Frank G. Riddell

Membrane permeabilisation and antimycoplasmic activity of the 18-residue peptaibols, trichorzins PA

The membrane permeabilisation properties of six linear natural 18-residue peptaibols, termed trichorzins PA, have been assessed on liposomes and on mollicutes (trivial name, mycoplasmas), a class of parasitic bacteria characterized by a small genome, the lack of a cell wall, a minute cell size, and the incorporation in their plasma membrane of exogenously supplied cholesterol. The trichorzins PA used in this study (PA II, PA IV-VI, PA VIII, and PA IX) differ between them by amino acid or amino alcohol substitutions at positions 4, 7, and 18, and form slightly amphipathic alpha-helices. They proved bactericidal for mollicutes belonging to the genera Acholeplasma, Mycoplasma, and Spiroplasma, with minimal inhibitory concentrations (3.12</=MICs</=50 microM) generally 2 to 4 fold higher than those of alamethicin F50, a related 20-residue peptide (1.56</=MICs</=12.5 microM). Spiroplasma cells were apparently not protected by the presence of spiralin on their surface. The activities of the six trichorzins PA were not influenced by their sequence variations and no synergistic effect was observed. Consistent with the marginal effect of cholesterol on the incorporation of the trichorzins PA into liposome bilayers, the antibiotic activity was independent of the amount of cholesterol in the membranes of the different mollicutes. The trichorzins PA and alamethicin inhibited the motility of Spiroplasma melliferum, the helical cells being deformed and split into coccoid forms. Membrane potential measurements in Acholeplasma laidlawii and S. melliferum showed that trichorzin PA V and alamethicin F50 very efficiently depolarized the plasma membrane of mollicutes. This was consistent with fluorescence and 23Na NMR measurements on liposomes that revealed the permeabilisation of the lipid bilayer and the nonselective ionophoric activity of the trichorzins PA. These data suggest that the bactericidal activity exhibited by the trichorzins PA on mollicutes is due to the permeabilisation of the plasma membrane.

The transport of Na+ and K+ ions through phospholipid bilayers mediated by the antibiotics salinomycin and narasin studied by 23Na- and 39K-NMR spectroscopy

Addition of the ionophoric antibiotics salinomycin or narasin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine in sodium or potassium chloride solutions gives rise to dynamic effects in the 23Na- and 39K-NMR spectra. The dynamic spectra arise from the ionophore-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of the concentrations of ionophore and the metal ion and are compatible in all cases with a model in which one ionophore molecule transports one metal ion. For both ionophores the transport of potassium ions is appreciably faster than that of sodium and in both cases the rate-limiting step for sodium transport is dissociation of the ionophore-metal complex. Assuming dissociation to be rate limiting in all four cases it is shown that the transport rate differences between the pairs of complexes of each metal arise solely from differences in the rates of formation. The stability constants for ionophore-metal complex formation in the membrane/water interface are evaluated.

The transport of Li+, Na+ and K+ ions through phospholipid bilayers mediated by the antibiotic M139603 studied by7Li-, 23Na- and 39K-NMR

Addition of the ionophoric antibiotic M139603 to preparations of large unilamellar vesicles made from egg-yolk phosphatidylcholine in lithium, sodium or potassium chloride solutions gives rise to dynamic effects in the 7 Li-, 23 na- and 39 K-NMR spectra. The dynamic spectra arise from M139603-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of the concentrations of M139603 and the metal ion and are compatible in all cases with a model in which one M139603 molecule transports one metal ion. The transport of metal ions mediated by M139603 is appreciably faster than that of monensin and nigericin. The results show that diffusion of the complex through the membrane cannot be (wholly) rate-limiting for Na + and K + transport. For Li + transport dissociation is the rate-limiting step. The stability constants for complex formation are evaluated. M139603 shows some selectivity in its complexes with the alkali metal ions with the Na + complex being the most stable.

Synthesis and ring—Chain tautomerism of angularly substituted cycloalkane-fused tetrahydro-1,3-oxazines

Abstract Starting from 1-methyl-1-cyclopentene and -cyclohexene, regioisomeric 1,3-amino alcohols 6a,b and 7a,b were prepared by regio- and stereospecific reactions. When the amino alcohols were condensed with aromatic aldehydes, well-defined products 8–11 were obtained, which exist as three-component tautomeric mixtures in CDCl3 solution. When equation (1) was applied in all four sets 8–11, good linear correlations were obtained. For regioisomeric compounds, introduction of the methyl group in the bridgehead position resulted in reverse effects on the stability.

A solid-state NMR study of ring-chain tautomerism in 1,3-O,N-Heterocycles☆

The solid-state 13C CP/MAS nmr spectra of nineteen tetrahydrooxazines and oxazolidines have been recorded to determine their preferences for the ring or chain tautomeric form in the solid state. The chain form is preferred for those cases in which <80% of the ring tautomer is found in solution. The ring form is exclusively found only in derivatives with ⪢93% preference for the ring tautomer in solution. In one case the solid-state reaction converting the open chain to the ring was observed to take place over a time-scale of several days. An investigation of the tautomeric equilibrium in tetrahydrooxazines and oxazolidines in the solid state is reported.

Measurement of temperature in 13C CP/MAS NMR

A method for temperature calibration of CP/MAS NMR probes and their temperature controllers is described that relies upon known phase changes in the compounds adamantane, d‐camphor, pivalic acid and DABCO. The method appears to give a convenient and reliable calibration over the temperature range ca. 173–373 K. In a separate experiment, making use of a well defined lineshape coalescence, it was shown that the temperature effect of spinning in a 4 mm Bruker MAS probe with zirconia rotors could be fitted by a squared relationship but that if treated as approximately linear the variation was ca. 1 K kHz−1

The conformations of monensin-A metal complexes in solution determined by NMR spectroscopy

The structures in solution of the Na+, K+ and Rb+ salts of monensin have been determined by the use of NOESY distance restraints and molecular modelling. The structures are similar to those obtained by X-ray diffraction with the exception of a close carboxylate oxygen-to-metal distance and only five observed close metal-to-oxygen ligating interactions. Molecular dynamics involving the derived structure of the sodium salt at increasing relative permittivity sheds light on the mechanism by which monensin binds to and unbinds from sodium ions in the membrane surface. The series of structures as the size of the metal ion increases also shows that the whole monensin molecule adapts to incorporate changes in the ionic radius including changes in torsion angles, changes in heterocyclic ring conformations and changes in the ligation and hydrogen bonding patterns.

Five-component equilibria of ring-chain tautomeric mixtures derived from 3-amino-1,2-propanediol and aromatic aldehydes

Abstract In the reactions of 3-amino-1,2-propanediol with aromatic aldehydes, five-component ring-chain tautomeric mixtures were formed, involving oxazolidine and oxazine C-2 epimers and the corresponding Schiff base. These multicomponent tautomeric equilibria could be described by the equation log K = ϱσ+ + log Ko, used earlier for the two- and three-component equilibria relating to only oxazine or oxazolidine rings. In the reactions of 3-amino,1,2-propanediol with aromatic aldehydes, five-component tautomeric mixtures were formed, involving oxazolidine and oxazine C-2 epimers and the corresponding Schiff base.

FIVE-COMPONENT EQUILIBRIA OF RING-CHAIN TAUTOMERIC MIXTURES DERIVED FROM 2-AMINO-1-PHENYL-1,3-PROPANEDIOL DIASTEREOMERS

Abstract In the reactions of (1R ∗ ,2S ∗ )- or (1S,2S)-2- amino-1-phenyl-1,3-propanediol with aromatic aldehydes, five-component ring-chain tautomeric mixtures were formed, involving C-2 epimers of structurally isomeric oxazolidines and the corresponding Schiff base. These multicomponent tautomeric equilibria could be described by the equation log KX = ϱσ′ + log KX=H.

The effect of lithium therapy upon the composition of the human erythrocyte membrane

The membrane phospholipids and membrane proteins from the erythrocytes of manic depressive patients undergoing lithium therapy and of normal controls were examined by high performance TLC and gel electrophoresis, respectively. The phospholipid composition of the erythrocyte membranes of the normal controls was close to that reported previously. The lipid composition of the erythrocyte membranes of the patients was very similar but differed from the normal controls in that the proportion of sphingomyelin and cholesterol appeared to be lower, whereas phosphatidylserine appeared to be higher in the patients by amounts comparable with the sums of the 95% confidence limits on the measurements. No major differences in the protein composition of the erythrocyte membranes were detected.