An observational study of cognitive function in patients with irritable bowel syndrome and inflammatory bowel disease.

BACKGROUND: Irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) are associated with several risk factors for developing cognitive impairment. These include altered cytokine levels, concurrent mood disorders, and the presence of chronic pain. This observational study aimed to explore the cognitive profile of patients with these conditions. METHODS: Participants completed the Cardiff Cognitive Battery, a series of computerized neuropsychological performance tests that examine a range of cognitive function including psychomotor speed, memory, and intelligence. A progressive analysis of covariance model was used with demographic details, anxiety and depression scores entered as covariates. Fecal calprotectin levels were measured in IBD patients to determine disease activity. KEY RESULTS: In total 231 participants were recruited (150 IBD patients, 40 IBS patients, and 41 healthy controls). IBD patients had significantly lower scores on fluid (p = 0.01) and crystalline intelligence tests (p = 0.028) compared to healthy volunteers, however, this reflected differences in concurrent mood disorder and level of education. When these factors were added as covariates, there was no significant difference between the groups. Duration and activity of disease did not affect cognitive function in IBD patients. Severity of symptoms had no impact on cognition in patients with IBS. CONCLUSIONS & INFERENCES: The results of this observational study do not support the hypothesis that IBS or IBD have an intrinsic disease process that is associated with cognitive dysfunction. It is possible that concurrent mood disorders, in particular depression, may affect the cognitive performance of patients with IBD in specific tasks.

Selectivity mechanism of the mechanosensitive channel MscS revealed by probing channel subconducting states.

The mechanosensitive channel of small conductance (MscS) has been characterized at both functional and structural levels and has an integral role in the protection of bacterial cells against hypoosmotic shock. Here we investigate the role that the cytoplasmic domain has in MscS channel function by recording wild-type and mutant MscS single-channel activity in liposome patches. We report that MscS preferentially resides in subconducting states at hyperpolarising potentials when Ca(2+) and Ba(2+) ions are the major permeant cations. In addition, our results indicate that charged residues proximal to the seven vestibular portals and their electrostatic interactions with permeating cations determine selectivity and regulate the conductance of MscS and potentially other channels belonging to the MscS subfamily. Furthermore, our findings suggest a role for mechanosensitive channels in bacterial calcium regulation, indicative of functions other than protection against osmolarity changes that these channels possibly fulfil in bacteria.

Symptoms of irritable bowel syndrome in patients with inflammatory bowel disease: examining the role of sub-clinical inflammation and the impact on clinical assessment of disease activity.

BACKGROUND: Symptoms compatible with irritable bowel syndrome (IBS) are frequently present in patients with inflammatory bowel disease (IBD); however, the cause of this phenomenon is unclear. AIM: To determine the different contributions of 'true IBS' and sub-clinical inflammation in producing IBS-type symptoms in IBD patients, and to ascertain the impact these symptoms have on the clinical assessment of IBD activity. METHODS: In this cross-sectional study, 169 IBD patients completed questionnaires to assess disease activity, presence of IBS-type symptoms, and levels of anxiety and depression. Stool samples were collected for analysis of faecal calprotectin (FC). RESULTS: IBS-type symptoms were significantly more common in female patients (OR = 4.64, 1.55-13.88) and were associated with higher levels of anxiety (OR = 1.11, 1.01-1.21). There was no statistical difference between the FC levels of patients in clinical remission with IBS-type symptoms compared with those without (median values = 111 µg/g vs. 45.5 µg/g respectively, P = 0.171). The prevalence of IBS-type symptoms in patients with a normal FC level was 31%. CONCLUSIONS: A substantial number of IBD patients with normal faecal calprotectin level experience IBS-type symptoms. These patients exhibit similar features to people diagnosed with IBS in the general community, suggesting that the conditions are not mutually exclusive and may coexist in a considerable number of IBD patients. A systematic diagnostic approach is required to assess IBD patients with IBS-type symptoms as sub-clinical inflammation may play a role in a proportion of cases.

Enzymatic activity of albumin shown by coelenterazine chemiluminescence.

Bioluminescence, the emission of light from live organisms, occurs in 18 phyla and is the major communication system in the deep sea. It has appeared independently many times during evolution but its origins remain unknown. Coelenterazine bioluminescence discovered in luminous jellyfish is the most common chemistry causing bioluminescence in the sea, occurring in seven phyla. Sequence similarities between coelenterazine luciferases and photoproteins from different phyla are poor (often < 5%). The aim of this study was to examine albumin that binds organic substances as a coelenterazine luciferase to test the hypothesis that the evolutionary origin of a bioluminescent protein was the result of the formation of a solvent cage containing just a few key amino acids. The results show for the first time that bovine and human albumin catalysed coelenterazine chemiluminescence consistent with a mono-oxygenase, whereas gelatin and haemoglobin, an oxygen carrier, had very weak activity. Insulin also catalysed coelenterazine chemiluminescence and was increased by Zn(2+). Albumin chemiluminescence was heat denaturable, exhibited saturable substrate characteristics and was inhibited by cations that bound these proteins and by drugs that bind to human albumin drug site I. Molecular modelling confirmed the coelenterazine binding site and identified four basic amino acids: lys195, arg222, his242 and arg257, potentially important in binding and catalysis similar to naturally occurring coelenterazine bioluminescent proteins. These results support the 'solvent cage' hypothesis for the evolutionary origin of enzymatic coelenterazine bioluminescent proteins. They also have important consequences in diseases such as diabetes, gut disorders and food intolerance where a mono-oxygenase could affect cell surface proteins.

Association of lactose sensitivity with inflammatory bowel disease--demonstrated by analysis of genetic polymorphism, breath gases and symptoms.

BACKGROUND: Sensitivity to lactose has been reported in Crohn's disease, but its true role in inflammatory bowel disease (IBD) is unclear. The genetic marker CC13910, on chromosome2, with measurement of breath hydrogen and methane, and gut and systemic symptoms, are now the most comprehensive tests for evaluating sensitivity to lactose. AIM: To investigate, for the first time, the prevalence of lactose sensitivity in IBD, using the most comprehensive tests for diagnosing this condition. METHODS: Prevalence of CC13910 genotype was investigated using RT-PCR in 165 patients (Crohn's disease = 70, ulcerative colitis = 95), and 30 healthy volunteers. Genotype was correlated with breath hydrogen and methane up to 6 h after 50 g of oral lactose, all symptoms being recorded for up to 48 h. Critically, Crohn's disease and ulcerative colitis patients were selected with no record of lactose sensitivity, in remission at the time of the test. RESULTS: Lactose sensitivity occurred in a much higher proportion of patients, (approximately 70%), with IBD than previously thought. Seventeen per cent had raised methane, without raised breath hydrogen; those with ulcerative colitis exhibiting most symptoms. All CC patients were lactose sensitive. There was no correlation between genetic phenotype and IBD. As substantial numbers of IBD patients were CT or TT, and were lactose sensitive, this polymorphism cannot explain full down-regulation of the lactase gene. CONCLUSIONS: Our results have implications for the clinical management of IBD. The high breath methane raised the possibility of a pathogenic role for methanogenic archaebacteria (Archaea) in IBD. This needs to be investigated.

Bacterial metabolic 'toxins': a new mechanism for lactose and food intolerance, and irritable bowel syndrome.

Lactose and food intolerance cause a wide range of gut and systemic symptoms, including gas, gut pain, diarrhoea or constipation, severe headaches, severe fatigue, loss of cognitive functions such as concentration, memory and reasoning, muscle and joint pain, heart palpitations, and a variety of allergies (Matthews and Campbell, 2000; Matthews et al., 2005; Waud et al., 2008). These can be explained by the production of toxic metabolites from gut bacteria, as a result of anaerobic digestion of carbohydrates and other foods, not absorbed in the small intestine. These metabolites include alcohols, diols such as butan 2,3 diol, ketones, acids, and aldehydes such as methylglyoxal (Campbell et al., 2005, 2009). These 'toxins' induce calcium signals in bacteria and affect their growth, thereby acting to modify the balance of microflora in the gut (Campbell et al., 2004, 2007a,b). These bacterial 'toxins' also affect signalling mechanisms in cells around the body, thereby explaining the wide range of symptoms in people with food intolerance. This new mechanism also explains the most common referral to gastroenterologists, irritable bowel syndrome (IBS), and the illness that afflicted Charles Darwin for 50 years (Campbell and Matthews, 2005a,b). We propose it will lead to a new understanding of the molecular mechanism of type 2 diabetes and some cancers.

Quantifying the 'hidden' lactose in drugs used for the treatment of gastrointestinal conditions.

BACKGROUND: Lactose intolerance affects 70% of the world population and may result in abdominal and systemic symptoms. Treatment focuses predominantly on the dietary restriction of food products containing lactose. Lactose is the most common form of excipient used in drug formulations and may be overlooked when advising these patients. AIM: To identify and quantify the amount of lactose in medications used for the treatment of gastrointestinal disorders and to identify 'lactose-free' preparations. METHODS: Medications used for the treatment of gastrointestinal disorders were identified from the British National Formulary (BNF). Their formulation including excipients was obtained from the Medicines Compendium. The lactose content and quantity in selected medications was measured using high-performance liquid chromatography (HPLC). RESULTS: A wide range of medications prescribed for the treatment of gastrointestinal conditions contain lactose. We have quantified the lactose content in a selection of medications using HPLC. Lactose is present in amounts that may contribute towards symptoms. Lactose-free alternatives were also identified. CONCLUSIONS: Lactose is present in a range of medications and may contribute towards symptoms. This may not be recognized by the prescribing doctor as excipients are not listed in the BNF, and the quantity of lactose is not listed on the label or in the accompanying manufacturer's leaflet.

Systemic lactose intolerance: a new perspective on an old problem.

Intolerance to certain foods can cause a range of gut and systemic symptoms. The possibility that these can be caused by lactose has been missed because of "hidden" lactose added to many foods and drinks inadequately labelled, confusing diagnosis based on dietary removal of dairy foods. Two polymorphisms, C/T13910 and G/A22018, linked to hypolactasia, correlate with breath hydrogen and symptoms after lactose. This, with a 48 hour record of gut and systemic symptoms and a six hour breath hydrogen test, provides a new approach to the clinical management of lactose intolerance. The key is the prolonged effect of dietary removal of lactose. Patients diagnosed as lactose intolerant must be advised of "risk" foods, inadequately labelled, including processed meats, bread, cake mixes, soft drinks, and lagers. This review highlights the wide range of systemic symptoms caused by lactose intolerance. This has important implications for the management of irritable bowel syndrome, and for doctors of many specialties.

Low erythrocyte folate, but not plasma vitamin B-12 or homocysteine,is associated with dementia in elderly Latinos.

The relationship between B vitamin status and cognitive function has been of interest for many years. There is evidence of relationships between intake and status of folate and vitamin B-12 with neurological, cognitive, and memory impairment, but results have been inconsistent. Plasma B-12, erythrocyte folate, methylmalonic acid,and homocysteine were evaluated as predictors of cognitive function in a large population based sample of Latino elderly living in the Sacramento, California region. The hypothesis tested was that low folate and/or B-12 status predicts cognitive function impairment and dementia. Logistic regression was used to examine the differences in B-vitamin status by cognitive function category. Erythrocyte folate was related to dementia after controlling for age, gender, education, income, diabetes diagnosis, serum creatinine, and depressive symptoms. The highest prevalence of low erythrocyte folate occurred in the Dementia group and was significantly higher than in the Normal group. Plasma B-12, MMA, Hcy, and prevalence of a normal values for these variables, were not significantly different among the cognitive function classes. We conclude that folate status is associated with dementia but that more research is needed on the relationship between vitaminB-12 status, Hcy and cognitive function to explore possible associations with these parameters.

Direct measurement of free Ca(2+) shows different regulation of Ca(2+) between the periplasm and the cytosol of Escherichia coli.

As in eukaryotes, bacterial free Ca(2+) can play an important role as an intracellular signal. However, because free Ca(2+) is difficult to measure in live bacteria, most of the evidence for such a role is indirect. Gram-negative bacteria also have an outer membrane separating the external fluid from the periplasm as well as the cytosol where most bacterial metabolism takes place. Here we report, for the first time, direct measurement of free Ca(2+) in the periplasmic space of living Escherichia coli. Periplasmic free Ca(2+) was measured by targeting the Ca(2+)-activated photoprotein aequorin to this compartment using the N-terminal OmpT signal sequence. Cytosolic free Ca(2+) was determined using aequorin alone. We show that, under certain conditions, the periplasm can concentrate free Ca(2+), resulting in the inner membrane being exposed to free Ca(2+) concentrations several fold higher than in the bulk external fluid. Manipulation of periplasmic membrane-derived oligosaccharides (MDOs) altered the free Ca(2+) as predicted by the Donnan potential. With micromolar concentrations of external free Ca(2+), the periplasm concentrated free Ca (2+) some three to sixfold with respect to the external medium. A Ca(2+) gradient also existed between the periplasm and the cytosol under these conditions, the periplasmic free Ca(2+) being some one to threefold higher. At millimolar levels of external free Ca(2+), a similar concentration was detected in the periplasm, but the bacteria still maintained tight control of cytosolic free Ca(2+) in the micromolar range. We propose that the highly anionic MDOs in the periplasmic space generate a Donnan potential, capable of concentrating Ca(2+) in this compartment, where it may constitute a sink for regulation of Ca(2+)-dependent processes in the cytoplasm.

Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin.

Homogeneous assays, without a separation step, are essential for measuring chemical events in live cells and for drug discovery screens, and are desirable for making measurements in cell extracts or clinical samples. Here we demonstrate the principle of chemiluminescence resonance energy transfer (CRET) as a homogeneous assay system, using two proteases as models, one extracellular (alpha-thrombin) and the other intracellular (caspase-3). Chimaeras were engineered with aequorin as the chemiluminescent energy donor and green fluorescent protein (GFP) or enhanced GFP as the energy acceptors, with a protease linker (6 or 18 amino acid residues) recognition site between the donor and acceptor. Flash chemiluminescent spectra (20--60 s) showed that the spectra of chimaeras matched GFP, being similar to that of luminous jellyfish, justifying their designation as 'Rainbow' proteins. Addition of the protease shifted the emission spectrum to that of aequorin in a time- and dose-dependent manner. Separation of the proteolysed fragments showed that the ratio of green to blue light matched the extent of proteolysis. The caspase-3 Rainbow protein was able to provide information on the specificity of caspases in vitro and in vivo. It was also able to monitor caspase-3 activation in cells provoked into apoptosis by staurosporine (1 or 2 microM). CRET can also monitor GFP fluor formation. The signal-to-noise ratio of our Rainbow proteins is superior to that of fluorescence resonance energy transfer, providing a potential platform for measuring agents that interact with the reactive site between the donor and acceptor.

[Validation of the use of aequorin for cytoplasmic free calcium determination by chemiluminescence in Streptococcus pneumoniae]. / Validation de l'outil aequorine pour le dosage du calcium libre cytoplasmique par chimioluminescence chez Streptococcus pneumoniae.

In the extracellular pathogen Streptococcus pneumoniae, transformable by soluble DNA, calcium transport is shown to play a key role for vegetative growth, developement of competence for genetic transformation and experimental virulence. To get a more precise localisation of Ca2+ in the cell, we cloned the cDNA of apoaequorine in the chromosome of Streptococcus pneumoniae. This allowed the reconstitution of the acquorine system and chemoluminescence measurements of the cytoplasmic free calcium concentration in the bacteria. Intracellular free Ca2+ is 2 microM at the steady state and can reach 14 microM when calcium is added to the bacterial suspension. Increase in free Ca2+ in response to an imposed Ca2+ gradient depends on the initial velocity (Vi) of the DMB-sensitive Ca2+ transport, showing that changes in cytoplasmic Ca2+ involve active transport.

Phorbol ester-induced phosphoinositide hydrolysis in rat aorta: role of cyclooxygenase products.

This study investigates whether phorbol esters increase phosphoinositide hydrolysis in intact vascular smooth muscle, and the mechanism underlying the hydrolysis. Phorbol myristate acetate induced time- and concentration-dependent increases in phosphoinositide hydrolysis, as demonstrated by elevated inositol monophosphate levels, in deendothelialized rat aorta. The phorbol ester-elevated inositol monophosphate levels were abolished by indomethacin, a cyclooxygenase inhibitor, but were only partially decreased by SQ29548, a thromboxane A2/prostaglandin H2 receptor antagonist. SQ29548 also only partially decreased elevated inositol monophosphate levels due to prostaglandin E2, prostaglandin F2alpha, prostaglandin I2 and carbacyclin, a stable prostaglandin I2 analog. SQ29548 abolished elevated inositol monophosphate levels due to U46619, a stable thromboxane A2/prostaglandin H2 receptor agonist. These studies demonstrate that phorbol esters increase phosphoinositide hydrolysis in intact vascular smooth muscle, and that the increase is due, at lease in part, to endogenously released prostaglandins other than prostaglandin H2.

Differential regulation of norepinephrine- and prostaglandin F2alpha-induced contraction by extracellular Na+ in rat aorta.

The purpose of this study was to test whether extracellular Na+ differentially regulates agonist-induced contraction in vascular smooth muscle. Exposure of rat aorta to 20 nM extracellular Na+ by substitution of 123 mM Na+ with N-methyl-D-glucamine or choline, inhibited norepinephrine-induced contraction to a greater magnitude than contraction to prostaglandin F2alpha. In the absence of extracellular Ca2+ and in 20 mM Na+ solution containing 123 mM N-methyl-D-glucamine, the norepinephrine and prostaglandin F2alpha contraction remained unaltered. In contrast, in the absence of extracellular Ca2+ and in 20 mM Na+ solution containing 123 mM choline, the norepinephrine and prostaglandin F2alpha contraction were decreased and increased, respectively. Contraction to the phorbol ester, phorbol dibutyrate, was inhibited in 20 mM extracellular Na+ solution containing N-methyl-D-glucamine. Removal of extracellular Ca2+ inhibited the phorbol dibutyrate contraction, and 20 mM extracellular Na+ solution containing N-methyl-D-glucamine did not inhibit the phorbol dibutyrate contraction elicited in the absence of extracellular Ca2+. Complete replacement of extracellular Na+ with choline, and concomitant treatment with nifedipine to reduce the elevated basal tone after Na+ replacement, also resulted in greater inhibition of norepinephrine- as compared with prostaglandin F2alpha-induced contraction. Ethylisopropylamiloride, a Na+/H+ exchange inhibitor, did not alter norepinephrine contraction, as determined in the presence of nifedipine to reduce the elevated basal tone due to ethylisopropylamiloride. Acidification, which may result from decreased Na+/H+ exchange, inhibited the prostaglandin F2alpha-induced contraction to a greater magnitude than contraction to norepinephrine. These results demonstrate that extracellular Na+ selectively regulates agonist-induced contraction. The study further suggests that the selectivity may be related to an extracellular Na+-dependent process that is activated by protein kinase C, such as Na+/Ca2+ exchange, and is unrelated to the release of intracellular Ca2+ and Na+/H+ exchange.

Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level.

Monitoring calcium fluxes in real time could help to understand the development, the plasticity, and the functioning of the central nervous system. In jellyfish, the chemiluminescent calcium binding aequorin protein is associated with the green fluorescent protein and a green bioluminescent signal is emitted upon Ca(2+) stimulation. We decided to use this chemiluminescence resonance energy transfer between the two molecules. Calcium-sensitive bioluminescent reporter genes have been constructed by fusing green fluorescent protein and aequorin, resulting in much more light being emitted. Chemiluminescent and fluorescent activities of these fusion proteins have been assessed in mammalian cells. Cytosolic Ca(2+) increases were imaged at the single-cell level with a cooled intensified charge-coupled device camera. This bifunctional reporter gene should allow the investigation of calcium activities in neuronal networks and in specific subcellular compartments in transgenic animals.

Cloning and expression of the bioluminescent photoprotein pholasin from the bivalve mollusc Pholas dactylus.

Pholasin is the photoprotein responsible for luminescence in the bivalve Pholas dactylus and consists of a luciferin tightly bound to a glycosylated protein. It is a sensitive indicator of reactive oxygen species. A full-length clone encoding apopholasin was isolated from a P. dactylus light organ cDNA library. The unprocessed apoprotein contained 225 amino acids, starting with a signal peptide of 20 amino acids, 3 predicted N-linked glycosylation sites, 1 O-linked site, no histidines, and 7 cysteines. The recombinant apoprotein was expressed in cell extracts and insect cells. The size of the apoprotein expressed in cell extracts and the cytosol of insect cells was 26 kDa but that of the fully processed protein was 34 kDa, as was native pholasin. Both the processed and unprocessed recombinant apoproteins were recognized by a polyclonal antibody raised against native pholasin. Acid methanol extracts from Pholas added to recombinant apoprotein resulted in chemiluminescence triggered by sodium hypochlorite but not photoprotein formation. These results have important implications in understanding the molecular evolution of bioluminescence and will allow the development of recombinant pholasin as an intracellular indicator of reactive oxygen species.

Apoaequorin monitors degradation of endoplasmic reticulum (ER) proteins initiated by loss of ER Ca(2+).

Apoaequorin was targeted to the cytosol, nucleus, and endoplasmic reticulum of HeLa cells in order to determine the effect of Ca(2+) release from the ER on protein degradation. In resting cells apoaequorin had a rapid half-life (ca. 20-30 min) in the cytosol or nucleus, but was relatively stable for up to 24 h in the ER (t(1/2) > 24 h). However, release of Ca(2+) from the ER, initiated by the addition of inhibitors of the ER Ca(2+)/Mg(2+) ATPase such as 2 microM thapsigargin or 1 microM ionomycin, initiated rapid loss of apoaequorin in the ER, but had no detectable effect on apoaequorin turnover in the cytosol nor the nucleus. This loss of apoprotein was not the result of secretion into the external fluid, and could not be inhibited by inhibitors of protein degradation by proteosomes. Proteolysis of apoaequorin in cell extracts (t(1/2) < 20 min) was completely inhibited in the presence of 1 mM Ca(2+), and this effect was independent of the ER retention signal KDEL at the C-terminus. Proteolysis was unaffected by the presence of selected serine protease inhibitors, or 10 microM Zn(2+), a known caspase-3 inhibitor. The results show that apoaequorin can monitor proteolysis of ER proteins activated by loss of ER Ca(2+). Several Ca(2+)-binding proteins exist in the ER, acting as the Ca(2+) store and chaperones. Our results have important implications both for the role of ER Ca(2+) in cell activation and stress and when using aequorin for monitoring free ER Ca(2+) over long time periods.

An assessment of the role of intracellular free Ca2+ in E. coli.

We have previously proposed that fluctuations in Ca(2+) levels should play an important role in bacteria as in eukaryotes in regulating cell cycle events (Norris et al., J. Theor. Biol. 134 (1998) 341-350). This proposal implied the presence of Ca(2+) uptake systems in bacteria, cell cycle mutants simultaneously defective in Ca(2+)-homeostasis, and perturbation of cell cycle processes when cellular Ca(2+) levels are depleted. We review the properties of new cell cycle mutants in E. coli and B. subtilis resistant to inhibitors of calmodulin, PKC or Ca(2+)-channels; the evidence for Ca(2+)-binding proteins including Acp and FtsZ; and Ca(2+)-transporters. In addition, the effects of EGTA and verapamil (a Ca(2+) channel inhibitor) on growth, protein synthesis and cell cycle events in E. coli are described. We also describe new measurements of free Ca(2+)-levels, using aequorin, in E. coli. Several new cell cycle mutants were obtained using this approach, affecting either initiation of DNA replication or in particular cell division at non-permissive temperature. Several of the mutants were also hypersensitive to EGTA and or Ca(2+). However, none of the mutants apparently involved direct alteration of a drug target and surprisingly in some cases involved specific tRNAs or a tRNA synthetase. The results also indicate that the expression of several genes in E. coli may be regulated by Ca(2+). Cell division in particular appears very sensitive to the level of cell Ca(2+), with the frequency of division clearly reduced by EGTA and by verapamil. However, whilst the effect of EGTA was clearly correlated with depletion of cellular Ca(2+) including free Ca(2+), this was not the case with verapamil which appears to change membrane fluidity and the consequent activity of membrane proteins. Measurement of free Ca(2+) in living cells indicated levels of 200-300 nM, tightly regulated in wild type cells in exponential phase, somewhat less so in stationary phase, with apparently La(2+)-sensitive PHB-polyphosphate complexes involved in Ca(2+) influx. The evidence reviewed increasingly supports a role for Ca(2+) in cellular processes in bacteria, however, any direct link to the control of cell cycle events remains to be established.