Cell Wall Analogues Research Articles

An NMR study of eremomycin and its derivatives.

Complete 1H and 13C NMR assignments are presented for eremomycin (1) and some of its desglycosylated derivatives 2, 3 and compared to the structurally closely related glycopeptide vancomycin. Primary structure and stereochemistry of eremomycin is corroborated by the present high field total correlation spectroscopy, NOESY and heteronuclear multiple-bond correlation NMR methods. A rough motional characterization of the title compound is attempted by 13C-T1 and 13C-[1H] NOE measurements. Dimerization of eremomycin is observed both in DMSO-d6-CCl4 and D2O solutions. Complexation with cell wall analogue dipeptide Ac-D-Ala-D-Ala is also demonstrated.

INDUCTION AND REGULATION OF MONOCYTE PROCOAGULANT ACTIVITY

Monocyte (MO) procoagulant activity (PCA) is induced by various stimuli including allogeneic stimulation, immunocomplexes, and bacterial products. Antigen-antibody complex stimulation therefore represents a pathway for MO PCA induction. Activation of MO PCA has been demonstrated in immunocomplex disease and could represent a major pathology in transplanted immunocomplex disease patients. Stimulation of monocytes via their FcRI receptor has been demonstrated to induce TNF and PGE2. This report demonstrates that stimulation of the high-density FcRI receptor-bearing (FcRI+) MO by resetting with anti-Rh coated erythrocytes also induces significant PCA levels (P less than 0.001). Muramyl dipeptide (MDP), a Gram-positive bacterial cell wall analogue, further increased PCA levels in the FcRI stimulated MO subpopulation (P less than 0.003). Although increased PCA levels were also induced in the FcRI- MO subpopulation by MDP (P less than 0.003), the FcRI+ MO responded with much greater levels of PCA and PGE2 (P less than 0.001). Greater PCA levels in the FcRI-positive MO subpopulation may indicate that stimulation of MO through their FcRI represents a different pathway from allogenic PCA activation, which can be augmented by subsequent bacterial challenge. A novel inhibitory effect of IL-4 on MO PCA induction is also demonstrated. IL-4 downregulated MO PCA levels either after isolation stimulation (55 +/- 19%), FcRI stimulation (57 +/- 12%), or FcRI plus MDP stimulation (60 +/- 13%). PCA and PGE2 levels were concomitantly downregulated by IL-4 both in the FcRI-stimulated, FcRI+ and in the MDP-stimulated FcRI- MO subpopulations. Since indomethacin blocked MDP induced MO PGE2 production without affecting MO PCA levels, PGE2 production is not required for FcRI-stimulated PCA induction.

Molecular basis of the activity of antibiotics of the vancomycin group

Abstract

Molecular basis of the activity of antibiotics of the vancomycin group

The formation of a carboxylate anion binding pocket in the antibiotics of the vancomycin group is made energetically more favorable by the S stereochemistry of residue 3 relative to the R stereochemistries of residues 1, 2, and 4. Pocket formation is further favoured by the covalent cross-linking of the sidechains of residues 2 and 4, and in some cases, also of residues 1 and 3. In the absence of the latter cross-link in vancomycin itself, the pocket is only partially formed in the free antibiotic, and is in equilibrium with a second conformation in which the peptide backbone alternates as in a P-pleated sheet. The sugar vancosamine plays a role in the thermodynamics of binding of vancomycin to cell wall analogues. This role is based upon a hydrophobic interaction between the 6-methyl group of the sugar and the methyl group of the C-terminal alanine of the cell-wall analogue. The hydrophobic interaction is however only significant in the presence of the nearby charged amino group of the sugar. It is believed that the charged group, by local ordering of the water structure, strengthens the hydrophobic interaction. The sugar mannose in ristocetin A is able, by partially shielding the non-C-terminal alanine methyl group of the dipeptide cell wall analogue from water, to increase the binding of ristocetin A to this analogue. However, in the binding of the tripeptide cell-wall analogue, this same methyl group is shielded from water by the sidechain of lysine, and mannose loses a significant role in the thermodynamics of binding. This observation accounts for the much larger increase in binding, on passing from di- to tri-peptide, observed for vancomycin (which lacks mannose) relative to ristocetin A (which possesses mannose).

Structure elucidation of a glycopeptide antibiotic, OA-7653

The structure of a vancomycin-type antibiotic, OA-7653, isolated from Streptomyces hygroscopicus subsp. hiwasaensis subsp. nov. Nishida, has been elucidated by a combination of classical chemical methods, mass spectrometry, and nuclear magnetic resonance spectroscopy. The interaction of OA-7653 with the peptide cell wall analogues N-acetyl-D-alanyl-D-alanine, and di-N-acetyl-L-lysyl-D-alanyl-D-alanine, in solution in aqueous dimethyl sulphoxide, has been examined by n.m.r. and u.v. difference spectroscopy.

1H n.m.r. studies of the structure of ristocetin A and of its complexes with bacterial cell wall analogues in aqueous solution

The conformation of ristocetin A in aqueous solution is shown to be very similar to that previously found in dimethyl sulphoxide solution. In particular, there is a cleft on one face which binds the carboxylate anion of bacterial cell wall analogues, and which is not fully accessible to the solvent. Binding studies with the analogues N-acetyl-D-alanyl-D-alanine and N,N-diacetyl-L-lysyl-D-alanyl-D-alanine indicate that the structures of the complexes with ristocetin A are also very similar to those previously found in dimethyl sulphoxide solution; again, there is very unusual behaviour of some amide protons. Two-dimensional nuclear Overhauser effect studies in D2O show that the Iysyl side chain is in a different orientation in aqueous solution to that found in dimethyl sulphoxide.

Structure of chitin in the cell walls of newly formed and mature conidia of Fusarium sulphureum

Samples of 7-day-old septate conidia (mature conidia) and newly released aseptate ones (immature conidia) of Fusarium sulphureum were hydrolyzed successively in KOH, acetic acid – H2O2, and H2SO4. The cell wall residue of the mature conidia remained intact throughout the hydrolysis but that of immature conidia dissolved in the H2SO4. Thus, the immature conidial cell wall is substantially different from that of mature conidial cells or growing hyphae and the cell wall undergoes a structural transformation following conidium release from the conidiogenous cell. X-ray diffraction analyses of the wall residues following KOH and acetic acid – H2O2 hydrolysis showed that the mature conidial wall residue had a crystalline chitin component, while the residue of the immature conidial wall was more amorphous and had smaller crystals. An X-ray diffraction pattern of the dissolved immature conidial wall that was recovered from the H2SO4 hydrolysate showed that it contained crystalline chitin.Infrared spectroscopic analysis of the mature conidial cell wall residue showed absorption bands due to inter- and intra-molecular hydrogen bonding and for hydrogen stretching associated with crystalline chitin. Such bands were lacking in the immature cell wall analogue.

Components of mycobacteria and muramyl dipeptide with adjuvant activity induce lymphocyte activating factor

Several components of mycobacteria including a water-soluble extract (WSA) and an interphase material (IPM) as well as the synthetic cell wall analog muramyl dipeptide (MDP) all stimulated human mononuclear cells (MNL) to produce a factor which was mitogenic for murine thymocytes. The mediator induced by MDP is probably lymphocyte-activating factor (LAF) because it was produced by adherent but not nonadherent MNL and yields two characteristic peaks of activity in the 16,000–22,000 and 60,000–70,000 molecular weight range when eluted from Bio-Gel P-100 columns. The 6- O-stearoyl derivative of MDP was an active inducer of MNL LAF production, whereas, the d-alanine analog of MDP was somewhat less potent. Unfractionated as well as adherent, but not nonadherent, mouse peritoneal cells also produced LAF in response to WSA, IPM, and MDP. P388D 1 cell line macrophages, which are completely devoid of lymphocytes, could be stimulated by WSA, IPM, and MDP to produce LAF after prolonged incubation. These adjuvants did not stimulate nonadherent Balb/C or human blood cells to produce a mitogenic factor. However, when the P388D 1 macrophages were stimulated with these adjuvants in the presence of nonadherent murine or human peripheral blood cells, a mitogenic activity was produced in a shorter period of incubation suggesting that activated lymphocytes can facilitate the production of LAF by macrophages.