Ruminal transport and metabolism of short-chain fatty acids (SCFA) in vitro: effect of SCFA chain length and pH

The unidirectional transport and metabolism of 14C-labeled acetate, propionate and butyrate across the isolated bovine rumen epithelium was measured in vitro by the Ussing chamber technique. There was a significant, but relatively small, net secretion of acetate and propionate, and a large and significant net absorption of butyrate. The results demonstrate that the mucosal-serosal (MS) pathway for short-chain fatty acids (SCFA) is different from the serosal-mucosal (SM) pathway, and that butyrate is treated differently from acetate and propionate by the epithelium. The results support that the main route for epithelial SCFA transport is transcellular. The correlation between SCFA lipophility and the flux rate was positive but weak at both pH 7.3 and 6.0. Decreasing pH increased all SCFA fluxes significantly, but not proportionally to the increase of protonized SCFA in the bathing solution. There was a significant and apparently non-competitive interaction between the transport of acetate, propionate and butyrate. It seems that mediated transport mechanisms must be involved in epithelial SCFA transport in the bovine rumen, but the data do not exclude that passive diffusion could account for a significant part of the flux. The metabolism of SCFA in the Ussing chamber system was considerable, and there was a clear preference for excretion of CO2 from this metabolism to the mucosal side, while side preference for non-CO2 metabolite excretion was not studied. Of the propionate and butyrate transported in the MS direction, 78 and 95% was metabolised, while only 37 and 38% was metabolised in the SM direction (acetate metabolism could not be measured). There was, however, no simple relation between the degree of metabolism and the transport rate or the transport asymmetry of the SCFA.     

Identification of carbohydrates binding to lectins by using surface plasmon resonance in combination with HPLC profiling

A new, powerful method is presented for screening the binding in real time and taking place under dynamic conditions of oligosaccharides to lectins. The approach combines an SPR biosensor and HPLC profiling with fluorescence detection, and is applicable to complex mixtures of oligosaccharides in terms of ligand-fishing. Labeling the oligosaccharides with 2-aminobenzamide ensures a detection level in the fmol range. In an explorative study the binding of RNase B-derived oligomannose-type N-glycans to biosensor-immobilized concanavalin A (Con A) was examined, and an affinity ranking could be established for Man(5)GlcNAc(2) to Man(9)GlcNAc(2), as monitored by HPLC. In subsequent experiments and using well-defined labeled as well as nonlabeled oligosaccharides, it was found that the fluorescent tag does not interfere with the binding and that the optimum epitope for the interaction with Con A comprises the tetramannoside unit Manalpha2Manalpha6(Manalpha3)Man[D(3)B(A)4'], rather than the generally accepted trimannoside Manalpha6 (Manalpha3)Man [B(A)4' or 4(4')3]. In a similar experimental setup, the interaction of various fucosylated human milk oligosaccharides with the fucose-binding lectin from Lotus tetragonolobus purpureaus was studied, and it appeared that oligosaccharides containing blood group H could selectively be retained and eluted from the lectin-coated surface. Finally, using the same lectin and a mixture of O-glycans derived from bovine submaxillary gland mucin, minor constituents but containing fucose could selectively be picked from the analyte solution as demonstrated by HPLC profiling.     

Metabolomic analysis of Strychnos nux-vomica, Strychnos icaja and Strychnos ignatii extracts by 1H nuclear magnetic resonance spectrometry and multivariate analysis techniques

1H Nuclear magnetic resonance spectrometry and multivariate analysis techniques were applied for the metabolic profiling of three Strychnos species: Strychnos nux-vomica (seeds, stem bark, root bark), Strychnos ignatii (seeds), and Strychnos icaja (leaves, stem bark, root bark, collar bark). The principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between all samples, using the three first components. The key compounds responsible for the discrimination were brucine, loganin, fatty acids, and Strychnos icaja alkaloids such as icajine and sungucine. The method was then applied to the classification of several "false angostura" samples. These samples were, as expected, identified as S. nux-vomica by PCA, but could not be clearly discriminated as root bark or stem bark samples after further statistical analysis.     

HPLC-based bioactivity profiling of plant extracts: a kinetic assay for the identification of monoamine oxidase-A inhibitors using human recombinant monoamine oxidase-A

An assay for the HPLC-based search for monoamine oxidase-A (MAO-A) inhibitors in plant extracts was established. It combines human recombinant MAO-A, expressed as GST-fusion protein in yeast, with a kinetic measurement of the conversion of kynuramine to 4-hydroxyquinoline. Substrate selectivity and kinetic parameters of the GST-fusion protein were comparable to the wild-type enzyme. The applicability of the assay to HPLC-based activity profiling was tested with plant extracts spiked with small amounts of known MAO inhibitors.     

Molecular dynamics simulations of 14 HIV protease mutants in complexes with indinavir

The molecular mechanisms of HIV drug resistance were studied using molecular dynamics simulations of HIV-1 protease complexes with the clinical inhibitor indinavir. One nanosecond molecular dynamics simulations were run for solvated complexes of indinavir with wild type protease, a control variant and 12 drug resistant mutants. The quality of the simulations was assessed by comparison with crystallographic and inhibition data. Molecular mechanisms that contribute to drug resistance include structural stability and affinity for inhibitor. The mutants showed a range of structural variation from 70 to 140% of the wild type protease. The protease affinity for indinavir was estimated by calculating the averaged molecular mechanics interaction energy. A correlation coefficient of 0.96 was obtained with observed inhibition constants for wild type and four mutants. Based on this good agreement, the trends in binding were predicted for the other mutants and discussed in relation to the clinical data for indinavir resistance. Figure Poincare map representation for WT protease-indinavir complex. The side chain of Tyr 59 showing the positions of hydrogen atoms.This revised version was published online in October 2004 with corrections to the Graphical Abstract.     

Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co-regulation in biochemical networks

A novel extraction protocol is described with which metabolites, proteins and RNA are sequentially extracted from the same sample, thereby providing a convenient procedure for the analysis of replicates as well as exploiting the inherent biological variation of independent samples for multivariate data analysis. A detection of 652 metabolites, 297 proteins and clear RNA bands in a single Arabidopsis thaliana leaf sample was validated by analysis with gas chromatography coupled to a time of flight mass spectrometer for metabolites, two-dimensional liquid chromatography coupled to mass spectrometry for proteins, and Northern blot analysis for RNA. A subset of the most abundant proteins and metabolites from replicate analysis of different Arabidopsis accessions was merged to form an integrative dataset allowing both classification of different genotypes and the unbiased analysis of the hierarchical organization of proteins and metabolites within a real biochemical network.