Research progress on gut health of farmers teleost fish: a viewpoint concerning the intestinal mucosal barrier and the impact of its damage

Reviews in Fish Biology and Fisheries - The intestinal mucosal barrier plays a critical role in the maintenance of host health. In farmed teleost fish, the intestinal epithelium is challenged by a...     

Epithelium-associated bacteria in the gastrointestinal tract of Arctic charr (Salvelinus alpinus L.). An electron microscopical study

AIMS: The primary aim was to use transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to define the location of epithelium-associated bacteria in the digestive tract of the salmonid fish, Arctic charr (Salvelinus alpinus). METHODS AND RESULTS: TEM and SEM examination of the gastrointestinal tract demonstrated substantial numbers of ovoid and rod-shaped bacterial cells associated with the microvillous brush borders of enterocytes. Bacteria were found at the tips of microvilli as well as between adjacent microvilli. Endocytosis of bacteria by epithelial cells was observed in two regions (pyloric caeca and midgut). CONCLUSION: Electron microscope examination of the gut is an important tool for evaluating the microbial ecology of the fish digestive tract ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the current study clearly demonstrate that the intestine is involved in bacterial endocytosis.     

Membrane binding of SRP pathway components in the halophilic archaea Haloferax volcanii.

Across evolution, the signal recognition particle pathway targets extra-cytoplasmic proteins to membranous translocation sites. Whereas the pathway has been extensively studied in Eukarya and Bacteria, little is known of this system in Archaea. In the following, membrane association of FtsY, the prokaryal signal recognition particle receptor, and SRP54, a central component of the signal recognition particle, was addressed in the halophilic archaea Haloferax volcanii. Purified H. volcanii FtsY, the FtsY C-terminal GTP-binding domain (NG domain) or SRP54, were combined separately or in different combinations with H. volcanii inverted membrane vesicles and examined by gradient floatation to differentiate between soluble and membrane-bound protein. Such studies revealed that both FtsY and the FtsY NG domain bound to H. volcanii vesicles in a manner unaffected by proteolytic pretreatment of the membranes, implying that in Archaea, FtsY association is mediated through the membrane lipids. Indeed, membrane association of FtsY was also detected in intact H. volcanii cells. The contribution of the NG domain to FtsY binding in halophilic archaea may be considerable, given the low number of basic charges found at the start of the N-terminal acidic domain of haloarchaeal FtsY proteins (the region of the protein thought to mediate FtsY-membrane association in Bacteria). Moreover, FtsY, but not the NG domain, was shown to mediate membrane association of H. volcanii SRP54, a protein that did not otherwise interact with the membrane.     

Flight behaviour of Cadra cautella males in rapidly pulsed pheromone plumes

Abstract Airborne pheromone plumes in wind comprise filaments of odour interspersed with gaps of clean air. When flying moths intercept a filament, they have a tendency to surge upwind momentarily, and then fly crosswind until another filament is intercepted. Thus, the moment-to-moment contact with pheromone mediates the shape of a flight track along the plume. Within some range of favourable interception rates, flight tracks become straighter and are headed more due upwind. However, as the rate of interception increases, there comes a point at which the moth should not be able to discern discreet filaments but, rather, should perceive a 'fused signal'. At the extreme, homogeneous clouds of pheromone inhibit upwind progress by representative tortricids. In a wind tunnel, Cadra cautella (Walker) (Lepidoptera: Pyralidae) were presented with 10 ms pulses of pheromone at a repetition rate of 5, 10, 17 and 25/s and a continuous, internally turbulent plume. Pulse size and concentrations were verified with a miniature photoionization detector sampling surrogate odour, propylene, at 100 Hz. Male moths maintain upwind progress even at plumes of 25 filaments/s. Furthermore, moths exhibited greater velocities and headings more due upwind at 17 and 25 Hz than at the lower frequencies or with the continuous plume. It is hypothesized that either C. cautella possesses a versatile sensory system that allows the resolution of these rapidly pulsed pheromone plumes, or that this species does not require a 'flickering' signal to fly upwind.     

Isoprenoids Are Essential for Fruiting Body Formation in Myxococcus xanthus

It was recently shown that Myxococcus xanthus harbors an alternative and reversible biosynthetic pathway to isovaleryl coenzyme A (CoA) branching from 3-hydroxy-3-methylglutaryl-CoA. Analyses of various mutants in these pathways for fatty acid profiles and fruiting body formation revealed for the first time the importance of isoprenoids for myxobacterial development.Myxobacteria are unique among the prokaryotes as (i) they can form highly complex fruiting bodies under starvation conditions, even up to microscopic tree-like structures (28); (ii) they can move on solid surfaces using different motility mechanisms (16); (iii) they produce some of the most cytotoxic secondary metabolites, with epothilone already in clinical use against cancer (2, 3); and (iv) they harbor the largest prokaryotic genomes found so far (15, 27). The large genome might be directly related to their complex life-style and the diverse secondary (3) and primary (9) metabolisms. Already in 2002 we found that myxobacteria are able to produce isovaleryl coenzyme A (IV-CoA) and compounds derived thereof via a new pathway that branches from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is the central intermediate of the well-known mevalonate-dependent isoprenoid biosynthesis (Fig. ​(Fig.1)1) (22, 23). Usually IV-CoA is derived from leucine degradation via the branched-chain keto acid dehydrogenase (BKD) complex (24), which is also the preferred pathway to IV-CoA in the myxobacteria Myxococcus xanthus and Stigmatella aurantiaca (Fig. ​(Fig.2A).2A). However, in bkd mutants, where no or only residual leucine degradation is possible (30), the alternative pathway is induced (Fig. ​(Fig.2B),2B), presumably to ensure the production of iso-fatty acids (iso-FAs) (5). A possible reason for this alternative pathway is the importance of IV-CoA-derived compounds in the complex myxobacterial life cycle, which is the starvation-induced formation of fruiting bodies in which the cells differentiate into myxospores. We showed that this pathway is induced during fruiting body formation in M. xanthus when leucine is limited. Under these conditions, this pathway might be more important for protein synthesis than for lipid remodeling, as lipids are present in excess during development due to the surface reduction from vegetative rods to round myxospores as described previously (29). Examples of IV-CoA-derived compounds are the unusual iso-branched ether lipids, which are almost exclusively produced in the developing myxospores. They might serve as structural lipids and signaling compounds during fruiting body formation (26).Open in a separate windowFIG. 1.Biosynthesis of IV-CoA and compounds derived thereof and biosynthesis of isoprenoids in M. xanthus. Broken arrows indicate multistep reactions; supplementation (double-lined arrows) with MVL and IVA can be used to complement selected mutants.Open in a separate windowFIG. 2.Short representations of proposed metabolic fluxes through the IV-CoA/isoprenoid network. Broken arrows indicate no metabolic flux. (A) DK1622 (wild type); (B) DK5643 (Δbkd); (C) DK5624 (Δbkd mvaS::kan); (D) HB002 (Δbkd liuC::kan); (E) HB002 with 1 mM IVA; (F) HB002 with 1 mM MVL. Ac-CoA, acetyl-CoA; MVA, mevalonic acid.In M. xanthus, we could recently identify candidate genes involved in the alternative pathway from HMG-CoA to IV-CoA. We also described the genes required for the degradation pathway of leucine and subsequently also those involved in the transformation of IV-CoA to HMG-CoA (4). In myxobacteria leucine is an important precursor for isoprenoid biosynthesis, as was already shown elsewhere for the biosynthesis of steroids (7) and prenylated secondary metabolites like aurachin (22) or leupyrrins (6), as well as volatiles like geosmin or germacradienol in M. xanthus and S. aurantiaca (11, 13). The interconnection of iso-FAs and isoprenoid biosynthesis made it difficult to assign functions to these compound classes during fruiting body formation in M. xanthus because it cannot be excluded that reduced leucine degradation also impairs isoprenoid biosynthesis. A mutant strain of M. xanthus that was blocked in the degradation of leucine and the alternative pathway had a deletion in the bkd locus as well as a plasmid insertion in the mvaS gene encoding the HMG-CoA synthase (strain DK5624). This double mutation severely affected isoprenoid biosynthesis (5), and cultures of DK5624 must be supplemented with mevalonolactone (MVL; the cyclized form of mevalonic acid) in order to enable growth (Fig. ​(Fig.2C).2C). Since we have identified the genes involved in IV-CoA biosynthesis and the mevalonate pathway (4), we can now start to identify differences between strains that show deficiencies in iso-FAs and strains that show deficiencies in isoprenoids via simple analysis of the FA profile and analysis of the myxobacterial development of selected mutants.All mutants used in this study (HB002 [Δbkd liuC::kan], HB015 [Δbkd MXAN_4265::kan], DK5624 [Δbkd mvaS::kan], HB019 [Δbkd mvaS::kan mvaS⁺], and HB020 [Δbkd MXAN_4265::kan mvaS⁺]) have been published previously (4), and FA analysis as well as myxobacterial fruiting body formation has also been described previously (26).M. xanthus HB002 (Δbkd liuC) shows only residual amounts of iso-FAs, as both leucine degradation and the alternative pathway to IV-CoA are blocked (Fig. ​(Fig.2D)2D) and its capability to form fruiting bodies is strongly reduced (Fig. ​(Fig.3).3). The residual amount of iso-FAs results from a second BKD activity in M. xanthus that has been identified by residual leucine incorporation as well as by residual enzymatic activity in bkd mutants (23, 30). This second BKD activity might be a side activity of the pyruvate dehydrogenase or a related chemical oxidative decarboxylation, as no second bkd locus could be identified in the genome (unpublished results). Moreover, growth of HB002 is not MVL dependent because the block in the alternative pathway does not affect isoprenoid biosynthesis, as liuC encodes a dehydratase/hydratase that is involved in the conversion of HMG-CoA to 3-methylglutaconyl-CoA and vice versa (4). As expected, the FA profile (4) as well as the developmental phenotype (data not shown) can be complemented (Fig. ​(Fig.2E)2E) by the addition of isovaleric acid (IVA), the free acid of IV-CoA, indicating the importance of iso-branched compounds for development in M. xanthus. Unexpectedly, addition of MVL (Fig. ​(Fig.2F)2F) also partially restored fruiting body formation without restoring the FA profile (Fig. ​(Fig.3).3). Similarly, M. xanthus HB015 (Δbkd MXAN_4265::kan) can produce only traces of iso-FAs, as both pathways to IV-CoA are blocked. MXAN_4265 encodes a protein with similarity to a glutaconyl-CoA transferase subunit, but from our previous results, we postulated it to be involved in the alternative pathway to IV-CoA (Fig. ​(Fig.1)1) (4). The respective mutant shows a severely impaired developmental phenotype, which can be complemented not only by the addition of IVA (not shown) but also by the addition of MVL (Fig. ​(Fig.3).3). Again, no change in the FA profile was observed after the addition of MVL. However, a plasmid insertion into MXAN_4265 has a polar effect on mvaS, which is the last gene in this five-gene operon and which is crucial for HMG-CoA formation from acetoacetyl-CoA and acetyl-CoA. Therefore, we assume that both pathways to HMG-CoA are blocked in HB015: no HMG-CoA can be made from acetyl-CoA and hardly any can be made via leucine degradation. In order to prove this hypothesis, we complemented HB015 with an additional copy of mvaS under the constitutive T7A1 promoter as described previously, using the plasmid pCK4267exp (4). The resulting strain, HB020 (Δbkd MXAN_4265::kan mvaS⁺), showed a restored developmental phenotype but still produced only trace amounts of iso-FAs.Open in a separate windowFIG. 3.Fruiting body formation on TPM agar in selected mutants at 24, 48, and 72 h after starvation. Numbers refer to the relative amounts (in percentages) of the most abundant iso-FA, iso-15:0, which is indicative of iso-FAs in general. Strains were DK1622 (wild type), HB002 (Δbkd liuC::kan), HB015 (Δbkd MXAN_4265::kan), DK5624 (Δbkd mvaS::kan), HB019 (Δbkd mvaS::kan mvaS⁺), and HB020 (Δbkd MXAN_4265::kan mvaS⁺). DK5624 was grown with 0.3 mM MVL prior to starvation, and the cells were washed and plated on TPM with or without 1 mM of MVL.The data from HB002, HB015, and HB020 indicate an important function of the mevalonate-dependent isoprenoid pathway for fruiting body formation in M. xanthus. Therefore, MVL addition can at least partially complement the developmental phenotype of DK5624, which cannot form fruiting bodies without MVL (Fig. ​(Fig.3).3). However, genetic complementation with mvaS in HB019 resulted in the expected complementation of the fruiting body formation and the FA profile (Fig. ​(Fig.3,3, bottom row).Leucine is one of the most abundant proteinogenic amino acids. It is also an essential amino acid for M. xanthus (8), which has a predatory life-style (1), as it lives on other bacteria and fungi that contain a lot of leucine. Moreover, leucine is very efficiently incorporated into isoprenoids like geosmin and aurachin (10, 22). Thus, one can conclude that in fact leucine degradation is the major pathway for HMG-CoA biosynthesis instead of the usual formation via acetoacetyl-CoA and acetyl-CoA by the HMG-CoA synthase MvaS as indicated in Fig. ​Fig.2A.2A. No difference in growth was observed between culture with and culture without MVL for HB002 (Δbkd liuC::kan) and HB015 (Δbkd MXAN_4265::kan) in rich medium (data not shown), probably due to the complete MvaS activity (in HB002) or residual BKD activity (in HB002 and HB015), resulting in all precursors for the mevalonate-dependent isoprenoid biosynthesis still being present in excess under these conditions. However, under starvation conditions a small reduction in HMG-CoA biosynthesis caused by completely blocked leucine degradation (as in HB002 due to the mutation in liuC [Fig. ​[Fig.2D])2D]) or reduced leucine degradation and a mutation in mvaS (as in HB015) might each result in a reduced isoprenoid level, which can be complemented at least partially by the addition of MVL. This would also explain the difference in the developmental phenotypes of HB002 and HB015, with the phenotype being more severe in HB002 (Fig. ​(Fig.3).3). The fact that complementation with IVA is in all cases more efficient than that with MVL can be explained by the role of the already-mentioned isolipids. They can be produced only after IVA addition, which also complements the (developmental) phenotype of some of these mutants (26).As isoprenoids represent probably the most diverse class of natural products (14), it is very hard to predict which particular isoprenoids might be responsible for the observed effects. Several isoprenoids (7, 11-13), prenylated secondary metabolites (6, 22), and carotenoids (18-21) are known from myxobacteria in general, and a major volatile compound from M. xanthus is the terpenoid geosmin (13). In order to test whether geosmin might be required for fruiting body formation, we constructed a plasmid insertion mutant in MXAN_6247, which is involved in the cyclization of farnesyl diphosphate to geosmin, following published procedures (4, 5). The resulting strain, HB022, showed the expected loss in geosmin production but no developmental phenotype (data not shown).Additionally, it cannot be excluded that prenylated proteins, sugars, or quinones from the respiratory chain are important for fruiting body formation. Moreover, stigmolone has been described as a pheromone involved in fruiting body formation in S. aurantiaca (25). Although its biosynthesis has not been elucidated yet, stigmolone could be an isoprenoid as well, which is deducible from the two iso-branched residues within its chemical structure (17). Nevertheless, the importance of isoprenoids for M. xanthus is evident from the data presented, and clearly more work is needed to identify the compound(s) involved.     

Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.      

EFFECT OF VITAMIN E SUPPLEMENTATION AND PARTIAL SUBSTITUTION OF POLY- WITH MONO-UNSATURATED FATTY ACIDS IN PIG DIETS ON MUSCLE,AND MICROSOME EXTRACT a-TOCOPHEROL CONCENTRATION AND LIPID OXIDATION

The experiment was organized in a 3×2 factorial arrangement with three dietary fat blends and a basal (20 mg kg^?1 diet) or supplemented (220 mg kg^?1) level of α-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle α-tocopherol concentration (α-tocopherol [log μg g^?1]=0.18 (±0.105)+0.0034 (±0.0003)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.39 (±0.122)·dietary MUFA/PUFA (P<0.0036)). An interaction between dietary α-tocopherol and dietary MUFA/PUFA exists for microsome α-tocopherol concentration (α-tocopherol [log μg g^?1]=1.14 (±0.169) (P<0.0001)+0.0056 (±0.00099)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.54 (±0.206)·dietary MUFA/PUFA (P<0.0131)?0.0033 (±0.0011)·dietary α-tocopherol [mg kg^?1)]×dietary MUFA/PUFA (P<0.0067)), and hexanal concentration in meat (hexanal [ng·g^?1]=14807.9 (±1489.8)?28.8 (±10.6) dietary α-tocopherol [mg·kg^?1] (P<0.01)?8436.6 (±1701.6)·dietary MUFA/PUFA (P<0.001)+24.0 (±11.22)·dietary α-tocopherol·dietary MUFA/PUFA (P<0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of α-tocopherol in muscle and microsome extracts. An interaction between dietary α-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of α-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values.     

Differences in mortality from acute myocardial infarction between coronary care unit and medical ward: treatment or bias?

To analyse the effectiveness of coronary care units in reducing mortality from myocardial infarction 18 hospitals ranging from large urban teaching hospitals to small country hospitals were stratified into four levels of care. Previous analysis had failed to show significant differences in the overall mortality in hospital among levels. There were significant differences in mortality, however, between those patients allocated to be cared for in the coronary care unit and those in the medical wards in the more advanced hospitals. The differences were largest in the hospitals with the most elaborate facilities (level 1) and non-existent in those with the least (level 4). Several analytical approaches to these observed differences indicated that they were: (a) reduced by adjustment for age and severity of infarction; (b) paralleled by differences in coexisting disease recorded on death certificates; (c) no longer significant at level 1 after allowing for differences in coexisting disease; and (d) not significant at any level after exclusion of patients first diagnosed at necropsy. These findings suggest that the observed differences in mortality between coronary care units and medical wards are largely due to bias in selection and diagnosis.     

Changes in electrical thresholds for evoking movements from the cat cerebral cortex following lesions of the sensori-motor area

We evaluated motor maps in the cerebral cortex and motor performance in cats before and after lesions of the forelimb representation in the primary motor area. After the lesion there was a reduction in the use of the affected forelimb and loss of accuracy in prehension tasks using the forelimb; some recovery occurred during the mapping study. Electrode tracts and lesion sites were located in cytoarchitectonically identified cortical areas 4gamma, 4delta, 6aalpha, 6agamma, 3a. The lesions were mainly in area 4gamma. In the lesioned hemisphere there were many points around the lesion site (in areas 4gamma and 3a) from which movements could not be evoked. In some areas distant from the lesion site (e.g. area 6agamma) the mean thresholds for evoking forelimb movements were significantly elevated. Mean thresholds for evoking hindlimb and facial movements were not different from before. In the contralateral hemisphere mean thresholds for evoking forelimb, but not hindlimb or facial movements, were significantly elevated in several sensorimotor areas (area 4gamma, 6agamma and 3a). Mean thresholds for evoking forelimb movements appeared to progressively increase during the time of study. Minimal currents required to evoke forelimb movements from the cerebral cortex increase (possibly progressively) following a lesion of the forelimb representation in the primary motor area, affecting many interconnected motor areas in the hemispheres ipsilateral and contralateral to the lesioned site. This increase in thresholds may play a role in the changes in cortical control of the affected and contralateral limbs following brain lesions and explain the increased sense of effort required to produce movements.     

Quirks of dye nomenclature. 2. Congo red

The history, origin, identity, chemistry and uses of Congo red are described. Originally patented in 1884, Congo red soon found applications in dyeing cotton, as a pH indicator for chemists and as a biological stain. Unlike the majority of the 19th century synthetic dyes, it still is available commercially.