EGFR and p38MAPK Contribute to the Apoptotic Effect of the Recombinant Lectin from Tepary Bean (Phaseolus acutifolius) in Colon Cancer Cells.

Previous works showed that a Tepary bean lectin fraction (TBLF) induced apoptosis on colon cancer cells and inhibited early colonic tumorigenesis. One Tepary bean (TB) lectin was expressed in Pichia pastoris (rTBL-1), exhibiting similarities to one native lectin, where its molecular structure and in silico recognition of cancer-type N-glycoconjugates were confirmed. This work aimed to determine whether rTBL-1 retained its bioactive properties and if its apoptotic effect was related to EGFR pathways by studying its cytotoxic effect on colon cancer cells. Similar apoptotic effects of rTBL-1 with respect to TBLF were observed for cleaved PARP-1 and caspase 3, and cell cycle G0/G1 arrest and decreased S phase were observed for both treatments. Apoptosis induction on SW-480 cells was confirmed by testing HA2X, p53 phosphorylation, nuclear fragmentation, and apoptotic bodies. rTBL-1 increased EGFR phosphorylation but also its degradation by the lysosomal route. Phospho-p38 increased in a concentration- and time-dependent manner, matching apoptotic markers, and STAT1 showed activation after rTBL-1 treatment. The results show that part of the rTBL-1 mechanism of action is related to p38 MAPK signaling. Future work will focus further on the target molecules of this recombinant lectin against colon cancer.

Comparative study, homology modelling and molecular docking with cancer associated glycans of two non-fetuin-binding Tepary bean lectins.

We present the purification and characterization of the two most abundant isoforms of lectins isolated from Tepary bean (Phaseolus acutifolius) seeds, which have been shown to differentially affect the survival of different cancer cells. They were separated by concanavalin A-affinity chromatography. After purification, to release the N-glycans, they were digested with the endoglycosidases PNGase and Glycanase A. Fractions resulted from the hydrolysis products were analyzed to determine their carbohydrate composition. Mass spectrometry data indicated that both isoforms contained high mannose glycans being mannose 6 the most abundant form. Furthermore, based on sequence Ans-X-Ser/Thr, where X is any amino acid except proline, a glycosylation site was determined on asparagine 36. When their metal requirement to preserve their biological activity was determined, the lectins showed differences. While lectin A (LA) agglutination activity was best in the presence of magnesium, lectin B (LB) was best with calcium. Additionally, only LA exhibited affinity to human type-A erythrocytes. Although both lectins showed small differences in their properties, an identical structure-model for both lectins was generated by the homology modelling process. Also, the analysis of ligand binding sites and in silico glycosylation were achieved. Molecular docking with colon adenocarcinoma associated-N-glycans revealed some highly possible interactions and, on the other hand, that N-glycan interaction zones of Tepary bean lectins is not restricted to the carbohydrate binding domain but to an extended part of their surface, which could lead new strategies to explain their biological activity.

Hybridization between Crotalus aquilus and Crotalus polystictus Species: A Comparison of Their Venom Toxicity and Enzymatic Activities.

Hybridization is defined as the interbreeding of individuals from two populations distinguishable by one or more heritable characteristics. Snake hybridization represents an interesting opportunity to analyze variability and how genetics affect the venom components between parents and hybrids. Snake venoms exhibit a high degree of variability related to biological and biogeographical factors. The aim of this work is to analyze the protein patterns and enzymatic activity of some of the main hemotoxic enzymes in snake venoms, such as serine proteases (trypsin-like, chymotrypsin-like, and elastase-like), metalloproteases, hyaluronidases, and phospholipase A2. The lethal dose of 50 (LD50) of venom from the Crotalus aquilus (Cabf) and Crotalus polystictus (Cpbm) parents and their hybrids in captivity was determined, and phenetic analysis is also conducted, which showed a high similarity between the hybrids and C. polystictus. The protein banding patterns and enzymatic activity analyze by zymography resulted in a combination of proteins from the parental venoms in the hybrids, with variability among them. In some cases, the enzymatic activity is higher in the hybrids with a lower LD50 than in the parents, indicating higher toxicity. These data show the variability among snake venoms and suggest that hybridization is an important factor in changes in protein concentration, peptide variability, and enzymatic activity that affect toxicity and lethality.

Bioaccessibility and In Vitro Intestinal Permeability of a Recombinant Lectin from Tepary Bean (Phaseolus acutifolius) Using the Everted Intestine Assay.

Tepary bean (Phaseolus acutifolius) lectins exhibit differential in vitro and in vivo biological effects, but their gastrointestinal interactions and digestion have not yet been assessed. This work aimed to evaluate the changes of a recombinant Tepary bean lectin (rTBL-1) through an in vitro and ex vivo gastrointestinal process. A polyclonal antibody was developed to selectively detect rTBL-1 by Western blot (WB) and immunohistochemical analysis. Everted gut sac viability was confirmed until 60 min, where protein bioaccessibility, apparent permeability coefficient, and efflux ratio showed rTBL-1 partial digestion and absorption. Immunoblot assays suggested rTBL-1 internalization, since the lectin was detected in the digestible fraction. The immunohistochemical assay detected rTBL-1 presence at the apical side of the small intestine, potentially due to the interaction with the intestinal cell membrane. The in silico interactions between rTBL-1 and some saccharides or derivatives showed high binding affinity to sialic acid (-6.70 kcal/mol) and N-acetylglucosamine (-6.10 kcal/mol). The ultra-high-performance liquid chromatography-electron spray ionization-quantitative time-of-flight coupled to mass spectrometry (UHPLC-ESI-QTOF/MS) analysis showed rTBL-1 presence in the gastric content and the non-digestible fraction after intestinal simulation conditions. The results indicated that rTBL-1 partially resisted the digestive conditions and interacted with the intestinal membrane, whereas its digestion allowed the absorption or internalization of the protein or the derivative peptides. Further purification of digestion samples should be conducted to identify intact rTBL-1 protein and digested peptides to assess their physiological effects.

Tepary bean (Phaseolus acutifolius) lectin fraction provokes reversible adverse effects on rats' digestive tract.

The Tepary bean (Phaseolus acutifolius) lectin fraction (TBLF) exhibits differential cytotoxicity on colon cancer cells and inhibition of early tumorigenesis in the colon (50 mg/kg, three times per week, for 6 weeks). TBLF showed low toxicity with the ability to activate the immune system; however, some adverse effects are the loss in body weight gain, intestinal atrophy, and pancreatic hyperplasia. After a recovery period of 2 weeks after treatment, reversion of pancreatic hyperplasia but no recovery of intestinal atrophy was observed. As TBLF has shown anticancer effects on the colon, it is important to characterize the adverse effects and how they can be reversed. Sprague Dawley rats were administered with TBLF (50 mg/kg) for 6 weeks, three times per week, and then allowed to recover for 6 weeks post-treatment. After TBLF administration, small intestine atrophy, villus atrophy, and cryptic hyperplasia were confirmed, as well as increased intestinal mucus production, increased permeability and a decrease in the apparent ileal digestibility of crude proteins. The colon showed damage in the simple prismatic tissue and decreased crypt depth, and changes in microbiota and a decrease in the apparent fecal digestibility of crude protein were determined. Our results show that the adverse effects provoked by TBLF were partially reversed after 6 weeks of recovery post-treatment, suggesting that increasing the recovery period it could be possible to reverse all adverse effects observed.

Agrobacterium rhizogenes-mediated transformation of grain (Amaranthus hypochondriacus) and leafy (A. hybridus) amaranths.

KEY MESSAGE: Transgenic A. hypochondriacus and A. hybridus roots were generated. Further, a distinct plant regeneration program via somatic embryos produced from hairy roots was established. Work was implemented to develop an optimized protocol for root genetic transformation of the three grain amaranth species and A. hybridus, their presumed ancestor. Transformation efficiency was species-specific, being higher in A. hypochondriacus and followed by A. hybridus. Amaranthus cruentus and A. caudatus remained recalcitrant. A reliable and efficient Agrobacteruim rhizogenes-mediated transformation of these species was established using cotyledon explants infected with the previously untested BVG strain. Optimal OD600 bacterial cell densities were 0.4 and 0.8 for A. hypochondriacus and A. hybridus, respectively. Hairy roots of both amaranth species were validated by the amplification of appropriate marker genes and, when pertinent, by monitoring green fluorescent protein emission or ß-glucuronidase activity. Embryogenic calli were generated from A. hypochondriacus rhizoclones. Subsequent somatic embryo maturation and germination required the activation of cytokinin signaling, osmotic stress, red light, and calcium incorporation. A crucial step to ensure the differentiation of germinating somatic embryos into plantlets was their individualization and subcultivation in 5/5 media containing 5% sucrose, 5 g/L gelrite, and 0.2 mg/L 2-isopentenyladenine (2iP) previously acidified to pH 4.0 with phosphoric acid, followed by their transfer to 5/5 + 2iP media supplemented with 100 mg/L CaCl2. These steps were strictly red light dependent. This process represents a viable protocol for plant regeneration via somatic embryo germination from grain amaranth transgenic hairy roots. Its capacity to overcome the recalcitrance to genetic transformation characteristic of grain amaranth has the potential to significantly advance the knowledge of several unresolved biological aspects of grain amaranths.

Recombinant Lectin from Tepary Bean (Phaseolus acutifolius) with Specific Recognition for Cancer-Associated Glycans: Production, Structural Characterization, and Target Identification.

Herein, we report the production of a recombinant Tepary bean lectin (rTBL-1), its three-dimensional (3D) structure, and its differential recognition for cancer-type glycoconjugates. rTBL-1 was expressed in Pichia pastoris, yielding 316 mg per liter of culture, and was purified by nickel affinity chromatography. Characterization of the protein showed that rTBL-1 is a stable 120 kDa homo-tetramer folded as a canonical leguminous lectin with two divalent cations (Ca2+ and Mn2+) attached to each subunit, confirmed in its 3D structure solved by X-ray diffraction at 1.9 Å resolution. Monomers also presented a ~2.5 kDa N-linked glycan located on the opposite face of the binding pocket. It does not participate in carbohydrate recognition but contributes to the stabilization of the interfaces between protomers. Screening for potential rTBL-1 targets by glycan array identified 14 positive binders, all of which correspond to ß1-6 branched N-glycans' characteristics of cancer cells. The presence of α1-6 core fucose, also tumor-associated, improved carbohydrate recognition. rTBL-1 affinity for a broad spectrum of mono- and disaccharides was evaluated by isothermal titration calorimetry (ITC); however, no interaction was detected, corroborating that carbohydrate recognition is highly specific and requires larger ligands for binding. This would explain the differential recognition between healthy and cancer cells by Tepary bean lectins.

Quantum Dot Labelling of Tepary Bean (Phaseolus acutifolius) Lectins by Microfluidics.

Lectins are bioactive proteins with the ability to recognize cell membrane carbohydrates in a specific way. Diverse plant lectins have shown diagnostic and therapeutic potential against cancer, and their cytotoxicity against transformed cells is mediated through the induction of apoptosis. Previous works have determined the cytotoxic activity of a Tepary bean (Phaseolus acutifolius) lectin fraction (TBLF) and its anti-tumorigenic effect on colon cancer. In this work, lectins from the TBLF were additionally purified by ionic-exchange chromatography. Two peaks with agglutination activity were obtained: one of them was named TBL-IE2 and showed a single protein band in two-dimensional electrophoresis; this one was thus selected for coupling to quantum dot (QD) nanoparticles by microfluidics (TBL-IE2-QD). The microfluidic method led to low sample usage, and resulted in homogeneous complexes, whose visualization was achieved using multiphoton and transmission electron microscopy. The average particle size (380 nm) and the average zeta potential (-18.51 mV) were determined. The cytotoxicity of the TBL-IE2 and TBL-IE2-QD was assayed on HT-29 colon cancer cells, showing no differences between them (p ≤ 0.05), where the LC50 values were 1.0 × 10-3 and 1.7 × 10-3 mg/mL, respectively. The microfluidic technique allowed control of the coupling between the QD and the protein, substantially improving the labelling process, providing a rapid and efficient method that enabled the traceability of lectins. Future studies will focus on the potential use of the QD-labelled lectin to recognize tumor tissues.

Tepary Bean (Phaseolus acutifolius) Lectins Induce Apoptosis and Cell Arrest in G0/G1 by P53(Ser46) Phosphorylation in Colon Cancer Cells.

A Tepary bean lectin fraction (TBLF) has been studied because it exhibits differential cytotoxic and anticancer effects on colon cancer. The present work focuses on the evaluation of the apoptotic mechanism of action on colon cancer cells. Initially, lethal concentrations (LC50) were obtained for the three studied cell lines (HT-29, RKO and SW-480). HT-29 showed the highest LC50, 10 and 100 times higher than that of RKO and SW-480 cells, respectively. Apoptosis was evaluated by flow cytometry, where HT-29 cells showed the highest levels of early and total apoptosis, caspases activity was confirmed and necrosis was discarded. The effect on cell cycle arrest was shown in the G0/G1 phase. Specific apoptosis-related gene expression was determined, where an increase in p53 and a decrease in Bcl-2 were observed. Expression of p53 gene showed the maximum level at 8 h with an important decrease at 12 and 24 h, also the phosphorylated p53(ser46) increased at 8 h. Our results show that TBLF induces apoptosis in colon cancer cells by p-p53(ser46) involvement. Further studies will focus on studying the specific signal transduction pathway.

Properties of a Non-canonical Complex Formed Between a Tepary Bean (Phaseolus acutifolius) Protease Inhibitor and α-Chymotrypsin.

Protease inhibitors are crucial for the control of proteolytic activity in different physiological processes. However, some inhibitors do not show canonical enzyme recognition of the enzyme under certain conditions. In this work, we present evidence that indicates the formation of an active complex between the protease bovine α-chymotrypsin and the Tepary bean protease inhibitor (TBPI). The composition of the active chymotrypsin-TBPI complex (AC) was confirmed by three different methods: size-exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry. The kinetic parameters for the AC were similar to those of the enzyme alone, indicating that TBPI binding does not produce any large changes in chymotrypsin. The molecular model proposed here postulates that TBPI binds outside the active cleft of the protease, but near enough to hinder the binding of high molecular weight substrates into the active site. This model was experimentally supported by the inhibitory effect on casein as a substrate, and the unaltered protease activity when a small synthetic substrate was used. We also found that the formation of this complex provided the enzyme with extra stability in denaturing conditions or in the presence of a reducing agent. The chymotrypsin-TBPI complex exhibited higher stability, indicating that autolysis can be partially prevented. When the enzyme was first inactivated followed by the addition of the inhibitor, the activity of the protease was restored. We described a possible mechanism where a plant protease inhibitor binds outside the active site of the enzyme while increasing its stability.

Snake Venom Hemotoxic Enzymes: Biochemical Comparison between Crotalus Species from Central Mexico.

Snakebite envenoming is a serious medical problem in different areas of the world. In Latin America, the major prevalence is due to snakes of the family Viperidae, where rattlesnakes (Crotalus) are included. They produce hemotoxic venom which causes bleeding, tissue degradation and necrosis. Each venom has several enzymatic activities, producing different effects in the envenoming, doing its clinical effects difficult to study. Comparison between venom molecules is also difficult when different techniques are used, and therefore, their identification/characterization using the same methodology is necessary. In this work, a general biochemical characterization in snake venom of serine proteases (SVSP), phospholipases A2 (PLA2), metalloproteases (SVMP) and hyaluronidases (SVH) of Crotalus aquilus (Ca), Crotalus polystictus (Cp) and Crotalus molossus nigrescens (Cmn) was done. Differences in protein pattern, enzyme content and enzymatic activities were observed. All the venoms showed high PLA2 activity, high molecular weight SVSP, and a wide variety of SVMP and SVH forms. Ca and Cp showed the highest enzymatic activities of SVMP and SVSP trypsin-like and chymotrypsin-like, whereas Cmn showed the highest SVH and similar PLA2 activity with Ca. All the venoms showed peptides with similar molecular weight to crotamine-like myotoxins. No previous biochemical characterization of C. aquilus has been reported and there are no previous analyses that include these four protein families in these Crotalus venoms.

Rhizosecretion of a cisgenic lectin by genetic manipulation of Tepary bean plants (Phaseolus acutifolius).

Tepary bean (Phaseolus acutifolius) lectin fraction (TBLF) has been shown to specifically bind and induce cell death of different types of cancer cells and also has exhibited an effect on early colon tumorigenesis. However, the development of a pharmaceutical formula is not possible yet because the production process is expensive and slow and provides low yields. Therefore, the purpose of the present work was to develop a strategy to produce one bioactive lectin by rhizosecretion through root exudates on genetically modified plants. Amplification of Tepary bean transcripts was performed using degenerate primers, and the products obtained were sequenced. Multiple alignments of sequences led to elucidating one of the lectins present in TBLF. Its coding sequence was flanked by an N-terminal secretion signal peptide and a 6xHis-tail. This construction was introduced into P. acutifolius plants using Agrobacterium tumefaciens to subsequently carry out the in vitro growth of the plants. When roots grew, plants were transferred to hydroponic conditions and root exudates were analyzed. Results showed the presence of a glycosylated cisgenic lectin with biological activity, confirming that the strategy followed provides an alternative for the synthetic production and purification of this lectin.

Expression of Lectins in Heterologous Systems.

Lectins are proteins that have the ability to recognize and bind in a reversible and specific way to free carbohydrates or glycoconjugates of cell membranes. For these reasons, they have been extensively used in a wide range of industrial and pharmacological applications. Currently, there is great interest in their production on a large scale. Unfortunately, conventional techniques do not provide the appropriate platform for this purpose and therefore, the heterologous production of lectins in different organisms has become the preferred method in many cases. Such systems have the advantage of providing better yields as well as more homogeneous and better-defined properties for the resultant products. However, an inappropriate choice of the expression system can cause important structural alterations that have repercussions on their biological activity since the specificity may lay in their post-translational processing, which depends largely on the producing organism. The present review aims to examine the most representative studies in the area, exposing the four most frequently used systems (bacteria, yeasts, plants and animal cells), with the intention of providing the necessary information to determine the strategy to follow in each case as well as their respective advantages and disadvantages.

Effects of intragastrically-administered Tepary bean lectins on digestive and immune organs: Preclinical evaluation.

Previous work showed that Tepary bean (Phaseolus acutifolius) lectins exhibit differential cytotoxic effects on cancer cell lines by apoptosis induction. In vivo studies using a Tepary bean lectin fraction (TBLF, 50 mg/kg of body weight) after colon cancer induction in rats showed that TBLF inhibited early precancerous lesions without systemic toxicity however, loss of body weight gain and activation of immune cells were observed. In order to know more about the possible adverse effects, we evaluated the administration of TBLF on digestive and immune organs. Sprague Dawley rats were administered TBLF for six weeks and allowed to recover for two weeks. Immune activation was observed through an increased lymphocyte-granulocyte ratio, an increased number of lymphoid follicles in intestinal Peyer's patches and a slight expansion of the splenic white pulp. Atrophy was observed in small intestine villi and crypt foci of the colon without normalization after the recovery period. Pancreas histopathology showed hypertrophy after the six-week administration period, particularly vacuolation and trabecular widening; but after the two-week recovery period atrophy was observed, suggesting a partial compensatory type process. Our results show that TBLF activates the immune system and affects digestive organs through direct interaction with intestinal epithelium, and indirectly by producing pancreatic hyperfunction. Further work will focus in longer recuperation periods after TBLF treatment.

Phaseolus acutifolius Lectin Fractions Exhibit Apoptotic Effects on Colon Cancer: Preclinical Studies Using Dimethilhydrazine or Azoxi-Methane as Cancer Induction Agents.

Phaseolus acutifolius (Tepary bean) lectins have been studied as cytotoxic molecules on colon cancer cells. The toxicological profile of a Tepary bean lectin fraction (TBLF) has shown low toxicity in experimental animals; exhibiting anti-nutritional effects such as a reduction in body weight gain and a decrease in food intake when using a dose of 50 mg/kg on alternate days for six weeks. Taking this information into account, the focus of this work was to evaluate the effect of the TBLF on colon cancer using 1,2-dimethylhydrazine (DMH) or azoxy-methane/dextran sodium sulfate (AOM/DSS) as colon cancer inductors. Rats were treated with DMH or AOM/DSS and then administered with TBFL (50 mg/kg) for six weeks. TBLF significantly decreased early tumorigenesis triggered by DMH by 70%, but without any evidence of an apoptotic effect. In an independent experiment, AOM/DSS was used to generate aberrant cryptic foci, which decreased by 50% after TBLF treatment. TBLF exhibited antiproliferative and proapoptotic effects related to a decrease of the signal transduction pathway protein Akt in its activated form and an increase of caspase 3 activity, but not to p53 activation. Further studies will deepen our knowledge of specific apoptosis pathways and cellular stress processes such as oxidative damage.

Plant Lectins as Medical Tools against Digestive System Cancers.

Digestive system cancers-those of the esophagus, stomach, small intestine, colon-rectum, liver, and pancreas-are highly related to genetics and lifestyle. Most are considered highly mortal due to the frequency of late diagnosis, usually in advanced stages, caused by the absence of symptoms or masked by other pathologies. Different tools are being investigated in the search of a more precise diagnosis and treatment. Plant lectins have been studied because of their ability to recognize and bind to carbohydrates, exerting a variety of biological activities on animal cells, including anticancer activities. The present report integrates existing information on the activity of plant lectins on various types of digestive system cancers, and surveys the current state of research into their properties for diagnosis and selective treatment.

Common Bean Leaves as a Source of Dietary Iron: Functional Test in an Iron-Deficient Rat Model.

Recent findings made by our group indicate that the iron content in Phaseolus vulgaris leaves is at least four times greater than in grains therefore, we evaluated the effect of supplementation with bean leaf (iron content of 275 mg/kg on a dry basis) in iron-deficient rats. Anemia was induced by feeding rats with an iron-deficient diet (IDD) for 11 days and iron-recovery diets were subsequently tested for 14 days using a normal diet, a 10 % bean leaf-supplemented IDD (BLSD) or a ferrous sulfate-supplemented IDD. Decreased levels of leukocytes (64 %), erythrocytes (30 %), lymphocytes (62 %), granulocytes (72 %), hematocrit (34 %), hemoglobin (35 %), and ferritin (34 %) were observed in the iron-deficient rats compared to the control rats. BLSD supplementation showed the highest recovery values relative to those recorded for control rats: leukocytes (40 %), erythrocytes (24 %), lymphocytes (33 %), granulocytes (88 %), hematocrit (17 %), and hemoglobin (18 %), suggesting that common bean leaves could be a good source of bioavailable iron with possible immunomodulatory effects.

Tolerability assessment of a lectin fraction from Tepary bean seeds (Phaseolus acutifolius) orally administered to rats.

Our previous studies have shown that a lectin rich fraction (TBLF) extracted from Tepary bean seeds differentially inhibits cancer cells proliferation in vitro. Before testing the in vivo anticancer effect, the acute and subchronic toxicological assays in rats were conducted, where an oral dose of 50 mg/body weight kg was determined as the NOAEL. This study evaluated the resistance to digestion and complete blood count (CBC) after 24 h of the orally administered 50 mg/kg TBLF. The digestion resistance test showed lectins activity retention after 72 h and the CBC study showed a high level of eosinophils, suggesting an allergic-like response. Tolerability was assayed after 6 weeks of treatment by dosing with an intragastric cannula every third day per week. It was observed a transient reduction in food intake and body weight in the first weeks, resulting in body weight gain reduction of 10% respect to the control group at the end of the study. Additionally, organs weight, histopathological analysis and blood markers for nutritional status and for liver, pancreas and renal function were not affected. Our results suggest that 50 mg/kg TBLF administered by oral route, exhibit no toxicity in rats and it was well tolerated. Further studies will focus on long-term studies.

Exclusive rewards in mutualisms: ant proteases and plant protease inhibitors create a lock-key system to protect Acacia food bodies from exploitation.

Myrmecophytic Acacia species produce food bodies (FBs) to nourish ants of the Pseudomyrmex ferrugineus group, with which they live in an obligate mutualism. We investigated how the FBs are protected from exploiting nonmutualists. Two-dimensional gel electrophoresis of the FB proteomes and consecutive protein sequencing indicated the presence of several Kunitz-type protease inhibitors (PIs). PIs extracted from Acacia FBs were biologically active, as they effectively reduced the trypsin-like and elastase-like proteolytic activity in the guts of seed-feeding beetles (Prostephanus truncatus and Zabrotes subfasciatus), which were used as nonadapted herbivores representing potential exploiters. By contrast, the legitimate mutualistic consumers maintained high proteolytic activity dominated by chymotrypsin 1, which was insensitive to the FB PIs. Larvae of an exploiter ant (Pseudomyrmex gracilis) taken from Acacia hosts exhibited lower overall proteolytic activity than the mutualists. The proteases of this exploiter exhibited mainly elastase-like and to a lower degree chymotrypsin 1-like activity. We conclude that the mutualist ants possess specifically those proteases that are least sensitive to the PIs in their specific food source, whereas the congeneric exploiter ant appears partly, but not completely, adapted to consume Acacia FBs. By contrast, any consumption of the FBs by nonadapted exploiters would effectively inhibit their digestive capacities. We suggest that the term 'exclusive rewards' can be used to describe situations similar to the one that has evolved in myrmecophytic Acacia species, which reward mutualists with FBs but safeguard the reward from exploitation by generalists by making the FBs difficult for the nonadapted consumer to use.

Effects of Tepary bean (Phaseolus acutifolius) protease inhibitor and semipure lectin fractions on cancer cells.

Some natural and synthetic protease inhibitors (PI), such as the Bowman-Birk PI from soybean, have anticancer effects. We previously purified and characterized a Bowman-Birk-type PI from Tepary bean (Phaseolus acutifolius) seeds (TBPI). A semipure protein fraction containing this inhibitor, when tested its in vitro effect on transformed cells, showed a differential cytotoxic effect, as well as an increase in cell attachment to culture dishes. In this article we report that lectins were responsible for the cytotoxic effect previously observed, exhibiting a differential, antiproliferative effect on nontransformed cells and on different lineages of cancer cells. Although the purified TBPI lacked cytotoxicity, it was found to be responsible for the increase in cell adhesion, decreasing culture dishes' extracellular matrix degradation, leading to a decrease of the in vitro cell invasion capacity. This effect coincided with the suppression of Matrix Metalloproteinase-9 activity. These results indicate that Tepary bean seeds contain at least 2 different groups of bioactive proteins with distinct effects on cancer cells.