Evaluation of the inclusion of a mixture of organic acids or lactulose into the feed of pigs experimentally challenged with Salmonella Typhimurium

A mixture of organic acids and lactulose for preventing or reducing colonization of the gut by Salmonella Typhimurium was evaluated in pigs. A total of 63 4-week-old commercial piglets were randomly distributed into three different experimental dietary groups: a plain diet without additives (PD) and the same diet supplemented with either 0.4% (w/v) formic acid and 0.4% lactic acid (w/v) (AC) or 1% (w/v) lactulose (LC). After 7 days of adaptation, two-thirds of the pigs (14 from each diet) were challenged with a 2-mL oral dose of 10(8) CFU/mL of Salmonella Typhimurium, leaving the remaining animals unchallenged (UC). After 4 and 10 days post-challenge, pigs were euthanized and the ileum and caecum content were aseptically sampled to (a) quantify lactic, formic, and short-chain fatty acids (SCFA), (b) quantify bacterial populations and Salmonella by fluorescence in situ hybridization and (c) qualitatively analyse bacterial populations through denaturing gradient gel electrophoresis (DGGE). Modification of fermentation products and counts of some of the bacterial groups analysed in the challenged pigs receiving the treatments AC and LC were minimal. Treatments only influenced the bacterial diversity after 10 days post-challenge, with AC generating a lower number of DGGE bands than UC(P < 0.05). Neither the inclusion of a mixture of 0.4% (w/v) formic and 0.4% (w/v) lactic acids nor of 1% (w/v) lactulose in the feed influenced numbers of Salmonella in the ileum and caecum of experimentally challenged pigs. (C) 2009 Elsevier B.V. All rights reserved.

Synergistic effect of BSA on antioxidant activities in model food emulsions

Sunflower oil-in-water emulsions containing TBHQ, caffeic acid, epigallocatechin gallate (EGCG), or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), both with and without BSA, were stored at 50 and 30degreesC. Oxidation of the oil was monitored by determination of the PV, conjugated diene content, and hexanal formation. Emulsions containing EGCG, caffeic acid, and, to a lesser extent, Trolox were much more stable during storage in the presence of BSA than in its absence even though BSA itself did not provide an antioxidant effect. BSA did not have a synergistic effect on the antioxidant activity of TBHQ. The BSA structure changed, with a considerable loss of fluorescent tryptophan groups during storage of solutions containing BSA and antioxidants, and a BSA-antioxidant adduct with radical-scavenging activity was formed. The highest radical-scavenging activity observed was for the isolated protein from a sample containing EGCG and BSA incubated at 30degreesC for 10 d. This fraction contained unchanged BSA as well as BSA-antioxidant adduct, but 95.7% of the initial fluorescence had been lost, showing that most of the BSA had been altered. It can be concluded that BSA exerts its synergistic effect with antioxidants because of formation of a protein-antioxidant adduct during storage, which is concentrated at the oil-water interface owing to the surface-active nature of the protein.

Evidence that GABA rho subunits contribute to functional ionotropic GABA receptors in mouse cerebellar Purkinje cells

Ionotropic gamma-amino butyric acid (GABA) receptors composed of heterogeneous molecular subunits are major mediators of inhibitory responses in the adult CNS. Here, we describe a novel ionotropic GABA receptor in mouse cerebellar Purkinje cells (PCs) using agents reported to have increased affinity for rho subunit-containing GABA(C) over other GABA receptors. Exogenous application of the GABA(C)-preferring agonist cis-4-aminocrotonic acid (CACA) evoked whole-cell currents in PCs, whilst equimolar concentrations of GABA evoked larger currents. CACA-evoked currents had a greater sensitivity to the selective GABA(C) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) than GABA-evoked currents. Focal application of agonists produced a differential response profile; CACA-evoked currents displayed a much more pronounced attenuation with increasing distance from the PC soma, displayed a slower time-to-peak and exhibited less desensitization than GABA-evoked currents. However, CACA-evoked currents were also completely blocked by bicuculline, a selective agent for GABA(A) receptors. Thus, we describe a population of ionotropic GABA receptors with a mixed GABA(A)/GABA(C) pharmacology. TPMPA reduced inhibitory synaptic transmission at interneurone-Purkinje cell (IN-PC) synapses, causing clear reductions in miniature inhibitory postsynaptic current (mIPSC) amplitude and frequency. Combined application of NO-711 (a selective GABA transporter subtype 1 (GAT-1) antagonist) and SNAP-5114 (a GAT-(2)/3/4 antagonist) induced a tonic GABA conductance in PCs; however, TPMPA had no effect on this current. Immunohistochemical studies suggest that rho subunits are expressed predominantly in PC soma and proximal dendritic compartments with a lower level of expression in more distal dendrites; this selective immunoreactivity contrasted with a more uniform distribution of GABA(A) alpha 1 subunits in PCs. Finally, co-immunoprecipitation studies suggest that rho subunits can form complexes with GABA(A) receptor alpha 1 subunits in the cerebellar cortex. Overall, these data suggest that rho subunits contribute to functional ionotropic receptors that mediate a component of phasic inhibitory GABAergic transmission at IN-PC synapses in the cerebellum.

Investigation of liquid MALDI and optimization for instrument tuning and quantitative measurements

The success of Matrix-assisted laser desorption / ionisation (MALDI) in fields such as proteomics has partially but not exclusively been due to the development of improved data acquisition and sample preparation techniques. This has been required to overcome some of the short comings of the commonly used solid-state MALDI matrices such as - cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). Solid state matrices form crystalline samples with highly inhomogeneous topography and morphology which results in large fluctuations in analyte signal intensity from spot to spot and positions within the spot. This means that efficient tuning of the mass spectrometer can be impeded and the use of MALDI MS for quantitative measurements is severely impeded. Recently new MALDI liquid matrices have been introduced which promise to be an effective alternative to crystalline matrices. Generally the liquid matrices comprise either ionic liquid matrices (ILMs) or a usually viscous liquid matrix which is doped with a UV lightabsorbing chromophore [1-3]. The advantages are that the droplet surface is smooth and relatively uniform with the analyte homogeneously distributed within. They have the ability to replenish a sampling position between shots negating the need to search for sample hot-spots. Also the liquid nature of the matrix allows for the use of additional additives to change the environment to which the analyte is added.

Influence of end-capping on the self-assembly of model amyloid peptide fragments

The influence of charge and aromatic stacking interactions on the self-assembly of a series of four model amyloid peptides has been examined. The four model peptides are based on the KLVFF motif from the amyloid Beta peptide, ABeta(16-20) extended at the N terminus with two Beta-alanine residues. We have studied NH2-BetaABetaAKLVFF-COOH (FF), NH2-BetaABetaAKLVFCOOH (F), CH3CONH-BetaABetaAKLVFF-CONH2 (CapF), and CH3CONH-BetaABetaAKLVFFCONH2 (CapFF). The former two are uncapped (net charge plus 2) and differ by one hydrophobic phenylalanine residue; the latter two are the analogous capped peptides (net charge plus 1). The self-assembly characteristics of these peptides are remarkably different and strongly dependent on concentration. NMR shows a shift from carboxylate to carboxylic acid forms upon increasing concentration. Saturation transfer measurements of solvent molecules indicate selective involvement of phenylalanine residues in driving the self-assembly process of CapFF due presumably to the effect of aromatic stacking interactions. FTIR spectroscopy reveals beta-sheet features for the two peptides containing two phenylalanine residues but not the single phenylalanine residue, pointing again to the driving force for self-assembly. Circular dichroism (CD) in dilute solution reveals the polyproline II conformation, except for F which is disordered. We discuss the relationship of this observation to the significant pH shift observed for this peptide when compared the calculated value. Atomic force microscopy and cryogenic-TEM reveals the formation of twisted fibrils for CapFF, as previously also observed for FF. The influence of salt on the self-assembly of the model beta-sheet forming capped peptide CapFF was investigated by FTIR. Cryo-TEM reveals that the extent of twisting decreases with increased salt concentration, leading to the formation of flat ribbon structures. These results highlight the important role of aggregation-induced pKa shifts in the self-assembly of model beta-sheet peptides.

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes, A series of 10 C5-modified analogues of 2'-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality, For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties, The imidazole function was conjugated to these CS-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid), The substrate properties of the nucleotides (fully replacing dTTP) with Taq polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments, 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates, In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates, The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XbaI and by mass spectrometry of the PCR products.

Gut microbial catabolism of grape seed flavan-3-ols by human faecal microbiota. Targetted analysis of precursor compounds, intermediate metabolites and end-products.

In vitro batch culture fermentations were conducted with grape seed polyphenols and human faecal microbiota, in order to monitor both changes in precursor flavan-3-ols and the formation of microbial-derived metabolites. By the application of UPLC-DAD-ESI-TQ MS, monomers, and dimeric and trimeric procyanidins were shown to be degraded during the first 10 h of fermentation, with notable inter-individual differences being observed between fermentations. This period (10 h) also coincided with the maximum formation of intermediate metabolites, such as 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone and 4-hydroxy-5-(3′,4′-dihydroxyphenyl)-valeric acid, and of several phenolic acids, including 3-(3,4-dihydroxyphenyl)-propionic acid, 3,4-dihydroxyphenylacetic acid, 4-hydroxymandelic acid, and gallic acid (5–10 h maximum formation). Later phases of the incubations (10–48 h) were characterised by the appearance of mono- and non-hydroxylated forms of previous metabolites by dehydroxylation reactions. Of particular interest was the detection of γ-valerolactone, which was seen for the first time as a metabolite from the microbial catabolism of flavan-3-ols. Changes registered during fermentation were finally summarised by a principal component analysis (PCA). Results revealed that 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone was a key metabolite in explaining inter-individual differences and delineating the rate and extent of the microbial catabolism of flavan-3-ols, which could finally affect absorption and bioactivity of these compounds.

In vitro fermentation of a red wine extract by human gut microbiota: changes in microbial groups and formation of phenolic metabolites

An in vitro batch culture fermentation experiment was conducted with fecal inocula from three healthy volunteers in the presence and absence of a red wine extract. Changes in main bacterial groups were determined by FISH during a 48 h fermentation period. The catabolism of main flavonoids (i.e., flavan-3-ols and anthocyanins) and the formation of a wide a range of phenolic microbial metabolites were determined by a targeted UPLC-PAD-ESI-TQ MS method. Statistical analysis revealed that catechol/pyrocatechol, as well as 4-hydroxy-5-(phenyl)-valeric, 3- and 4-hydroxyphenylacetic, phenylacetic, phenylpropionic, and benzoic acids, showed the greatest increases in concentration during fermentation, whereas 5-(3′-hydroxyphenyl)-γ-valerolactone, its open form 4-hydroxy-5-(3′-hydroxyphenyl)-valeric acid, and 3,4-dihydroxyphenylacetic acid represented the largest interindividual variations in the catabolism of red wine polyphenols. Despite these changes, microbial catabolism did not produce significant changes in the main bacterial groups detected, although a slight inhibition of the Clostridium histolyticum group was observed.

Sample preparation: a crucial factor for the analytical performance of rationally designed MALDI matrices

Evidence is presented that the performance of the rationally designed MALDI matrix 4-chloro-α-cyanocinnamic acid (ClCCA) in comparison to its well-established predecessor α-cyano-4-hydroxycinnamic acid (CHCA) is significantly dependent on the sample preparation, such as the choice of the target plate. In this context, it becomes clear that any rational designs of MALDI matrices and their successful employment have to consider a larger set of physicochemical parameters, including sample crystallization and morphology/topology, in addition to parameters of basic (solution and/or gas-phase) chemistry.

Coupling liquid MALDI MS to liquid chromatography

Matrix-assisted laser desorption/ionisation (MALDI) coupled with time-of-flight (TOF) mass spectrometry (MS) is a powerful tool for the analysis of biological samples, and nanoflow high-performance liquid chromatography (nanoHPLC) is a useful separation technique for the analysis of complex proteomics samples. The off-line combination of MALDI and nanoHPLC has been extensively investigated and straightforward techniques have been developed, focussing particularly on automated MALDI sample preparation that yields sensitive and reproducible spectra. Normally conventional solid MALDI matrices such as α-cyano-4-hydroxycinnamic acid (CHCA) are used for sample preparation. However, they have limited usefulness in quantitative measurements and automated data acquisition because of the formation of heterogeneous crystals, resulting in highly variable ion yields and desorption/ ionization characteristics. Glycerol-based liquid support matrices (LSM) have been proposed as an alternative to the traditional solid matrices as they provide increased shot-to-shot reproducibility, leading to prolonged and stable ion signals and therefore better results. This chapter focuses on the integration of the liquid LSM MALDI matrices into the LC-MALDI MS/MS approach in identifying complex and large proteomes. The interface between LC and MALDI consists of a robotic spotter, which fractionates the eluent from the LC column into nanoliter volumes, and co-spots simultaneously the liquid matrix with the eluent fractions onto a MALDI target plate via sheath flow. The efficiency of this method is demonstrated through the analysis of trypsin digests of both bovine serum albumin (BSA) and Lactobacillus plantarum WCFS1 proteins.

Temperature- and Pressure-induced Proton Transfer in the 1:1 Adduct Formed between Squaric Acid and 4,4 '-Bipyridine

We have applied a combination of spectroscopic and diffraction methods to study the adduct formed between squaric acid and bypridine, which has been postulated to exhibit proton transfer associated with a single-crystal to single-crystal phase transition at ca. 450 K. A combination of X-ray single-crystal and very-high flux powder neutron diffraction data confirmed that a proton does transfer from the acid to the base in the high-temperature form. Powder X-ray diffraction measurements demonstrated that the transition was reversible but that a significant kinetic energy barrier must be overcome to revert to the original structure. Computational modeling is consistent with these results. Modeling also revealed that, while the proton transfer event would be strongly discouraged in the gas phase, it occurs in the solid state due to the increase in charge state of the molecular ions and their arrangement inside the lattice. The color change is attributed to a narrowing of the squaric acid to bipyridine charge-transfer energy gap. Finally, evidence for the possible existence of two further phases at high pressure is also presented.

Short Communication: effects of 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester on milk production and composition of lactating Holstein dairy cows

Sixteen multiparous Holstein cows were used to determine the effects of 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester (HMBi: 0 vs. 1.26 g/kg of total ration dry matter (DM) and dietary crude protein (CP) concentration [14.7% (low) vs. 16.9% (standard), DM basis] on milk yield and composition using a replicated 4 x 4 Latin square design experiment with 4-wk periods. Cows were fed ad libitum a total mixed ration with a 1: 1 forage-to-concentrate ratio (DM basis), and diets provided an estimated 6.71 and 1.86% lysine and methionine, respectively, in metabolizable protein for the low-protein diet and 6.74 and 1.82% in the standard protein diet. Dry matter intake, milk yield, and composition were measured during wk 4 of each period. There were no effects on DM intake, which averaged 24.7 kg/d. There was an interaction between dietary CP and HMBi for milk yield and 3.5% fat-corrected milk (FCM). Feeding HMBi decreased milk and FCM yield when fed with the low-CP diet but did not affect milk or FCM yield when fed with the standard CP diet. Feeding HMBi increased milk protein concentration regardless of diet CP concentration and increased milk protein yield when added to the standard CP diet but not the low-CP diet. The positive effect of HMBi on milk protein yield was only observed at the standard level of dietary CP, suggesting other factors limited the response to HMBi when dietary protein supply was restricted.

Extraction behavior of the synergistic system 2,6-bis-(benzoxazolyl)-4-dodecyloxylpyridine and 2-bromodecanoic acid using Am and Eu as radioactive tracers

In this study, the extraction properties of a synergistic system consisting of 2,6-bis-(benzoxazolyl)-4-dodecyloxylpyridine (BODO) and 2-bromodecanoic acid (HA) in tert-butyl benzene (TBB) have been investigated as a function of ionic strength by varying the nitrate ion and perchlorate ion concentrations. The influence of the hydrogen ion concentration has also been investigated. Distribution ratios between 0.03-12 and 0.003-0.8 have been found for Am(III) and Eu(HI), respectively, but there were no attempts to maximize these values. It has been shown that the distribution ratios decrease with increasing amounts of ClO4-, NO3-, and H+. The mechanisms, however, by which the decrease occurs, are different. In the case of increasing perchlorate ion concentration, the decrease in extraction is linear in a log-log plot of the distribution ratio vs. the ionic strength, while in the nitrate case the complexation between nitrate and Am or Eu increases at high nitrate ion concentrations and thereby decreases the distribution ratio in a non-linearway. The decrease in extraction could be caused by changes in activity coefficients that can be explained with specific ion interaction theory (SIT); shielding of the metal ions, and by nitrate complexation with Am and Eu as competing mechanism at high ionic strengths. The separation factor between Am and Eu reaches a maximum at similar to1 M nitrate ion concentration. Thereafter the values decrease with increasing nitrate ion concentrations.

Acid dissociation of 2,2 '-dipyridylamine in non-aqueous medium when chelated to some Ru(II)N-4 cores

[Ru(2,2'-bipyridine)(2)(Hdpa)](BF4)(2) center dot 2H(2)O (1), [Ru(1,10-phenanthroline)(2)(Hdpa)] (PF6)(2) center dot CH2Cl2 (2) and [Ru(4,4,4',4'-tetramethyl-2,2'- bisoxazoline)(2)(Hdpa)] (PF6)(2) (3) are synthesized where Hdpa is 2,2'-dipyridylamine. The X-ray crystal structures of 1 and 2 have been determined. Hdpa in 1 and 2 is found to bind the metal via the two pyridyl N ends. Comparing the NMR spectra in DMSO-d(6), it is concluded that 3 has a similar structure. The pK(a) values (for the dissociation of the NH proton in Hdpa) of free Hdpa and its complexes are determined in acetonitrile by exploiting molar conductance. These correlate linearly with the chemical shift of the NH proton in the respective entities. (C) 2007 Elsevier B.V. All rights reserved.