteins share extensive homology with the analogous proteins found in Canavalia ensiformis seeds. The changes in CNTX-CRM and Con A-CRM levels throughout the maturation process were assayed by rocket immunoelectrophoresis. Synthesis of Con A-CRM was detectable at 30 days post-anthesis (DPA) while its hemagglutinating activity appeared only at 35 DPA. The CNTX-CRM was detected as a biologically active protein from 30 DPA onwards. The behavior of CNTX-CRM during maturation of C. brasiliensis seeds was quite distinct from that of Con A-CRM, pointing to different biological roles of these proteins in the seed.
  • Article
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  • Article
    The research was conducted with two different recently released Brazilian soybean cultivars (Rio Balsas and Bays) to evaluate whether there is any correlation between the different levels of antinutritional and/or toxic proteins in the cultivars and their nutritive value as sources of protein for monogastric animals (rats). Furthermore, it is discussed, for the first time, the role of the dietary soyatoxin on the performance of rats fed on diets containing soyatoxin-rich (cv. Bays) and soyatoxin-free (cv. Rio Balsas) soybean cultivars. Feeding rats with diets containing raw soybean cultivars showed a lower growth rate, net protein utilization and digestibility, a much higher dry matter and nitrogen excretion and macroscopic alterations in internal organs when compared to rats fed on egg-white protein. The nutritional parameters measured for the diet based on raw Bays cultivar were poorer than those of the diet prepared with Rio Balsas. In the raw soybeans, trypsin inhibitor and lectin, and urease to a lesser extent, significantly affected at different fashion the soybean protein utilization. Heating treatment of the Bays seeds increased the growth rate, NPU, in vivo protein digestibility and practically eliminated or attenuated all the organ alterations observed. This study might be helpful in the choice of safe and nutritious soybean cultivars.
  • Article
    The effects of various processing methods on the hemagglutinating and antitryptic activities of the seeds of two species of the genus Canavalia, C. ensiformis and C. braziliensis, were investigated. Raw C. ensiformis and C. braziliensis contained 13 532 and 10 204 hemagglutinating units (HU), respectively, and also 1682 and 1662 trypsin inhibitor units (TIU) g-1 of seed, respectively. Moist heat proved more effective than dry heat as a method of inactivating the two antinutritional factors in the seeds. Whereas 90 min of cooking at 96 °C was required to completely eliminate antitryptic activities of both species, 3 h of cooking was required to completely eliminate their hemagglutinating activities. Pressure cooking eliminated their antitryptic activities in 30 min and their hemagglutinating activities in 45 min. Soaking in water for even up to 96 h before cooking had no effect on hemagglutinating and antitryptic activities of C. braziliensis. Soaking in water for 72 h, however, completely eliminated the antitryptic activity of C. ensiformis in 30 min of cooking and the hemagglutinating activity in 60 min of cooking. Breaking the seeds before cooking proved to be most effective, resulting in complete elimination of antitryptic and hemagglutinating activities of both species in 30 and 60 min of cooking, respectively, and is therefore most highly recommended. Keywords: Canavalia ensiformis; Canavalia braziliensis; antitryptic/hemagglutinating activities; processing methods
  • Article
    The amounts of glycinin and β-conglycinin were measured in 12 soybean varieties by using rocket J immunoelectrophoresis. These two proteins constitute 55-75% of the soluble protein in the soybean seed. When 10 varieties (Corsoy, Hodgson, Kitamusume, Tokachi-nagaha, Toyosuzu, Vinton, Wasekogane, Weber, and Yuuzuru) were grown in a uniform environment in 1980 and 1981, the glycinin content (as seed protein) was 46.9-54.4% and 46.7-57.2%, respectively. The average glycinin content was 51.0%. β-Conglycinin content for the 2 years examined averaged 18.5%, with a range of 16.8-20.1% and 16.5-20.9%, respectively. Vinton and Weber soybeans from several growing seasons and different environments had glycinin contents with a range of 11.8% and 14.5%, respectively. β-Conglycinin content of these soybeans varied by 5.0%. There seemed to be no relationship between glycinin and β-conglycinin content in these soybeans. Environmental influences seem to have a much greater impact on glycinin concentration in soybeans than on β-conglycinin content. Genetics also has an influence on the expression of these two proteins but to a lesser extent than environment.
  • Article
    The content and heat stability of protease inhibitors of a standard cultivar (Williams 82) and an isoline (L81-4590) lacking the Kunitz trypsin inhibitor (KTI) were measured by using enzyme inhibition and enzyme-linked immunosorbent assays (ELISA). The KTI content of the isoline was less than 0.2% compared to Williams 82, with the exact content depending on the extent of cross-pollination of the soybeans. Steam heating of the isoline flour (121 °C, 20 min) resulted in a near-zero level of trypsin inhibitory activity, while 20 % remained in the Williams 82 sample. The raw soy flour prepared from the isoline was nutritionally superior to the raw flour prepared from the standard variety, as measured by PER and pancreatic weights. The increased PER was likely due to the lower level of trypsin inhibitory activity in the isoline. Steam heating the flours for up to 30 min at 121 °C progressively increased the PER for both strains. Screening of several accessions from the USDA Soybean Germplasm Collection showed variation in the content of trypsin inhibitor, sulfur amino acids, and lectins, indicating that further screening studies could lead to the discovery of soybeans which yield flour that is safe and nutritious, with minimal heating.
  • Article
    The Berthelot color reaction has been investigated with the particular aim of presenting a simple, reliable analytical procedure. The combination of two reagents prepared readily, phenol plus nitroprusside and alkali plus hypochlorite, gave excellent reproducibility with great convenience. After the ammonium sulfate sample was mixed with the phenol reagent the hypochlorite reagent could be added up to 30 minutes later without change of absorbance, whereas if this sample were mixed with a reagent containing hypochlorite or with alkaline phenate plus nitroprusside, there was a decrease in absorbance unless the second reagent was added immediately. Satisfactory color development with minimal precautions was given by a variety of reagent concentrations and by reaction temperatures of 20°, 25°, 37°, and 75° C. Sensitivity was increased at 75° C although the time to reach maximum absorbance was longer than at 37° C. The absorbance maximum was reached quickly at 100° C but this temperature is not recommended because the absorbance decreased rapidly thereafter.
  • Article
    The effects on organ relative weights (g per 100 g bw) and plasma amino acid concentrations of diets based in legume (faba bean (Vicia faba), lupin (Lupinus angustifolius), chickpea (Cicer arietinum) and soybean (Glycine max)) seed meals as the only source of protein were studied with growing rats using lactalbumin or casein as controls. Also, legume seed meal extracted globulins were included in control diets replacing lactalbumin to produce legume globulin-based diets, and legume residue fractions, containing most of the starch and/or insoluble fibre (NSP+lignin) from the seed meals, were included in control diets to reach the same amounts present in whole legume seed meal diets. All experimental diets were formulated to contain the same amounts of protein (100 g kg−1) and energy (15.5 kJ g−1), and were supplemented with essential amino acids. Compared with lactalbumin, higher relative weights of gastrointestinal sections were determined in rats fed legume seed meals or their corresponding residue fractions. On the contrary, spleen relative weight was lower in rats fed diets containing lupin, chickpea or soybean meals or extracted globulins, while residue fractions had no effect on it. Thymus relative weight was also lower in rats fed whole chickpea seed meal or any of the extracted legume globulins. Except for chickpea meal, animals fed legume- or legume protein-based diets had lower liver relative weights than controls. Lower proportions (mg g−1 tissue) of glycogen, and lower total protein and RNA, were also determined in the livers of rats fed lupin seed meal. Free plasma concentrations (mM litre −1) of glycine, histidine and arginine were higher, and threonine, leucine and lysine were lower, in rats fed diets based in all legume seed meals of their respective globulin proteins. The possible reasons and implications of these results are discussed.© 1999 Society of Chemical Industry
  • Article
    Soybean quality depends on growing, harvesting and storage conditions. The amount of broken or otherwise damaged seeds tends to increase during storage and may affect the quality of products. Protein isolates from altered seeds have physicochemical properties less useful than those of normal seeds. The effect is substantial on viscosity, gel forming ability and emulsion stability. Isolated proteins from damaged seeds mixed with those of normal seeds altered proportionally their gelation and water retention capacity of the gels. Viscosity or water absorbing capacity of protein isolates may be used as simple indicators to evaluate quality.
  • Article
    Full-text available
    Protease inhibitor and lectin concentrations and N and lipid levels were evaluated in 27 soya bean samples. All contained trypsin and chymotrypsin inhibiting activity and lectin. Shinano midori, Shinano wasediro, Shinano-oomame and Tsuronoku had high protein contents and average lipid levels, and their lectin and trypsin and chymotrypsin inhibitory activities were not unduly high. These lines, in particular, merit further nutritional evaluation. Food intake, feed conversion efficiency and growth by rats was reduced when soya bean was included in their diet, but the extent to which these parameters were affected did not appear to depend only on the protease inhibitor or lectin content. In contrast, pancreatic and small intestinal growth did seem to reflect the levels of these factors in the diet. Chymotrypsin inhibitor activity in soya bean was more readily abolished by heat-treatment than was trypsin inhibitory activity. Lectin activity was also relatively heat-resistant. However, all of these activities could be abolished by aqueous heat-treatment of fully imbibed seeds at 100°C for 10 min. The effects of a soya diet on pancreas and small intestine weights were also abolished by this pre-treatment, and food intake and nutrient utilisation by rats was greatly improved. © 1998 Society of Chemical Industry
  • Article
    Five different recently released Brazilian soybean cultivars (Bays, BR-10, Rio Balsas, Serido and Tropical) were compared for their proximate analyses and presence of antinutritional or toxic factors. As expected, the seeds are rich in proteins, varying from 360·7 to 485·4 g kg−1 flour, and they also have a high amount of fat (from 183·0 to 215·3 g kg−1 flour). Crude extracts from seeds of Bays, BR-10, Serido and Tropical were highly toxic to mice within 1–12 h, depending on the administration route (intraperitoneal, intramuscular or subcutaneous) and dose used while Rio Balsas was not. These acute effects were very similar to those produced by the soytoxin, a neurotoxin that has been recently purified from the commercial soybean sold in Brazil. The amount of trypsin inhibited in the presence of crude extracts ranged from 28·5 to 62·5 g kg−1 flour. Urease was also present and the seed lectin agglutinated preferentially rabbit erythrocytes. A heat treatment at 92°C for 1 min destroyed completely the toxic activity while the haemagglutinating and trypsin inhibitor activities were abolished within 5 min. At these conditions urease was still active. Due to its high protein content, lack of soytoxin, and low levels of trypsin inhibitor, lectin, and urease it is suggested that Rio Balsas could be an alternative for breeding programmes aimed to improve the nutritional quality of soybeans. ©1997 SCI
  • Article
    The soybean (Glycine max [L.] Merr.) contains two urease isozymes whose expression is regulated in a tissue-specific and temporal manner. The ubiquitous urease is expressed in all tissues examined (leaf, embryo, seed coat, cell culture); the embryo-specific urease is synthesized exclusively in the developing embryo. The embryo-specific urease accumulates during seed development while the ubiquitous urease is found in highest levels during early development of both leaves and seeds. We have isolated mutants which fall in three phenotypic classes lacking one or both urease isozyme activities. Genetic analysis has thus far identified three unlinked loci which control the expression of urease(s). Genomic and cDNA clones of urease structural genes have also been recovered and we are working to assign these to genetic loci by sequence and RFLP analyses. That the ubiquitous urease isozyme is expressed in cell culture makes it possible to include cell culture in physiological and developmental studies. Additionally, we have developed direct selections for urease-negative mutants, and their revertants, in cell culture. These selections will facilitate the study of the expression of cloned urease genes in genetically transformed tissue.
  • Article
    ABSTRACTA lectin was purified from seeds of Dioclea guianensis by Sephadex G-50 affinity chromatography. Apparent homogeneity of the lectin was demonstrated by native polyacrylamide gel electrophoresis, chromatography on DEAE- and CM-Sepharose, and immunochemistry. The lectin showed a carbohydrate specificity for D-mannose (D-glucose)-binding, had a requirement for Ca−2 and Mn−2, contained no covalently bound carbohydrate and had an amino acid composition characterized by high content of aspartic acid, serine and threonine, and low levels of sulfur-containing amino acids. At pH 7.5 it exists as two species of molecular weight about 100 and 47 kD and in dissociating solvents three subunits of approximate size of 30, 18 and 12 kD were obtained. The lectin agglutinated erythocytes from rabbit and chicken but not from human, cow, sheep, goat or pig and was toxic to brine shrimp (Artemia salina) larvae. It was relatively heat-stable, retaining half of its original activity even after 90 min at 70°C.
  • Article
    This study reports on the performance of rats fed with diets based on Canavalia brasiliensis seed proteins. The raw seeds are not a good regime, causing the animals to lose weight, exhibiting low net protein utilization (NPU) values, high nitrogen excretion and macroscopic alterations of key internal organs. Heat treatment, but not dialysis of the seed meal, had a beneficial effect on the nutritional parameters. Feeding rats with a diet containing pure lectin showed a lower digestibility and NPU, a much higher dry matter and nitrogen excretion, impaired growth rate and small-intestine enlargement in comparison with rats pair-fed on egg-white protein. In addition, lectin was detected fully active against rabbit erythrocytes in the faeces of rats fed lectin-containing diets. It was suggested that the seed lectin acts as an antinutritive dietary component.
  • Article
    A substance which inhibits the growth-promoting properties of proteins in rats is present in raw soybeans. The hubstance is non-dialyzable, precipitated by d t, and inactivated by heat. I t appears, therefore, to be a protein, which has been 5homn to be concentrated in the acid-soluble (pH 4.2) fraction of the soybean protein. These results are in agreement with those presented by Ham, Sandstedt, and Musseh for the chick
  • Article
    Full-text available
    A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
  • Article
    Full-text available
    Helicobacter pylori produces a potent urease that is believed to play a role in the pathogenesis of gastroduodenal diseases. Four genes (ureA, ureB, ureC, and ureD) were previously shown to be able to achieve a urease-positive phenotype when introduced into Campylobacter jejuni, whereas Escherichia coli cells harboring these genes did not express urease activity (A. Labigne, V. Cussac, and P. Courcoux, J. Bacteriol. 173:1920-1931, 1991). Results that demonstrate that H. pylori urease genes could be expressed in E. coli are presented in this article. This expression was found to be dependent on the presence of accessory urease genes hitherto undescribed. Subcloning of the recombinant cosmid pILL585, followed by restriction analyses, resulted in the cloning of an 11.2-kb fragment (pILL753) which allowed the detection of urease activity (0.83 +/- 0.39 mumol of urea hydrolyzed per min/mg of protein) in E. coli cells grown under nitrogen-limiting conditions. Transposon mutagenesis of pILL753 with mini-Tn3-Km permitted the identification of a 3.3-kb DNA region that, in addition to the 4.2-kb region previously identified, was essential for urease activity in E. coli. Sequencing of the 3.3-kb DNA fragment revealed the presence of five open reading frames encoding polypeptides with predicted molecular weights of 20,701 (UreE), 28,530 (UreF), 21,744 (UreG), 29,650 (UreH), and 19,819 (UreI). Of the nine urease genes identified, ureA, ureB, ureF, ureG, and ureH were shown to be required for urease expression in E. coli, as mutations in each of these genes led to negative phenotypes. The ureC, ureD, and ureI genes are not essential for urease expression in E. coli, although they belong to the urease gene cluster. The predicted UreE and UreG polypeptides exhibit some degree of similarity with the respective polypeptides encoded by the accessory genes of the Klebsiella aerogenes urease operon (33 and 92% similarity, respectively, taking into account conservative amino acid changes), whereas this homology was restricted to a domain of the UreF polypeptide (44% similarity for the last 73 amino acids of the K. aerogenes UreF polypeptide). With the exception of the two UreA and UreB structural polypeptides of the enzyme, no role can as yet be assigned to the nine proteins encoded by the H. pylori urease gene cluster.
  • Article
    Ribosome-inactivating plant toxic proteins and ADP-ribosylating microbial toxins share a common structural organization. These proteins present domains displaying different biological properties: a target cell membrane-binding component (B-subunit or haptomer) and an enzymatically active component (A-subunit or effectomer). Interactions of these toxins with the target cells are mediated by the hemilectin-like haptomer, which recognizes and specifically binds to a given glycoderivative present at the cell surface. After binding the holoprotein is internalized via endocytosis. Inside the endocytic compartment the toxin is processed to release its effectomer moiety which catalytically modifies a cytoplasmic component, and this step accounts for its toxic effect. The structural relationships between toxic hemilectins and plant lectins are discussed, with emphasis on the example of canatoxin and concanavalin A, both present in the seeds of the jack bean Canavalia ensiformis. Contrary to other plant toxic proteins, which inhibit protein synthesis, canatoxin-induced toxicity includes central nervous system-mediated effects. In vivo as well as in vitro canatoxin acts as lipoxygenase-mediated secretagogue in several types of cells: blood platelets, mast cells, pancreatic islets and synaptosomes. Elucidation of structure vs biological activity relationships of canatoxin and other toxic proteins may provide data for their utilization as pharmacological tools and as therapeutic agents.
  • Article
    Full-text available
    The effects on the small intestine and the growth of rats of six pure plant lectins: PHA (Phaseolus vulgaris); SBL (Glycine maxima); SNA-I and SNA-II (Sambucus nigra); GNA (Galanthus nivalis) and VFL (Vicia faba), covering most sugar specificities found in nature, were studied in vivo. Variable amounts, 25% (VFL) to 100% (PHA, GNA) of the lectins administered intragastrically, remained in immunochemically intact form in the small intestine after 1 h. All lectins, except GNA, showed binding to the brush border on first exposure, although this was slight with VFL. Thus, binding to the gut wall was not obligatory for resistance to proteolysis. Exposure of rats to lectins, except VFL, for 10 days, retarded their growth but induced hyperplastic growth of their small intestine. The two activities were directly related. PHA and SNA-II, whose intestinal binding and endocytosis was appreciable after 10 days of feeding the rats with diets containing these lectins and similar to that found on acute (1 h) exposure, were powerful growth factors for the small intestine. GNA, which did not bind at the start but was reactive after 10 days, and SNA-I, which behaved in the opposite way, induced changes in receptor expression in the gut. As they were bound to the brush border transiently, they were less effective growth factors. VFL was not bound or endocytosed, was non-toxic and did not promote gut growth.
  • Article
    Full-text available
    Inclusion of raw soyabean in diets considerably inhibits the growth of young animals. This is due to interference with normal gut and systemic metabolism, particularly of pancreas, liver and muscle. Pancreatic hypertrophy and hyperplasia occur in the young of a number of species given soyabean. In the rat, this enlargement, which is primarily a result of interference with CCK-mediated feedback control of exocrine pancreatic secretion, persists upon prolonged feeding and leads to a susceptibility of the pancreas to carcinogens and an increased incidence of neoplasia. In contrast, with pigs or dogs, in which feedback regulation is primarily mediated via secretin, no increase in pancreas enlargement results from consumption of soyabean. Dietary soyabean or trypsin inhibitors do however alter pancreatic secretion in humans. It is at present unclear how this response is mediated. The growth inhibition and interference with intestinal and systemic metabolism observed upon soyabean feeding is due to the presence of trypsin inhibitors, lectin and anti-nutritional factors, devoid of trypsin inhibitory or lectin activity, in the seed meal. The effects of these dietary factors are additive and possibly synergistic. Most of the anti-nutritional effects of soyabean can be abolished by proper aqueous heart-treatment. However, with a proportion of calves, pigs, lambs and humans even heat-treated soyabean has deleterious effects. These can only be eliminated by hot aqueous-ethanol extraction of the meal.
  • Article
    Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.
  • Article
    1. alpha(2)-Macroglobulin is known to bind and inhibit a number of serine proteinases. We show that it binds thiol and carboxyl proteinases, and there is now reason to believe that alpha(2)-macroglobulin can bind essentially all proteinases. 2. Radiochemically labelled trypsin, chymotrypsin, cathepsin B1 and papain are bound by alpha(2)-macroglobulin in an approximately equimolar ratio. Equimolar binding was confirmed for trypsin by activesite titration. 3. Pretreatment of alpha(2)-macroglobulin with a saturating amount of one proteinase prevented the subsequent binding of another. We conclude that each molecule of alpha(2)-macroglobulin is able to react with one molecule of proteinase only. 4. alpha(2)-Macroglobulin did not react with exopeptidases, non-proteolytic hydrolases or inactive forms of endopeptidases. 5. The literature on binding and inhibition of proteinases by alpha(2)-macroglobulin is reviewed, and from consideration of this and our own work several general characteristics of the interaction can be discerned. 6. A model is proposed for the molecular mechanism of the interaction of alpha(2)-macroglobulin with proteinases. It is suggested that the enzyme cleaves a peptide bond in a sensitive region of the macroglobulin, and that this results in a conformational change in the alpha(2)-macroglobulin molecule that traps the enzyme irreversibly. Access of substrates to the active site of the enzyme becomes sterically hindered, causing inhibition that is most pronounced with large substrate molecules. 7. The possible physiological importance of the unique binding characteristics of alpha(2)-macroglobulin is discussed.
  • Article
    The specificity of the purified soybean agglutinin has been studied immunochemically by quantitative precipitin and quantitative precipitin inhibition assays. The lectin is precipitated by human A and Lea blood-group substance, by the products of the second, third, fourth, and fifth stages of periodate oxidation of a human H blood-group substance (JS), and by precursor blood-group substances, as well as by a pig-submaxillary mucin having blood-group A activity, by partially hydrolyzed blood-group B substances (Pl fraction), and by group C streptococcal polysaccharide. The activity is attributable to terminal α-linked 2-acetamido-2-deoxy-d-galactopyranosyl or to α- or β-d-galactopyranosyl residues. The lectin did not precipitate with human blood-group H substances, with the product of the first stage of periodate oxidation (JS), with streptococcal group A polysaccharide, or with pig-submaxillary mucin devoid of blood-group A activity, and is poorly precipitated by blood-group B substances. Inhibition of precipitation with various monosaccharides indicated that the lectin is strongly specific for 2-acetamido-2-deoxy-d-galactose and for its oligosaccharides, and to a lesser extent for d-galactose and its oligosaccharides; the α-glycosides of both sugars were slightly more reactive than the β-glycosides of 2-acetamido-2-deoxy-d-galactose, and both α- and β-glycosides were more active than the free monosaccharides. Aromatic α- and β-glycosides of 2-acetamido-2-deoxy-d-galactose and d-galactose were better inhibitors than the corresponding methyl or ethyl compounds. The blood-group A trisaccharide α-d-GalNAcp-(1→3)-β-d-Galp-(1→3)-d-GlcNAc was more active than the disaccharide lectins by the use of precipitation with polysaccharides, as well as inhibition reactions, is essential to the understanding of their reactivity with cell-surface receptors.
  • Article
    Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
  • Article
    Full-text available
    Feeding trials have been done with rats to assess the effects of long-term (700 d) consumption of diets based on raw cowpeas (Vigna unguiculata; moderate Bowman-Birk inhibitor content, low lectin content), lupin seeds (Lupinus angustifolius; low lectin and protease inhibitor content) or soya beans (Glycine max; high Kunitz inhibitor content, moderate Bowman-Birk inhibitor content, moderate lectin content) or diets containing low levels of raw kidney bean (Phaseolus vulgaris; high lectin content, low Bowman-Birk inhibitor content) on body weight and composition and organ weights. All the legume-based diets reduced feed conversion efficiency and growth rates during the initial 250 d. However, after 250 d the weight gains by rats given legume-based diets were similar to those of controls given the same daily feed intake. Long-term consumption of diets containing low levels of kidney bean significantly altered body composition of rats. The levels of lipid in the body were significantly reduced. As a result, carcasses of these rats contained a higher proportion of muscle/protein than did controls. Small-intestine relative weight was increased by short- and long-term consumption of the kidney-bean-based diet. However, the increase in relative pancreatic weight observed at 30 d did not persist long term. None of the other legume-based diets caused any significant changes in body composition. However, long-term exposure to a soya-bean- or cowpea-based diet induced an extensive increase in the relative and absolute weights of the pancreas and caused an increase in the incidence of macroscopic pancreatic nodules and possibly pancreatic neoplasia. Long-term consumption of the cowpea-, kidney-bean-, lupin-seed- or soya-bean-based diets by rats resulted in a significant increase in the relative weight of the caecum and colon.
  • Article
    Soybeans are high in protein but also contain a number of minor constituents traditionally considered to be antinutritional factors. These include trypsin inhibitors, phytic acid, saponins and isoflavones. These compounds are now thought to have beneficial biological effects in the diet, such as lowering blood cholesterol or preventing cancer. Soybean processing changes the content of these minor constituents in various ways. This review discusses the changes in content of trypsin inhibitors, phytic acid, saponins and isoflavones as soybeans are processed into the conventional protein ingredients, flours, concentrates and isolates, as well as some of the traditional Oriental soybean foods.
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    We sought to determine the genetic basis of expression of the ubiquitous (metabolic) urease of soybean. This isozyme is termed the metabolic urease because its loss, in eu4/eu4 mutants, leads to accumulation of urea, whereas loss of the embryo-specific urease isozyme does not. The eu4 lesion eliminated the expression of the ubiquitous urease in vegetative and embryonic tissues. RFLP analysis placed urease clone LC4 near, or within, the Eu4 locus. Sequence comparison of urease proteins (ubiquitous and embryo-specific) and clones (LC4 and LS1) indicated that LC4 and LS1 encode ubiquitous and embryo-specific ureases, respectively. That LC4 is transcribed into poly(A)+ RNA in all tissues was indicated by the amplification of its transcript by an LC4-specific PCR primer. (The LS1-specific primer, on the other hand, amplified poly(A)+ RNA only from developing embryos expressing the embryo-specific urease.) These observations are consistent with Eu4 being the ubiquitous urease structural gene contained in the LC4 clone. In agreement with this notion, the mutant phenotype of eu4/eu4 callus was partially corrected by the LC4 urease gene introduced by particle bombardment.
  • Article
    Physicochemical characterization and biological properties of a new toxic protein isolated from soybeans (Glycine max) is reported. The purification procedure consisted basically of ammonium sulfate fractionation, ion exchange, and affinity chromatographies, the latter being used for the removal of the seed's lectin and of its trypsin inhibitor. The highly purified protein, designated soyatoxin, is a single chain acidic protein (pI 4.4-4.6) of 21 kDa, dependent on reduced thiol groups to maintain its solubility and biological activities. The toxin is a metalloprotein containing iron, calcium, zinc, and magnesium. Soyatoxin is highly toxic to mice (LD50 7-8 mg/kg mouse body wt upon intraperitoneal injection). It produces dyspnoea, tonic-clonic convulsions, and flaccid paralysis prior to death of intraperitoneally injected mice. Furthermore, soyatoxin is immunologically related to another toxic protein (canatoxin), isolated from Canavalia ensiformis seeds, which is distinct from soyatoxin in containing 18 x 10 kDa noncovalently bound subunits. Some biological properties including acute intraperitoneal toxicity, canatoxin-like immunoreactivity, hemagglutination, trypsin inhibitory activity, induction of platelet release reaction, and aggregation displayed by soyatoxin were studied and used to differentiate soyatoxin from soybean lectin and trypsin inhibitors.
  • Article
    There are a number of components present in soybeans that exert a negative impact on the nutritional quality of the protein. Among those factors that are destroyed by heat treatment are the protease inhibitors and lectins. Protease inhibitors exert their antinutritional effect by causing pancreatic hypertrophy/hyperplasia, which ultimately results in an inhibition of growth. The lectin, by virtue of its ability to bind to glycoprotein receptors on the epithelial cells lining the intestinal mucosa, inhibits growth by interfering with the absorption of nutrients. Of lesser significance are the antinutritional effects produced by relatively heat stable factors, such as goitrogens, tannins, phytoestrogens, flatus-producing oligosaccharides, phytate, and saponins. Other diverse but ill-defined factors appear to increase the requirements for vitamins A, B12, D, and E. The processing of soybeans under severe alkaline conditions leads to the formation of lysinoalanine, which has been shown to damage the kidneys of rats. This is not generally true, however, for edible soy protein that has been produced under milder alkaline conditions. Also meriting consideration is the allergenic response that may sometimes occur in humans, as well as calves and piglets, on dietary exposure to soybeans.
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    Animal feeding studies were conducted with rats, broiler chickens, catfish and dairy cows as part of a safety assessment program for a soybean variety genetically modified to tolerate in-season application of glyphosate. These studies were designed to compare the feeding value (wholesomeness) of two lines of glyphosate-tolerant soybeans (GTS) to the feeding value of the parental cultivar from which they were derived. Processed GTS meal was incorporated into the diets at the same concentrations as used commercially; diary cows were fed 10 g/100 g cracked soybeans in the diet, a level that is on the high end of what is normally fed commercially. In a separate study, laboratory rats were fed 5 and 10 g unprocessed soybean meal 100 g diet. The study durations were 4 wk (rats and dairy cows), 6 wk (broilers) and 10 wk (catfish). Growth, feed conversion (rats, catfish, broilers), fillet composition (catfish), and breast muscle and fat pad weights (broilers) were compared for animals fed the parental and GTS lines. Milk production, milk composition, rumen fermentation and nitrogen digestibility were also compared for dairy cows. In all studies, measured variables were similar for animals fed both GTS lines and the parental line, indicating that the feeding value of the two GTS lines is comparable to that of the parental line. These studies support detailed compositional analysis of the GTS seeds, which showed no meaningful differences between the parental and GTS lines in the concentrations of important nutrients and antinutrients. They also confirmed the results of other studies that demonstrated the safety of the introduced protein, a bacterial 5-enolpyruvyl-shikimate-3-phosphate synthase from Agrobacterium sp. strain CP4.
  • Article
    One important aspect of the safety assessment of genetically engineered crops destined for food and feed uses is the characterization of the consumed portion of the crop. One crop currently under development, glyphosate-tolerant soybeans (GTS), was modified by the addition of a glyphosate-tolerance gene to a commercial soybean cultivar. The composition of seeds and selected processing fractions from two GTS lines, designated 40-3-2 and 61-67-1, was compared with that of the parental soybean cultivar, A5403. Nutrients measured in the soybean seeds included macronutrients by proximate analyses (protein, fat, fiber, ash, carbohydrates), amino acids and fatty acids. Antinutrients measured in either the seed or toasted meal were trypsin inhibitor, lectins, isoflavones, stachyose, raffinose and phytate. Proximate analyses were also performed on batches of defatted toasted meal, defatted nontoasted meal, protein isolate, and protein concentrate prepared from GTS and control soybean seeds. In addition, refined, bleached, deodorized oil was made, along with crude soybean lecithin, from GTS and control soybeans. The analytical results demonstrated the GTS lines are equivalent to the parental, conventional soybean cultivar.
  • Article
    Bothrojaracin, a 27-kDa C-type lectin from Bothrops jararaca venom, is a selective and potent thrombin inhibitor (K(d) = 0.6 nM) which interacts with the two thrombin anion-binding exosites (I and II) but not with its catalytic site. In the present study, we analyzed the allosteric effects produced in the catalytic site by bothrojaracin binding to thrombin exosites. Opposite effects were observed with alpha-thrombin, which possesses both exosites I and II, and with gamma-thrombin, which lacks exosite I. On the one hand, bothrojaracin altered both kinetic parameters K(m) and k(cat) of alpha-thrombin for small synthetic substrates, resulting in an increased efficiency of alpha-thrombin catalytic activity. This effect was similar to that produced by hirugen, a peptide based on the C-terminal hirudin sequence (residues 54-65) which interacts exclusively with exosite I. On the other hand, bothrojaracin decreased the amidolytic activity of gamma-thrombin toward chromogenic substrates, although this effect was observed with higher concentrations of bothrojaracin than those used with alpha-thrombin. In agreement with these observaions, bothrojaracin produced opposite effects on the fluorescence intensity of alpha- and gamma-thrombin derivatives labeled at the active site with fluorescein-Phe-Pro-Arg-chloromethylketone. These observations support the conclusion that bothrojaracin binding to thrombin produces two different structural changes in its active site, depending on whether it interacts exclusively with exosite II, as seen with gamma-thrombin, or with exosite I (or both I and II) as observed with alpha-thrombin. The ability of bothrojaracin to evoke distinct modifications in the thrombin catalytic site environment when interacting with exosites I and II make this molecule an interesting tool for the study of allosteric changes in the thrombin molecule.
  • Article
    Full-text available
    Ubiquitous soybean urease, as opposed to the seed-specific urease, designates the seemingly identical ureolytic activities of suspension cultures and leaves. It also appears to be the basal urease in developing seeds of a variety, Itachi, which lacks the seed-specific urease (Polacco, Winkler 1984 Plant Physiol 74: 800-804). On native polyacrylamide gels the ureolytic activities in crude extracts of these three tissues comigrate as determined by assays of gel slices. At this level of resolution the ubiquitous urease also migrates with or close to the fast (trimeric) form of the seed-specific urease.The ubiquitous urease was purified approximately 100-fold from suspension cultures of two cultivars (Itachi and Prize) as well as from developing seeds of Itachi. These partially purified preparations allowed visualization of native urease on polyacrylamide gels by activity staining and of urease subunits on denaturing lithium dodecyl sulfate gels by electrophoretic transfer to nitrocellulose and immunological detection ("Western Blot"). The ubiquitous urease holoenzyme migrates slightly less rapidly than the fast seed urease in native gels; its subunit migrates slightly less rapidly than the 93.5 kilodaltons subunit of either the fast or slow (hexameric) seed enzyme. The ubiquitous urease elutes from an agarose A-0.5 meter column with the fast form of the seed urease species suggesting that the ubiquitous urease, like the fast seed urease, exists as a trimeric holoenzyme. The soybean cultivar, Prize, produces the hexameric seed urease; yet its ubiquitous urease (from leaf and suspension culture) is trimeric.The pH dependence of the ureolytic activity of seed coats of both seed urease-negative (Itachi) and seed urease-positive (Williams) cultivars suggests that this activity is exclusively the ubiquitous urease. Its relatively higher levels in seed coats than in embryos of Itachi suggests that the ubiquitous urease is involved in degradation of urea derived from ureides. Consistent with a ureide origin for urea is the observation that addition of a urease inhibitor, phenylphosphordiamidate, to extracts of developing Itachi seeds (seed coat plus embryo) results in accumulation of urea from allantoic acid.
  • Article
    Full-text available
    The soybean (Glycine max L. [Merrill]) var Itachi has 0.2 to 0.3% the urease activity found in developing embryos of a normal line, Prize. The hydroxyurea sensitivity and pH preference of this basal seed urease indicate that it represents a unique enzyme rather than an unusually low level of the normal seed urease. Itachi's seed urease is less sensitive to hydroxyurea inhibition (65-80% inhibition) than Prize seed urease (85-95% inhibition) and is more active at pH 6.1 and 8.8 than at 7.4, whereas the normal seed urease is least active at pH 8.8. Both properties of the basal seed urease are in agreement with the behavior of the leaf urease in extracts of Prize and Itachi leaves.Neither the leaf urease nor the Itachi seed urease is immuneprecipitated by affinity-purified seed urease antibodies. However, when antibody is in excess, Staphylococcus aureus (Cowan) cell walls containing protein A can precipitate soluble antibody-urease complexes (47-68% of total enzyme) from both leaf (Itachi and Prize) and Itachi seed extracts. Under identical conditions, greater than 90% of Prize seed urease is precipitated. At a 100-fold dilution of antibody, 60% of Prize seed urease is still antibody-complexed while the antibody recognition of the leaf or Itachi seed urease is reduced to 2 to 24%.The cell culture urease also resembles leaf urease by the criteria of pH preference, hydroxyurea sensitivity, and recognition by seed urease antibodies. In the presence of cycloheximide, nickel stimulates cell culture urease levels (14- or 35-fold depending on assay pH) indicating that cell cultures make a preponderance of apourease under nickel-limiting conditions.Inasmuch as the ureases of leaf, cell culture, and Itachi seeds are more closely related to each other than they are to the abundant (Prize) seed urease, suggests that the three tissues either contain an identical urease or related tissue-specific isozymes. This second form of urease may have an assimilatory role since it is found in both leaf and seed sink tissues and is required for urea assimilation in cell culture (Polacco 1977 Plant Physiol 59: 827-830).
  • Lectins as growth factors for the small intestine and the gut, in Lectins—Biomedical Perspectives
    • S Bardocz
    • Ewen
    • Swb
    • Grant
    • Pusztai
    51 Bardocz S, Ewen SWB, Grant G and Pusztai A, Lectins as growth factors for the small intestine and the gut, in Lectins—Biomedical Perspectives, Ed by Pusztai A and Bardocz S. Taylor and Francis, London, pp 103–116 (1995).