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Highlights: [•] A simple and quick method for low TIA soymilk is supplied. [•] Heat-induced KTI incorporation into protein aggregates causes KTI’s TIA loss. [•] Heat-induced peptide bond cleavage of BBI causes BBI’s TIA/CIA loss. [•] NaCl accelerates protein aggregate formation and peptide bond cleavage of BBI. [ABSTRACT FROM AUTHOR]

Highlights: [•] First demonstration of magnetic extraction in flow through mode. [•] Purification of Bowman-Birk protease inhibitor from pretreated soy feedstock. [•] New, easily cleanable stainless steel Continuous Magnetic Extraction equipment. [•] Scalable process and therefore adaptable to virtually any volume. [•] Easily separable, DEAP functionalized magnetic PVA beads for contaminant adsorption. [ABSTRACT FROM AUTHOR]

In previously performed animal studies and Phase I-II human trials, Bowman-Birk inhibitor concentrate (BBIC) appeared to be a promising cancer chemopreventive agent. The present study describes the results of two phase I randomized double-blind placebo-controlled trials performed in male subjects to assess the safety and toxicity of the original and new formulations of BBIC administered in a single dose as a suspension in orange juice. The dose of BBIC varied from 800-2,000 chymotrypsin inhibitor (CI) units. The BBI concentration in the serum samples collected from the subjects was analyzed by a dot-blot analysis procedure using the 5G2 monoclonal antibody, which is specific for reduced BBI. A total of 41 subjects were enrolled, 20 in the initial BBIC study and 21 in the second BBIC study. In these human trials, no clinically relevant changes in hematological or biochemical parameters were observed. Overall, BBIC was found to be well-tolerated. For these BBIC single-dose phase I trials, there was no dose-limiting toxicity for BBIC, even at the highest dose evaluated, and there were no apparent differences between the clinical trial results for the two formulations of BBIC. The bioavailability of BBI in the second clinical trial, which used the new BBIC formulation, was approximately 40 to 43% of the BBI bioavailability reached in the first clinical trial, which used the original BBIC formulation. The observed bioavailability difference was attributed to the different BBIC formulations used in these two clinical trials. These trials demonstrated that BBIC is safe when administered in a single dose of up to 2,000 CI units. Therefore, the results from the two trials indicate that a multi-dose trial of BBIC may be safely performed with doses of up to 2,000 CI units per day. [ABSTRACT FROM AUTHOR]

Proteasome inhibitors have been described as an important target for cancer therapy due to their potential to regulate the ubiquitin-proteasome system in the degradation pathway of cellular proteins. Here, we reported the effects of a Bowman-Birk-type protease inhibitor, the Black-eyed pea Trypsin/Chymotrypsin Inhibitor (BTCI), on proteasome 20S in MCF-7 breast cancer cells and on catalytic activity of the purified 20S proteasome from horse erythrocytes, as well as the structural analysis of the BTCI-20S proteasome complex. In vitro experiments and confocal microscopy showed that BTCI readily crosses the membrane of the breast cancer cells and co-localizes with the proteasome in cytoplasm and mainly in nucleus. Indeed, as indicated by dynamic light scattering, BTCI and 20S proteasome form a stable complex at temperatures up to 55°C and at neutral and alkaline pHs. In complexed form, BTCI strongly inhibits the proteolytic chymotrypsin-, trypsin- and caspase-like activities of 20S proteasome, indicated by inhibition constants of 10−7 M magnitude order. Besides other mechanisms, this feature can be associated with previously reported cytostatic and cytotoxic effects of BTCI in MCF-7 breast cancer cells by means of apoptosis. [ABSTRACT FROM AUTHOR]

The peptide leucine arginine (pLR)belongs to a new class of cyclicpeptides isolated from frog skin. Its primary sequence is similarto the reactive loop of plant Bowman–Birk inhibitors (BBI),and the recently discovered circular sunflower trypsin inhibitor-1(SFTI-1). The conformational properties of pLR in solution were determinedby NMR spectroscopy and revealed excellent structural similarity toBBI and SFTI-1. Moreover, pLR is a highly potent trypsin inhibitor,with Kivalues in the nanomolar range,and, due to its small size, a potential inhibitor of the serine proteasetryptase. Since tryptase plays a crucial role in the development ofallergic airway inflammation, the therapeutic potential of pLR ina murine asthma model was investigated. Treatment of ovalbumin-sensitizedmice with pLR during allergen challenge reduced the acute asthma phenotype.Most importantly, application even at the end of a long-lasting chronicasthma model decreased the development of chronic airway inflammationand tissue remodeling. [ABSTRACT FROM AUTHOR]

Highlights: [•] Legume Bowman-Birk inhibitors contain seven conserved disulfide bonds and are stable to heat. [•] Two intradomain disulfides in the trypsin subdomain contribute profoundly to thermal stability. [•] A disulfide in the trypsin subdomain plays a key role in structural and functional stability. [•] Overall the study demonstrates that the two subdomains in BBIs are not independent of each other. [ABSTRACT FROM AUTHOR]

Abstract: The seeds of the legume horsegram (Dolichos biflorus), a protein rich pulse (bean), contain multiple forms of Bowman-Birk inhibitors (protease inhibitors). The major inhibitor HGI-III contains seven interweaving disulfides and is extremely stable to high temperatures. A soluble HGI-III (rHGI) with the native N-terminus was produced using a pTWIN IMPACT™ purification system. Yield of rHGI was improved by introducing a trypsin sepharose affinity chromatography step resulting in ∼670 fold purification. The biochemical characteristics of rHGI point to its close similarity to seed HGI-III not only in its structure but also in its inhibitory characteristics toward bovine trypsin and chymotrypsin. The expression and purification strategy presented here promises to produce BBIs in their natural form for pharmacological and therapeutic use. [Copyright &y& Elsevier]

The present study examines the resistance of the α-lactalbumin to α-chymotrypsin (EC 3.4.21.1) digestion under various experimental conditions. Whey protein isolate (WPI) was hydrolysed using randomised hydrolysis conditions (5 and 10% of WPI; pH 7·0, 7·8 and 8·5; temperature 25, 37 and 50 °C; enzyme-to-substrate ratio, E/S, of 0·1%, 0·5 and 1%). Reversed-phase high performance liquid chromatography (RP-HPLC) was used to analyse residual proteins. Heat, pH adjustment and two inhibitors (Bowman–Birk inhibitor and trypsin inhibitor from chicken egg white) were used to stop the enzyme reaction. While operating outside of the enzyme optimum it was observed that at pH 8·5 selective hydrolysis of β-lactoglobulin was improved because of a dimer-to-monomer transition while α-la remained relatively resistant. The best conditions for the recovery of native and pure α-la were at 25 °C, pH 8·5, 1% E/S ratio, 5% WPI (w/v) while the enzyme was inhibited using Bowman–Birk inhibitor with around 81% of original α-la in WPI was recovered with no more β-lg. Operating conditions for hydrolysis away from the chymotrypsin optimum conditions offers a great potential for selective WPI hydrolysis, and removal, of β-lg with production of whey protein concentrates containing low or no β-lg and pure native α-la. This method also offers the possibility for production of β-lg-depleted milk products for sensitive populations. [ABSTRACT FROM PUBLISHER]

Abstract: Bowman–Birk inhibitor (BBI) genes encode serine protease inhibitors well known for their anticarcinogenic properties and roles in plant defense against insects and pathogens. Here we investigated the expression of a BBI gene in response to water deficit, recovery and phytohormones. A full length cDNA encoding a novel BBI (AhBBI) was isolated from peanut (Arachis hypogaea L.) leaves. The deduced protein is a polypeptide of 11.5kDa containing a signal peptide of 20 amino acids which is missing from peanut seed full-length BBI. Sequence analysis showed that AhBBI presents the characteristic features of BBIs but its first inhibitory loop is unique among the Fabaceae species. Real-time PCR analyses indicated that in peanut leaves, AhBBI is upregulated by water deficit and exogenous jasmonic acid (JA) but repressed by abscissic acid (ABA) after 24h of treatment. The transcripts accumulation patterns during water deficit differed between two cultivars studied in relation to their tolerance levels to drought. AhBBI transcripts accumulated earlier and stronger in the tolerant cultivar (cv. Fleur11) compared to the susceptible one (cv. 73–30) suggesting that BBI genes are involved in drought stress tolerance. Subsequent rehydration reversed the accumulation of AhBBI transcripts in both cultivars but at different levels. The overall role of BBI in abiotic stress tolerance and the possible mechanisms of action are discussed. [Copyright &y& Elsevier]

Abstract: Soybean proteins offer exceptional promise in the area of cancer prevention and treatment. Specifically, Bowman-Birk Inhibitor (BBI) has the ability to suppress carcinogenesis in vivo, which has been attributed to BBI’s inhibition of serine protease (trypsin and chymotrypsin) activity. The lack of molecular probes for the isolation of this protein has made it difficult to work with, limiting its progress as a significant candidate in the treatment of cancer. This study has successfully identified a set of novel synthetic peptides targeting the BBI, and has demonstrated the ability to bind BBI in vitro. One of those probes has been covalently immobilised on superparamagnetic microbeads to allow the isolation of BBI from soy whey mixtures in a single step. Our ultimate goal is the use of the described synthetic probe to facilitate the isolation of this potentially therapeutic protein for low cost, scalable analysis and production of BBI. [Copyright &y& Elsevier]

A pool of twelve cDNA sequences coding for Bowman–Birk inhibitors (BBIs) was identified in the legume grass pea (Lathyrus sativus L.). The corresponding amino acid sequences showed a canonical first anti-trypsin domain, predicted according to the identity of the determinant residue P1. A more variable second binding loop was observed allowing to identify three groups based on the identity of residue P1: two groups (Ls_BBI_1 and Ls_BBI_2) carried a second reactive site specific for chymotrypsin, while a third group (Ls_BBI_3) was predicted to inhibit elastase. A fourth variant carrying an Asp in the P1 position of the second reactive site was identified only from genomic DNA. A phylogenetic tree constructed using grass pea BBIs with their homologs from other legume species revealed grouping based on taxonomy and on specificity of the reactive sites. Five BBI sequences, representing five different second reactive sites, were heterologously expressed in the yeast Pichia pastoris. The recombinant proteins demonstrated to be active against trypsin, while three of them were also active against chymotrypsin, and one against human leukocyte elastase. Comparative modeling and protein docking were used to further investigate interactions between two grass pea BBI isoforms and their target proteases. Thus two reliable 3D models have been proposed, representing two potential ternary complexes, each constituted of an inhibitor and its target enzymes. [ABSTRACT FROM AUTHOR]

Soybean [Glycine max (L.) Merr.] contains several health promoting compounds including phytosterols, isoflavones, phytic acid, and protease inhibitors. The two abundant protease inhibitors of soybean seeds are the Kunitz trypsin inhibitor and the Bowman-Birk inhibitor (BBI). Bowman-Birk inhibitor has been touted as a potential cancer chemopreventive agent for humans. Little information is available on the effect of growing location on the accumulation of this cancer chemopreventive agent. In this study we have examined the protein profile of eight soybean varieties that were grown in three Missouri locations in 2009 and 2010. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that soybean varieties that were grown in Grand Pass contained elevated levels of the β subunit of β-conglycinin and reduced amounts of BBI. This observation was further confirmed by Western blot analysis. This difference in the levels of BBI was also reflected in the chymotrypsin inhibitor activity. Growing location also influenced the overall S content of soybean seeds as evidenced by inductively coupled plasma mass spectrometry analysis. Seeds grown in Grand Pass had lower amounts of total S content in both 2009 and 2010. Our results demonstrate that growing location has a profound effect on the accumulation of BBI and it is possible to modulate the concentration of this cancer chemopreventive agent by simple changes in agronomic practices. [ABSTRACT FROM AUTHOR]

Abstract: Bowman-Birk inhibitors (BBIs) are protein molecules containing two inhibitory domains for enzymes similar to trypsin and chymotrypsin. Interest in these inhibitors arose from their properties against the cancer chemically induced by 1,2-dimethylhydrazine (DMH). In this study the effect of two BBI preparations (from Glycine max and Macrotyloma axillare) were evaluated for the prevention of colorectal neoplasia induced by intraperitoneal injections of DMH, given at a dose of 30mg/kg, during 12weeks. Mice treated with DMH presented histopathological alterations consistent with tumor development, augmented CD44 expression and increased proteasome peptidase activities. Lysosomal fractions, obtained from the intestines, were chromatographed in a Sepharose-BBI column and increased activity for trypsin and chymotrypsin-like proteases recovered from DMH-treated animals. In parallel, mice treated for eight weeks with BBIs showed a decrease in the chymotrypsin and trypsin-like proteasome activities compared to animals fed on normal diet. For the groups receiving simultaneous treatment with DMH and BBIs, dysplasic lesions were not observed and proteasome peptidase activities were similar to the control group after the 24th week. These results suggest that the mechanism by which BBIs could prevent the appearance of pre neoplastic lesions is associated with inhibition of both the lysosomal and proteasome-dependent proteolytic pathways. [Copyright &y& Elsevier]

Abstract: The aims of this work were 1) to determine the bioactivities resulting from the hydrolysis of soy (Glycine max) proteins isolate (SPI) by a double enzymatic treatment of pepsin and pancreatin and 2) to investigate whether fractionation of the protein hydrolysates by two ultrafiltration techniques using either hollow fiber (HF) or spiral-wounded (SW) membrane (10kDa cut-off), increases biological activity of the recovered peptides. The focus was on the screening of bioactivity of the peptide fractions using multiple in vitro bioassays such as the proliferation of cancer cells, destruction of murine norovirus-1 (MNV-1), anti-oxidative and immuno-modulating properties, and finally glucose metabolism in a muscle cell line. Significant biological effects of the peptides for both antioxidant capacity (ORAC assay) and metabolism (muscle glucose uptake assay) were found. These results show that ultrafiltration of SPI hydrolysates is a convenient process for the recovery of large amount of bioactive peptides. [Copyright &y& Elsevier]

Abstract: The Bowman–Birk inhibitor (BBI) is a soybean-derived serine protease inhibitor with anti-inflammatory properties. Experimental autoimmune encephalomyelitis (EAE) serves as an animal model of the central nervous system (CNS) inflammatory disorder multiple sclerosis (MS). EAE is mediated by Th1 and Th17 cells which migrate into the CNS and initiate inflammation directed against myelin components, resulting in CNS pathology and neurological clinical deficit. We have shown previously that oral treatment with BBI delays onset of EAE and reduces its severity. These beneficial effects were associated with an increase in IL-10 secretion by immune cells of BBI-treated mice. It is not known, however, whether this was a causal relationship or simply an epiphenomenon. In the present study we provide evidence that BBI regulates CD4+ T cell immune responses in EAE. BBI administration delayed the onset of EAE and reduced its severity in an IL-10-dependent manner, as BBI-mediated suppression of EAE was abrogated in IL-10 knockout mice. The beneficial effects were accompanied by reduced IFN-γ, IL-17 and increased IL-10 production, as well as increased Foxp3 expression. CD4+ T cells were the major source of IL-10 in the periphery and in the CNS during BBI treatment. Furthermore, BBI-treated mice had reduced numbers of infiltrated cells in the CNS, including Th17 cells, as compared with PBS-treated control animals. In conclusion, our data provide clear evidence for the essential role of IL-10 in BBI-mediated suppression in EAE, and indicate that BBI may be a promising candidate for the development of a novel MS therapy. [Copyright &y& Elsevier]

Bowman-Birk Inhibitor (BBI), a serine protease inhibitor derived from soybeans, has anti-inflammatory properties and is able to suppress the development of central nervous system (CNS) autoimmunity in animal models. Experimental autoimmune encephalomyelitis (EAE), a widely used animal model of multiple sclerosis (MS), is characterized by breakdown of the blood-brain barrier and infiltration of inflammatory cells into the CNS, resulting in pathology. In this study, we observed that BBI-treated mice showed delayed onset of EAE and reduced disease severity compared to control mice. BBI-treated mice had fewer inflammatory cells in the CNS including significantly reduced numbers of Th1 and Th17 cells. In the periphery, BBI treatment suppressed the development of encephalitogenic Th1 and Th17 responses early on [day 7 post-immunization (p.i.)], while after disease onset (day 14 p.i.) BBI-treated mice had stronger Th responses, as determined by antigen-specific proliferation and cytokine production. These results demonstrate that BBI treatment temporarily suppressed the development of encephalitogenic responses, but these responses eventually attained normal magnitude. Given that BBI-treated mice exhibited stronger encephalitogenic responses in the periphery during clinically manifesting EAE, delayed disease onset, and reduced numbers of CNS-infiltrating cells, it appears likely that BBI impedes the exit of pathogenic Th1 and Th17 cells from lymphoid organs, thereby delaying their migration into the CNS. [ABSTRACT FROM AUTHOR]

Abstract: Chronic wounds are the result of excessive amounts of tissue destructive proteases such as human neutrophil elastase (HNE). The high levels of this enzyme found on those types of wounds inactivate the endogenous inhibitor barrier thus, the search for new HNE inhibitors is required. This work presents two new HNE inhibitor peptides, which were synthesized based on the reactive-site loop of the Bowman–Birk inhibitor protein. The results obtained indicated that these new peptides are competitive inhibitors for HNE and, the inhibitory activity can be modulated by modifications introduced at the N- and C-terminal of the peptides. Furthermore, these peptides were also able to inhibit elastase from a human wound exudate while showing no cytotoxicity against human skin fibroblasts in vitro, greatly supporting their potential application in chronic wound treatment. [Copyright &y& Elsevier]

Soybean (Glycine max) is a major protein source for animal and human nutrition. The Bowman- Birk protease inhibitor (BBI), ranking 3rd of protein contents among soybean seed storage proteins, is a major antinutritional factor. BBI was incubated with 1 mM DTT at 37°C for 2 h and loaded directly onto a Sephadex G-25 gel column for purification. The molecular mass of the reduced form of BBI is ca. 8 kDa determined by SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis). The methodology we used includes total antioxidant status, (1,1-diphenyl-2-picryl hydrazyl) DPPH staining, DPPH radical scavenging activity, reducing power method, Fe2+-chelating ability, FTC (ferric thiocyanate) method, and protection calf thymus DNA against hydroxyl radical-induced damage. The oxidized and reduced form of BBI with a concentration of 200 μg/mL exhibited the highest activity (expressed as 4.74 ± 0.36 and 7.20 ± 0.20 mM Trolox equivalent antioxidative value, TEAC) in total antioxidant status test. In the DPPH staining the reduced form of BBI appeared as white spots when it was diluted to 12.5 μg/mL (a final amount of 0.6 μg). Like total antioxidant status, the reducing power, Fe2+-chelating ability, FTC activity and protection against hydroxyl radical-induced calf thymus DNA damage all showed that the reduced BBI exhibited higher antioxidative activities than the oxidized BBI. The results suggested that the reduced BBI exhibited higher antioxidative activities than the oxidized BBI in a series of in vitro tests. These findings provide one of the molecular bases for BBI applications to treat various serious diseases. [ABSTRACT FROM AUTHOR]

Abstract: The present study assessed the insecticidal potential of purified Bowman–Birk proteinase inhibitors from red gram (RgPI) and black gram (BgPI) towards lepidopteran insect pests. Both RgPI and BgPI showed remarkable inhibitory activity against midgut trypsin-like proteinases of Achaea janata. While, BgPI showed moderate to low inhibitory activity towards the midgut trypsin-like proteinases of Helicoverpa armigera, Spodoptera litura, Papilio demoleus, Amsacta albistriga, Corcyra cephalonica, Bombyx mori and Daphnis nerii, RgPI showed low to negligible inhibitory activity. The isoinhibitors of RgPI and BgPI were stable in presence of trypsin and chymotrypsin of bovine pancreatic origin, as well as larval midgut proteinases of A. janata, H. armigera and S. litura. The midgut trypsin-like proteinases of A. janata and H. armigera as well as midgut trypsin- and chymotrypsin-like proteinases of S. litura were susceptible to inhibition by RgPI and BgPI. Feeding assays displayed a dose-dependent decrease in body weight and survival rate of these larvae with increasing concentration of inhibitors supplemented in the diet. RgPI displayed significant reduction in body weight and survival rate of A. janata larvae compared with BgPI. Conversely, BgPI showed significant reduction in body weight of S. litura larvae compared with RgPI. [Copyright &y& Elsevier]

Abstract: The objective was to determine the effect of time and temperature on the concentration of bioactive compounds during germination of Brazilian soybean cultivar BRS 258. The concentration of bioactive compounds was optimized using Response Surface Methodology, with a 22 central composite rotational design and germination time and temperature as independent variables. Germination was carried out in a germination chamber, using paper in trays containing 500g of seeds. Germination temperature and time modified the concentrations of bioactive compounds within the ranges studied. An increase in germination time at 25°C decreased the concentration of Bowman–Birk inhibitor, lectin and lipoxygenase. After 63h of germination, a temperature increase from 20°C to 30°C resulted in a decrease of lipoxygenase activity by 22.5%. Optimal increases in the concentrations of isoflavone aglycones (daidzein and genistein) and saponin glycosides were observed with a 63h germination time at 30°C. Both germination time and temperature had an influence on the composition and concentration of bioactive compounds in germinated soybean flour. Equations determined can be used to predict concentrations of bioactive compounds in germinated soybeans in relation to climate changes and thus optimize their potential use in human health and nutrition. [Copyright &y& Elsevier]

We present the in vivo biosynthesis of wild-type sunflower trypsin inhibitor 1 (SFTI-1) inside E. coli cells using an intramolecular native chemical ligation in combination with a modified protein splicing unit. SFTI-1 is a small backbone cyclized polypeptide with a single disulfide bridge. A small library containing multiple Ala mutants was also biosynthesized and its activity was assayed using a trypsin-binding assay. This study clearly demonstrates the exciting possibility of generating large cyclic peptide libraries in live E. coli cells, and is a critical first step for developing in vivo screening and directed evolution technologies using the cyclic peptide SFTI-1 as a molecular scaffold. [ABSTRACT FROM AUTHOR]

Abstract: The objective was to optimise the effect of germination time and temperature on the concentration of soluble protein, lunasin, BBI, lectin, saponins and isoflavones in soybean seeds from cultivar BRS 133. Isoflavone and saponin concentrations were analysed by HPLC. Lunasin, Bowman-Birk inhibitor and lectin were analysed by ELISA and western blot. The effects of the variations in germination time and temperature on bioactive compounds were analysed using the response surface methodology (RSM), with a 22 central composite rotational design. Germination of soybean for 42h at 25°C resulted in an increase of 61.7% of lunasin, decrease of 58.7% in lectin and 70.0% in lipoxygenase activity. Optimal increases in the concentrations of isoflavone aglycones were observed in combination of 63h of germination and 30°C. A significant increase of 32.2% in the concentration of soy saponins was observed in combination of 42h of germination at 25°C. [Copyright &y& Elsevier]

Abstract: A proteinase inhibitor (BgPI) was purified from black gram, Vigna mungo (cv. TAU-1) seeds by using ammonium sulfate fractionation, followed by ion-exchange, affinity and gel-filtration chromatography. BgPI showed a single band in SDS–PAGE under non-reducing condition with an apparent molecular mass of ∼8kDa correlating to the peak 8041.5Da in matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum. BgPI existed in different isoinhibitor forms with pI values ranging from 4.3 to 6.0. The internal sequence “SIPPQCHCADIR” of a peak 1453.7 m/z, obtained from MALDI-TOF-TOF showed 100% similarity with Bowman-Birk inhibitor (BBI) family. BgPI exhibited non-competitive-type inhibitory activity against both bovine pancreatic trypsin (K i of 309.8nM) and chymotrypsin (K i of 10.7μM), however, with a molar ratio of 1:2 with trypsin. BgPI was stable up to a temperature of 80°C and active over a wide pH range between 2 and 12. The temperature-induced conformational changes in secondary structure are reversed when BgPI was cooled from 90 to 25°C. Further, upon reduction with dithiothreitol, BgPI lost both its inhibitory activity as well as secondary structural conformation. Lysine residue(s) present in the reactive site of BgPI play an important role in inhibiting the bovine trypsin activity. The present study provides detailed biochemical characteristic features of a BBI type serine proteinase inhibitor isolated from V. mungo. [Copyright &y& Elsevier]

The soybean-derived serine protease inhibitor, Bowman–Birk inhibitor (BBI), has been reported as a potent chemoprevention agent against several types of tumors. The present study was undertaken to evaluate the effects of BBI on androgen-sensitive/dependent prostate cancers using a human prostate cancer cell (LNCaP) and the transgenic rats developing adenocarcinoma of the prostate (TRAP) model. Treatment of LNCaP prostate cancer cells with 500 µg/mL BBI resulted in inhibition of viability measured on WST-1 assays, with induction of connexin 43 (C×43) and cleaved caspase-3 protein expression. Feeding of 3% roughly prepared BBI (BBIC) to TRAP from the age 3 weeks to 13 weeks resulted in significant reduction of the relative epithelial areas within the acinus and multiplicity of the adenocarcinomas in the lateral prostate lobes. C×43- and terminal deoxynucleotidyl transferase mediated dUTP-biotin end labeling of fragmented DNA (TUNEL)-positive apoptotic cancer cells were more frequently observed in the lateral prostates treated with BBIC than in the controls. These in vivo and in vitro results suggest that BBI possesses chemopreventive activity associated with induction of C×43 expression and apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: The inverse association between the intake of soybean foods and cancer incidence and mortality rates supported by published literature has led to studies on identifying bioactive components. The cancer preventive properties of the soybean peptides lunasin and Bowman-Birk protease inhibitor (BBI) have been demonstrated by in vitro and in vivo assays. Since there is no comprehensive information on the concentrations of these two peptides, US commercially available soy foods, including soy milk, soy-based infant formula, tofu, bean curd, soybean cake, tempeh, natto, miso and su-jae samples, were analyzed for lunasin and BBI. Both peptides were present in most of the products, in varying concentrations, depending mainly on the soybean variety and the manufacturing process. Lunasin and BBI were absent in the fermentation products natto and miso, suggesting that fermentation destroys both peptides. To study the bioavailability of lunasin and BBI, three soy milk samples with different concentrations of these peptides were subjected to an enzymatic hydrolysis process simulating physiological digestion. The results confirm the important role BBI plays in the protection of lunasin from digestion by pepsin and pancreatin. [Copyright &y& Elsevier]

A BAC library from common bean has been used in order to isolate the entire multigene Bowman–Birk serine protease inhibitor family and to study its genome organization. Using a previously isolated trypsin/chymotrypsin inhibitor nucleotide sequence as probe, two positive BAC clones were identified. The P2B8 BAC clone, of about 135 kbp and containing the complete BBI family, was chosen and partially sequenced. Our results confirm that a small multigene family codes for three double-headed inhibitors named: tc-BBI-1, tc-BBI-2 and et-BBI. They contain the binding loop trypsin/chymotrypsin (tc-BBI-1 and tc-BBI-2) and the elastase/trypsin one (et-BBI), respectively. Genes coding for tc-BBI-1 and et-BBI, were found to be very close to each other and arranged in a head to head fashion. Southern blot hybridisation on genomic DNA digested with PstI enzyme suggests that all three genes are present in a fragment of 19 kbp. Northern blot analyses on RNA isolated from various common bean organs showed that the expression of tc-BBI-1 and et-BBI was restricted to the developing cotyledons. [ABSTRACT FROM AUTHOR]

Abstract: The Bowman–Birk inhibitor (BBI) is a soybean-derived serine protease inhibitor. BBI concentrate (BBIC) is an extract enriched with BBI, but predominantly contains other ingredients including several protease inhibitors. We previously found that BBIC administration to Lewis rats with experimental autoimmune encephalomyelitis (EAE) significantly suppresses disease. In the present study we determined whether BBI mediates the suppressive effects of BBIC in EAE, evaluated its potential neuroprotective effects, and investigated mechanisms of BBI action. We tested effects of purified BBI on clinical and histopathological parameters of EAE in two models (relapsing/remitting EAE in SJL/J mice and chronic EAE in C57BL/6 mice). Effects of BBI were compared to BBIC in relapsing/remitting EAE, and effects of BBI on neuronal survival were examined during acute optic neuritis. Treatment with BBI in both EAE models significantly improved EAE disease parameters (onset, severity, weight loss, inflammation and demyelination). BBI significantly reduced the incidence of optic neuritis and prevented loss of retinal ganglion cells. In most experiments proliferation of immune cells derived from BBI-treated mice was significantly lower relative to control groups. Using Boyden''s chamber assay we found that BBI inhibited invasiveness of activated splenocytes through the matrigel barrier. BBI also induced higher production of EAE-suppressive cytokine IL-10 by immune cells. These results demonstrate that BBI is the active component of BBIC that ameliorates clinical EAE. BBI reduces inflammation and attenuates neuronal loss, making it an excellent candidate for oral therapy in MS. BBI likely ameliorates EAE by inhibiting multiple pathways involved in disease pathogenesis. [Copyright &y& Elsevier]

Abstract: The paper describes the purification, structural characterization and inhibitory properties of a trypsin inhibitor from Lupinus albus L., a leguminous plant believed to be devoid of any protease inhibitor. The protein has been isolated by a newly set-up procedure and characterized by direct amino acid sequencing, MALDI-TOF mass spectroscopy and circular dichroism. Inhibitory properties toward bovine trypsin and chymotrypsin, as well as its thermal and pH stabilities, have been also assessed. The inhibitor is 63 amino acid long (M r 6858; pI 8.22) and it is capable to inhibit two trypsin molecules simultaneously, with a Kd of 4.2±0.4nM, but not chymotrypsin. BLAST search against UniProtKB/TrEMBL database indicates that the inhibitor belongs to the Bowman–Birk inhibitor (BBI) family. The interest in these serine-protease inhibitors arises from the ability to prevent or suppress carcinogen-induced transformation, as shown in various in vitro and in vivo model systems. [Copyright &y& Elsevier]

The Bowman-Birk inhibitor (BBI) is a small water-soluble protein present in soybean and almost all monocotyledonous and dicotyledonous seeds. The molecular size of BBI ranges from 1,513 Da to about 20,000 Da. BBI is to seeds what alpha(1)-antitrypsin is to humans. Soy-based food products rich in BBI include soybean grits, soymilk, oilcake, soybean isolate, and soybean protein concentrate. BBI is stable within the pH range encountered in most foods, can withstand boiling water temperature for 10 min, resistant to the pH range and proteolytic enzymes of the gastrointestinal tract, bioavailable, and not allergenic. BBI reduces the proteolytic activities of trypsin, chymotrypsin, elastase, cathepsin G, and chymase, serine protease-dependent matrix metalloproteinases, urokinase protein activator, mitogen activated protein kinase, and PI3 kinase, and upregulates connexin 43 (Cx43) expression. Several studies have demonstrated the efficacy of BBI against tumor cells in vitro, animal models, and human phase IIa clinical trials. FDA considers BBI as a drug. In 1999, FDA allowed a health claim on food labels stating that a daily diet containing 25 grams of soy protein, also low in saturated fat and cholesterol, may reduce the risk of heart disease [corrected] This review highlights the biochemical and functional food properties of the Bowman-Birk inhibitor. [ABSTRACT FROM AUTHOR]

Abstract: The structure of the ternary complex of the anticarcinogenic Bowman–Birk protease inhibitor purified from snail medic (Medicago scutellata) seeds (MSTI) and two molecules of bovine trypsin has been solved by X-ray diffraction analysis of single crystals to a resolution of 2.0Å. This is the highest resolution model of a ternary complex of this type currently available. The two binding loops of the MSTI differ in only one amino acid and have in both cases an arginine in position P1. The distances between the residues of the inhibitor at the binding interface and the trypsin side chains that recognize them are almost identical in the two sites. When compared to the NMR model of the uncomplexed MSTI, the inhibitor in the functional assembly with trypsin shows the largest differences in the two P2′ residues. Compared with the similar ternary complex of the soybean trypsin inhibitor, this model shows very small differences in the polypeptide chain of the trypsin binding sites and its largest difference in the area between Asp 26 and His 32 of the MSTI which in the soybean inhibitor has an extra Leu inserted in position 29. [Copyright &y& Elsevier]

Bowman–Birk serine protease inhibitors are a family of small plant proteins, whose physiological role has not been ascertained as yet, while chemopreventive anticarcinogenic properties have repeatedly been claimed. In this work we present data on the isolation of a lentil ( Lens culinaris, L., var. Macrosperma) seed trypsin inhibitor (LCTI) and its functional and structural characterization. LCTI is a 7448 Da double-headed trypsin/chymotrypsin inhibitor with dissociation constants equal to 0.54 nm and 7.25 nm for the two proteases, respectively. The inhibitor is, however, hydrolysed by trypsin in a few minutes timescale, leading to a dramatic loss of its affinity for the enzyme. This is due to a substantial difference in the kon and k*on values (1.1 µm−1·s−1 vs. 0.002 µm−1·s−1), respectively, for the intact and modified inhibitor. A similar behaviour was not observed with chymotrypsin. The twenty best NMR structures concurrently showed a canonical Bowman–Birk inhibitor (BBI) conformation with two antipodal β-hairpins containing the inhibitory domains. The tertiary structure is stabilized by ion pairs and hydrogen bonds involving the side chain and backbone of Asp10-Asp26-Arg28 and Asp36-Asp52 residues. At physiological pH, the final structure results in an asymmetric distribution of opposite charges with a negative electrostatic potential, centred on the C-terminus, and a highly positive potential, surrounding the antitryptic domain. The segment 53–55 lacks the anchoring capacity found in analogous BBIs, thus rendering the protein susceptible to hydrolysis. The inhibitory properties of LCTI, related to the simultaneous presence of two key amino acids (Gln18 and His54), render the molecule unusual within the natural Bowman–Birk inhibitor family. [ABSTRACT FROM AUTHOR]

Epidemiologic data suggest that soy consumption may protect against cancer induction in several tissues in humans. Although the soy components responsible for this activity remain unidentified, isoflavones (e.g., genistein) and protease inhibitors (e.g., Bowman-Birk inhibitor complex [BBIC]) demonstrate chemopreventive activity in several animal cancer models. As part of their preclinical development for cancer prevention, PTI G-2535 (a soy isoflavone mixture containing 45% genistein, 23% daidzein, and 4% glycitein) and BBIC were evaluated for oncogenicity in p53 (+/-) mice. In separate studies, groups of 25 p53 (+/-) mice/sex received daily gavage exposure to PTI G-2535 (0, 250, 1000, or 2500 mg/kg/day) or BBIC (0, 500, 1000, or 2000 mg/kg/day) for 6 months. The high doses of both PTI G-2535 and BBIC were limited by viscosity. p -Cresidine (400 mg/kg/day) served as a positive-control article in both studies. PTI G-2535 induced no gross toxicity in any animal, but did induce a dose-related suppression of body weight gain in male mice. Modest hematologic alterations and increased liver and spleen weights were seen in both sexes exposed to the isoflavone mixture. BBIC had no significant effect on body weight, food consumption, clinical pathology, or organ weights in either sex. Histopathologic evaluations demonstrated no increases in the incidence of either benign or malignant tumors in any group of p53 (+/-) mice exposed to PTI G-2535 or to BBIC. By contrast, the positive-control article, p -cresidine, induced urinary bladder cancers in both studies. Neither PTI G-2535 nor BBIC demonstrates any evidence of oncogenicity in the p53 (+/-) mouse model. [ABSTRACT FROM AUTHOR]

Epidemiologic data suggest that soy consumption may protect against cancer induction in several tissues in humans. Although the soy components responsible for this activity remain unidentified, isoflavones (e.g., genistein) and protease inhibitors (e.g., Bowman-Birk inhibitor complex [BBIC]) demonstrate chemopreventive activity in several animal cancer models. As part of their preclinical development for cancer prevention, PTI G-2535 (a soy isoflavone mixture containing 45%genistein,23%daidzein, and 4%glycitein) and BBIC were evaluated for oncogenicity in p 53(+/-) mice. In separate studies, groups of 25 p53(+/-) mice/sex received daily gavage exposure to PTI G-2535 (0, 250, 1000, or 2500 mg/kg/day) or BBIC (0, 500, 1000, or 2000 mg/kg/day) for 6 months. The high doses of both PTI G-2535 and BBIC were limited by viscosity. p -Cresidine (400 mg/kg/day) served as a positive-control article in both studies. PTI G-2535 induced no gross toxicity in any animal, but did induce a dose-related suppression of body weight gain in male mice. Modest hematologic alterations and increased liver and spleen weights were seen in both sexes exposed to the isoflavone mixture. BBIC had no significant effect on body weight, food consumption, clinical pathology, or organ weights in either sex. Histopathologic evaluations demonstrated no increases in the incidence of either benign or malignant tumors in any group of p53(+/-) mice exposed to PTI G-2535 or to BBIC. By contrast, the positive-control article, p -cresidine, induced urinary bladder cancers in both studies. Neither PTI G-2535 nor BBIC demonstrates any evidence of oncogenicity in the p53(+/-) mouse model. [ABSTRACT FROM PUBLISHER]

Rice Bowman-Birk inhibitors (RBBI), with one (8 kD) or two homologous domains (16 kD), were found to be effective trypsin inhibitors in vitro. In this study, we demonstrate that the 25-kD protein corresponding to the three-domain RBBI indeed exists in rice in planta, and that the RBBIs are regulated by development and wounding. We also found by inhibitory activity assay that the 3:13 disulfide bond, but not the 4:5 disulfide bond, suppresses the trypsin-inhibitory activity, and the D3 domain of RBBI3-1 has no inhibitory activity against trypsin, chymotrypsin, paparin or subtilisin. Mutation analyses showed that conversion from Lys to Leu or Tyr in the N-terminal P1 site in D1 domain did not create chymotrypsin-inhibitory activity, suggesting that the structure of the reactive loop in D1 domain hinder the new inhibitory specificity at P1 site, and the chymotrypsin-inhibitory activity might need the participation of other structures, e.g. 3:13 disulfide bond. [ABSTRACT FROM AUTHOR]

Skeletal muscle atrophy in response to a number of muscle wasting conditions, including disuse, involves the induction of increased protein breakdown, decreased protein synthesis, and likely a variable component of apoptosis. The increased activation of specific proteases in the atrophy process presents a number of potential therapeutic targets to reduce muscle atrophy via protease inhibition. In this study, mice were provided with food supplemented with the Bowman-Birk inhibitor (BBI), a serine protease inhibitor known to reduce the proteolytic activity of a number of proteases, such as chymotrypsin, trypsin, elastase, cathepsin G, and chymase. Mice fed the BBI diet were suspended for 3-14 days, and the muscle mass and function were then compared with those of the suspended mice on a normal diet. The results indicate that dietary supplementation with BBI significantly attenuates the normal loss of muscle mass and strength following unloading. Furthermore, the data reveal the existence of yet uncharacterized serine proteases that are important contributors to the evolution of disuse atrophy, since BBI inhibited serine protease activity that was elevated following hindlimb unloading and also slowed the loss of muscle fiber size. These results demonstrate that targeted reduction of protein degradation can limit the severity of muscle mass loss following hindlimb unloading. Thus BBI is a candidate therapeutic agent to minimize skeletal muscle atrophy and loss of strength associated with disuse, cachexia, sepsis, weightlessness, or the combination of age and inactivity. [ABSTRACT FROM AUTHOR]

A novel bifunctional bicyclic inhibitor has been created that combines features both from the Bowman–Birk inhibitor (BBI) proteins, which have two distinct inhibitory sites, and from sunflower trypsin inhibitor-1 (SFTI-1), which has a compact bicyclic structure. The inhibitor was designed by fusing together a pair of reactive loops based on a sequence derived from SFTI-1 to create a backbone-cyclized disulfide-bridged 16-mer peptide. This peptide has two symmetrically spaced trypsin binding sites. Its synthesis and biological activity have been reported in a previous communication [Jaulent and Leatherbarrow, 2004, PEDS 17, 681]. In the present study we have examined the three-dimensional structure of the molecule. We find that the new inhibitor, which has a symmetrical 8-mer half-cystine CTKSIPP′I′ motif repeated through a C2 symmetry axis also shows a complete symmetry in its three-dimensional structure. Each of the two loops adopts the expected canonical conformation common to all BBIs as well as SFTI-1. We also find that the inhibitor displays a strong and unique structural identity, with a notable lack of minor conformational isomers that characterise most reactive site loop mimics examined to date as well as SFTI-1. This suggests that the presence of the additional cyclic loop acts to restrict conformational mobility and that the deliberate introduction of cyclic symmetry may offer a general route to locking the conformation of β-hairpin structures. [ABSTRACT FROM AUTHOR]

The Bowman-Birk inhibitors (BBI) are found widespread in legume species. The primary structure of 23 BBI sequences, isolated from genomic DNA of eight Italian landraces of common bean (Phaseolus vulgaris L.), is presented in this study. The similarity among the analysed sequences ranged from 92 to 99%. The sequences, though highly similar to those previously described for common bean, showed some distinct features. Differences were detected in number of cysteine residues and in both trypsin and chymotrypsin binding loops. Two variants of trypsin binding loop, differing for the residue at P2′ position (Ile or Arg) and two variants of chymotrypsin binding loop showing at the P1 position Leu or Phe were found. Although P2′-Ile is the most frequent residue observed in natural BBI sequences as well as in this study, the detection of the variant P2′-Arg in four landraces make them very interesting for further investigations. In fact, sources of different BBI are very interesting due to the different inhibitory activity associated to them. [Copyright &y& Elsevier]

The WIP1-2 gene was cloned from rice. It belongs to the Bowman-Birk inhibitor gene family. Northern blot showed that expression of this gene was induced by wounding and jasmonic acid (JA). It indicates that the OsWIP1 gene plays an important role in the rice defense system. The OsWIP1-2 was cloned into pET28a and expressed in E. coli. Its expressed product was purified in the form of fusion protein and tested for the inhibitory activities against trypsin and chymotrypsin. It was found that the fusion protein could inhibit chymotrypsin, but not trypsin. It was also found that the His tag at its C-terminal affected its inhibitory activity significantly. The fusion protein with a natural C-terminal had the inhibitory activity, while no inhibitory activity was detected in the fusion protein with a (His)6-tag at its C-terminal. This implies that extra amino acid residues at the C-terminal of OsWIP1-2 may interfere with its correct folding. The inhibitory assay indicated that the members of rice Bowman-Birk inhibitor gene family probably differentiated both in their structure and function. [ABSTRACT FROM AUTHOR]

The effect of acylation of Bowman–Birk soybean proteinase inhibitor (BBI) by derivatives of various unsaturated fatty acids on inhibition of trypsin, α-chymotrypsin, and human leukocyte elastase was investigated. Inhibition ( Ki) and kinetic ( kass, kdiss) constants of interaction between proteases and acylated BBI derivatives were determined. For mono-, di-, and triacylated BBI derivatives, insertion of two oleic residues into the BBI molecule was demonstrated to be more potent for exhibiting antiproteinase activity. [ABSTRACT FROM AUTHOR]

A procedure was developed for acylation of Bowman–Birk soybean proteinase inhibitor (BBI) by N-hydroxysuc-cinimide esters of oleic, linoleic, and α-linolenic acids in a dimethyl sulfoxide–dioxane–pyridine mixture. BBI derivatives containing two acylated amino groups were prepared with high yield. The use of the reversible modifier citraconic anhydride in the first stage of synthesis permitted the synthesis of hydrophobized BBI derivatives retaining high antitrypsin and antichymotrypsin activities. It was found that the insertion of two long chain moieties in the BBI molecule decreases its thermostability. [ABSTRACT FROM AUTHOR]

Conjugates of the classical soybean Bowman–Birk inhibitor (BBI) with clinical dextran were synthesized. Clinical dextran was preliminarily oxidized with periodate to dialdehydedextran (DAD). The effect of the degree of oxidation of DAD on coupling of the inhibitor was evaluated. The binding of the protein was shown to increase with increasing degree of DAD oxidation (5,10,20%). Total coupling of the inhibitor occurred when the degree of oxidation of the dextran was 20%. The BBI–DAD (20%) conjugate contained 13% protein with BBI/DAD molar ratio 1 :1. The conjugates retained the ability to inhibit trypsin ( Ki = 0.2-0.3 nM) and α-chymotrypsin ( Ki = 15-30 nM). Thus, the coupling of BBI with the polymeric carrier caused practically no decrease in the antiproteolytic activity of the inhibitor. [ABSTRACT FROM AUTHOR]

Bowman-Birk inhibitor concentrate (BBIC) is a soybean extract enriched in the Bowman-Birk inhibitor, a protein protease inhibitor. The Bowman-Birk inhibitor can inhibit proteases released from inflammation mediating cells and suppress superoxide anion radical secretion from immunocytes. This study investigates the ability of Bowman-Birk inhibitor concentrate to inhibit colon inflammation in the dextran sulfate sodium model of ulcerative colitis, an inflammatory bowel disease. When compared to mice on a standard diet, mice given food supplemented with 0.5% BBIC during and after dextran sulfate sodium treatment showed suppression of three of four scored histopathological inflammation criteria (P < 0.01), total histopathological score (P < 0.01), a 15% lower mortality rate (P < 0.01), and a delayed onset of mortality. We conclude that dietary Bowman-Birk inhibitor concentrate can beneficially affect dextran sulfate sodium-treated mice and may be useful in the treatment of human inflammatory bowel diseases, particularly ulcerative colitis. [ABSTRACT FROM AUTHOR]

The binding of α-chymotrypsin to black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) has been studied using the inhibitory activity against the enzyme and the formation of the complex enzyme/inhibitor followed by measurements of fluorescence polarization. Apparent equilibrium constants were estimated for several temperatures and the values obtained range from 0.32 × 107 to 1.36 × 107 M−1. The following values were found from van't Hoff plots: Δ H = 10.8 kcal mol-1 (from inhibitory assays) and 11.1 kcal mol−1 (from fluorescence polarization); Δ S° = 67.9 and = 67.8 kcal K−1 mol−1, respectively. Calorimetric binding enthalpy was determined (corrected for the ionization heat of the buffer) and the resulting value was ΔH = 4.9 kcal mol-1. These results indicate that the binding of chymotrypsin to BTCI is an entropically driven process. [ABSTRACT FROM AUTHOR]

The Bowman­Birk inhibitor from soybean is a small protein that contains a binary arrangement of trypsin-reactive and chymotrypsin-reactive subdomains. In this report, the crystal ,structure of this anticarcinogenic protein has been determined to 0.28-nm resolution by molecular replacement from crystals grown at neutral pH. The crystal structure differs from a previously determined NMR structure [Werner, M. H. & Wemmer, D. E. (1992) Biochemisoy 31, 999–10101 in the relative orientation of the two enzyme-insertion loops, in some details of the main chain trace, in the presence of favourable contacts in the trypsin-insertion loop, and in the orientation of several amino acid chains. The proximity of Met27 and Gin48 in the X-ray structure contradicts the solution structure, in which these two side chains point away from each other. The significant effect of a Met27→Ile replacement on the inhibitory activity of the chymotrypsin-reactive subdomain agrees with the X-ray structure. Exposed hydrophobic patches, the presence of charged amino acid residues, and the presence of water molecules in the protein interior are in contrast to standard proteins that comprise a hydrophobic core and exposed polar amino acids. [ABSTRACT FROM AUTHOR]

Rat mast cell tryptase was purified to homogeneity from rat tongue by a series of standard chromatographic procedures. Since the enzyme gave band corresponding to molecular mass of 32–35 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and exhibited a molecular mass of 135 kDa on gel filtration, it was presumed to be a noncovalently associated tetramer. The N-terminal amino acid sequence of 50 residues of the enzyme showed the highest degree of homology with the same region in mouse mast cell protease 7 (92%), and less homology to those of tryptases from man and dog, and peritoneal cells of rats and Mongolian gerbils. The inhibitor specificity of rat tongue tryptase was similar to that of rat peritoneal mast cell tryptase free from trypstatin: it was inhibited by α1-antitrypsin, Kunitz-type soybean trypsin inhibitor and Bowman-Birk soybean trypsin inhibitor, but these inhibitors do not inhibit the tryptases from rat skin, human lung, and dog mast cells. Judging from these results, together with other enzymatic properties, the enzyme may be a novel isoform of tryptase in rat tongue. Analysis by differential staining with peroxidaselabeled lectins of the enzyme suggested that it has tri- and/or tetraantennary complex-type oligosaccharides containing a relatively high amount of sialic acid. The immunohistochemical distribution of this enzyme indicated that the reactive antigen was specific in connective tissue but not in mucosal mast cells. [ABSTRACT FROM AUTHOR]

Certain protease inhibitors are effective at preventing or suppressing carcinogen-induced transformation in vitro and carcinogenesis in animal model systems. One protease inhibitor, the soybean-derived Bowman-Birk inhibitor (BBI) is particularly effective in suppressing carcinogenesis. BBI is a protein of a molecular weight of 8000 with a well-characterized ability to inhibit trypsin and chymotrypsin. BBI has been extensively studied, both as purified BBI and as an extract of soybeans enriched in BBI called BBI concentrate (BBIC). Purified BBI and BBIC have comparable suppressive effects on the carcinogenic process in a variety of in vivo and in vitro systems. BBI appears to be a universal cancer preventive agent. Purified BBI and BBIC suppress carcinogenesis as follows: in 3 different species (mice, rats, and hamsters); in several organ systems and tissue types [eg, colon, liver, lung, esophagus, cheek pouch (oral epithelium), and cells of hematopoietic origin]; and in cells of epithelial and connective tissue origin when given to animals by several different routes of administration, including the diet, leading to different types of cancer (eg, squamous cell carcinomas, adenocarcinomas, and angiosarcomas), and induced by various chemical and physical carcinogens. About half of an oral dose of BBI is taken up into the bloodstream and distributed throughout the body, with excretion via the urine. Pharmacokinetic studies of BBI have been performed in animals with radioactively labeled BBI, whereas antibodies that react with reduced BBI are being used in pharmacokinetic studies in humans. The calculated serum half-life is 10 h in both rats and hamsters. BBIC achieved Investigational New Drug status from the FDA in April 1992 (IND no. 34671; sponsor, Ann R Kennedy), and studies to evaluate BBIC as an anticarcinogenic agent in human populations began. Both BBI and BBIC prevent and suppress malignant transformation in vitro and carcinogenesis in vivo without toxicity. [ABSTRACT FROM AUTHOR]

ATP sulfurylase, an enzyme which catalyzes the conversion of sulfate to adenosine 5′-phosphosulfate (APS), plays a significant role in controlling sulfur metabolism in plants. In this study, we have expressed soybean plastid ATP sulfurylase isoform 1 in transgenic soybean without its transit peptide under the control of the 35S CaMV promoter. Subcellular fractionation and immunoblot analysis revealed that ATP sulfurylase isoform 1 was predominantly expressed in the cell cytoplasm. Compared with that of untransformed plants, the ATP sulfurylase activity was about 2.5-fold higher in developing seeds. High-resolution 2-D gel electrophoresis and immunoblot analyses revealed that transgenic soybean seeds overexpressing ATP sulfurylase accumulated very low levels of the β-subunit of β-conglycinin. In contrast, the accumulation of the cysteine-rich Bowman–Birk protease inhibitor was several fold higher in transgenic soybean plants when compared to the non-transgenic wild-type seeds. The overall protein content of the transgenic seeds was lowered by about 3% when compared to the wild-type seeds. Metabolite profiling by LC–MS and GC–MS quantified 124 seed metabolites out of which 84 were present in higher amounts and 40 were present in lower amounts in ATP sulfurylase overexpressing seeds compared to the wild-type seeds. Sulfate, cysteine, and some sulfur-containing secondary metabolites accumulated in higher amounts in ATP sulfurylase transgenic seeds. Additionally, ATP sulfurylase overexpressing seeds contained significantly higher amounts of phospholipids, lysophospholipids, diacylglycerols, sterols, and sulfolipids. Importantly, over expression of ATP sulfurylase resulted in 37–52% and 15–19% increases in the protein-bound cysteine and methionine content of transgenic seeds, respectively. Our results demonstrate that manipulating the expression levels of key sulfur assimilatory enzymes could be exploited to improve the nutritive value of soybean seeds. [ABSTRACT FROM AUTHOR]