Nigel J. Belshaw

Profiling CpG island field methylation in both morphologically normal and neoplastic human colonic mucosa

Aberrant CpG island (CGI) methylation occurs early in colorectal neoplasia. Quantitative methylation-specific PCR profiling applied to biopsies was used to quantify low levels of CGI methylation of 18 genes in the morphologically normal colonic mucosa of neoplasia-free subjects, adenomatous polyp patients, cancer patients and their tumours. Multivariate statistical analyses distinguished tumour from mucosa with a sensitivity of 78.9% and a specificity of 100% (P=3 × 10−7). In morphologically normal mucosa, age-dependent CGI methylation was observed for APC, AXIN2, DKK1, HPP1, N33, p16, SFRP1, SFRP2 and SFRP4 genes, and significant differences in CGI methylation levels were detected between groups. Multinomial logistic regression models based on the CGI methylation profiles from normal mucosa correctly identified 78.9% of cancer patients and 87.9% of non-cancer (neoplasia-free+polyp) patients (P=4.93 × 10−7) using APC, HPP1, p16, SFRP4, WIF1 and ESR1 methylation as the most informative variables. Similarly, CGI methylation of SFRP4, SFRP5 and WIF1 correctly identified 61.5% of polyp patients and 78.9% of neoplasia-free subjects (P=0.0167). The apparently normal mucosal field of patients presenting with neoplasia has evidently undergone significant epigenetic modification. Methylation of the genes selected by the models may play a role in the earliest stages of the development of colorectal neoplasia.

Nutritional factors and gender influence age-related DNA methylation in the human rectal mucosa.

Aberrant methylation of CpG islands (CGI) occurs in many genes expressed in colonic epithelial cells, and may contribute to the dysregulation of signalling pathways associated with carcinogenesis. This cross‐sectional study assessed the relative importance of age, nutritional exposures and other environmental factors in the development of CGI methylation. Rectal biopsies were obtained from 185 individuals (84 male, 101 female) shown to be free of colorectal disease, and for whom measurements of age, body size, nutritional status and blood cell counts were available. We used quantitative DNA methylation analysis combined with multivariate modelling to investigate the relationships between nutritional, anthropometric and metabolic factors and the CGI methylation of 11 genes, together with LINE‐1 as an index of global DNA methylation. Age was a consistent predictor of CGI methylation for 9/11 genes but significant positive associations with folate status and negative associations with vitamin D and selenium status were also identified for several genes. There was evidence for positive associations with blood monocyte levels and anthropometric factors for some genes. In general, CGI methylation was higher in males than in females and differential effects of age and other factors on methylation in males and females were identified. In conclusion, levels of age‐related CGI methylation in the healthy human rectal mucosa are influenced by gender, the availability of folate, vitamin D and selenium, and perhaps by factors related to systemic inflammation.

Flavonoids and intestinal cancers

Cancers of the gastrointestinal tract are amongst the most common causes of death from cancer, but there is substantial variation in incidence across populations. This is consistent with a major causative role for diet. There is convincing evidence that fruits and vegetables protect against cancers of the upper alimentary tract and the large bowel, and this has focused attention on biologically active phytochemicals, and on flavonoids in particular. Many flavonoids exert anticarcinogenic effects in vitro and in animals, and many of these effects occur via signalling pathways known to be important in the pathogenesis of colorectal, gastric and oesophageal cancers. However dietary flavonoid intakes are generally low and their metabolism in humans is extremely complex. The advent of new post-genomic technologies will do much to address these problems by making it possible to monitor patterns of gene expression in humans to provide essential molecular biomarkers of early disease. By combining such data with knowledge of the dietary exposure and bioavailability of the most effective compounds it will be possible to predict the most effective dietary sources and to properly evaluate the potential role of flavonoids in clinical nutrition.

Production and purification of a granular‐starch‐binding domain of glucoamylase 1 from Aspergillus niger

A domain of glucoamylase 1 from Aspergillus niger which binds to granular starch was produced by proteolytic digestion and purified to apparent homogeneity by extraction with corn starch followed by anion‐exchange chromatography and gel filtration. The peptide has a molecular weight of 25100, contains approximately 38% carbohydrate ( ) and corresponds to residues 471–616 at the C‐terminus of glucoamylase 1. The peptide bound to granular corn starch maximally at 1.08 starch. It inhibited the hydrolysis of granular starch by glucoamylase 1 but had no effect on the hydrolysis of starch in solution.

Hydrolysis of A- and B-type crystalline polymorphs of starch by α-amylase, β-amylase and glucoamylase 1

The hydrolysis of A-type and B-type spherulitic polycrystalline amylose (degree of polymerisation ∼ 20) by glucoamylase 1 (Aspergillus niger), by β-amylase (sweet potato) and by α-amylase (Aspergillus oryzae) has been examined by differential scanning calorimetry, X-ray powder diffraction and microscopy (polarised light and scanning electron). B-type structure is hydrolysed more slowly than A-type by all enzymes probably due to surface area effects. A-type shows no discernible spherulitic structure after partial hydrolysis, whereas the B-type spherulites retain a large amount of visible spherical structure. Glucoamylase l-treated B-type crystals retain their positive birefringence, appear smooth, exhibit a 40% decrease in melting energy and retain crystallinity. On the other hand, α- and β-amylase treatment of B-type spherulites results in spherulites that are not birefringent, show extensive small pits (β-amylase) or several very large pits (α-amylase), exhibit an 83% (β-amylase) and 87% (α-amylase) decrease in melting energy and lose a substantial amount of crystallinity. A-type crystals show no change in melting energy of the solid material after partial hydrolysis by any enzyme, and fully retain crystallinity of the remaining material. The results demonstrate that the pattern of enzyme attack depends on the crystal type, and that each enzyme has a specific mode of attack on B-type amylose even in chemically homogeneous spherulites of a single crystalline type.

A family 11 xylanase from Penicillium funiculosum is strongly inhibited by three wheat xylanase inhibitors.

Steady-state kinetic approaches were used to investigate the binding of a novel Penicillium funiculosum xylanase, XYNC, with three known xylanase inhibitor proteins from wheat (Triticum aestivum). The xylanase gene (xynC) was cloned from a P. funiculosum genomic library and the deduced amino acid sequence of XYNC exhibited high sequence similarity with fungal family 11 xylanases. xynC was overexpressed in P. funiculosum and the product (XYNC: M(r)=23.6 kDa; pI=3.7) purified and shown to efficiently degrade birchwood xylan [K(m)=0.47% w/v, Vmax=2540 micromol xylose min(-1) (mg protein)(-1) at pH 5.5 and 30 degrees C] and soluble wheat arabinoxylans [K(m)=1.45% w/v, Vmax=7190 micromol xylose min(-1) mg protein)(-1) at pH 5.5 and 30 degrees C]. The xylanase activity of XYNC was inhibited strongly by three xylanase inhibitor proteins from wheat; XIP-I, TAXI I and TAXI II. The inhibition for each was competitive, with very tight binding (K(i)=3.4, 16 and 17 nM, respectively) equivalent to free energy changes (deltaG degrees ) of -49, -45 and -45 kJ mol(-1). This is the first report describing a xylanase that is inhibited by all three wheat xylanase inhibitor proteins described to date.

Procyanidin effects on oesophageal adenocarcinoma cells strongly depend on flavan-3-ol degree of polymerization

Epidemiological studies have shown that the risk of developing oesophageal adenocarcinoma (OA) is inversely correlated to consumption of fruits and vegetables. Flavan-3-ols are the most abundant subclass of flavonoids in these types of foods. Three apple-derived procyanidin fractions with different average degrees of polymerization (aDP) were characterized and the effects of these fractions and of pure flavan-3-ol monomers ((-)-epicatechin and (+)-catechin) and dimers (B1, B2) on two OA cell lines were investigated. Flavan-3-ol monomers and dimers had no effect on the two cell lines, while apple-derived flavan-3-ol oligomers and polymers induced a time-dependent reduction of cell viability. The reduction in the cell viability was due to the induction of caspase-mediated apoptosis and an arrest of the cell cycle in G0/G1. The magnitude of the reduction in cell viability and induction of apoptosis after exposure to flavan-3-ol oligomeric/polymeric fractions positively correlated with their aDP. These results indicate that only flavan-3-ol oligomers and polymers, but not monomers and dimers, have an effect on the proliferation of OA cells in vitro. As tested flavan-3-ol concentrations are achievable through diet, this study suggests that apple-derived PA may possess chemotherapeutic effects against OA.

The effect of cadaverine on the formation of anabasine from lysine in hairy root cultures of Nicotiana hesperis

Unlabelled cadaverine did not diminish the incorporation into anabasine of 14C from L-[U-14C] lysine supplied to hairy root cultures of Nicotiana nesperis, despite causing a stimulation of anabasine production. The finding is discussed in the context of previous observations indicating that free cadaverine is not an intermediate in the biosynthesis of anabasine from lysine.

Interaction of β-cyclodextrin with the granular starch binding domain of glucoamylase

The granular starch binding domain of glucoamylase 1 (EC 3.2.1.3 1,4-alpha-D-glucan glucohydrolase) binds two molecules of beta-cyclodextrin, with a dissociation constant (Kd) for the second ligand of 1.68 microM. The catalytic domain showed no interaction with beta-cyclodextrin. Beta-cyclodextrin competitively inhibited the adsorption of the binding domain onto granular starch with an inhibition constant (Ki) of 11.0 +/- 1.9 microM. The results show that beta-cyclodextrin binds to the binding domain of glucoamylase at the same site(s) as granular starch.

Recent advances in understanding the role of diet and obesity in the development of colorectal cancer

Colorectal cancer (CRC) is a major cause of premature death in the UK and many developed countries. However, the risk of developing CRC is well recognised to be associated not only with diet but also with obesity and lack of exercise. While epidemiological evidence shows an association with factors such as high red meat intake and low intake of vegetables, fibre and fish, the mechanisms underlying these effects are only now being elucidated. CRC develops over many years and is typically characterised by an accumulation of mutations, which may arise as a consequence of inherited polymorphisms in key genes, but more commonly as a result of spontaneously arising mutations affecting genes controlling cell proliferation, differentiation, apoptosis and DNA repair. Epigenetic changes are observed throughout the progress from normal morphology through formation of adenoma, and the subsequent development of carcinoma. The reasons why this accumulation of loss of homoeostatic controls arises are unclear but chronic inflammation has been proposed to play a central role. Obesity is associated with increased plasma levels of chemokines and adipokines characteristic of chronic systemic inflammation, and dietary factors such as fish oils and phytochemicals have been shown to have anti-inflammatory properties as well as modulating established risk factors such as apoptosis and cell proliferation. There is also some evidence that diet can modify epigenetic changes. This paper briefly reviews the current state of knowledge in relation to CRC development and considers evidence for potential mechanisms by which diet may modify risk.