Peter Speers

Influence of morphometric factors on quantitation of paracellular permeability of intestinal epithelia in vitro.

AbstractPurpose. The relative contribution of the small and large intestine to paracellular absorption is a subject of some controversy. Direct comparison of paracellular permeability in different epithelia is complicated by variations in junctional density and/or the absorptive surface area. Methods. This study used a combination of morphometric analyses and in vitro absorption studies to define permeability characteristics in relation to the amount of paracellular pathway present in rat ileum, colon and the model epithelium, Caco-2. Results. Mucosal to serosal amplification was higher in ileum (3.9) than colon (1.9) or Caco-2 (1). Tight junctional density (1p) of ileal crypts was ≈3 fold greater (91 m/cm2) than that measured in ileal villi, colonic surface and crypt cells or Caco-2 monolayers (34−37 m/ cm2). However, when the relative contributions of the crypts and villi was taken into account there was no significant difference in the mean 1p per mucosal area for the three epithelia studied. Using these data to correct for morphometric differences the permeabilities of a range of small hydrophilic molecules (atenolol, D-PheAsp and PEG oligomers MW 282-634) was measured. Permeability of rat ileum and colon were virtually identical for all compounds studied. In contrast, Caco-2 monolayers showed a significantly lower permeability than intestinal tissues with the difference increasing markedly with molecular size. Conclusions. These studies suggest the importance of accounting for morphological variation when comparing the permeability characteristics of different epithelial systems.

The potential role of glycine-160 of human O6-alkylguanine-DNA alkyltransferase in reaction with O6-benzylguanine as determined by site-directed mutagenesis and molecular modelling comparisons

O6-Alkylguanine DNA-alkyltransferase (ATase) repairs toxic, mutagenic and carcinogenic O6-alkylguanine (O6-alkG) lesions in DNA by a highly conserved reaction involving the stoichiometric transfer of the alkyl group to the active centre cysteine residue of the ATase protein. In the Escherichia coli Ada ATase, which is effectively refactory to inhibition by O6-benzylguanine (O6-BzG), the residue corresponding to glycine-160 (G160) for the mammalian proteins of this class is replaced by a tryptophan (W). Therefore, to investigate the potential role of the G160 of the human ATase (hAT) protein in determining sensitivity to O6-BzG, site-directed mutagenesis was used to produce a mutant protein (hATG160W) substituted at position 160 with a W residue. The hATG160W mutant was found to be stably expressed and was 3- and 5-fold more sensitive than hAT to inactivation by O6-BzG, in the absence and presence of additional calf-thymus DNA respectively. A similar, DNA dependent increased sensitivity of the hATG160W mutant relative to wild-type was also found for O6-methylguanine mediated inactivation. The potential role of the W160 residue in stabilising the binding of the O6-alkG to the protein is discussed in terms of a homology model of the structure of hAT. The region occupied by G/W-160 forms the site of a putative hinge that could be important in the conformational change that is likely to occur on DNA binding. Three sequence motifs have been identified in this region which may influence O6-BzG access to the active site; YSGG or YSGGG in mammals (YAGG in E. coli Ogt, YAGS in Dat from Bacillus subtilis), YRWG in E. coli Ada and Salmonella typhimurium (but YKWS in Saccharomyces cerevisiae) or YRGGF in AdaB from B. Subtilis. Finally,conformational and stereoelectronic analysis of the putative transition states for the alkyl transfer from a series of inactivators of hAT, including O6-BzG was undertaken to rationalise the unexpected weak inhibition shown by the alpha-pi-unsaturated electrophiles.

Theoretical studies of protonation and lithiation of first- and second-row aldehydes

We investigate the effects of protonation and lithiation upon the atomic apd molecular properties of formaldehyde and thioformaldehyde. At the HF/6-311 ++ G** level, protonation is much more exothermic than lithiation in both systems; protonation of the thioformaldehyde is favoured over formaldehyde, while lithiation of the aldehyde is favoured over the thioaldehyde. Lithiation results in a much larger dipole polarization and quadrupole depolarization of the H2CX| fragment than does protonation. On the atomic level, protonation is favoured by the large transfer of charge to the proton and its subsequent stabilization, while lithiation is driven by the inter-atomic stabilization afforded X by the proximity of Li+. Lithiation of either aldehyde is well approximated by an electrostatic model, but protonation is not.

Synthesis and characterisation of D-amino acid-based oligopeptides for use as probes of the influence of molecular structure on the paracellular route of gastrointestinal drug uptake

SummaryA series of peptides based on D-amino-acids, and with an N-terminal D-phenylalanine residue, was synthesised by solution methods using t-butoxycarbonyl amino protection. These peptides were designed to resist metabolism in the gastrointestinal tract and to serve as probes of the effects of molecular shape and charge on the paracellular route of drug uptake in the gut. The peptides were characterised by NMR spectroscopy, FAB mass spectrometry, optical rotation and purified by HPLC.

Synthesis and comparative conformational energetics of D-phenylalanylsarcosine and its cyclic dehydration product, (R)-1-methyl-3-(phenylmethyl)-2,5-piperazinedione

SummarySynthetic protocols are presented both for D-PheSar and the corresponding cyclised diketopiperazine, prepared from N-t-butoxycarbonylprotected D-PheSar. Deprotection conditions could be manipulated to yield either D-Phenylalanylsarcosine or (R)-1-methyl-3-(phenylmethyl)-2,5-piperazinedione. Molecular modelling revealed several low energy conformers which contained a Z-peptide bond and which were readily amenable to cyclisation. Cyclisation was found by HPLC to be fastest in strongly acidic conditions.

Kinetics and mechanism of the addition of triphenylphosphoniocyclopentadienide to tetrahalogeno-p-benzoquinones. Part 4. The substitution reactions of fluoranil

Fluoranil reacts with excess triphenylphosphoniocyclopentadienide to yield the monosubstituted and 2,6-disubstituted quinone derivatives through two parallel, irreversible, second-order reactions which are well separated in time. The rate of formation of the monosubstitution product was resolved using the stopped-flow technique and its half-life was found to be in the millisecond range. Kinetic data for the mono- and di-substitution products indicate that both reactions proceed in two steps involving rate-limiting addition to form a polar betaine intermediate followed by elimination of hydrogen halide through an E1 mechanism. The empirical rate law was established by carrying out a multi-response non-linear least-square analysis of the absorbance of the reaction mixtures.