Bernard M. Corfe

Dysbiosis of the gut microbiota in disease

There is growing evidence that dysbiosis of the gut microbiota is associated with the pathogenesis of both intestinal and extra-intestinal disorders. Intestinal disorders include inflammatory bowel disease, irritable bowel syndrome (IBS), and coeliac disease, while extra-intestinal disorders include allergy, asthma, metabolic syndrome, cardiovascular disease, and obesity. In many of these conditions, the mechanisms leading to disease development involves the pivotal mutualistic relationship between the colonic microbiota, their metabolic products, and the host immune system. The establishment of a ‘healthy’ relationship early in life appears to be critical to maintaining intestinal homeostasis. Whilst we do not yet have a clear understanding of what constitutes a ‘healthy’ colonic microbiota, a picture is emerging from many recent studies identifying particular bacterial species associated with a healthy microbiota. In particular, the bacterial species residing within the mucus layer of the colon, either through direct contact with host cells, or through indirect communication via bacterial metabolites, may influence whether host cellular homeostasis is maintained or whether inflammatory mechanisms are triggered. In addition to inflammation, there is some evidence that perturbations in the gut microbiota is involved with the development of colorectal cancer. In this case, dysbiosis may not be the most important factor, rather the products of interaction between diet and the microbiome. High-protein diets are thought to result in the production of carcinogenic metabolites from the colonic microbiota that may result in the induction of neoplasia in the colonic epithelium. Ever more sensitive metabolomics methodologies reveal a suite of small molecules produced in the microbiome which mimic or act as neurosignallers or neurotransmitters. Coupled with evidence that probiotic interventions may alter psychological endpoints in both humans and in rodent models, these data suggest that CNS-related co-morbidities frequently associated with GI disease may originate in the intestine as a result of microbial dysbiosis. This review outlines the current evidence showing the extent to which the gut microbiota contributes to the development of disease. Based on evidence to date, we can assess the potential to positively modulate the composition of the colonic microbiota and ameliorate disease activity through bacterial intervention.

Damage-induced Bax N-terminal change, translocation to mitochondria and formation of Bax dimers/complexes occur regardless of cell fate.

Sequential steps in the activation of the pro‐apoptotic protein Bax are described for cells with different sensitivity to cytotoxins. SH‐EP1 and SH‐SY5Y human neuroblastoma cells, derived from a single precursor cell line, differed in their sensitivity to taxol but showed the same sensitivity to cisplatin. Both drugs, in both cell lines, induced exposure of a constitutively occluded N‐terminal epitope of Bax. This was reversible and occurred before the translocation of cytosolic Bax to mitochondria. The N‐terminal change in Bax, its subsequent movement to mitochondria and its dimerization/complex formation were insufficient for commitment to death, occurring in the same proportion of cells that either maintained (SH‐SY5Y) or lost (SH‐EP1) clonogenic survival after taxol treatment. Suppression of taxol‐induced apoptosis occurred upstream of cytochrome c release from mitochondria in SH‐SY5Y cells. The data suggest that a further drug damage‐induced event occurs after Bax dimerization/complex formation but prior to cytochrome c release. This event was absent in the taxol‐resistant cells.

Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore.

The GerAA, -AB, and -AC proteins of the Bacillus subtilis spore are required for the germination response to L-alanine as the sole germinant. They are likely to encode the components of the germination apparatus that respond directly to this germinant, mediating the spores response; multiple homologues of the gerA genes are found in every spore former so far examined. The gerA operon is expressed in the forespore, and the level of expression of the operon appears to be low. The GerA proteins are predicted to be membrane associated. In an attempt to localize GerA proteins, spores of B. subtilis were broken and fractionated to give integument, membrane, and soluble fractions. Using antibodies that detect Ger proteins specifically, as confirmed by the analysis of strains lacking GerA and the related GerB proteins, the GerAA protein and the GerAC+GerBC protein homologues were localized to the membrane fraction of fragmented spores. The spore-specific penicillin-binding protein PBP5*, a marker for the outer forespore membrane, was absent from this fraction. Extraction of spores to remove coat layers did not release the GerAC or AA protein from the spores. Both experimental approaches suggest that GerAA and GerAC proteins are located in the inner spore membrane, which forms a boundary around the cellular compartment of the spore. The results provide support for a model of germination in which, in order to initiate germination, germinant has to permeate the coat and cortex of the spore and bind to a germination receptor located in the inner membrane.

Clinical trial: a multistrain probiotic preparation significantly reduces symptoms of irritable bowel syndrome in a double-blind placebo-controlled study

Background  The efficacy of probiotics in alleviating the symptoms of irritable bowel syndrome (IBS) appears to be both strain‐ and dose‐related.

Classification and functions of enteroendocrine cells of the lower gastrointestinal tract

With over thirty different hormones identified as being produced in the gastrointestinal (GI) tract, the gut has been described as ‘the largest endocrine organ in the body’ (Ann. Oncol., 12, 2003, S63). The classification of these hormones and the cells that produce them, the enteroendocrine cells (EECs), has provided the foundation for digestive physiology. Furthermore, alterations in the composition and function of EEC may influence digestive physiology and thereby associate with GI pathologies. Whilst there is a rapidly increasing body of data on the role and function of EEC in the upper GI tract, there is a less clear‐cut understanding of the function of EEC in the lower GI. Nonetheless, their presence and diversity are indicative of a role. This review focuses on the EECs of the lower GI where new evidence also suggests a possible relationship with the development and progression of primary adenocarcinoma.

Daily ingestion of alginate reduces energy intake in free-living subjects

Sodium alginate is a seaweed-derived fibre that has previously been shown to moderate appetite in models of acute feeding. The mechanisms underlying this effect may include slowed gastric clearance and attenuated uptake from the small intestine. In order to assess whether alginate could be effective as a means of appetite control in free-living adults, 68 males and females (BMI range: 18.50-32.81 kg/m(2)) completed this randomised, controlled two-way crossover intervention to compare the effects of 7 day daily ingestion of a strong-gelling sodium alginate formulation against a control. A sodium alginate with a high-guluronate content was chosen because, upon ingestion, it forms a strong gel in the presence of calcium ions. Daily preprandial ingestion of the sodium alginate formulation produced a significant 134.8 kcal (7%) reduction in mean daily energy intake. This reduced energy intake was underwritten by significant reductions in mean daily carbohydrate, sugar, fat, saturated fat and protein intakes. The absence of any significant interaction effects between the main effect of preload type and those of gender, BMI classification and/or timing of preload delivery indicates the efficacy of this treatment for individuals in different settings. These findings suggest a possible role for a strong-gelling sodium alginate formulation in the future management of overweight and obesity.

Cellular damage signals promote sequential changes at the N-terminus and BH-1 domain of the pro-apoptotic protein Bak.

The pro-apoptotic protein Bak is converted from a latent to an active form by damage-induced signals. This process involves an early exposure of an occluded N-terminal epitope of Bak in intact cells. Here we report a subsequent damage-induced change in Bak, detected using an antibody to the central BH-1 domain. Bak co-immunoprecipitated with Bc1-xL both in undamaged cells and early after damage, when the N-terminal epitope was exposed but the BH-1 epitope remained occluded. A subsequent decrease in binding of Bak to Bc1-xL correlated with exposure of an epitope in the Bak BH-1 domain. Overexpression of Bc1-xL did not affect the kinetics of exposure of the Bak N-terminal epitope but delayed exposure of the BH-1 domain. Cytochrome c release from mitochondria facilitates the activation of apoptotic caspases. The majority of cells with exposed Bak BH-1 domains contained cytosolic cytochrome c. However, a small proportion of cells exhibited exposed Bak BH-1 domains that co-localized with mitochondrial cytochrome c. The data are consistent with a two-step model for the activation of Bak by drug-induced damage signals where dissociation of Bc1-xL from the BH-1 domain of Bak occurs immediately prior to or concomitantly with cytochrome c release.

Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line

Butyrate, a known histone deacetylase inhibitor (HDACi) and product of fibre fermentation, is postulated to mediate the protective effect of dietary fibre against colon cancer. The transcription factor Sp1 is a target of acetylation and is known to be associated with class I HDACs, including HDAC1. Sp1 is a ubiquitous transcription factor and Sp1-regulated genes include those involved in cell cycle regulation, apoptosis and lipogenesis: all major pathways in cancer development. The only known acetylated residue of Sp1 is lysine703 which resides in the DNA binding domain. Here we show that acetylated Sp1 loses p21- and bak-promoter -binding function in vitro. Furthermore treatment with a panel of HDAC inhibitors showed clustering of activities for a subset of inhibitors, causing G2 cell cycle arrest, Sp1 acetylation, p21 and Bak over-expression, all with very similar EC50 concentrations. These HDACi activities were not distributed according to the molecular class of compound. In order to mimic loss of binding, an siRNA strategy was used to reduce Sp1 expression. This resulted in altered expression of multiple elements of the p53/p21 pathway. Taken together our data suggest a mechanistic model for the chemopreventive actions of butyrate in colon epithelial cells, and provide new insight into the differential activities some classes of HDAC inhibitors.

Upregulation of BAK by butyrate in the colon is associated with increased Sp3 binding.

Butyrate is a key bioactive product of dietary fibre fermentation thought to play a key role in cancer prevention. One contributory mechanism in this role is the regulation of apoptosis by butyrate. As butyrate shows low levels of toxicity, the mechanisms by which it triggers or regulates apoptosis are of great interest. We and others have shown that the proapoptotic protein BAK is upregulated by butyrate. We show here that this observation is conserved across multiple cell lines, that it occurs in all cells in a population and is at the transcriptional level. We have used a promoter-reporter construct to identify the regulatory regions of the BAK promoter and found that much of the transcriptional activity occurs via a single Sp1/Sp3 binding site. We have shown that both Sp1 and Sp3 bind, but upon butyrate treatment Sp1 binding decreases in favour of Sp3 binding. We speculate that this may be an acetylation-mediated event.

The gerB region of the Bacillus subtilis 168 chromosome encodes a homologue of the gerA spore germination operon

Spores of gerB spore germination mutants of Bacillus subtilis 168 are defective in response to the germinative mixture of L-asparagine, glucose, fructose and potassium ions (AGFK), but are normal in the L-alanine (ALA) triggered germination response. A lambda clone of 15 kbp carrying the gerB region has been identified. Sequencing of the gerB region of the clone revealed a cluster of three ORFs encoding putative proteins of 53.3, 41.3 and 42.4 kDa (GerBA, GerBB and GerBC, respectively). The first two of these proteins have substantial hydrophobic regions and the third is a possible lipoprotein. At least two, and probably all three products are required for normal germination in AGFK. The three proteins form a set of homologues of the products of the gerA operon, mutations in which cause a defect in the ALA germination pathway, but cause no defect in AGFK. The GerB proteins show 42%, 31% and 35% identity at the amino-acid level to the corresponding GerA proteins, and the homologues occur in the same order in both operons.