Persistence of anticancer activity in berry extracts after simulated gastrointestinal digestion and colonic fermentation.

Fruit and vegetable consumption is associated at the population level with a protective effect against colorectal cancer. Phenolic compounds, especially abundant in berries, are of interest due to their putative anticancer activity. After consumption, however, phenolic compounds are subject to digestive conditions within the gastrointestinal tract that alter their structures and potentially their function. However, the majority of phenolic compounds are not efficiently absorbed in the small intestine and a substantial portion pass into the colon. We characterized berry extracts (raspberries, strawberries, blackcurrants) produced by in vitro-simulated upper intestinal tract digestion and subsequent fecal fermentation. These extracts and selected individual colonic metabolites were then evaluated for their putative anticancer activities using in vitro models of colorectal cancer, representing the key stages of initiation, promotion and invasion. Over a physiologically-relevant dose range (0-50 µg/ml gallic acid equivalents), the digested and fermented extracts demonstrated significant anti-genotoxic, anti-mutagenic and anti-invasive activity on colonocytes. This work indicates that phenolic compounds from berries undergo considerable structural modifications during their passage through the gastrointestinal tract but their breakdown products and metabolites retain biological activity and can modulate cellular processes associated with colon cancer.

Thorn-dwelling ants provide antiherbivore defence for camelthorn trees, Vachellia erioloba, in Namibia

Ants are widely employed by plants as an antiherbivore defence. A single host plant can associate with multiple, symbiotic ant species, although usually only a single ant species at a time. Different plant-ant species may vary in the degree to which they defend their host plant. In Kenya, ant–acacia interactions are well studied, but less is known about systems elsewhere in Africa. A southern African species, Vachellia erioloba, is occupied by thorn-dwelling ants from three different genera. Unusually, multiple colonies of all these ants simultaneously and stably inhabit trees. We investigated if the ants on V. erioloba (i) deter insect herbivores; (ii) differ in their effectiveness depending on the identity of the herbivore; and (iii) protect the tree against an important herbivore, the larvae of the lepidopteran Gonometa postica. We show that experimental exclusion of ants leads to greater levels of herbivory on trees. The ants inhabiting V. erioloba are an effective deterrent against hemipteran and coleopteran, but not lepidopteran herbivores. Defensive services do not vary among ant species, but only Crematogaster ants exhibit aggression towards G. postica. This highlights the potential of the V. erioloba–ant mutualism for studying ant–plant interactions that involve multiple, simultaneously resident thorn-dwelling ant species.

Expression of cyclooxygenase-2 in intestine of pigs of different ages and hygiene status

Prostaglandins (PG) are bioactive lipids derived from the metabolism of membrane polyunsaturated fatty acids (PUFA), and play important roles in a number of biological processes including cell division, immune responses and wound healing. Cyclooxygenase (COX) is the key enzyme in PG synthesis from arachidonic acid. The hypothesis of the present study was that expression of COX-2 in porcine intestine was dependent on the microbial load and the age of piglets. Piglets were obtained from sows raised either on outdoor free-range farms or on indoor commercial farms, and littermates were divided into three treatments: One group of piglets suckled the sow, a second group was put into an isolator and fed a milk formula, and a third group was put into the isolator fed milk formula and injected with broad spectrum antibiotics. Samples were collected from the 75% level of the small intestine at day 5, 28 and 56 of age. Tissue section from four piglets from each of these six treatment groups was analysed by immunofluorescence for COX-2 and type-IV collagen (basement membrane, defining lamina propria (LP)). Image analysis was used to determine the number of positive pixels expressing LP and epithelial COX-2. COX-2 expressing cells were observed in LP and epithelium in all porcine intestinal samples. When analysing images obtained on day 28, injection of antibiotics seemed to reduce the COX-2 expression in intestinal samples of piglets when compared to other treatments (P=0.053). No significant effect of farm, treatments or age of piglets was observed on COX-2 expressing data when analysing all data of images obtained at day 28 and 56. By double-labelling experiments, COX-2 was found not to be expressed on cell co-expressing CD45, CD16, CD163 or CD2, thus indicating that mucosal leukocytes, including dendritic cells, macrophages and NK cells did not express COX-2. Future research should investigate the role of COX-2 expression in the digestive tract in relation to pig health.

Biased oviposition and biased survival together help resolve a fig-wasp conflict

We report evidence that helps resolve two competing explanations for stability in the mutualism between Ficus racemosa fig trees and the Ceratosolen fusciceps wasps that pollinate them. The wasps lay eggs in the tree's ovules, with each wasp larva developing at the expense of a fig seed. Upon maturity, the female wasps collect pollen and disperse to a new tree, continuing the cycle. Fig fitness is increased by producing both seeds and female wasps, whereas short-term wasp fitness increases only with more wasps, thereby resulting in a conflict of interests. We show experimentally that wasps exploit the inner layers of ovules first (the biased oviposition explanation), which is consistent with optimal-foraging theory. As oviposition increases, seeds in the middle layer are replaced on a one-to-one basis by pollinator offspring, which is also consistent with biased oviposition. Finally, in the outer layer of ovules, seeds disappear but are only partially replaced by pollinator offspring, which suggests high wasp mortality (the biased survival or ‘unbeatable seeds’ explanation). Our results therefore suggest that both biased oviposition and biased survival ensure seed production, thereby stabilizing the mutualism. We further argue that biased oviposition can maintain biased survival by selecting against wasp traits to overcome fig defenses. Finally, we report evidence suggesting that F. racemosa balances seed and wasp production at the level of the tree. Because figs are probably selected to allocate equally to male and female function, a 1:1 seed:wasp ratio suggests that fig trees are in control of the mutualism.

Protease-activated receptors: protease signaling in the gastrointestinal tract

Serine proteases from the circulation, inflammatory cells, digestive glands and microorganisms can signal to cells by cleaving protease-activated receptors (PARs), a family of four G-protein-coupled receptors. Proteases cleave PARs at specific sites to expose tethered ligand domains that bind to and activate the cleaved receptors. Despite this irreversible mechanism of activation, PAR signaling is tightly regulated to prevent the uncontrolled stimulation of cells. Although PARs are found in all organ systems, protease signaling is of particular interest in the gastrointestinal tract, where proteases regulate neurotransmission, secretion, motility, epithelial permeability and intestinal inflammation, and can thus contribute to disease.

Direct anthelmintic effects of condensed tannins from diverse plant sources against Ascaris suum

Ascaris suum is one of the most prevalent nematode parasites in pigs and causes significant economic losses, and also serves as a good model for A. lumbricoides, the large roundworm of humans that is ubiquitous in developing countries and causes malnutrition, stunted growth and compromises immunity to other pathogens. New treatment options for Ascaris infections are urgently needed, to reduce reliance on the limited number of synthetic anthelmintic drugs. In areas where Ascaris infections are common, ethno-pharmacological practices such as treatment with natural plant extracts are still widely employed. However, scientific validation of these practices and identification of the active compounds are lacking, although observed effects are often ascribed to plant secondary metabolites such as tannins. Here, we extracted, purified and characterised a wide range of condensed tannins from diverse plant sources and investigated anthelmintic effects against A. suum in vitro. We show that condensed tannins can have potent, direct anthelmintic effects against A. suum, as evidenced by reduced migratory ability of newly hatched third-stage larvae and reduced motility and survival of fourth-stage larvae recovered from pigs. Transmission electron microscopy showed that CT caused significant damage to the cuticle and digestive tissues of the larvae. Furthermore, we provide evidence that the strength of the anthelmintic effect is related to the polymer size of the tannin molecule. Moreover, the identity of the monomeric structural units of tannin polymers may also have an influence as gallocatechin and epigallocatechin monomers exerted significant anthelmintic activity whereas catechin and epicatechin monomers did not. Therefore, our results clearly document direct anthelmintic effects of condensed tannins against Ascaris and encourage further in vivo investigation to determine optimal strategies for the use of these plant compounds for the prevention and/or treatment of ascariosis.

O6-carboxymethylguanine DNA adduct formation and lipid peroxidation upon in vitro gastrointestinal digestion of haem-rich meat

Scope Epidemiological and clinical studies have demonstrated that the consumption of red haem-rich meat may contribute to the risk of colorectal cancer. Two hypotheses have been put forward to explain this causal relationship, i.e. N-nitroso compound (NOC) formation and lipid peroxidation (LPO). Methods and Results In this study, the NOC-derived DNA adduct O6-carboxymethylguanine (O6-CMG) and the LPO product malondialdehyde (MDA) were measured in individual in vitro gastrointestinal digestions of meat types varying in haem content (beef, pork, chicken). While MDA formation peaked during the in vitro small intestinal digestion, alkylation and concomitant DNA adduct formation was observed in seven (out of 15) individual colonic digestions using separate faecal inocula. From those, two haem-rich meat digestions demonstrated a significantly higher O6-CMG formation (p < 0.05). MDA concentrations proved to be positively correlated (p < 0.0004) with haem content of digested meat. The addition of myoglobin, a haem-containing protein, to the digestive simulation showed a dose–response association with O6-CMG (p = 0.004) and MDA (p = 0.008) formation. Conclusion The results suggest the haem-iron involvement for both the LPO and NOC pathway during meat digestion. Moreover, results unambiguously demonstrate that DNA adduct formation is very prone to inter-individual variation, suggesting a person-dependent susceptibility to colorectal cancer development following haem-rich meat consumption.

Gastrointestinal microbiota

Bacteria are associated with all areas of the human body from the skin to the genitourinary, respiratory and gastrointestinal (GI) tracts. The GI tract is the most heavily populated, with the majority of the total bacterial population of humans residing therein. The GI tract has evolved to become a functional organ comprising anatomically distinct areas. The digestive process starts in the oral cavity, then moves through the stomach, small and large intestine and finally the rectum. This chapter summarizes the functions of the human gastrointestinal tract. A main function of the GI microbiota is modulation of the immune system. The chapter focues on the factors influencing composition of the microbiota.

Interactions between nutrition and infections with Haemonchus contortus and related gastro-intestinal nematodes in small ruminants

Interactions between host nutrition and feeding behaviour are central to understanding the pathophysiological consequences of infections of the digestive tract with parasitic nematodes. The manipulation of host nutrition provides useful options to control gastrointestinal nematodes as a component of an integrated strategy. Focused mainly on the Hameonchus contortus infection model in small ruminants, this chapter (i) illustrates the relationship between quantitative (macro- and micro-nutrients) and qualitative (plant secondary metabolites) aspects of host nutrition and nematode infection, and (ii) shows how basic studies aimed at addressing some generic questions can help provide solutions, despite the considerable diversity of epidemiological situations and breeding systems.

Are tortoises important seed dispersers in Amazonian forests?

According to most studies on seed dispersal in tropical forests, mammals and birds are considered the main dispersal agents and the role played by other animal groups remains poorly explored. We investigate qualitative and quantitative components of the role played by the tortoise Chelonoidis denticulata in seed dispersal in southeastern Amazon, and the influence of seasonal variation in tortoise movement patterns on resulting seed shadows. Seed shadows produced by this tortoise were estimated by combining information on seed passage times through their digestive tract, which varied from 3 to 17 days, with a robust dataset on movements obtained from 18 adult C. denticulata monitored with radio transmitters and spoon-and-line tracking devices. A total of 4,206 seeds were found in 94 collected feces, belonging to 50 seed morphotypes of, at least, 25 plant genera. Very low rates of damage to the external structure of the ingested seeds were observed. Additionally, results of germination trials suggested that passage of seeds through C. denticulata`s digestive tract does not seem to negatively affect seed germination. The estimated seed shadows are likely to contribute significantly to the dispersal of seeds away from parent plants. During the dry season seeds were dispersed, on average, 174.1 m away from the location of fruit ingestion; during the rainy season, this mean dispersal distance increased to 276.7 m. Our results suggest that C. denticulata plays an important role in seed dispersal in Amazonian forests and highlight the influence of seasonal changes in movements on the resulting seed shadows.

A Plant Needs Ants like a Dog Needs Fleas: Myrmelachista schumanni Ants Gall Many Tree Species to Create Housing

Hundreds of tropical plant species house ant colonies in specialized chambers called domatia. When, in 1873, Richard Spruce likened plant-ants to fleas and asserted that domatia are ant-created galls, he incited a debate that lasted almost a century. Although we now know that domatia are not galls and that most ant-plant interactions are mutualisms and not parasitisms, we revisit Spruce`s suggestion that ants can gall in light of our observations of the plant-ant Myrmelachista schumanni, which creates clearings in the Amazonian rain forest called ""supay-chakras,"" or ""devil`s gardens."" We observed swollen scars on the trunks of nonmyrmecophytic canopy trees surrounding supay-chakras, and within these swellings, we found networks of cavities inhabited by M. schumanni. Here, we summarize the evidence supporting the hypothesis that M. schumanni ants make these galls, and we hypothesize that the adaptive benefit of galling is to increase the amount of nesting space available to M. schumanni colonies.

Atrophy and Neuron Loss: Effects of a Protein-Deficient Diet on Sympathetic Neurons

Protein deficiency is one of the biggest public health problems in the world, accounting for about 30-40% of hospital admissions in developing countries. Nutritional deficiencies lead to alterations in the peripheral nervous system and in the digestive system. Most studies have focused on the effects of protein-deficient diets on the enteric neurons, but not on sympathetic ganglia, which supply extrinsic sympathetic input to the digestive system. Hence, in this study, we investigated whether a protein-restricted diet would affect the quantitative structure of rat coeliac ganglion neurons. Five male Wistar rats (undernourished group) were given a pre- and postnatal hypoproteinic diet receiving 5% casein, whereas the nourished group (n = 5) was fed with 20% casein (normoproteinic diet). Blood tests were carried out on the animals, e.g., glucose, leptin, and triglyceride plasma concentrations. The main structural findings in this study were that a protein-deficient diet (5% casein) caused coeliac ganglion (78%) and coeliac ganglion neurons (24%) to atrophy and led to neuron loss (63%). Therefore, the fall in the total number of coeliac ganglion neurons in protein-restricted rats contrasts strongly with no neuron losses previously described for the enteric neurons of animals subjected to similar protein-restriction diets. Discrepancies between our figures and the data for enteric neurons (using very similar protein-restriction protocols) may be attributable to the counting method used. In light of this, further systematic investigations comparing 2-D and 3-D quantitative methods are warranted to provide even more advanced data on the effects that a protein-deficient diet may exert on sympathetic neurons. (C) 2009 Wiley-Liss, Inc.

Effects of Ischemia and Reperfusion on P2X(2) Receptor Expressing Neurons of the Rat Ileum Enteric Nervous System

Purpose We investigated the effects of ischemia/reperfusion in the intestine (I/R-i) on purine receptor P2X(2)-immunoreactive (IR) neurons of the rat ileum. Methods The superior mesenteric artery was occluded for 45 min with an atraumatic vascular clamp and animals were sacrificed 4 h later. Neurons of the myenteric and submucosal plexuses were evaluated for immunoreactivity against the P2X(2) receptor, nitric oxide synthase (NOS), choline acetyl transferase (ChAT), calbindin, and calretinin. Results Following I/R-i, we observed a decrease in P2X(2) receptor immunoreactivity in the cytoplasm and surface membranes of neurons of the myenteric and submucosal plexuses. These studies also revealed an absence of calbindin-positive neurons in the I/R-i group. In addition, the colocalization of the P2X(2) receptor with NOS, ChAT, and calretinin immunoreactivity in the myenteric plexus was decreased following I/R-i. Likewise, the colocalization between P2X(2) and calretinin in neurons of the submucosal plexus was also reduced. In the I/R-i group, there was a 55.8% decrease in the density of neurons immunoreactive (IR) for the P2X(2) receptor, a 26.4% reduction in NOS-IR neuron, a 25% reduction in ChAT-IR neuron, and a 47% reduction in calretinin-IR neuron. The density of P2X(2) receptor and calretinin-IR neurons also decreased in the submucosal plexus of the I/R-i group. In the myenteric plexus, P2X(2)-IR, NOS-IR, ChAT-IR and calretinin-IR neurons were reduced in size by 50%, 49.7%, 42%, and 33%, respectively, in the I/R-i group; in the submucosal plexus, P2X(2)-IR and calretinin-IR neurons were reduced in size by 56% and 72.6%, respectively. Conclusions These data demonstrate that ischemia/reperfusion of the intestine affects the expression of the P2X(2) receptor in neurons of the myenteric and submucosal plexus, as well as density and size of neurons in this population. Our findings indicate that I/R-i induces changes in P2X(2)-IR enteric neurons that could result in alterations in intestinal motility.

Early weaning accelerates the differentiation of mucous neck cells in rat gastric mucosa: Possible role of TGF alpha/EGFR

The development of the gastric mucosa is controlled by hormones, growth factors and feeding behavior. Early weaning (EW), which means the abrupt interruption of suckling, increases proliferation and differentiation in the rat gastric epithelium. Transforming growth factor alpha(TGF alpha) is secreted in the stomach, binds to the epidermal growth factor receptor( EGFR) and may control cell proliferation, differentiation and migration. Here, we investigated the influence of suckling-weaning transition on the differentiation of mucous neck cells in the stomach and its association to the expression of TGF alpha and EGFR. Fifteen-day-old Wistar rats were divided into two groups: suckling( control), in which pups were kept with the dam, and early weaning( EW), in which rats were separated from their mother and fed with hydrated powdered chow. TGF alpha and EGFR levels were increased at 18 days in EW animals compared to control ones (p<0.05). Histochemical reactions with Periodic Acid-Schiff reagent+Alcian Blue or Bandeiraea simplicifolia II lectin were used to stain the mucous neck cells and showed an increase in this cell population throughout EW, which was more pronounced at 17 days when compared to suckling pups (p<0.05). These morphological results were confirmed by RT-PCR for mucin 6. The levels of mucin 6 mRNA were higher in EW animals from the 16th to the 18th day(1-3 days post-weaning) when compared to the respective control group. Inhibition of EGFR through AG1478 administration to EW animals prevented the expansion of mucous neck cell population induced by EW (p<0.05). Therefore, early weaning up regulated TGF alpha/EGFR expression and induced differentiation of mucous neck cells. Moreover, we showed that EGFR takes part in the maturation of this cell population. We conclude that regular suckling-weaning transition is crucial to guarantee the development of the gastric mucosa. (C) 2009 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.

A novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombin coagulant inhibitory activity

Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. In this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI (A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity with insect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptase inhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized the recombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant (K(i)) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. The rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquito`s development. (C) 2010 Elsevier Masson SAS. All rights reserved.