Gut microbial metabolism of polyphenols from black tea and red wine/grape juice is source-specific and colon-region dependent.

The colonic microbial degradation of a polyphenol-rich black tea extract (BTE) and red wine/grape juice extract (RWGE) was compared in a five-stage in vitro gastrointestinal model (TWINSHIME). Microbial metabolism of BTE and RWGE polyphenols in the TWINSHIME was studied subsequently in single- and continuous-dose experiments. A combination of liquid or gas chromatography with mass spectrometry (LC-MS or GC-MS) and NMR-based metabolic profiling was used to measure selected parent polyphenols, their microbial degradation into phenolic acids, and the production of short-chain fatty acids (SCFAs) in different colon compartments. Acetate production was increased by continuous feeding of BTE but not RWGE. During RWGE feeding, gallic acid and 4-hydroxyphenylpropionic acid remained elevated throughout the colon, while during BTE feeding, they were consumed in the distal colon, while 3-phenylpropionic acid was strongly produced. Gut microbial production of phenolics and SCFAs is dependent on colon location and polyphenol source, which may influence potential health benefits.

Rapid phase-compositional assessment of lipid-based food products by time domain NMR.

In food science and technology, assessment of phase-compositional behaviour of lipids is critical for understanding many product properties and for effective process control. Time Domain NMR is a rapid and easy-to-handle technique, and is already well appreciated as a tool for phase-compositional assessment in foods. The phase-compositional detail that can be obtained with the established methodology is limited, however. In this work, we set out to obtain more phase-compositional details of lipids as currently feasible with the already established 'classical' NMR methods. We deployed a combined FID-CPMG experiment, and analyzed the Transversal Relaxation Decays by Deconvolution (TRDD) with semi-empirical mathematical functions for solid, semi-solid and liquid components. Within the solid component, different lipid crystal polymorphs can be discerned in a quantitative manner, i.e. alpha, beta and beta'. The TRDD method was validated against established NMR SFC methods, in terms of accuracy ('trueness') and precision. The solid fat content (SFC) of a large collection of fat blends was measured by the commonly employed NMR Direct and Indirect SFC methods and a good correlation with the results of the TRDD was observed, thereby demonstrating accuracy. Furthermore, TRDD was found to be equally precise as the established NMR SFC methods.

MRI of hip prostheses using single-point methods: in vitro studies towards the artifact-free imaging of individuals with metal implants.

Use of magnetic resonance imaging (MRI) in individuals with orthopedic implants is limited because of the large distortions caused by metallic components. As a possible solution for this problem, we suggest the use of single-point imaging (SPI) methods, which are immune to the susceptibility artifacts observed with conventional MRI methods. A further advantage of SPI, based on the fact that signal encoding is achieved in ultra-short times (as short as tens of microseconds), is that they enable the direct visualization of the polymeric elements of the implants, allowing the detection of possible implant failures. We present in vitro SPI images of polymeric sockets of two hip prostheses together with artifact-free images of gelatin phantoms containing their respective metallic stems. These data underscore the great potential of the SPI technique for obtaining artifact-free images of individuals with large metal implants.

Difftrain: a novel approach to a true spectroscopic single-scan diffusion measurement.

Diffusion-ordered spectroscopy (DOSY) has gained considerable attention over the past decade as a useful tool for calculating diffusion-related parameters or in the analysis of complex (reaction) mixtures. A major drawback of the established methods are the relatively long recording times needed to acquire the spectra, excluding the monitoring of rapidly progressing reactions and (hence) measurements of less stable components. In order to overcome these shortcomings a new pulse sequence, Difftrain, has been developed. The sequence involves stimulated echo attenuation, multilow flip angle excitation, and multiple sampling of the FID during the longitudinal storage. The calculated diffusion parameters obtained by Difftrain were compared with those obtained by the established sequence BPPSTE (bipolar pulse pair stimulated echo) and were in good agreement. For systems with moderate to good signal-to-noise ratios the Difftrain building block yields significant saving in recording time (single-shot acquisition instead of acquiring n-different gradients strengths), thus opening up new applications in nonequilibrium systems and screening of compositions and/or interactions of (larger) compound arrays.

Measurement of Oil Droplet Size Distributions in Food Oil/Water Emulsions by Time Domain Pulsed Field Gradient NMR.

A new method for measuring oil droplet size distributions by means of a benchtop pulsed field gradient NMR spectrometer operating in the time domain is presented. The continuous water phase is successfully suppressed by gradient pulses in order to measure the dispersed oil phase. Simulations show that for most common oil/water food emulsions the influence of droplet diffusion is negligible due to a rather large droplet size or a high viscosity of the continuous water phase. The merits of the NMR method relative to other methods are discussed in terms of sample preparation, sensitivity to cluster phenomena, and matrix effects. Preliminary results of a short validation study show a good correlation with conventional reference techniques. Copyright 2001 Academic Press.

Mitosene-DNA adducts. Characterization of two major DNA monoadducts formed by 1,10-bis(acetoxy)-7-methoxymitosene upon reductive activation.

Reductive activation of racemic 1,10-bis(acetoxy)-7-methoxymitosene WV15 in the presence of DNA, followed by enzymatic digestion and HPLC analysis, revealed the formation of various DNA adducts. Reduction is a necessary event for adduct formation to occur. This reductive activation was performed under hypoxic conditions in various ways: (1) chemically, using a 2-fold excess of sodium dithionite (Na2S2O4), (2) enzymatically using NADH-cytochrome c reductase, (3) electrochemically on a mercury pool working electrode, and (4) catalytically, using a H2/PtO2 system. Five different mitosene-DNA adducts were detected. These adducts were also present when poly(dG-dC) was used instead of DNA, but were absent with poly(dA-dT). All were shown to be adducts of guanine. Reduction of 1, 10-dihydroxymitosene WV14 in the presence of DNA did not result in detectable adduct formation, demonstrating the importance of good leaving groups for efficient adduct formation by these mitosenes. Finally, two of the adducts were isolated and their structures elucidated, using mass spectrometry, 1H NMR and circular dichroism (CD). The structures were assigned as the diastereoisomers N2-(1"-n-hydroxymitosen-10"-yl), 2'-deoxyguanosine (n = alpha or beta). These type of adducts, in which the mitosene C-10 is covalently bonded to the N-2 of a guanosylic group, are different from the well-known mitomycin C 2'-deoxyguanosine monoadducts, that is linked via the mitomycin C C-1 position, demonstrating that the order of reactivity of the C-1 and C-10 in these mitosenes is reversed, as compared to mitomycin C. The 7-methoxy substituent of WV15 is a likely factor causing this switch. Evidence is presented that the 7-substituent of mitosenes also influences their DNA alkylation site. Adducts 4 and 5 represent the first isolated and structurally characterized covalent adducts of DNA and a synthetic mitosene.

Exploring the DNA binding domain of gene V protein encoded by bacteriophage M13 with the aid of spin-labeled oligonucleotides in combination with 1H-NMR.

The DNA binding domain of the single-stranded DNA binding protein gene V protein encoded by the bacteriophage M13 was studied by means of 1H nuclear magnetic resonance, through use of a spin-labeled deoxytrinucleotide. The paramagnetic relaxation effects observed in the 1H-NMR spectrum of M13 GVP upon binding of the spin-labeled ligand were made manifest by means of 2D difference spectroscopy. In this way, a vast data reduction was accomplished which enabled us to check and extend the analysis of the 2D spectra carried out previously as well as to probe the DNA binding domain and its surroundings. The DNA binding domain is principally situated on two beta-loops. The major loop of the two is the so-called DNA binding loop (residues 16-28) of the protein where the residues which constitute one side of the beta-ladder (in particular, residues Ser20, Tyr26, and Leu28) are closest to the DNA spin-label. The other loop is part of the so-called dyad domain of the protein (residues 68-78), and mainly its residues at the tip are affected by the spin-label (in particular, Phe73). In addition, a part of the so-called complex domain of the protein (residues 44-51) which runs contiguous to the DNA binding loop is in close vicinity to the DNA. The NMR data imply that the DNA binding domain is divided over two monomeric units of the GVP dimer in which the DNA binding loop and the tip of the dyad loop are part of opposite monomers. The view of the GVP-ssDNA binding interaction which emerges from our data differs from previous molecular modeling proposals which were based on the GVP crystal structure (Brayer & McPherson, 1984; Hutchinson et al., 1990). These models implicate the involvement of one or two tyrosines (Tyr34, Tyr41) of the complex loop of the protein to participate in complex formation with ssDNA. In the NMR studies with the spin-labeled oligonucleotides, no indication of such interactions has been found. Other differences between the models and our NMR data are related to the structural differences found when solution and crystal structures are compared.

Exploration of the single-stranded DNA-binding domains of the gene V proteins encoded by the filamentous bacteriophages IKe and M13 by means of spin-labeled oligonucleotide and lanthanide-chelate complexes.

Scrutiny of NOE data available for the protein encoded by gene V of the filamentous phage IKe (IKe GVP), resulted in the elucidation of a beta-sheet structure which is partly five stranded. The DNA-binding domain of IKe GVP was investigated using a spin-labeled deoxytrinucleotide. The paramagnetic-relaxation effects observed in the 1H-NMR spectrum of IKe GVP, upon binding of this DNA fragment, could be visualized using two-dimensional difference spectroscopy. In this way, the residues present in the DNA-binding domain of IKe GVP can be located in the structure of the protein. They exhibit a high degree of identity with residues in the gene V protein encoded by the distantly related phage M13 (M13 GVP), for which similar spectral perturbations are induced by such a spin-labeled oligonucleotide. Binding studies with negatively charged lanthanide-1,4,7,10-tetraazacyclodecanetrayl-1,4,7-10- tetrakis(methylene)tetrakisphosphonic acid (DOTP) complexes, showed that these complexes bind to IKe and M13 GVP at two spatially remote sites whose affinities have different pH dependencies. Above pH 7, there is one high-affinity binding site for Gd(DOTP)5-/M13 GVP monomer, which coincides with the single-stranded DNA-binding domain as mapped with the aid of spin-labeled oligonucleotide fragments. The results show that single-stranded DNA binds to conserved (phosphate binding) electropositive clusters at the surface of M13 and IKe GVP. These positive patches are interspersed with conserved or conservatively replaced hydrophobic residues. At pH 5, a second Gd(DOTP)(5-)-binding site becomes apparent. The corresponding pattern of spectral perturbations indicates the accommodation of patches of conserved, or conservatively replaced, hydrophobic residues in the cores of the M13 and IKe dimers.

Assignment of the 1H NMR spectrum and secondary structure elucidation of the single-stranded DNA binding protein encoded by the filamentous bacteriophage IKe.

By means of 2D NMR techniques, all backbone resonances in the 1H NMR spectrum of the single-stranded DNA binding protein encoded by gene V of the filamentous phage IKe have been assigned sequence specifically (at pH 4.6, T = 298 K). In addition, a major part of the side chain resonances could be assigned as well. Analysis of NOESY data permitted the elucidation of the secondary structure of IKe gene V protein. The major part of this secondary structure is present as an antiparallel beta-sheet, i.e., as two beta-loops which partly combine into a triple-stranded beta-sheet structure, one beta-loop and one triple-stranded beta-sheet structure. It is shown that a high degree of homology exists with the secondary structure of the single-stranded DNA binding protein encoded by gene V of the distantly related filamentous phage M13.

Sequence-specific 1H-NMR assignment and secondary structure of the Tyr41----His mutant of the single-stranded DNA binding protein, gene V protein, encoded by the filamentous bacteriophage M13.

Sequence-specific 1H-NMR assignments are reported for the Tyr41----His (Y41H) mutant of the single-stranded DNA binding protein, encoded by gene V of the filamentous bacteriophage M13 (GVP). The mutant protein was chosen for this purpose because it exhibits significantly improved solubility characteristics over wild-type GVP [Folkers et al. (1991) Eur. J. Biochem. 200, 139-148]. The secondary structure elements present in the protein are deduced from a qualitative interpretation of the nuclear Overhauser enhancement spectra and amide exchange data. The protein is entirely composed of antiparallel beta-structure. It is shown that identical structural elements are present in wild-type GVP. Previously, we have demonstrated that the secondary structure of the beta-loop, encompassing residues 13-31 which is present in GVP in solution, deviates from that proposed for the same amino acid sequence on the basis of X-ray diffraction data [van Duynhoven et al. (1990) FEBS Lett. 261, 1-4]. Now that we have arrived at a complete description of the secondary structure of the protein in solution, other deviations with respect to the crystallographically determined structure became apparent as well. The N-terminal part of the protein is, in solution, part of a triple-stranded beta-sheet while, in the crystal, it is an extended strand pointing away from the bulk of the protein dimer. One of the antiparallel beta-sheets in the protein which had been designated earlier as the complex loop has, in the solution structure, a different pairwise arrangement of the residues in its respective beta-ladders. Residues 30 and 48 are opposite to one another in the solution structure while in the crystal structure residues 32 and 48 are paired. A similar observation is made for the so-called dyad domain of the protein of which the beta-sheet in the solution structure is shifted by one residue with respect to that of the crystal structure.

Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR.

Recording of good quality NMR spectra of the single-stranded DNA binding protein gene V of the bacteriophage M13 is hindered by a specific protein aggregation effect. Conditions are described for which NMR spectra of the protein can best be recorded. The aromatic part of the spectrum has been reinvestigated by means of two-dimensional total correlation spectroscopy. Sequence-specific assignments were obtained for all of the aromatic amino acid residues with the help of a series of single-site mutant proteins. The solution properties of the mutants of the aromatic amino acid residues have been fully investigated. It has been shown that, for these proteins, either none or only local changes occur compared to the wild-type molecule. Spin-labeled oligonucleotide-binding studies of wild-type and mutant gene V proteins indicate that tyrosine 26 and phenylalanine 73 are the only aromatic residues involved in binding to short stretches of single-stranded DNA. The degree of aggregation of wild-type gene V protein is dependent on both the total protein and salt concentration. The data obtained suggest the occurrence of specific protein-protein interactions between dimeric gene V protein molecules in which the tyrosine residue at position 41 is involved. This hypothesis is further strengthened by the observation that the solubility of tyrosine 41 mutants of gene V protein is significantly higher than that of the wild-type protein. The discovery of the so-called 'solubility' mutants of M13 gene V protein has finally made it possible to study the solution structure of gene V protein and its interaction with single-stranded DNA by means of two-dimensional NMR.

Structure of the DNA binding wing of the gene-V encoded single- stranded DNA binding protein of the filamentous bacteriophage M13.

The structure in solution of a beta-loop in mutant Y41H of the single-stranded DNA binding protein encoded by gene-V of the filamentous phage M13 has been elucidated using 2-dimensional 1H-nuclear magnetic resonance techniques. Furthermore, these studies enabled us to demonstrate that an identical structural element is present in wild-type gene-V-protein and that this element intimately is involved in the binding of gene-V-protein to single-stranded DNA. It is shown that the structure of the DNA binding wing deviates from that proposed for the same amino acid sequence on the basis of X-ray diffraction data. The structure is, however, identical to that of the DNA binding wing present in the single-stranded DNA binding protein encoded by the genome of the evolutionary distantly related filamentous phage IKe. The latter observations support our current view that in the binding of these proteins to single-stranded DNA a common structural motif is involved.

Two-dimensional 1H nuclear magnetic resonance studies on the gene V-encoded single-stranded DNA-binding protein of the filamentous bacteriophage IKe. I. Structure elucidation of the DNA-binding wing.

Two-dimensional nuclear magnetic resonance techniques were used to obtain residue- and sequence-specific assignments in the 1H spectrum of the single-stranded DNA-binding protein encoded by gene V of the filamentous phage IKe (IKe GVP). The residue-specific assignments are based on the analysis of J-correlated spectra, i.e. correlated spectroscopy and homonuclear-Hartmann-Hahn total correlated spectroscopy. Complete assignments of side-chain spin systems, e.g. long side-chains, were, to a major part, derived from two-dimensional spectra obtained by means of the latter technique. Sequence-specific residue assignments were obtained for the two neighbouring residues V41 and Y42, and the amino acid sequence segment encompassing residues S17 through I29. The structure of this segment, a beta-loop, was deduced from the interresidue nuclear Overhauser effect pattern. Residues S17 through V19 and P26 through I29 form an anti-parallel beta-ladder segment, whereas residues Q21 to K25 constitute the loop region. The beta-loop is expected to project into the solution and is intimately involved in binding to single-stranded DNA; it is therefore designated the "DNA-binding wing". By analogy with the structure of the DNA-binding wing deduced from IKe GVP, a similar structure is proposed for the corresponding domain of the gene V protein encoded by the filamentous phage Ff for which, from X-ray diffraction studies, a three-dimensional structure has been deduced. Essential differences appear to exist between the DNA-binding domain in the X-ray structure and that proposed in this paper. Possible reasons for these differences are discussed.

Two-dimensional 1H nuclear magnetic resonance studies on the gene V-encoded single-stranded DNA-binding protein of the filamentous bacteriophage IKe. II. Characterization of the DNA-binding wing with the aid of spin-labelled oligonucleotides.

The DNA-binding domain of the single-stranded DNA-binding protein IKe GVP was studied by means of 1H nuclear magnetic resonance, through use of oligonucleotides of two and three adenyl residues in length, that were spin-labelled at their 3' and/or 5' termini. These spin-labelled ligands were found to cause line broadening of specific protein resonances when bound to the protein, although they were present in small quantities, i.e. of the order of 0.04 molar equivalent and less. The line broadening of protein resonances was made manifest by means of difference one and two-dimensional spectroscopy. Difference one-dimensional experiments revealed line broadening of the same protein resonances upon binding of either 3' or 5' spin-labelled oligonucleotides. Evidence in favour of the existence of a fixed 5' to 3' orientation in the binding of oligonucleotides to the protein surface was therefore not obtained from the spin-labelled oligonucleotide binding studies. Residue-specific assignments of broadened resonances could not, or could only sparsely, be derived from the difference one-dimensional spectra, because of the tremendous overlap in the aliphatic region of the spectrum. In contrast, such assignments were easily obtained from the difference two-dimensional spectra, which were recorded by means of both total correlated spectroscopy and nuclear Overhauser effect spectroscopy. Difference signals were detected for 15 spin systems; ten out of these were assigned to the residues I29, Y27, S20, G18, R16, T28, K22, Q21, V19 and S17 in the amino acid sequence of IKe GVP; the other five spin systems could be assigned to a phenylalanyl residue, an arginyl or lysyl residue, an aspartic acid or asparagyl residue, a glycyl residue and a glutamic acid or glutamyl residue. From the evaluation of the relative difference signals, it was concluded that the direct surroundings of the spin-label group of the labelled oligonucleotide in the bound state is composed of the first five residues in the former group of residues and the five residues in the latter group.(ABSTRACT TRUNCATED AT 400 WORDS)

Eosin, a fluorescent marker for the high-affinity ATP site of (K+ + H+)-ATPase.

Eosin has been used as a fluorescent probe for studying conformational states in (K+ + H+)-ATPase. The eosin fluorescence level is increased by Mg2+ (K0.5 = 0.2 mM). This increase is counteracted by K+ (I0.5 = 1.3 mM) and choline (I0.5 = 17.2 mM) and by ATP. Binding studies with eosin indicate that the increase and decrease in fluorescence is due to changes in binding of eosin to the enzyme. The Mg2+-induced specific binding has a Kd of 0.7 microM and a maximal capacity of 3.5 nmol per mg enzyme, which is equivalent to 2.5 site per phosphorylation site. These experiments and the fact that eosin competitively inhibits (K+ + H+)-ATPase towards ATP, suggest that eosin binds to ATP binding sites.