J.R. Bacon

Broccoli Consumption Interacts with GSTM1 to Perturb Oncogenic Signalling Pathways in the Prostate

Background Epidemiological studies suggest that people who consume more than one portion of cruciferous vegetables per week are at lower risk of both the incidence of prostate cancer and of developing aggressive prostate cancer but there is little understanding of the underlying mechanisms. In this study, we quantify and interpret changes in global gene expression patterns in the human prostate gland before, during and after a 12 month broccoli-rich diet. Methods and Findings Volunteers were randomly assigned to either a broccoli-rich or a pea-rich diet. After six months there were no differences in gene expression between glutathione S-transferase mu 1 (GSTM1) positive and null individuals on the pea-rich diet but significant differences between GSTM1 genotypes on the broccoli-rich diet, associated with transforming growth factor beta 1 (TGFβ1) and epidermal growth factor (EGF) signalling pathways. Comparison of biopsies obtained pre and post intervention revealed more changes in gene expression occurred in individuals on a broccoli-rich diet than in those on a pea-rich diet. While there were changes in androgen signalling, regardless of diet, men on the broccoli diet had additional changes to mRNA processing, and TGFβ1, EGF and insulin signalling. We also provide evidence that sulforaphane (the isothiocyanate derived from 4-methylsuphinylbutyl glucosinolate that accumulates in broccoli) chemically interacts with TGFβ1, EGF and insulin peptides to form thioureas, and enhances TGFβ1/Smad-mediated transcription. Conclusions These findings suggest that consuming broccoli interacts with GSTM1 genotype to result in complex changes to signalling pathways associated with inflammation and carcinogenesis in the prostate. We propose that these changes may be mediated through the chemical interaction of isothiocyanates with signalling peptides in the plasma. This study provides, for the first time, experimental evidence obtained in humans to support observational studies that diets rich in cruciferous vegetables may reduce the risk of prostate cancer and other chronic disease. Trial Registration ClinicalTrials.gov NCT00535977

Quantitative variability inPisum seed globulins: its assessment and significance

Five different techniques have been used with sevenPisum (pea) genotypes to demonstrate the existence of extensive genotypic variability in the proportions of the major pea seed globulins, legumin, vicilin and convicilin. The accuracy and reproducibility of each method have been assessed and the significance of the observed variability discussed both in relation to studies of the control of globulin synthesis and to breeding for pea seed protein with higher nutritional quality.

Quantitative analysis of flavour precursors and pyruvate levels in different tissues and cultivars of onion (Allium cepa)

Onions of three different cultivars were dissected as in commercial peeling operations to give four tissue types: the dry, brown skin, the top/bottom sections, the outer fleshy leaf bases and the inner fleshy leaf bases. All tissue types were assayed for alliinase-produced pyruvate and for flavour precursors (alk(en)yl cysteine sulphoxides) and a positive correlation was observed between the levels of these classes of compounds. In all cultivars, the top and bottom onion sections have the highest levels of both pyruvate and flavour precursors with levels in the dry, brown skin being low or absent. Significant increases in levels of both pyruvate and flavour precursors were observed in inner tissues and top/bottom sections after storage under commercial conditions.

Characterization of the foaming properties of lysozymes and α-lactalbumins: a structural evaluation

The foaming properties of three lysozymes (human, turkey-egg and hen-egg) and two α-lactalbumins (bovine and human) have been assessed using a micro-conductivity method. Despite the very close structural similarities between these five proteins, major differences in foaming characteristics were found. For the three lysozymes, the order of increasing foam expansion and stability was turkey < hen < human. Although the two α-lactalbumins displayed similar foaming properties, these contrasted sharply with those of the lysozymes. For example, in the case of the lactalbumins measurable foams were obtained at concentrations as low as 0.05 mg/ml, whereas the best lysozyme required a concentration of 1 mg/ml. The foam expansion and foam stability of α-lactalbumin were largely independent of protein concentration, but the reverse was true for lysozyme. The effect of pH and protein conformation on the foaming of bovine α-lactalbumin and hen-egg lysozyme was, also studied. An explanation for the differences in foaming behaviour between the two protein families is proposed, based on our present understanding of the mechanism of protein foaming, and the structure of these proteins. The interpretation is extended to the “molecular level”. Inspection of the aligned sequences of the homologous lysozymes and α-lactalbumins highlighted 25 positions in the consensus sequence that are postulated tentatively as playing a central role in the foaming properties of these proteins.

Hydroxytyrosyl ethyl ether exhibits stronger intestinal anticarcinogenic potency and effects on transcript profiles compared to hydroxytyrosol

The anticarcinogenic activity of hydroxytyrosyl ethyl ether (HTy-Et) compared to its precursor hydroxytyrosol (HTy) has been studied in human Caco-2 colon adenocarcinoma cells. 451 and 977 genes were differentially expressed in Caco-2 cells exposed to HTy or HTy-Et for 24h, respectively, compared with untreated cells (P<0.005; FDR=0), using Affymetrix microarrays. Results showed that both HTy and HTy-Et inhibited cell proliferation and arrested the cell cycle by up-regulating p21 and CCNG2 and down-regulating CCNB1 protein expression. HTy and HTy-Et also altered the transcription of specific genes involved in apoptosis, as suggested by the up-regulation of BNIP3, BNIP3L, PDCD4 and ATF3 and the activation of caspase-3. Moreover, these polyphenols up-regulated xenobiotic metabolizing enzymes UGT1A10 and CYP1A1, enhancing carcinogen detoxification. In conclusion, these results highlight that HTy and its derivative HTy-Et modulate molecular mechanisms involved in colon cancer, with HTy-Et being more effective than HTy.

A study of the quaternary structure of glycinin

Abstract A range of six- and twelve-subunit models for glycinin, in which the subunits are assumed to be identical and spherical, has been tested by comparing experimental sedimentation, translational diffusion and small-angle X-ray scattering data with theoretical predictions for each model. None of the models considered, including the stacked parallel hexagon model described by Badley et al. (Badley, R.A.; Atkinson, D., Hauser, H., Oldani, D., Green, J.P. and Stubbs, J.M. (1975) Biochim. Biophys. Acta 412, 214–228), or the trigonal antiprism proposed by Plietz et al. (Pleitz, P., Damaschun, G., Muller, J.J. and Schwenke, K.-D. (1983) Eur. J. Biochem. 130, 315–320), provides acceptable interpretations of the sedimentation, diffusion and scattering data.

Synchrotron radiation SAXS of 11S soya globulin

Abstract Small-angle X-ray scattering using the Daresbury synchrotron source has been employed to obtain scattering curves from a 5% solution of the 11S soya globulin. The high intensity of the source allowed exposure times to be reduced by up to 1000 times compared with those for a conventional X-ray generator. Submaxima at higher angles were recorded which have not been reported previously. This improved resolution appears to result from reduced aggregation and/or denaturation of the protein due to the very short exposure times. Such detail in the scattering curve should be of importance for structural modelling of the proteins, particularly in the case of the 11S soya globulin for which intact individual subunits cannot be isolated.

Synchrotron radiation wide-angle X-ray scattering studies of glycinin solutions

X-ray scattering studies of glycinin solutions have been extended to higher scattering angles of 2θ ∼ 0.15 rad. Reproducible, high quality data in the angular range 0.38–6.29 nm−1 have been obtained. Scattering curves show features hitherto unreported that reveal details not only of overall shape and size of the protein but also its internal structure. Analysis suggests that currently accepted models for this protein based on the assumptions of equal-sized, touching spheres are inadequate to describe experimental observations.

Temperature-dependent binding of monoclonal antibodies to C hordein

The consensus octapeptide repeat motif of the barley seed storage protein C hordein, Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln, forms the epitope of two anti-prolamin monoclonal antibodies (Mabs), IFRN 0061 and 0614. The Mabs were found to exhibit unusual temperature-dependent binding characteristics, recognising C hordein and a peptide corresponding to the consensus repeat at 5 degrees C but not at 37 degrees C, as determined by enzyme-linked immunosorbent assay (ELISA). The K(d) of IFRN 0614 for the consensus peptide was found to be 1.2x10(12) mol(-1) at 12 degrees C, but no constant could be calculated at 37 degrees C due to a lack of binding. Similar ELISA binding characteristics were observed with an anti-C hordein polyclonal antiserum and a Mab raised to the consensus peptide. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy showed that the protein and the consensus peptide exist in a temperature-dependent equilibrium of poly-L-proline II type structures and beta-turn conformations. Whilst thermodynamic and kinetic effects may reduce antibody binding at higher temperatures, they cannot account for the complete loss of Mab recognition at higher temperatures. It seems likely that the Mabs preferentially recognise the Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motif when presented in a conformation which may correspond to the poly-L-proline II type conformation which dominates the CD and FTIR spectra at 4-12 degrees C.

Structure of the 7S globulin (vicilin) from pea (Pisum sativum)

Abstract The Daresbury synchrotron has been used to obtain X-ray scattering data on solutions of the pea 7S globulin (vicilin) to an angular resolution corresponding to the modulus of the scattering vector | k | ⪅ 14.5 nm −1 . The angular range 0.45 nm −1 ⩽| k | ⩽ 6.5 nm −1 reveals scattering details characteristic of the subunit size, shape and arrangement. The protein can be modelled in terms of three touching non-spherical subunits each containing two spherical scattering domains of unequal radii. Circular dichroism studies indicate that the ordered protein secondary structure contains mainly β-sheet and little α-helix. Structure-prediction calculations on the primary sequence of the subunit suggest that the domains may correspond to regions of β-sheet.